Le Thuc

C Cansell

K Stobbe

O Le Thuc

S Ben-Fradj

J Leredde

C Lebeaupin

D Debayle

L Fleuriot

F Brau

N Devaux

A Benani

E Audinat

N Blondeau

J L Nahon

author. Dr Carole Rovère
email: rovere@ipmc.cnrs.fr

Lipid-rich diet consumption induce thermal pain through the activation

Keywords: Obésité, Comportement alimentaire, Homéostasie glucidique, Régimes hyperlipidiques, Inflammation, Cytokine, Chimiokine, Signalisation de CCL5, Hypothalamus, Neuropeptides Obesity, Feeding behavior, Glucose homeostasis, High fat diet, Inflammation, Cytokine, Chemokine, CCL5 signaling, Hypothalamus, Neuropeptides

Le mode de vie occidental favorise le développement de l'obésité et du diabète de type 2. Le lien entre l'obésité et le diabète de type 2 est bien établi au niveau épidémiologique. Toutefois, ce lien reste encore mal défini au niveau étiologique. Les marqueurs du diabète de type 2, tels que l'hyperglycémie et la résistance à l'insuline et à la leptine, peuvent résulter d'un état pro-inflammatoire chronique du tissu adipeux, associé à une sécrétion de cytokines (IL-1β, IL-6, TNF-a) et chimiokines (RANTES/CCL5, MCP-1/CCL2) diabétogènes. Ces médiateurs pro-inflammatoires sécrétés seraient les promoteurs de l'inflammation systémique et de la neuroinflammation dans l'hypothalamus, région importante du cerveau, qui contient des réseaux neuronaux impliqués dans le contrôle du métabolisme énergétique et du comportement alimentaire.

Nous nous sommes intéressés à l'impact de régimes riches en lipides sur le développement de l'obésité nutritionnelle et les paramètres métaboliques associés, d'autre part sur la réponse inflammatoire dans l'hypothalamus et le tissu adipeux. Nous nous sommes focalisés sur le rôle de la chimiokine CCL5 et de son récepteur CCR5. Notre hypothèse est que la chimiokine CCL5 régule l'activité des neurones de l'hypothalamus impliqués dans le contrôle de l'homéostasie énergétique et de l'homéostasie glucidique. Nous proposons d'étudier le rôle de la chimiokine CCL5 dans le contrôle de la balance énergétique, l'obésité, le diabète de type 2 et la neuropathie périphérique diabétique, une des complications diabétiques les plus fréquentes, affectant jusqu'à la moitié des patients atteints de diabète de type 2.

Dans ma thèse, nous avons testé les effets à long terme (16 semaines) du régime hyperlipidique apportant 40% de lipides, comparé au régime standard (contenant 5% de lipides), sur le développement de l'obésité chez les souris invalidées pour le gène de CCL5 (souris CCL5 -/-) ou le gène de son récepteur CCR5 (CCR5 -/-) en comparaison aux souris sauvages (contrôles). Dans un premier temps, nous avons confirmé la présence de CCL5 et de son récepteur CCR5 dans l'hypothalamus par hybridation in situ (technologie RNAScope®) chez les souris contrôles. Nous avons alors montré que les souris CCL5 -/-et CCR5 -/-semblent plus résistantes à l'obésité et avoir une homéostasie du glucose moins perturbée que les souris sauvages. Pour évaluer l'implication du CCL5 dans la douleur neuropathique associée au diabète, la sensibilité à la douleur thermique des souris CCL5 -/-et CCR5 -/-a été mesurée chez chaque groupe de souris. Les souris CCL5 -/-sous régime hyperlipidique semblent moins sensibles à la douleur thermique que les souris contrôles. De plus, les souris CCL5 -/-semblent présenter une expression génique des neuropeptides hypothalamiques modifiée par rapport aux souris contrôles.

Nos résultats suggèrent d'une part que l'absence de CCL5 et de son récepteur CCR5 protège contre le développement de l'obésité et le diabète de type 2, d'autre part que l'absence du CCL5 abolit la sensibilité accrue à la douleur thermique observée sous régime hyperlipidique. Ainsi, la cascade de signalisation CCL5/CCR5 pourrait représenter une nouvelle cible thérapeutique pour lutter contre l'obésité.

I would like to kindly thank the jury members, Professor Jaques Noël for accepting to be the president of my committee, as well as the reporters, Dr. Nicolas Chartrel and Professor Denis Richard, for taking the time to evaluate my manuscript. Furthermore, I would like to kindly thank Dr. Annabelle Reaux-Le Goazigo and Dr. Ariane Sharif who agreed to be my examiners, and evaluate my work. I would also like to thank warmly my co-director and team leader Dr. Jean Lois Nahon to have welcomed me to be part of his team and gave me access to the laboratory and research facilities.

I would like to thank LABEX SIGNALIFE and the University of Côte d'Azur for their finanancial support to undertake this project.

I would like to express my sincere gratitude to Dr. Carole Rovere, my thesis director, who has accepted to be my thesis supervisor along this journey and warmly and kindly welcomed me to be part of this team. Thank you for all the opportunities that you have given me, from being part of this exciting project, to the many national and international conferences that I have had the opportunity to participate and learn from as well as all the great memories that I have made. Thank you equally for all the support and patience, especially in the last and most stressful part of my PhD, as well as for all the help with French administration that I had trouble adjusting to. I am also grateful for the possibility you gave me to learn and improve my French within these years and who made me discover and raise my mood with many French delights.

Thank you equally to the other members of this team to François and Nadege, for their support and help throughout these years, to have been there with an open ear for my questions. Thank you Nadege, also for not letting me forget my german language.

In this sense, I would like also to thank all the BTS students Myriam, Melanie and Léa, whom I had the pleasure to meet and with whom I gained some first experiences in tutoring. Thank you also for all the help with my experience during your stay.

A special gratitude goes out to Celine, who as my colleague and friend taught me many things, not limited to scientific experiments. I would like to thank you for always being there for me, supporting me and believing in me and cheering me up in difficult moments.

To Clara, thank you for your help with some last experiments but also with your informatics skills that helped me a lot with formatting this manuscript. I am sorry I did not have more time to get to know you better. I wish you all the best for your PhD.

A special mention to Nicolas Blondeau for the stimulating discussions, his help with the project and his humor.

My special thanks are extended to the staff of the animal facility and the imaging platform for their help and support throughout these years.

I would also like to thank all the other people in the IPMC, who I had the pleasure to meet, talk and share moments of laughter with and for making my years of PhD pleasant everyday.

To Ahmed, for your help with experiments when I needed a hand, for your encouragement and the stimulating discussions.

To Wejdane, Alex, Ligia and Paula who either from the beginning or only recently have shared my journey as fellow PhD students, for sharing special moments in the institute during lunch or coffe breaks, or outside the institute and for sharing the stress of writing and the last few months of PhD with me and always being there with an open ear and patience.

A special gratitude goes equally out to Maria, whom I have had the pleasure to get to know through the SIGNALIFE program. Thank you for becoming my friend and being there for me during the amazing and the less amazing moments and for listening to me rambling and your encouraging words and chocolate wine.

I am also thankful for many of the amazing SIGNALIFE people, who have made this journey special and worthwile.

With a special mention to my dear friends, Doreen, Theodora, and Neil for always being there for me and believing in me. Thank you for your friendship and your support despite the distance and your positivity and kind words of encouragement.

Last but not the least, I would like to thank my family, in particular my parents, without whom all this would not be possible.

List of Figures

Introduction 1. Hypothalamus and the Regulation of Energy Balance

Energy, in the form of adenosine triphosphate (ATP), is the life force for every existing organism, whether they are unicellular or multicellular organisms.

Different from autotrophs, humans and animals are heterotrophs that require external organic sources for energy production which they acquire through nutrition. Nutrition is mainly based on four macromolecules that can be divided into proteins, lipids, carbohydrates and nucleic acids and constitute sources of energy for cellular processes. While it seems that the decision to eat, what, when and how much to eat is made deliberately by humans, it is yet a highly complex and regulated process with the aim to maintain energy balance, and controlled by different areas of the brain, most notably and famous of which is the hypothalamus [START_REF] Morton | Neurobiology of food intake in health and disease[END_REF]. The brain is the center of regulation of many processes including energy balance.

The brain maintains energy balance by adjusting energy intake to match energy output, through regulating food intake and energy expenditure (EE).

EE can be subdivided into basal metabolic rate, thermogenesis and locomotor activity. To be able to do this complex task, it is dependent on both peripheral and central signals that keeps it informed about the nutritional or energy state of the body, previous experiences, taste, emotions associated with food and other factors that are encoded as memories (Fig. 1). The brain is constantly monitoring the energetic state and receives various information via different sensors, integrates them, compares it to the target set point and with previous experiences and elicits a response that results in adaptive behavior with the aim to keep the body in homeostasis (Fig. 1). In this sense, by adjusting feeding behavior and EE, the brain can maintain the body weight (BW) of an organism within a set range. Because of the dependence on external sources of energy, it is not surprising that the energy balance regulatory system consists of redundant pathways that assures energy intake without fail [START_REF] Schwartz | Central nervous system control of food intake[END_REF][START_REF] Zheng | Neural systems controlling the drive to eat: Mind versus metabolism[END_REF][START_REF] Yuan | Nutrient Sensing, Metabolism, and Cell Growth Control[END_REF][START_REF] Morton | Neurobiology of food intake in health and disease[END_REF]. The brain receives information about nutrient availability and stored energy in form of humoral, mechanical stimuli and neuronal signals from innervations of different organs via the enteric and autonomous nervous system (Fig. 1).

Ghrelin, a gut hormone, is the only known orexigenic or "hunger" signal that is secreted by the empty stomach to stimulate feeding in the central nervous system (CNS) (Fig. 1). It is secreted often in anticipation of food, with the highest increase in plasma levels detectable right before meals and decreases when nutrition reaches the duodenum [START_REF] Zheng | Neural systems controlling the drive to eat: Mind versus metabolism[END_REF].

In contrast, leptin is a satiety-inducing hormone or one among many adipokines released by white adipose tissue (WAT) in proportion to the state of adiposity, and thus informs the brain about the amount of long-term energy stores secured in the body (Fig. 1). However, although its serum level is dependent on fat mass, plasma leptin levels can vary during fasting and feeding. While fasting transiently but rapidly suppresses leptin, feeding rapidly increases leptin levels [START_REF] Zheng | Neural systems controlling the drive to eat: Mind versus metabolism[END_REF]. Leptin binds to leptin receptor that is expressed on neurons in the hypothalamus, brain endothelial cells, astrocytes and tanycytes. Leptin exerts its anorexigenic effect by activating anorexigenic-, and inhibiting orexigenic neurons in the hypothalamus to regulate energy balance as well as systemic lipid and glucose metabolism [START_REF] Freire-Regatillo | Non-neuronal cells in the hypothalamic adaptation to metabolic signals[END_REF].

Another anorexigenic hormone that induces satiety and a decrease in food intake is insulin, a hormone secreted by the endocrine pancreas in response to increases in blood glucose levels. Together with the incretins that are secreted by enteroendocrine cells and induce the secretion of insulin by the pancreas in proportion to incoming glucose, it maintains stable glucose levels and leads to glucose uptake by different metabolic tissues. In the brain, insulin binds to insulin receptor (IR) on neurons, astrocytes and endothelial cells to induce different effects including the increase in EE, a decrease in food intake and energy storage as well as the regulation of systemic glucose levels [START_REF] Freire-Regatillo | Non-neuronal cells in the hypothalamic adaptation to metabolic signals[END_REF]. (Adapted from [START_REF] Morton | Neurobiology of food intake in health and disease[END_REF]).

Furthermore, the stomach can transmit signals about incoming fats and proteins via the release of cholecystokinin (CCK) and peptide YY (PYY). In addition to that, it informs the brain about incoming food via receptors stimulated by mechanical distention of the stomach and transmitting the signal via vagal afferents. Glucose sensing is mediated by taste receptors in the mouth, incretins and 5-hydroxytryptamine (5-HT or serotonin) released by enteroendocrine cells in the intestines. While some of these signals act on vagal sensory neurons that innervate the portal vein and liver, that can sense the incretin glucagon-like peptide-1 (GLP-1) and circulating glucose levels, to transmit information to the brain, others can act directly in the brain [START_REF] Zheng | Neural systems controlling the drive to eat: Mind versus metabolism[END_REF]. Such sensors and receptors for nutrition-related molecules can also be found in several brain areas. The brain is protected by a blood-brain barrier (BBB) that limits the access to the brain. However, specific transport systems are in place to enable the passage of nutrients and important metabolic signals [START_REF] Zheng | Neural systems controlling the drive to eat: Mind versus metabolism[END_REF]. In addition to that, the brain contains a few areas, the so-called circumventricular organs, that are characterized by increased permeability and fenestrated capillaries, and include the median eminence (ME) in the hypothalamus and the area postrema in the medulla oblongata [START_REF] Ganong | Circumventricular Organs: Definition And Role In The Regulation Of Endocrine And Autonomic Function[END_REF]. Feeding is regulated by three interacting brain systems in the brain, two of which are responsible for sensing and integrating physiological signals and circulating humoral signals from the periphery (hypothalamus), and neuronal signals from nerves innervating the gut (brainstem) and thereby control satiety, while the third neuronal circuit, which receives inputs from the other two systems, controls motivation for food foraging and feeding [START_REF] Myers | SnapShot: Neural pathways that control feeding[END_REF].

The Hypothalamus

The hypothalamus is one of the most important regions of the brain that participates in the control of energy metabolism and feeding regulation by controlling adaptive behaviours like food intake or suppression and EE as well as adipose tissue (AT) function, in response to circulating nutritional and hormonal cues from the periphery [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF][START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF].

Being composed of highly heterogenic groups of neurons and glial cells such as tanycytes in the ME, astrocytes, microglia cells and some macrophages, it constitutes a highly interactive and complex neural-glia network [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF][START_REF] Morton | Neurobiology of food intake in health and disease[END_REF][START_REF] Freire-Regatillo | Non-neuronal cells in the hypothalamic adaptation to metabolic signals[END_REF]. With its placement around the 3V, it is well positioned for its task in regulating energy balance as it is in contact with the cerebrospinal fluid of the 3V and fenestrated microvessels in the ME where specialized neurons and glia cells can sense different nutrients, hormones and other important factors from the peripheral circulation and respond rapidly to metabolic changes and nutrient flux [START_REF] Murphy | Gut hormones and the regulation of energy homeostasis[END_REF][START_REF] Morton | Neurobiology of food intake in health and disease[END_REF]. In addition to these humoral factors it receives also neuronal input from other brain regions and via the vagal afferents that inform the brain about the nutritional state of the body, which is processed by the brai, which then elicits an adaptive response as behavioral, autonomic and endocrine output through changes in neuronal modulation [START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF].

The hypothalamus consists, beside a circumventricular organ at the base of the 3V termed the ME, of several nuclei, including the arcuate nucleus (ARC), ventromedial hypothalamic nucleus (VMH), paraventricular nucleus (PVN), the dorsomedial nucleus (DMN) and the lateral hypothalamus (LH) (Fig. 2) [START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF][START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF].

The Arcuate Nucleus

The ARC is one of the nuclei in the ventral part of the mediobasal hypothalamus (MBH) and lies adjacent to each side of the 3V. It contains mainly two populations of neurons, which co-express either the orexigenic neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY), or the anorexigenic neuropeptides pro-opiomelanocortin (POMC) and cocaineand amphetamine-regulated transcript (CART) (Fig. 2).

These two neural populations express leptin and IRs and thus, can receive peripheral signals like leptin, insulin and ghrelin as well as nutrients and transmit the information to second order neurons in the PVN and LH to regulate food intake and EE. They also send projections to other nulcei of the hypothalamus like the VMH and the perifornical area. AgRP/NPY neurons secrete both γ-aminobutyric acid (GABA) as well as the orexigenic neuroendocrine peptides AgRP and NPY to stimulate food intake and reduce EE [START_REF] Schwartz | Central nervous system control of food intake[END_REF]. In contrast to that, POMC neurons produce and secrete the neuroendocrine peptide called a-melanocyte-stimulating hormone (a-MSH), that is derived from the peptide POMC, and acts on second order neurons via melanocortin receptors (MCRs), to reduce food intake and induce EE [START_REF] Morton | Neurobiology of food intake in health and disease[END_REF]. Interestingly, both AgRP and a-MSH are competing for the same receptor, called melanocortin 4 receptor (MC4R), yet induce opposing effects by binding to the receptor on second order neurons [START_REF] Spiegelman | Obesity and the regulation of energy balance[END_REF]. Furthermore, ARC neurons have direct and rapid access to bloodborne and peripheral signals, as it is a vascularized area with fenestrated vessels forming the BBB, allowing the passage of hormones and other proteins or nutrients such as free fatty acids (FFA) [START_REF] Tran | Diet-induced cellular neuroinflammation in the hypothalamus: Mechanistic insights from investigation of neurons and microglia[END_REF]. It has been shown

that acute injection of palmitate-rich milk fat and 4 weeks of HFD leads to raised and accumulated FFAs and palmitate in the former case in the hypothalamus [START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF][START_REF] Tran | Diet-induced cellular neuroinflammation in the hypothalamus: Mechanistic insights from investigation of neurons and microglia[END_REF].

NPY and AgRP, peptides co-expressed in neurons of the ARC, are both very similar in their functions and effects, even though they bind to different receptors. NPY exerts its function by binding to different receptor subtypes, namely Y1R, Y5R, and potentially also Y2R, while AgRP belongs to the central melanocortin system and is an antagonist for the MC3R and MC4R receptors [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF].

These neurons are highly responsive to states of energy deficiencies or increased metabolic demands, such as food deprivation, increased exercise, cold, and pregnancy. Thus during these states, the expression and synthesis of both peptides is increased and mediate the stimulation of food intake to meet the energy demands of the body [START_REF] Hahn | Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons[END_REF][START_REF] Chen | Altered expression of agouti-related protein and its colocalization with neuropeptide Y in the arcuate nucleus of the hypothalamus during lactation[END_REF][START_REF] Mizuno | Hypothalamic agouti-related protein messenger ribonucleic acid is inhibited by leptin and stimulated by fasting[END_REF]. Food deprivation or negative energy balance is mediated by the stomach-derived hormone ghrelin, which can bind the growth hormone secretagogue receptor (GHSR) on NPY/AgRP neurons to increase the expression of both peptides, leading to increased food intake.

Experiments using either central or peripheral ghrelin administration in rodents found that it stimulates food intake and c-fos expression (marker for neuronal acitivty) in neurons of the PVN and leads to increased weight gain and fat mass through reduction in lipid utilization [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF].

Furthermore, NPY and AgRP are also stimulated by the adrenal steroid called corticosterone, which exerts its effect through glucocorticoid type II receptors to stimulate the expression and production of both peptides and ultimately leading to an increase in food intake [START_REF] Hisano | Localization of glucocorticoid receptor in neuropeptide Y-containing neurons in the arcuate nucleus of the rat hypothalamus[END_REF][START_REF] Stanley | Suppression of neuropeptide Y-elicited eating by adrenalectomy or hypophysectomy: reversal with corticosterone[END_REF][START_REF] Akabayashi | Hypothalamic neuropeptide Y, its gene expression and receptor activity: relation to circulating corticosterone in adrenalectomized rats[END_REF][START_REF] Tempel | Adrenal Steroid Receptors: Interactions with Brain Neuropeptide Systems in Relation to Nutrient Intake and Metabolism[END_REF][START_REF] Arvaniti | Effects of leptin and corticosterone on the expression of corticotropin-releasing hormone, agouti-related protein, and proopiomelanocortin in the brain of ob/ob mouse[END_REF][START_REF] Savontaus | Effects of adrenalectomy on AGRP, POMC, NPY and CART gene expression in the basal hypothalamus of fed and fasted rats[END_REF][START_REF] Makimura | Role of glucocorticoids in mediating effects of fasting and diabetes on hypothalamic gene expression[END_REF].

Interestingly, this steroid is implicated in circadian rhythm and thus rises at the onset of the nocturnal feeding period in rodents, but also during energy deficiency, stimulating NPY and AgRP expression [START_REF] Akabayashi | Hypothalamic neuropeptide Y, its gene expression and receptor activity: relation to circulating corticosterone in adrenalectomized rats[END_REF][START_REF] Tempel | Adrenal Steroid Receptors: Interactions with Brain Neuropeptide Systems in Relation to Nutrient Intake and Metabolism[END_REF][START_REF] Xin-Yun | Diurnal rhythm of agouti-related protein and its relation to corticosterone and food intake[END_REF][START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF].

NPY and AgRP are stimulated during states of negative energy balance, when ghrelin and corticosterone are increased and insulin and leptin, hormones correlated with adiposity and energy abundance, are low. In contrast, states of positive energy balance are characterized by low ghrelin and high levels of leptin and insulin. These hormones, in contrast to ghrelin and corticosterone, suppress the expression of NPY and AgRP peptides and inhibit food intake. The effect of these hormones was demonstrated by central injections of leptin and insulin, which decreased NPY and AgRP expression and synthesis, and resulted in a reduction of not only food intake but also meal size and weight gain, while at the same time increasing EE and sympathetic nervous system (SNS) activity [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF]. In accordance with this, the ablation of insulin and leptin in animals renders them hyperphagic and obese. Moreover, mice with genetic forms of obesity like ob/ob mice that lack leptin have strongly activated NPY/AgRP neurons as well as hyperphagia [START_REF] Schwartz | Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice[END_REF]. It thus seems like NPY/AgRP neurons are inhibited by leptin and insulin through binding to their respective receptors, which are expressed by those neurons.

Another interesting property of NPY/AgRP neurons was revealed in a series of experiments, which showed that NPY and AgRP respond to dietary nutrients, in particular to glucose levels. For example, both NPY and AgRP were decreased upon glucose injection, but increased when glucose levels returned to baseline. However, these peptides do not change to Intralipidinduced increases in circulating lipids. Similarly, pharmacological inhibiting glucose utilization with 2-deoxy-D-glucose (2-DG) increased the expression of both peptides. Strikingly, in rats with access to a low-energy diet high in carbohydrates, NPY and AgRP expression was consistently higher than when they were given a diet high in fat [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF]). Consistent with this, studies performing central injections of NPY or AgRP report increases in levels of corticosterone, food intake, a preferred intake of carbohydrates to fats, reduced EE and thermogenesis, increased lipogenesis and hence elevated body fat accumulation. Interestingly, the administration of both peptides stimulates food intake, the effect of AgRP seems to last longer than NPY [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF].

NPY/AgRP neurons that also express GABA can also interact directly with POMC neurons through local axon collaterals and inhibit their neuronal activity [START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF].

Studies have identified NPY and AgRP as primary inducers of food intake, because central injection of either of those peptides led to significant increases in food intake, while acute ablation of these peptides in adult rodents induced significant weight loss and hyperphagia. Interestingly, constitutive genetic deletion of either peptide has only a limited effect on weight regulation or leptin sensitivity, most likely due to compensation for their loss by other neuronal networks or pathways during early development [START_REF] Gropp | Agouti-related peptide-expressing neurons are mandatory for feeding[END_REF][START_REF] Luquet | NPY/AgRP neurons are essentials for feeding in adult mice but can be ablated in neonates[END_REF][START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF].

It seems that both peptides are redundant in that they act on parallel neuronal circuits inducing the same effect on feeding behavior in particular inducing feeding in situations of food or energy scarcity [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF].

CART and POMC belong to the peptide family known as the melanocortins that mediate the anorexigenic neuronal response to decrease food intake and increase EE. The expression of these neuropeptides increases in response to rising adiposity signals stimulating these neurons [START_REF] Singla | Metabolic effects of obesity: A review[END_REF].

POMC/CART neurons that are situated in the ARC represent likewise first-order neurons, which respond to blood-borne metabolic signals. There is a debate however, whether POMC/CART neurons are directly stimulated by metabolic signals or indirectly regulated through NPY/AgRP neurons via their inhibitory axonal collaterals [START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF].

It seems that POMC/CART neurons express, similar to their neighboring neurons, leptin and IRs and thus can be directly regulated through these metabolic signals.

Studies have shown that central leptin administration stimulates POMC expression and c-fos expression, while POMC expression is markedly reduced in mice with leptin-deficient (ob/ob) mice that develop obesity and hyperphagia, but become normal upon administration of leptin [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF]. Interestingly, while leptin has a stimulatory effect on POMC, insulin, serotonin and ghrelin lead to inhibition. Studies suggest that this might be due to increased inhibition by GABA-expressing axonal collaterals from NPY/AgRP neurons [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF].

Different from NPY/AgRP, POMC does not seem to be affected by dietary intake or components. CART, however, which is co-localized with POMC neurons increases with HFD feeding [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF].

In addition to that, they appear to be regulated by 5-HT through their 5HT-2C receptor to mediate their anorexigenic effect [START_REF] Heisler | Activation of central melanocortin pathways by fenfluramine[END_REF]. Similar to NPY/AgRP neurons, POMC/CART neurons target second-order neurons in different hypothalamic nuclei, such as the PVN, LH and VMH as well as autonomic preganglionic neurons in the brain stem and spinal cord [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF].

Apart from the ARC, POMC is expressed in many tissues such as the nucleus tractus solitarius (NTS) in the CNS but also outside the brain such as in the pituitary, the adrenal gland and the intestine among others. It is a polypeptide that upon cleavage by pro-hormone convertases (PC) gives rise to several different peptides of the melanocortin family, defined by which type of PC the tissue produces [START_REF] Harno | POMC: The Physiological Power of Hormone Processing[END_REF]. In the pituitary, the expression of PC1/3 but not PC2 results in the sequential cleavage of POMC to yield the peptides adrenocorticotropic hormone (ACTH) and β-lipotropin (β-LPH). In the hypothalamus however, PC1/3 and PC2 are expressed, that further process the resulting ACTH and β-LPH into a-, βand γ-MSH (melanocyte-stimulating hormone) and βendorphins.

a-MSH induces a reduction in food intake and increase in EE through binding to melanocortin receptors 3 and 4 (MC3R & MC4R) on neurons in the PVN and LH of the hypothalamus and the NTS in the brainstem [START_REF] Coll | Proopiomelanocortin and energy balance: Insights from human and murine genetics[END_REF][START_REF] Harno | POMC: The Physiological Power of Hormone Processing[END_REF].

The importance of MC4R in the regulation of feeding behavior and BW was shown in mice harboring a deletion in this gene because they develop obesity due to hyperphagia and reduced EE [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF].

Similarly, mutations in this gene lead to severe obesity with early-onset in humans and account for about 6% of obesity cases [START_REF] Tao | Molecular mechanisms of the neural melanocortin receptor dysfunction in severe early onset obesity[END_REF]. Furthermore, mice with deletions in the gene coding for POMC, develop similar to MC4R or leptin receptor deletion, hyperphagia and obesity, while central leptin injection has a reduced effect, when the melanocortin signaling is blocked in neurons [START_REF] Morton | Central nervous system control of food intake and body weight[END_REF].

Interestingly, POMC neurons mediate also the stimulating effect of leptin on locomotor activity and can thereby modulate EE.

CART is one of the anorexigenic neuropeptides abundantly expressed in the ARC and thus an important regulator of food intake and BW. In addition to that, CART is expressed also in the PVN, DMN, LH of the hypothalamus, but also many different neural circuits outside the hypothalamus as well as the adrenal and pituitary glands [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF][START_REF] Lau | CART in the regulation of appetite and energy homeostasis[END_REF]. Although no receptor has been identified that binds CART, it has been implicated in many different functions apart from energy balance such as reward and addiction, the stress response as well as psychostimulant and neuroendocrine functions [START_REF] Lau | CART in the regulation of appetite and energy homeostasis[END_REF].

The expression of CART in hypothalamic neurons has been found to be regulated by several hormones like leptin, insulin and also by glucocorticoids and food deprivation [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF].

While leptin administration, hyperleptinemia and chronic exposure to cold stimulate the expression of CART, leptin deficiency such as in ob/ob mice, reduces it. Similarly, CART expression falls in the ARC in states of food deprivation, which is also associated with decreases in serum leptin concentrations. It was suggested that CART might mediate SNS activation by leptin [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF]. Interestingly, injecting one of the peptides encoded by the gene for CART into the PVN or ARC stimulated the expression of uncoupling protein-1 (UCP-1) revealing its implication in the regulation of EE by modulating thermogenesis. Furthermore, CART has been

shown the be part of the regulation of the HPA axis in experiments where application of hypothalamic explants induced the production and secretion of corticotrophin-releasing and thyrotropin-releasing hormone (CRH and TRH) [START_REF] Lau | CART in the regulation of appetite and energy homeostasis[END_REF]. Intracerebroventricular (ICV) injection of CART revealed some contradicting results. While some studies reveal decreases in food intake and weight loss as well as inhibition of gastric acid secretion, gastric emptying and increased circulating FFAs after ICV injections, others injecting directly into the ARC report increases in food intake. Inhibiting central CART via antibodies in turn augments nocturnal food intake [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF].

In a few interesting studies, it was demonstrated that CART expression is sensitive to nutritional lipids. Studies have shown not only that CART expression is increased in the ARC and PVN in HFD-fed and obese rats, compared to low-diet-fed controls, but also increased CART expression in Intralipid administered animals, which raises blood triglycerides (TG) without changing leptin. Furthermore, HFD-feeding in CART-deficient mice leads to hyperphagia, increased weight gain, as well as fat mass, while SD-fed mice exhibit a normal BW. The increase in CART after HFD might be a compensatory mechanisms by the body to sense and react to high energy and dissipate excess energy by increasing EE [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF]).

The Ventromedial Hypothalamic Nucleus

The VMH of the hypothalamus that is situated just above the ARC is known as the satiety center of the hypothalamus because studies stimulating the VMH showed a decrease in food intake, while bilateral lesions in the VMH results in obesity and hyperphagia but also hyperglycemia [START_REF] Shimizu | Hyperphagia and obesity in rats with bilateral ibotenic acid-induced lesions of the ventromedial hypothalamic nucleus[END_REF][START_REF] Singla | Metabolic effects of obesity: A review[END_REF]. It reciprocally connects with the ARC and sends further projections to other areas such as the DMN, LH and areas of the brain stem [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF]. Similar to neurons of the ARC, subsets of cells in the VMH also express receptors for leptin and insulin and thus are implicated not only in energy metabolism regulation but also in glucose homeostasis [START_REF] Fei | Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues[END_REF][START_REF] Gonzàlez | Dissociation between sensing and metabolism of glucose in sugar sensing neurones[END_REF]. The VMH contains distinct populations of neurons, that can express apart from leptin receptor and IR also brain-derived neurotrophic factor (BNDF), estrogen receptor alpha (ERa), and steroidogenic factor-1 (SF-1) [START_REF] Fei | Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues[END_REF][START_REF] Xu | Brain-derived neurotrophic factor regulates energy balance downstream of melanocortin-4 receptor[END_REF][START_REF] Gonzàlez | Dissociation between sensing and metabolism of glucose in sugar sensing neurones[END_REF][START_REF] Choi | Revisiting the ventral medial nucleus of the hypothalamus: The roles of SF-1 neurons in energy homeostasis[END_REF]. Leptin administration in the VMH was shown to stimulate glucose uptake in peripheral tissues like skeletal muscle, heart and brown adipose tissue (BAT) via the SNS [START_REF] Haque | Role of the sympathetic nervous system and insulin in enhancing glucose uptake in peripheral tissues after intrahypothalamic injection of leptin in rats[END_REF][START_REF] Minokoshi | Microinjection of leptin into the ventromedial hypothalamus increases glucose uptake in peripheral tissues in rats[END_REF][START_REF] Toda | Distinct effects of leptin and a melanocortin receptor agonist injected into medial hypothalamic nuclei on glucose uptake in peripheral tissues[END_REF]. Interestingly, glucosesensing neurons in the VMH are activated by both low and high glucose and impairing IR signaling in the VMH in fasting or hypoglycemic state stimulates glucagon secretion [START_REF] Paranjape | Influence of insulin in the ventromedial hypothalamus on pancreatic glucagon secretion in vivo[END_REF][START_REF] Paranjape | Chronic reduction of insulin receptors in the ventromedial hypothalamus produces glucose intolerance and islet dysfunction in the absence of weight gain[END_REF]. Moreover, studies have shown that the VMH regulates BW and energy homeostasis via controlling the autonomic nervous system (ANS) [START_REF] Choi | Revisiting the ventral medial nucleus of the hypothalamus: The roles of SF-1 neurons in energy homeostasis[END_REF].

The Paraventricular Nucleus

The PVN is another nucleus of the dorsorostral part of the hypothalamus situated beside the 3V and contains many different neurons that are implicated in neuroendocrine, autonomic and behavioral functions [START_REF] Richard | Energy Homeostasis: Paraventricular Nucleus (PVN) System[END_REF]. It receives many neuronal projections such as from the brainstem, limbic system and the ARC and integrates visceral information about nutrition, emotional and reward-related signals as well as energy balance related signals to exert adaptive responses by sending projections towards different brain areas. For example, It harbors MC4R-expressing secondary neurons that receive terminals from both NPY/AgRP and POMC/CART neurons from the ARC and thus can mediate effects on appetite and thermogenesis [START_REF] Richard | Energy Homeostasis: Paraventricular Nucleus (PVN) System[END_REF][START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF]. As such it mediates the net catabolic effect of these neurons.

However, it can also control fatty acid (FA) oxidation and lipolysis in peripheral metabolic tissues via sympathetic efferents [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF].

Apart from that, it contains other neurons of the parvocellular neurosecretory system, such as CRH, and TRH as well as the magnocellular neurosecretory system including vasopressin and oxytocin expressing neurons among others.

It thus can also mediate different effects via the hypothalamic-pituitaryadrenal (HPA) and the hypothalamic-pituitary-thyroid axis (HPT) axis and is involved in regulating blood pressure, water balance and lactation [START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF][START_REF] Richard | Energy Homeostasis: Paraventricular Nucleus (PVN) System[END_REF].

The PVN receives many inputs from various areas including the ARC and has many downstream connections, for example it can regulate neuroendocrine functions via the hypothalamic pituitary axis or control the ANS [START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF]. Its role in energy balance has been shown in animals harboring lesions in the PVN or mutations in the sim1 gene that is a transcription factor, important for the correct development of the PVN leading to obesity and hyperphagia.

The Lateral Hypothalamus

Different from the VMH, the LH is known as the hunger center of the brain, because stimulation of the LH mediates feeding, while lesions leading to ablation of neurons within the LH have the opposite effect (hypophagia and weight loss) [START_REF] Singla | Metabolic effects of obesity: A review[END_REF][START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF].

The second-order neurons of the LH, which receive direct inputs from ARC neurons, express the neuropeptides orexin/hypocretin (ORX), melaninconcentrating hormone (MCH) (Figure 2) and 26RFa among others [START_REF] Chartrel | Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity[END_REF][START_REF] Chartrel | The Neuropeptide 26RFa (QRFP) and Its Role in the Regulation of Energy Homeostasis: A Mini-Review[END_REF][START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF][START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF].

Interestingly, 26RFa has been identified as a ligand for the orphan G protein coupled receptor (GPCR) GPR103 and its expression was found in neurons of the ARC, LH and VMH. Furthermore, this neuropeptide has been shown to act as an incretin but also as an important regulator of food intake and energy balance in the hypothalamus [START_REF] Prévost | Hypothalamic Neuropeptide 26RFa Acts as an Incretin to Regulate Glucose Homeostasis[END_REF][START_REF] Chartrel | The Neuropeptide 26RFa (QRFP) and Its Role in the Regulation of Energy Homeostasis: A Mini-Review[END_REF].

Additionally, neurons of the LH receive input from areas other than the ARC, namely areas transmitting sensory information like the insular and olfactory cortex as well as areas that integrate reward, motivation but also learning and memory (orbitofrontal cortex, nucleus accumbens, amygdala and ventral tegmental area). Furthermore, LH neurons are target for ascending neurons from brain stem areas such as the NTS, parabrachial nucleus (PBN) and locus coeruleus, which transmit vagal and visceral sensory information from the periphery [START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF].

The information that LH neurons receive from the various areas mentioned above is processed, integrated and further transmitted to different areas throughout the brain from the cortex to the spinal cord affecting many different neural networks to elicit the appropriate action [START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF].

Orexin is important in the regulation of glucose sensing and sleep-wake cycles and mutations of its receptor can lead to narcolepsy [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF].

Pharmacological and genetic studies have implicated MCH in development of obesity and the regulation of feeding behavior and identified it as a potent orexigenic peptide, mainly expressed in neurons of the posterior LH and zona incerta and projecting to different areas in the brain and hence controlling many different processes [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF]. It can bind the seven transmembrane (7TM) receptor melanin-concentrating hormone receptor 1 (MCHR1), which is widely expressed not only expressed in the ARC and VMH of the hypothalamus but also abundantly expressed in areas outside the hypothalamus that are related to olfactory and reward-related functions [START_REF] Leibowitz | Hypothalamic control of energy balance: Different peptides, different functions[END_REF]).

The Dorsomedial Nucleus

The DMN is interconnected with the ARC as it receives high amounts of terminals of NPY and a-MSH containing neurons and participates in the regulation of food intake as lesions in the DMN lead to hyperphagia and obesity [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF]. In addition to that, the DMN plays an important role in thermogenesis and thus contributes to EE. Downregulation of NPY neurons in the DMN has been shown to increase BAT activity and socalled browning of WAT, which is the development of brown-like adipocytes in WAT. This is further accompanied by higher rates of thermogenesis and EE.

Knockdown of NPY in DMN also ameliorates high fat diet (HFD)-induced obesity and glucose metabolism [START_REF] Bi | Dorsomedial hypothalamic NPY modulation of adiposity and thermogenesis[END_REF].

The hypothalamus thus contains different nuclei with distinct neurons that in concert adapt to peripheral signals informing the brain about nutritional and energy state, integrate these with information from other areas of the brain and elicit a behavioral response via the ANS or SNS or HPA to control food intake and EE in an attempt to maintain energy balance.

However, areas outside the hypothalamus such as the limbic system, the ventral tegmental area (VTA), NTS and PBN, are crucial in the regulation of energy balance and feeding behavior.

1.1.6. The Caudal Brainstem

The caudal brainstem contains important nuclei such as nucleus accumbens or raphe nucleus that are in contact with the hypothalamus and the periphery and together with the hypothalamus it plays an important role in homeostatic function. Furthermore, some appreciation has to be given to other areas in the cortex and the limbic system which play important roles, too, especially in the integration of past experience, emotions, reward and hedonic function as well as social and environmental information into the already complex system of energy balance regulation [START_REF] Berthoud | The Brain, Appetite, and Obesity[END_REF].

Strikingly, a recent study has identified another region of the brain, that is implicated in the control of feeding, namely the PBN.

In an interesting study using an optogenetic approach, they identified so called caltinonin gene-related peptide (CGRP) neurons, that receive inputs from AgRP neurons from the ARC of the hypothalamus, which when activated suppress feeding. They further found that the functions of these neurons seem to regulate meal duration [START_REF] Wu | Deciphering a neuronal circuit that mediates appetite[END_REF][START_REF] Roman | Genetically and functionally defined NTS to PBN brain circuits mediating anorexia[END_REF].

An important region for regulation of food intake, the NTS, receives signals from the gastrointestinal tract (GIT) about satiety by sensing the gut released hormone CCK or via the vagus nerve, which represents the major neuronal connection between the brain and the periphery and thus plays a big role in the regulation of many different processes [START_REF] Ellacott | Characterization of Leptin-Responsive Neurons in the Caudal Brainstem[END_REF][START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF]. Cutting this connection decreases meal size and duration. The NTS is also connected with the PVN of the hypothalamus and contains NPY, POMC as well as GLP-1 neurons that make the NTS another metabolic sensing area of the brain. Similarly, to the hypothalamus the NTS is in close proximity to another circumventricular area called the area postrema, which places it anatomically strategically for metabolite and signal sensing. POMC neurons in the NTS sense apart from CCK also leptin as indicated by upregulation of a leptin receptor marker called signal transducer and activator of transcription-3 (STAT3) [START_REF] Ellacott | Characterization of Leptin-Responsive Neurons in the Caudal Brainstem[END_REF].

Brain Regulation of Energy Expenditure

Leptin stimulation of hypothalamic POMC neurons as well as orexin-A from LH neurons both stimulate locomotor activity, while suprachiasmatic nucleus released tumor growth factor-a (TGF-a) inhibits locomotor activity via hypothalamic subparaventricular zone neurons in a circadian manner [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF].

BAT thermogenesis is regulated via SNS by the brain by inducing norepinephrine release, which stimulate β3-adrenergic receptors in BAT and inguinal fat pad cells. This triggers signaling pathways that lead to the increase in UCP-1 expression in BAT mitochondria.

Especially hypothalamic areas such as the preoptic area, VMH and ARC play an important role in thermogenic regulation by influencing the SNS according to the sensation of body temperature [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF]. While the preoptic area is involved in the control of body temperature, the VMH seems to regulate BAT activity.

ARC secreted a-MSH has a stimulatory role on BAT activity and sympathoexcitatory neurons of the DMN play a part in the regulation of thermogenic activity [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF]. Several metabolites contribute to the central regulation of BAT activity. For example, leptin, MC3/4R agonist, glucagon and GLP-1 can stimulate BAT activity when infused into the brain, while insulin has a dual role. At high concentrations, insulin seems to increase nerve activity in the BAT while low doses have the opposite effect.

Interestingly, BAT thermogenesis affects BW and fat mass, as it uses metabolic substrates like glucose and FAs as substrates and thereby dissipates excess energy after consumption of high amounts of calories or upon exposure to cold temperature to maintain body temperature [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF].

Interestingly, interleukin-6 (IL-6), which is secreted by skeletal muscle upon contraction during exercise, can cross the BBB and has been reported to stimulate EE in the brain [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF].

Obesity

Definition, Facts and Generalities

The physiological ability of vertebrates and humans to store energy as food has become one of the major burdens of the century for the human population. Obesity is a disorder that has become a huge economic and public health burden of global epidemic proportions in both developing and Obesity is a metabolic disease defined as an excess accumulation of energy in form of body fat in AT resulting in a risk to health (WHO -Obesity) and mainly assessed by the body mass index (BMI). The BMI is the BW in kilograms divided by the height in meters squared (Fig. 3). BMI thus classifies the state of the body as normal weight (BMI=18. 5-24.9), between 25 and 29.9 as overweight, obesity by a BMI of ≥30, and morbid obese with a BMI of 40 or higher (Fig. 3). However, as useful the BMI might be, it is but an estimate of body fat and health risk and has its limitations as it does not take into account the specific body composition such as muscles, body fat mass and distribution, lean mass, bone density and other factors and thus cannot accurately assess the state of the body.

Obesity is part of the metabolic syndrome, which is defined as a cluster of both chronic and complex disorders, that have in common at least one feature of metabolic deterioration (Hotamisligil, 2006). Others that are often associated with obesity are hyperglycemia, dyslipidemia, hypertension [START_REF] Oussaada | The pathogenesis of obesity[END_REF]. Metabolic syndrome is associated with an increased risk for insulin resistance, fatty liver disease, type 2 diabetes mellitus (T2DM), atherosclerosis, cardiovascular diseases but also with dementia, airway disease and cancer (Hotamisligil, 2006;[START_REF] Oussaada | The pathogenesis of obesity[END_REF].

Obese patients and mouse models of obesity often have increased serum levels of FFAs, leptin (hyperleptinemia), indicative of leptin resistance, and develop insulin resistance with hyperinsulinemia and glucose intolerance [START_REF] Klok | The role of leptin and ghrelin in the regulation of food intake and body weight in humans: A review[END_REF][START_REF] Dalmas | Variations in circulating inflammatory factors are related to changes in calorie and carbohydrate intakes early in the course of surgery-induced weight reduction1-3[END_REF][START_REF] Adamska | Intake of Meals Containing High Levels of Carbohydrates or High Levels of Unsaturated Fatty Acids Induces Postprandial Dysmetabolism in Young Overweight/Obese Men[END_REF][START_REF] González-Muniesa | Obesity[END_REF]. Interestingly, leptin and insulin resistance can also be seen at the central level [START_REF] Clegg | Consumption of a high-fat diet induces central insulin resistance independent of adiposity[END_REF][START_REF] Faulconbridge | Regulation of energy balance and body weight by the brain: a distributed system prone to disruption[END_REF][START_REF] Cheng | Palmitic acid induces central leptin resistance and impairs hepatic glucose and lipid metabolism in male mice[END_REF][START_REF] Tran | Diet-induced cellular neuroinflammation in the hypothalamus: Mechanistic insights from investigation of neurons and microglia[END_REF]. A few mechanisms have been proposed to explain leptin resistance: a reduced transport of leptin through the BBB as well as an impairment in the central leptin signaling pathway [START_REF] El-Haschimi | Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity[END_REF].

Obesity is a complex disorder and often caused by an interaction of various factors that can be genetic, environmental and psychosocial factors. Apart from monogenic forms of obesity, which are relatively rare and account for about only 2-3% of cases, obesity can be also caused by certain medication, depression, hormones and a disequilibrium between energy intake and EE, but also be due to a specific unhealthy diet [START_REF] González-Muniesa | Obesity[END_REF].

Genetic Causes of Obesity

Although genetic causes of obesity are accounting only for a small part of cases of obesity and cannot account for the tremendous rise of obesity prevalence in recent years, there are several cases of monogenic forms of obesity. One of the most famous and most studied forms of obesity are obesity induced by mutations in genes coding for both leptin and its receptor.

Leptin, a hormone or adipokine secreted by adipocytes of the AT has an important role in energy homeostasis. It is secreted in proportion to body fat mass and informs the brain about the energy state of the body. If leptin is missing due to a mutation in the gene for leptin, as was well researched in a model of obese mice (ob/ob), mice become hyperphagic and obese even on a normal diet [START_REF] Zhang | Positional cloning of the mouse obese gene and its human homologue[END_REF]. Such mutations in the gene for leptin were also found in children, who had no detectable serum levels of leptin and suffered from congenital early-onset obesity [START_REF] Montague | Congenital leptin deficiency is associated with severe earlyonset obesity in humans[END_REF][START_REF] Gibson | Congenital leptin deficiency due to homozygosity for the Δ133G mutation: Report of another case and evaluation of response to four years of leptin therapy[END_REF]. This autosomal recessive form of the disorder is heritable and leads to extreme forms of early-onset obesity, which produces a dysfunctional form of the protein leptin, that is either secreted in low amounts or fails to be secretion due to proteasomal degradation [START_REF] Montague | Congenital leptin deficiency is associated with severe earlyonset obesity in humans[END_REF][START_REF] Rau | Truncated human leptin (Δ133) associated with extreme obesity undergoes proteasomal degradation after defective intracellular transport[END_REF]. Leptin deficiency is also associated with other endocrine abnormalities like hypogonadotrophic hypogonadism in addition to alterations in the SNS as well as high infection rates, which are attributable to a lack of T cells but without impacts on EE [START_REF] Sadaf Farooqi | Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency[END_REF][START_REF] Dubern | Leptin and leptin receptor-related monogenic obesity[END_REF]. Other than leptin deficiency, which can be treated via injections of leptin to provide the body with the deficient protein, another monogenic form of obesity has no treatment options so far and is attributable to a loss of function mutation in the leptin receptor gene (db/db) [START_REF] Tartaglia | Identification and expression cloning of a leptin receptor[END_REF][START_REF] Chua | Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor[END_REF][START_REF] Anderson | Stearic acid accumulation in macrophages induces toll-like receptor 4/2-independent inflammation leading to endoplasmic reticulum stress-mediated apoptosis[END_REF]. The leptin receptor belongs to the family of cytokine receptors and the gene coding for the receptor seems to be inherited in an autosomal recessive manner causing human leptin receptor deficiency due to a mutation in this gene. Human leptin receptor deficiency leads similar to leptin deficiency to extreme obesity associated with hyperphagia but different from (ob/ob) to diabetes. Several models for this pathology have been produced in both mice (db/db mice) and rats (fatty Zucker rat). A deficiency in the leptin receptor similarly affects leptin signaling through phosphatidylinositol 3-kinase (PI3K) and STAT3, in particularly in the hypothalamus, where the receptor is expressed in normal patients or animals and affects appetite and feeding behavior [START_REF] Chua | Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor[END_REF][START_REF] Bates | The role of leptin receptor signaling in feeding and neuroendocrine function[END_REF].

Another form of monogenic obesity is due to an autosomal recessive inherited loss of function mutation in the gene encoding POMC, a neuropeptide of the melanocortin system in the hypothalamus responsible for regulating energy balance by suppressing food intake. This mutation leads to a POMC deficiency, found in a few human subjects, leading to severe and early-onset obesity, adrenal insufficiency, and red hair pigmentation and similar to leptin and its receptor deficiency, it is associated with hyperphagia [START_REF] Krude | Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans[END_REF][START_REF] Krude | Obesity Due to Proopiomelanocortin Deficiency: Three New Cases and Treatment Trials with Thyroid Hormone and ACTH4-10[END_REF][START_REF] Buono | Six Novel Mutations in the Proopiomelanocortin and Melanocortin Receptor 4 Genes in Severely Obese Adults Living in Southern Italy[END_REF]. Interestingly, POMC-expressing neurons in the ARC of the hypothalamus are a target of leptin, which upon binding induce POMC synthesis [START_REF] Coll | Proopiomelanocortin and energy balance: Insights from human and murine genetics[END_REF]. POMC is expressed in many tissue such as the ARC of the hypothalamus and the NTS in the CNS but also outside such as in the pituitary, the adrenal gland and the intestine among others. It is a polypeptide that upon cleavage by PC gives rise to several different peptides, defined by which type of PC the tissue produces [START_REF] Harno | POMC: The Physiological Power of Hormone Processing[END_REF]. In the pituitary, the expression of PC1/3 but not PC2 results in the sequential cleavage of POMC to yield the peptides ACTH and β-lipotropin (β-LPH). In the hypothalamus however, PC1/3 and PC2 are expressed, that further processes the resulting ACTH and β-LPH into a-, βand γ-MSH (melanocyte-stimulating hormone) and βendorphins.

a-MSH induces a reduction in food intake and increase in EE through binding to MC3R & MC4R on neurons in the PVN and LH of the hypothalamus and the NTS in the brainstem [START_REF] Coll | Proopiomelanocortin and energy balance: Insights from human and murine genetics[END_REF][START_REF] Harno | POMC: The Physiological Power of Hormone Processing[END_REF]. POMC deficient humans and mice thus develop severe cases of obesity but also other deficiencies related to the other peptides produced from the POMC precursor peptide, including pale skin color and red hair pigmentation and low cortisol levels [START_REF] Krude | Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans[END_REF][START_REF] Krude | Obesity Due to Proopiomelanocortin Deficiency: Three New Cases and Treatment Trials with Thyroid Hormone and ACTH4-10[END_REF][START_REF] Buono | Six Novel Mutations in the Proopiomelanocortin and Melanocortin Receptor 4 Genes in Severely Obese Adults Living in Southern Italy[END_REF][START_REF] Farooqi | Heterozygosity for a POMC-null mutation and increased obesity risk in humans[END_REF].

Similar to POMC deficiency, autosomal recessive forms of obesity can be induced by mutations in the gene for carboxypeptidase E (CPE) as was demonstrated in a fat/fat mouse model, that lead to less severe forms of obesity with later onset, compared to leptin or leptin receptor deficient forms of obesity. Interestingly, this enzyme is also responsible for the processing of prohormones including proinsulin in the pancreas and thus show an interesting genetic link between obesity and diabetes [START_REF] Naggert | Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity[END_REF].

In a similar example, a mutation in the PC1 gene that exerts its effect upstream of CPE caused obesity with elevated proinsulin levels and POMC levels with similar phenotype to the fat mouse in two human patients [START_REF] Jackson | Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene[END_REF][START_REF] Jackson | Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency[END_REF].

The most common form of monogenic human form of obesity is attributable to mutations in the gene for the MC4R. While deletion of murine MC4R leads to hyperphagia and reduced EE and ultimately obesity, human MC4R mutation is responsible for ~6% of severe and early-onset obesity cases and inherited in a co-dominant manner [START_REF] Tao | Molecular mechanisms of the neural melanocortin receptor dysfunction in severe early onset obesity[END_REF][START_REF] Chung | An overview of mongenic and syndromic obesities in humans[END_REF].

Furthermore, obesity can also appear as a consequence of other syndromes caused by mutations in several genes and include for example, Prader-Willi Syndrome and Cohen Syndrome among others.

Other factors that can lead to obesity are certain medications, depression and other environmental factors. One of the environmental causes of obesity and also one of the most common causes is a sedentary lifestyle and poor nutrition, especially a nutrition high in fat, leading to the dysfunction of energy balance regulation.

Obesity and Inflammation

It is characterized by a state of chronic low-grade systemic inflammation, the secretion of inflammatory cytokines and chemokines and an infiltration of immune cells including macrophages and T cells [START_REF] Gregor | Inflammatory Mechanisms in Obesity[END_REF].

Inflammation

The classical inflammatory response of the body´s immune system is a necessary and beneficial process to recognize and eliminate sources of infection or inflammation such as exogenous intruders like bacteria or other pathogens. However, inflammation can also take place in cases such as tissue injury to promote wound healing. In any case, these inflammatory processes are usually acute, and once the pathogenic intruder or wound has been successfully eliminated or healed, the inflammation subsides. The classical characteristics of inflammation are redness, swelling, heat and pain.

Furthermore, classical inflammation is also associated with an increased metabolic rate [START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF].

Inflammation mainly involves cells of the immune system that can be broadly subdivided and into lymphocytes and myeloid cell and functionally into proand anti-inflammatory immune cells [START_REF] Lee | Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance[END_REF]. Lymphocytes can be further classified into T, B, natural killer (NK), and natural killer T (NKT) cells, which belong to the adaptive immune system, while myeloid cells belong to the innate immune system and include macrophages, dendritic cells (DCs), mast cells, neutrophils, basophils and eosinophils. However, myeloid cells contribute to the adaptive immune system, too, for example as antigen presenting cells that activate lymphocytes, or through the production of cytokines and chemokines to help the development and activation of T and B cells and attract them to the target sites [START_REF] Lee | Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance[END_REF].

However, when the inflammation does not resolve, it can become of chronic nature and caused by an excess in triggers of inflammation, for example as seen in the case of obesity, where a metabolic trigger such as an excess consumption of nutrients or metabolic surplus is thought to be the cause of inflammatory processes. Moreover, it is suggested that metabolic cells such as adipocytes sustain the insult by the excess of nutrients and respond with initiating inflammatory processes, leading to a disturbed metabolic homeostasis.

Different from classical infection or inflammatory response, chronic inflammation often found in obesity but also other diseases, is although significant, rather modest and thus often called "low-grade" inflammation and can be local in certain tissues.

This inflammatory state, characterized by a chronic low-grade inflammation in metabolic tissues such as pancreas, muscles, AT but also liver and brain and often found in obesity, is triggered by metabolic stimuli like excess energy and nutrients and was termed "metaflammation". It is not an inflammation in the traditional meaning of the word, as none of the classical features of inflammation are present, although it does involve similar actors or inflammatory signaling pathways as does the classical inflammation (Hotamisligil, 2006). Hence, in this manuscript inflammation will be referred to the sub-class of metabolic inflammation. Chronic inflammation or meaflammation can further be linked to insulin resistance and metabolic dysfunction [START_REF] Gregor | Inflammatory Mechanisms in Obesity[END_REF].

Peripheral Inflammation in Obesity

Obesity is as mentioned previously associated with a cluster of disorders commonly referred to as metabolic syndrome. The disorders included in this syndrome all have in common the occurrence of chronic, low-grade inflammation that seems to be the cause of these disorders. Long-term overconsumption of HFD leads to an overpowering of the storage capacity of ATs and lipid/FA accumulation in metabolic tissues such as skeletal muscle, liver, pancreas, brain and gut and can result not only in lipotoxicity but also induce inflammation in these tissues including AT.

AT was long thought to have the sole purpose of energy storage of lipids as triacylglycerols in the body. However, since the discovery of leptin, a hormone secreted by WAT in proportion to the energy stored in the tissue, AT is viewed rather as the largest endocrine organ [START_REF] Calder | Dietary factors and low-grade inflammation in relation to overweight and obesity[END_REF][START_REF] Coelho | Biochemistry of adipose tissue: An endocrine organ[END_REF]. As an endocrine gland it is capable of synthesizing and releasing many bioactive compounds including hormones, cytokines and chemokines collectively referred to as adipokines. These adipokines like leptin have important regulatory functions like whole body metabolism and appetite control but also in immune function and others [START_REF] Coelho | Biochemistry of adipose tissue: An endocrine organ[END_REF]. AT can be broadly subdivided into two types, BAT and WAT.

The former has a lower storage capacity and different from WAT contains abundant mitochondria and more but smaller lipid vacuoles. Although BAT is more abundant in infants, adults also have BAT, which is located in supraclavicular and interscapular regions, around organs like kidneys, heart, pancreas but also aorta and the trachea [START_REF] Santhanam | Adiposity-related cancer and functional imaging of brown adipose tissue[END_REF][START_REF] Prodhomme | Imaging and identification of brown adipose tissue on CT scan[END_REF]. BAT contributes to energy balance regulation through its participation in nonshivering thermogenesis and diet-induced thermogenesis. It thereby contributes to EE and contains many mitochondria due to the high energy demand of these processes. [START_REF] Bartelt | Brown adipose tissue activity controls triglyceride clearance[END_REF][START_REF] Saely | Brown versus white adipose tissue: A minireview[END_REF]. A cold temperature setting induces the increased uptake of FAs and their oxidation for heat production [START_REF] Ertunc | Lipid signaling and lipotoxicity in metaflammation: indications for metabolic disease pathogenesis and treatment[END_REF]. BAT is highly innervated by sympathetic efferent fibers that link it to the hypothalamus and enables tight regulation by the brain [START_REF] Hankir | Loading and firing the brown adipocyte[END_REF]. In that manner, a cold temperature or energy surplus, sensed by afferent nerves of the hypothalamus lead to noradrenaline secretion via sympathetic efferent nerves that in turn activate BAT to produce heat and increase the corporal temperature [START_REF] Hankir | Loading and firing the brown adipocyte[END_REF]. Interestingly, while WAT is positively correlated with BMI, the inverse is true for BAT [START_REF] Saito | High incidence of metabolically active brown adipose tissue in healthy adult humans: Effects of cold exposure and adiposity[END_REF]. Moreover, BAT was implicated in the control of vascular lipoprotein homeostasis through TG-rich lipoprotein uptake from the circulation [START_REF] Bartelt | Brown adipose tissue activity controls triglyceride clearance[END_REF]. Furthermore, obese patients often have increased serum concentrations of leptin and decreased adiponectin, which is associated with higher adiposity [START_REF] Huber | CC chemokine and CC chemokine receptor profiles in visceral and subcutaneous adipose tissue are altered in human obesity[END_REF]. Depending on their location in the body, WAT can further be classified into subcutaneous and visceral AT, which in turn consists of intra-abdominal, perirenal and pericardial AT (Fig. 4) [START_REF] González-Muniesa | Obesity[END_REF]. WAT is composed of primarily adipocytes, a cell serving as a storage unit of energy as triacylglycerol, but contains also other cells like fibroblasts, mast cells, granulocytes, lymphocytes, macrophages, endothelial cells, pericytes, and precursors of adipocytes, and are commonly referred to as stroma-vascular fraction [START_REF] Coelho | Biochemistry of adipose tissue: An endocrine organ[END_REF]. FAs are stored in white adipocyte lipid droplets as esterified and compartmentalized FAs. Although AT is the primary storage unit for lipids and FAs, this form of lipid storage can occur in all cells of higher organisms [START_REF] Ertunc | Lipid signaling and lipotoxicity in metaflammation: indications for metabolic disease pathogenesis and treatment[END_REF].

When the body is in a situation of increased energy demand, adipocytes can liberate free FAs from lipid droplets via lipolysis and release them into the circulation to be accessible by other tissues for mitochondrial FA oxidation [START_REF] Ertunc | Lipid signaling and lipotoxicity in metaflammation: indications for metabolic disease pathogenesis and treatment[END_REF].

When the body undergoes metabolic stress like an excess consumption of energy dense food, AT can undergo changes like hypertrophy or hyperplasia, where adipocytes either increase in size or in number, in adaptation for the increased energy storage requirements of incoming lipids (Fig. 4). However, due to limited capacity of AT, chronic overnutrition might cause the saturated and hypertrophied adipocytes to rupture and cause inflammation and phagocytosis by macrophages. In addition, the saturation of AT capacity causes ectopic lipid accumulation in other undesired locations such as the liver, muscle, pancreas, heart, BAT, kidneys and the brain (Fig. 5) [START_REF] Posey | Hypothalamic proinflammatory lipid accumulation, inflammation, and insulin resistance in rats fed a high-fat diet[END_REF][START_REF] Guebre-Egziabher | Ectopic lipid accumulation: A potential cause for metabolic disturbances and a contributor to the alteration of kidney function[END_REF][START_REF] González-Muniesa | Obesity[END_REF]. This excess accumulation of intra-abdominal visceral fat during the development of diet-induced obesity (DIO) can be measured via waist circumference and used as a clinical determinant of obesity. Interestingly, while increased abdominal fat is associated with metabolic syndrome and inflammation, subcutaneous fat is less associated with metabolic dysfunction, insulin resistance and inflammation (Fig. 4) [START_REF] Koster | Body fat distribution and inflammation among obese older adults with and without metabolic syndrome[END_REF][START_REF] Esser | Obesity phenotype is related to NLRP3 inflammasome activity and immunological profile of visceral adipose tissue[END_REF].

This increase in AT as well as ectopic lipid accumulation gives rise to chronic low-grade inflammation, upregulation of inflammatory gene expression and acute phase proteins, infiltration of macrophages and production of cytokines and chemokines, which in turn can attract other immune cells to infiltrate the tissue (Fig. 5) [START_REF] Gregor | Inflammatory Mechanisms in Obesity[END_REF][START_REF] Ertunc | Lipid signaling and lipotoxicity in metaflammation: indications for metabolic disease pathogenesis and treatment[END_REF]. AT is an endocrine organ that secretes adipokines including hormones like leptin, adiponectin, visfatin, and omentin; acute CCL5 but also growth factors like tumor growth factor (TGF)-β as well as components of the complement system [START_REF] Calder | Dietary factors and low-grade inflammation in relation to overweight and obesity[END_REF]. Obesity and increased visceral adiposity is associated with increases in leptin secretion and leptin resistance, while adiponectin is negatively correlated with obesity and adiposity [START_REF] Calder | Dietary factors and low-grade inflammation in relation to overweight and obesity[END_REF][START_REF] Jung | Obesity and its metabolic complications: The role of adipokines and the relationship between obesity, inflammation, insulin resistance, dyslipidemia and nonalcoholic fatty liver disease[END_REF]. Furthermore, while leptin is considered to be pro-inflammatory and secreted like SAA in proportion to AT size, adiponectin appears to be anti-inflammatory [START_REF] Calder | Dietary factors and low-grade inflammation in relation to overweight and obesity[END_REF].

In the context of HFD-induced obesity, many studies have shown that cytokines and chemokines, in particular TNF-a, IL-1β, IL-6, IL-10, CCL2 and CCL5 are not only upregulated both systemically and in AT and other tissues but play important roles in metabolic dysfunction and insulin resistance [START_REF] Calder | Dietary factors and low-grade inflammation in relation to overweight and obesity[END_REF][START_REF] Gregor | Inflammatory Mechanisms in Obesity[END_REF]. Cytokine and chemokine levels were found to be higher in serum of obese patients and mice. For instance, one study found elevated serum levels of Creactive protein in addition to IL-6 in obese patients compared to lean controls as well as upregulated macrophage markers in not only visceral but also SC AT, which is indicative of increased immune cell infiltration [START_REF] Huber | CC chemokine and CC chemokine receptor profiles in visceral and subcutaneous adipose tissue are altered in human obesity[END_REF].

Cytokines

Several important studies have demonstrated not only an increase in the infiltration of macrophages into the expanding AT in both obese mice and humans [START_REF] Weisberg | Obesity is associated with macrophage accumulation in adipose tissue[END_REF]. In addition to that, it was also shown that macrophages shift their phenotype from an anti-inflammatory M2 profile ("classically activated") to pro-inflammatory M1 type ("alternatively activated") in obesity development [START_REF] Lumeng | Obesity induces a phenotypic switch in adipose tissue macrophage polarization[END_REF][START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF][START_REF] Chylikova | M1/M2 macrophage polarization in human obese adipose tissue[END_REF]. Chemokines like CCL2 and CCL5 have been shown to participate in the recruitment of immune cells and the polarization of macrophages to M1 types in AT in response to HFD [START_REF] Kanda | MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity[END_REF][START_REF] Wu | T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity[END_REF][START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF][START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF]. These macrophages can recruit other immune cells to the AT through the secretion of cytokines and chemokines as has been shown previously [START_REF] Chawla | Macrophage-mediated inflammation in metabolic disease[END_REF]. In fact, other immune cells like T cells have been shown to play an important role in the contribution of chronic low-grade inflammation in AT but also other tissues in obesity. Interestingly, infiltration of T cells has recently been reported as important factor in the regulation of macrophage trafficking and polarization in AT in the context of DIO, as TH1 CD4 + and CD8 + T cell count was raised in AT of obese rodents and humans [START_REF] Nishimura | CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity[END_REF][START_REF] Strissel | T-cell recruitment and Th1 polarization in adipose tissue during dietinduced obesity in C57BL/6 mice[END_REF]. [START_REF] Wu | T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity[END_REF][START_REF] Feuerer | Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters[END_REF][START_REF] Nishimura | CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity[END_REF][START_REF] Calder | Dietary factors and low-grade inflammation in relation to overweight and obesity[END_REF][START_REF] Esser | Obesity phenotype is related to NLRP3 inflammasome activity and immunological profile of visceral adipose tissue[END_REF]. Some studies found not only infiltration by T cells into AT in an obesity context, but also that T cell infiltration might precede recruitment of macrophages [START_REF] Wu | T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity[END_REF][START_REF] Kintscher | T-lymphocyte infiltration in visceral adipose tissue: A primary event in adipose tissue inflammation and the development of obesity-mediated insulin resistance[END_REF][START_REF] Rausch | Obesity in C57BL/6J mice is characterized by adipose tissue hypoxia and cytotoxic T-cell infiltration[END_REF].

Furthermore, AT macrophages can maintain and further contribute to the obesity associated low grade-inflammation through the secretion of cytokines and chemokines [START_REF] Curat | Macrophages in human visceral adipose tissue: Increased accumulation in obesity and a source of resistin and visfatin[END_REF].

Interestingly, weight loss interventions such as exercise, diet or surgery decreases recruitment of macrophages and pro-inflammatory markers in AT and plasma of obese patients [START_REF] Cancello | Reduction of macrophage infiltration and chemoattractant gene expression changes in white adipose tissue of morbidly obese subjects after surgery-induced weight loss[END_REF][START_REF] Bruun | Diet and exercise reduce low-grade inflammation and macrophage infiltration in adipose tissue but not in skeletal muscle in severely obese subjects[END_REF].

Obesity is associated with other impairments of AT like increased lipolysis and decreased lipogenesis, which adds to the lipotoxicity of lipds [START_REF] Gregor | Inflammatory Mechanisms in Obesity[END_REF][START_REF] Gao | Decreased lipogenesis in white adipose tissue contributes to the resistance to high fat diet-induced obesity in phosphatidylethanolamine N-methyltransferasedeficient mice[END_REF][START_REF] Ertunc | Lipid signaling and lipotoxicity in metaflammation: indications for metabolic disease pathogenesis and treatment[END_REF].

Interestingly, TNF-a treatment of adipocytes induces lipolysis, increasing the amount of FFAs, and inhibits the differentiation of pre-adipocytes into mature adipocytes [START_REF] Kawakami | Human recombinant TNF suppresses lipoprotein lipase activity and stimulates lipolysis in 3T3-L1 cells[END_REF][START_REF] Pape | Effect of Tumor Necrosis Factor on Acetyl-Coenzyme A Carboxylase Gene Expression and Preadipocyte Differentiation[END_REF][START_REF] Feingold | Stimulation of lipolysis in cultured fat cells by tumor necrosis factor, interleukin-1, and the interferons is blocked by inhibitionof prostaglandin synthesis[END_REF]. The latter effect might be mediated by peroxisome proliferatoractivated receptor γ (PPARγ), a transcription factor of the nuclear hormone receptor family, which plays a crucial role in adipogenesis, differentiation, AT function and gene expression and is downregulated by inflammation in AT [START_REF] Guilherme | Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes[END_REF]. PPARγ can be activated by molecules like FAs and eicosanoids, that can act as ligands, and is further implicated in peroxisome proliferation, lipid metabolism in neurons but also in insulin sensitivity in different tissues, energy balance regulation and feeding behavior [START_REF] Issemann | Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators[END_REF][START_REF] Dreyer | Positive regulation of the peroxisomal beta-oxidation pathway by fatty acids through activation of peroxisome proliferator-activated receptors (PPAR)[END_REF][START_REF] Lu | Brain PPAR-γ promotes obesity and is required for the insuling-sensitizing effect of thiazolidinediones[END_REF][START_REF] Ryan | A role for central nervous system PPAR-γ in the regulation of energy balance[END_REF][START_REF] Garretson | Peroxisome Proliferator-Activated Receptor Controls Ingestive Behavior, Agouti-Related Protein, and Neuropeptide Y mRNA in the Arcuate Hypothalamus[END_REF].

Interestingly, neuron-specific knockout (KO) of PPARγ in a DIO mouse model resulted in reduced food intake and increased EE and ultimately in BW loss in HFD-fed KO mice compared to controls. Furthermore, this study showed that PPARγ signaling is required for the insulin sensitizing effect of thioazolidinediones [START_REF] Lu | Brain PPAR-γ promotes obesity and is required for the insuling-sensitizing effect of thiazolidinediones[END_REF].

Experimental models of obesity and diabetes demonstrated increased TNF-a expression and accumulation of inflammatory cells in muscle tissue and showed a link between elevated circulating levels of FAs and TG with muscle insulin resistance [START_REF] Ertunc | Lipid signaling and lipotoxicity in metaflammation: indications for metabolic disease pathogenesis and treatment[END_REF]. Furthermore, the BBB increases its permeability, increasing the flux of nutrients into the brain as well as the infiltration of immune cells. The increased accumulation of lipids in the hypothalamus induces neuroinflammation, the increase in immune mediators as well as the activation of glia cells. Hypothalamic inflammation can ultimately result in changes in neuronal activity, as well as neuronal damage or apoptosis.

(Adapted from (Guillemot-Legris and Muccioli, 2017)).

Central Inflammation in Obesity

Many studies have shown that HFD induces chronic but low-grade central inflammation in the hypothalamus but also in other extra-hypothalamic areas such as the hippocampus, cortex, brainstem or amygdala [START_REF] Guillemot-Legris | Obesity-Induced Neuroinflammation: Beyond the Hypothalamus[END_REF]. It has been documented that hypothalamic inflammation occurs as early as 1 to 3 days after HFD consumption and is characterized by an upregulation of pro-inflammatory cytokine expression such as IL-1β and inhibitor of nuclear factor kappa-B kinase, but also a marker for neuronal injury, namely heat shock protein 72 [START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF]. This early, acute but transient immune response is followed by reactive gliosis, which is referring to the change in microglial and astrocyte activity, which then recedes in an adaptive response to avoid neuronal injury. However, with long-term exposure to HFD reinstates the inflammatory response, which becomes chronic [START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Berkseth | Hypothalamic gliosis associated with high-fat diet feeding is reversible in mice: A combined immunohistochemical and magnetic resonance imaging study[END_REF]. Recent results confirm the occurrence of hypothalamic gliosis also in human obese subjects via magnetic resonance imaging and link it with insulin resistance [START_REF] Schur | Radiologic evidence that hypothalamic gliosis is associated with obesity and insulin resistance in humans[END_REF]. Other studies have found not only hypothalamic inflammation in DIO rodent models, but also signs of apoptosis in the ARC and the LH and more specifically in neurons like AgRP and POMC of the ARC, which seemed to be dependent on diet composition more than caloric intake [START_REF] Moraes | High-fat diet induces apoptosis of hypothalamic neurons[END_REF]. Interestingly, the same study identified that the presence of toll-like receptor 4 (TLR4) seemed to be protective in the context of HFD-induced neuronal apoptosis and suggested a dual-function of TLR4 in hypothalamic inflammation.

Interestingly, another study demonstrated the importance of inhibitor of κB kinase β (IKKβ)/ nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway activation and endoplasmic reticulum (ER) stress in HFD or overnutrition-induced obesity and associated leptin and insulin resistance [START_REF] Zhang | Hypothalamic IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance and obesity[END_REF]. Forced activation of IKKβ/NF-κB impaired hypothalamic insulin and leptin signaling, they showed that inhibition thereof, including specific inhibition in AgRP neuron, protects against glucose intolerance and obesity. Authors suggested that this effect might be mediated through suppressor of cytokine signaling-3 (SOCS3), a common inhibitor of both insulin and leptin signaling pathways [START_REF] Zhang | Hypothalamic IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance and obesity[END_REF].

Other inflammatory pathways found to be activated in DIO-associated hypothalamic inflammation are IKKβ/NF-κB and mitogen-activated protein kinase (MAPK) pathways as well as c-jun N-terminal kinase (JNK) signaling [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF][START_REF] Benzler | Acute Inhibition of Central c-Jun N-terminal Kinase Restores Hypothalamic Insulin Signalling and Alleviates Glucose Intolerance in Diabetic Mice[END_REF][START_REF] Benzler | Central inhibition of IKKβ/NF-κB signaling attenuates high-fat dietinduced obesity and glucose intolerance[END_REF][START_REF] Rahman | Hypothalamic inflammation and malfunctioning glia in the pathophysiology of obesity and diabetes: Translational significance[END_REF]. NF-κB is a key mediator of cellular immune and inflammatory responses and mediates the expression of pro-inflammatory genes as a transcription factor. MAPK kinases are upstream of JNKs and induce their activation via phosphorylation in response to stress signals like nutritional components, hyperglycemia, ER stress or oxidative stress, resulting in the production of cytokines. JNKs are a lso inflammatory signaling molecules than can affect many metabolic processes because they can modulate the activity of many nuclear transcription factors like c-Jun, activator protein, forkhead box protein O4, nuclear hormone receptors and activating transcription factor 2 among others [START_REF] Rahman | Hypothalamic inflammation and malfunctioning glia in the pathophysiology of obesity and diabetes: Translational significance[END_REF].

The implication of inflammatory pathways in hypothalamic inflammation and its contribution to DIO was shown for example in a study that inhibited specifically IKKβ in the hypothalamus and observed a restoration of both leptin and insulin sensitivity of obese rodents [START_REF] Zhang | Hypothalamic IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance and obesity[END_REF]. TNF-a has been shown to induce the expression of other cytokines like IL-1β, IL-6 and anti-inflammatory molecules like IL-10 and SOCS3, as well as activate signal transduction pathways like JNK and NF-κB, and modulate the expression of neuropeptides involved in the regulation of energy balance and feeding regulation in the hypothalamus of rats [START_REF] Amaral | Tumor necrosis factor-α activates signal transduction in hypothalamus and modulates the expression of pro-inflammatory proteins and orexigenic/anorexigenic neurotransmitters[END_REF][START_REF] Romanatto | TNF-α acts in the hypothalamus inhibiting food intake and increasing the respiratory quotient-Effects on leptin and insulin signaling pathways[END_REF]. In addition to that blocking either TLR4 or TNF-a reduces hypothalamic inflammation as well as its resistance to leptin and restores hepatic glucose production [START_REF] Milanski | Inhibition of hypothalamic inflammation reverses diet-induced insulin resistance in the liver[END_REF]. Interestingly, also the inhibition of neuronal IKKβ/NF-κB signaling in ARC neurons reduces BW and fat gain in response to HFD feeding and increased expression of EE [START_REF] Benzler | Central inhibition of IKKβ/NF-κB signaling attenuates high-fat dietinduced obesity and glucose intolerance[END_REF]. Another study demonstrated that inhibiting TLR4 signaling pathway in the hypothalamus reduces hypothalamic inflammation, including IKKβ and improves insulin and leptin resistance as well as BW gain associated with HFD [START_REF] Kleinridders | MyD88 Signaling in the CNS Is Required for Development of Fatty Acid-Induced Leptin Resistance and Diet-Induced Obesity[END_REF].

Chemokines are important participants in the hypothalamic inflammation associated in DIO. For example, the chemokine fractalkine (CX3CL1) was found to be involved early on in the hypothalamic inflammation in HFD-fed mice [START_REF] Morari | Fractalkine (CX3CL1) is involved in the early activation of hypothalamic inflammation in experimental obesity[END_REF]. Its expression was induced early in neurons of HFDfed mic, while its inhibition decreased hypothalamic inflammation and the hypothalamic infiltration of bone marrow-derived monocytes.

It is still uncertain though what initiates that first inflammatory response in the hypothalamus. Several theories have been explored in an attempt to explain the as of yet uncovered mechanism.

Oxidative stress is one of the factors that was suggested to be an initiator of inflammatory events and metabolic abnormalities associated with DIO. It has been reported that oxidative stress and the production of reactive oxygen species (ROS) are upregulated in metabolic tissues in the periphery of DIO mice well before metabolic impairments such as insulin resistance become apparent [START_REF] Matsuzawa-Nagata | Increased oxidative stress precedes the onset of high-fat diet-induced insulin resistance and obesity[END_REF]. Similarly, hypothalamic neurons can be effected particularly by ROS as the brain consumes 15-20% of the total energy generated by the body [START_REF] Garbarino | Mechanisms of oxidative stress resistance in the brain: Lessons learned from hypoxia tolerant extremophilic vertebrates[END_REF]. Mitochondria use glucose or lipids and their metabolites for energy production in form of ATP, which as a side product yields ROS. Neurons in particular have many mitochondria due to their high demand in energy, and are thus more susceptible to ROS. Furthermore the brain has a high proportion of ω-3 polyunsaturated FAs (PUFA) containing phospholipids, which are highly susceptible to lipid peroxidation compared to less saturated lipids [START_REF] Garbarino | Mechanisms of oxidative stress resistance in the brain: Lessons learned from hypoxia tolerant extremophilic vertebrates[END_REF].

Some studies implicate ER stress and oxidative stress as an important component in the initiation of HFD-induced hypothalamic inflammation [START_REF] Özcan | Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes[END_REF][START_REF] Zhang | Hypothalamic IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance and obesity[END_REF][START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF]. A chronic excess of nutrients can lead to cellular stress like ER or oxidative stress, for example through an increase in mitochondrial ATP production, which in turn increases the production of ROS, for example during mitochondrial oxidation of FFAs [START_REF] Tran | Diet-induced cellular neuroinflammation in the hypothalamus: Mechanistic insights from investigation of neurons and microglia[END_REF]. ROS can lead to protein misfolding and thereby add more stress to the ER, which might activate the unfolded protein response (UPR) [START_REF] Özcan | Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes[END_REF][START_REF] Tran | Diet-induced cellular neuroinflammation in the hypothalamus: Mechanistic insights from investigation of neurons and microglia[END_REF].

Another interesting factor that was linked to hypothalamic dysfunction in the context of DIO is mitochondrial abnormalities and changes. It has been found

that changes in the number of mitochondria, their size and the number of contacts they have with ER seems in AgRP and POMC neurons play a role in the development of DIO [START_REF] Dietrich | Mitochondrial Dynamics Controlled by Mitofusins Regulate Agrp Neuronal Activity and Diet-Induced Obesity[END_REF][START_REF] Schneeberger | Mitofusin 2 in POMC neurons connects ER stress with leptin resistance and energy imbalance[END_REF]. Furthermore, it was shown that chronic microglial activation associated with DIO leads to secretion of TNF-a, which in turn induces mitochondrial stress in POMC neurons and thus contributes to obesity [START_REF] Yi | TNFα drives mitochondrial stress in POMC neurons in obesity[END_REF].

Interestingly, studies have found that acute hypertriglyceridemia as well as increased glucose increases mitochondrial respiration and transiently elevates ROS production in the hypothalamus without inducing any cytotoxic effects and rather play a part in hypothalamic nutrient signaling [START_REF] Leloup | Mitochondrial Reactive Oxygen Species Are Required for Hypothalamic Glucose Sensing[END_REF][START_REF] Benani | Role for mitochondrial reactive oxygen species in brain lipid sensing: Redox regulation of food intake[END_REF]. Systemic overload of lipids has not only been shown to promote the activity of hypothalamic neurons and alter their expression of neuropeptides but also hepatic insulin sensitivity. Moreover, it was shown that ICV injections of long-chain FAs has an inhibitory effect on food intake and whole body glucose production while promoting energy storage [START_REF] Obici | Central administration of oleic acid inhibits glucose production and food intake[END_REF][START_REF] Obici | Minireview: Nutrient Sensing and the Regulation of Insulin Action and Energy Balance[END_REF][START_REF] Obici | Minireview: Nutrient Sensing and the Regulation of Insulin Action and Energy Balance[END_REF]. The study by

Benani and colleagues further extended these results by demonstrating that hypothalamic ROS production is required for the reduction of food intake induced by hypertriglyceridemia, while fasting abolished the hypertriglyceridemia-induced ROS production [START_REF] Benani | Role for mitochondrial reactive oxygen species in brain lipid sensing: Redox regulation of food intake[END_REF]. The authors suggest that mitochondrial ROS production might thus be a physiological part of nutrient sensing in hypothalamic neurons [START_REF] Leloup | Mitochondrial Reactive Oxygen Species Are Required for Hypothalamic Glucose Sensing[END_REF][START_REF] Benani | Role for mitochondrial reactive oxygen species in brain lipid sensing: Redox regulation of food intake[END_REF]. It is thus not difficult to imagine that chronic overconsumption of nutrients such as glucose and lipids, could lead to and overproduction of ROS, and overload the cells antioxidant capacities and thus lead to damage induced by ROS, which might affect hypothalamic neurons regulating energy balance in the ARC.

Another interesting study investigated the time course of events in the hypothalamus like inflammation, ER stress and mitochondrial changes associated with HFD. The first event they observed in mice on a HFD after 3 hours was an increase in the hypothalamic expression of the chemokine fractalkine, followed by an inflammatory response characterized by an increase in cytokine expression. Subsequent to these events, they found increases in an ER chaperone after 6 hours although ER stress was not detected until 3 days after HFD. One day after HFD started, reductions in markers for mitochondrial fusion and tethering to ER were found, which however increased days after HFD consumption began [START_REF] Carraro | Hypothalamic mitochondrial abnormalities occur downstream of inflammation in diet-induced obesity[END_REF].

These experiments show that inflammation seems to be the earliest event after HFD consumption and might lead to other cellular event that could be damaging to the cell if HFD consumption persists. However, all events seem to have an important part to play in the development of hypothalamic dysfunction leading up to the development of DIO and associated metabolic impairments. Some studies have also investigated autophagy in this context.

Autophagy is a cellular mechanism that helps maintain homeostasis through the elimination of dysfunctional organelles or proteins but also lipids and can have pathological role during metabolic stress [START_REF] Kim | Autophagy -A key player in cellular and body metabolism[END_REF][START_REF] Rahman | Hypothalamic inflammation and malfunctioning glia in the pathophysiology of obesity and diabetes: Translational significance[END_REF]. Some studies found that HFD consumption can lead to defects in autophagy mechanisms in cells of the hypothalamus, which was associated with hypothalamic inflammation and a change in BW gain different susceptibility to DIO depending on the cell type that is affected [START_REF] Kaushik | Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance[END_REF][START_REF] Kaushik | Loss of autophagy in hypothalamic POMC neurons impairs lipolysis[END_REF][START_REF] Meng | Defective hypothalamic autophagy directs the central pathogenesis of obesity via the IκB kinase β(IKKβ)/NF-κB pathway[END_REF][START_REF] Portovedo | Saturated fatty acids modulate autophagy's proteins in the hypothalamus[END_REF]. It was further shown that inflammatory mediator production in part by microglia could decrease autophagy in neurons while chronic HFD seemed to increase autophagy and decrease the number of hypothalamic POMC neurons [START_REF] Alirezaei | Disruption of neuronal autophagy by infected microglia results in neurodegeneration[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF].

Firstly, it was suggested that elevated circulating FFAs, in particular saturated FAs (SFA), or certain lipids accumulate in the hypothalamus upon HFD consumption and could themselves induce the inflammatory response [START_REF] Posey | Hypothalamic proinflammatory lipid accumulation, inflammation, and insulin resistance in rats fed a high-fat diet[END_REF]. One of the hypothesis stated that hypothalamic neurons themselves could start the inflammatory process in response to sensing elevated FFAs [START_REF] Tran | Diet-induced cellular neuroinflammation in the hypothalamus: Mechanistic insights from investigation of neurons and microglia[END_REF]. Neurons of the hypothalamus can sense FFAs through either expression of TLR4, or FFA metabolism-dependent mechanisms to initiate inflammation [START_REF] Tran | Diet-induced cellular neuroinflammation in the hypothalamus: Mechanistic insights from investigation of neurons and microglia[END_REF]. Interestingly, a study has shown that inhibiting TLR4 or TNF-a via antibodies, pharmacologically or via mutation, abrogated hypothalamic inflammation as well as improved hypothalamic leptin resistance and protected not only from obesity but also abolished signs of liver steatosis and hyperglycemia [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF][START_REF] Milanski | Inhibition of hypothalamic inflammation reverses diet-induced insulin resistance in the liver[END_REF].

One study found that specific FFAs such as long-chain palmitoyl and stearoyl-CoA but not oleoyl-CoA are found accumulated in the hypothalamus in response to HFD and induce hypothalamic inflammation, leptin and insulin resistance [START_REF] Posey | Hypothalamic proinflammatory lipid accumulation, inflammation, and insulin resistance in rats fed a high-fat diet[END_REF]. Similarly, ICV injection of FFAs such as palmitate induced the upregulation of pro-inflammatory cytokines like TNF-a, IL-1β, and IL-6 and activated inflammatory pathways such as JNK, NF-κB and IKKβ in the hypothalamus of rodents. These events resulted in hypothalamic leptin and insulin resistance [START_REF] Arruda | Low-grade hypothalamic inflammation leads to defective thermogenesis, insulin resistance, and impaired insulin secretion[END_REF][START_REF] Cheng | Palmitic acid induces central leptin resistance and impairs hepatic glucose and lipid metabolism in male mice[END_REF]. Central inhibition of IKKB in HFD-fed rats was associated with reduced food intake and improved hypothalamic insulin sensitivity [START_REF] Posey | Hypothalamic proinflammatory lipid accumulation, inflammation, and insulin resistance in rats fed a high-fat diet[END_REF].

Interestingly, another study found that not HFD specifically, but rather overconsumption of single nutrients can induce the activation of inflammatory pathways like IKKβ/NF-κB in the hypothalamus as demonstrated in a model of hyperphagic obese mice on a normal diet, and ICV infusion of glucose or oleic acid into the 3V [START_REF] Zhang | Hypothalamic IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance and obesity[END_REF]. In contrast to other studies, this study also showed that the hypothalamic activation of IKKβ/NF-κB pathway is independent of cytokine upregulation but rather induced via elevated ER stress. Apart from neurons, FAs and their metabolites can induce inflammatory responses in non-neuronal cells like microglia and astrocytes and resident macrophages [START_REF] Lee | Reciprocal modulation of toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids[END_REF][START_REF] Anderson | Stearic acid accumulation in macrophages induces toll-like receptor 4/2-independent inflammation leading to endoplasmic reticulum stress-mediated apoptosis[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF]. Studies have shown that unlike unsaturated, SFAs like palmitic acid, lauric acid and stearic acid can induce inflammatory responses in cultured astrocytes from rat brains, such as the release of cytokines like TNF-a and IL-6 [START_REF] Gupta | Saturated long-chain fatty acids activate inflammatory signaling in astrocytes[END_REF]. However, there seem to be some variations, as another study in mice, did not find inflammatory responses in astrocytes, while another study confirmed no responses for astrocytes but found that microglia became "activated" in mice after palmitic acid treatment. Moreover, it has been found that microglia and resident macrophages react to palmitic and stearic acid with activation of proinflammatory signaling pathways dependent on TLR and lead to impairments in insulin signaling by phosphorylation of protein kinase C (PKC), Akt and insulin receptor substrate (IRS) 1 [START_REF] Lee | Reciprocal modulation of toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids[END_REF][START_REF] Benoit | Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents[END_REF][START_REF] Anderson | Stearic acid accumulation in macrophages induces toll-like receptor 4/2-independent inflammation leading to endoplasmic reticulum stress-mediated apoptosis[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF]. Interestingly, intracellular accumulation of FFA in macrophages can result in activation of inflammatory pathways independent of TLR, which mediate ER stress and ultimately lead to apoptosis. Furthermore, the polarization of macrophages to a proinflammatory state (M1), as was found in infiltrated macrophages in AT of obese mice and humans, renders them more susceptible to ER stress [START_REF] Anderson | Stearic acid accumulation in macrophages induces toll-like receptor 4/2-independent inflammation leading to endoplasmic reticulum stress-mediated apoptosis[END_REF][START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF][START_REF] Chylikova | M1/M2 macrophage polarization in human obese adipose tissue[END_REF].

Fitting to these results studies have suggested that a high ratio of ω-6 to ω-3 ratio is unfavorable and increases the risk of developing obesity [START_REF] Simopoulos | An increase in the Omega-6/Omega-3 fatty acid ratio increases the risk for obesity[END_REF]. While ω-3 PUFAs are considered to have anti-inflammatory properties, the opposite seems to be true for SFAs and ω-6, which are pro-inflammatory [START_REF] Simopoulos | The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases[END_REF][START_REF] Simopoulos | An increase in the Omega-6/Omega-3 fatty acid ratio increases the risk for obesity[END_REF][START_REF] Margioris | Fatty acids and postprandial inflammation[END_REF].

One study reported that SFAs and polyunsaturated ω-3 FAs both had a reciprocal modulating effect on the TLRs and its downstream signaling pathways. While the SFAs like lauric acid induced TLR4 activation, the PUFA docosahexaenoic acid (DHA) inhibited Akt, a downstream signaling component of TLR4, induced by lipopolysachharide (LPS) or lauric acid and suppressed TLR4-dependent NF-κB activation [START_REF] Lee | Reciprocal modulation of toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids[END_REF].

Data has previously shown that the ICV injection of arachidonic acid, an ω-6

PUFA) induces hypothalamic leptin resistance and impairs its downstream signaling regulating hepatic glucose and lipid metabolism [START_REF] Cheng | Arachidonic acid impairs hypothalamic leptin signaling and hepatic energy homeostasis in mice[END_REF].

Moreover, another theory suggests that diet-induced inflammation might be due to HFD causing a shift in the gut microbiota [START_REF] Everard | Responses of gut microbiota and glucose and lipid metabolism to prebiotics in genetic obese and diet-induced leptin-resistant mice[END_REF].

Changes in the composition of gut microbiota populations can in turn cause an increased permeability of the intestines, which enables facilitated entry of bacterial endotoxins like LPS into the blood stream leading to endotoxemia.

Endotoxemia was shown to activate the innate immune system in both humans and rodents in response to HFD (Fig. 5) [START_REF] Cani | Metabolic endotoxemia initiates obesity and insulin resistance[END_REF][START_REF] Cani | Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice[END_REF][START_REF] Pendyala | A high-fat diet is associated with endotoxemia that originates from the gut[END_REF].

Hypothalamic Gliosis

Apart from neurons the brain contains also glia cells that comprise about 50% of the total cells of the brain and can be broadly divided into astrocytes, microglia, tanycytes and oligodendrocytes [START_REF] Azevedo | Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain[END_REF]. Glia cells usually are reported as having a supporting function to neurons, regulate neuronal activity and play a critical role in regulating nutrient uptake and metabolism. In addition to that, microglia are often seen as the immune cells of the CNS [START_REF] Morari | Fractalkine (CX3CL1) is involved in the early activation of hypothalamic inflammation in experimental obesity[END_REF][START_REF] Foll | Hypothalamic Fatty Acids and Ketone Bodies Sensing and Role of FAT/CD36 in the Regulation of Food Intake[END_REF].

The HFD-induced inflammation is also associated with hypothalamic reactive gliosis -the change in glial activity such as astrocytes and microglia into a "reactive phenotype" characterized by a change in morphology, proliferation and an altered gene expression profile [START_REF] Horvath | Synaptic input organization of the melanocortin system predicts diet-induced hypothalamic reactive gliosis and obesity[END_REF]. This reactive state is characterized by the upregulation of cell specific structural proteins such as glial fibrillary acidic protein (GFAP) for astrocytes and ionized calcium binding adaptor molecule 1 (Iba1) for microglia. Furthermore, they produce cytokines such as TNF-a, IL-1β and IL-6 (De Souza et al., 2005) but also chemokines like CXCL1 by microglia [START_REF] Morari | Fractalkine (CX3CL1) is involved in the early activation of hypothalamic inflammation in experimental obesity[END_REF].

Glucose Homeostasis

Glucose belongs to the carbohydrates, which we take up with our nutrition, and is the major source of energy to sustain the functioning of the human organism. While other tissues or cells can use other metabolites like FAs, glucose is the main source of energy for the brain. It is of major importance that glucose levels remain stable throughout day and night, independent of activity, food intake or times of fasting. If plasma glucose levels get too low this can be dangerous, is referred to hypoglycemia, and can lead to apart from cognitive dysfunction, seizures, myocardial infarction, and retinal cell death to coma and death [START_REF] Kalra | Hypoglycemia: The neglected complication[END_REF]. Equally important, hyperglycemia, a condition of chronic elevation of plasma glucose levels can have damaging effects on the functioning of the organism, as seen in diseases such as diabetes, obesity and cardiovascular disease among others.

In healthy individuals, glucose is maintained between 4 to 7mM. To remain in this range glucose metabolism is tightly regulated by different mechanisms including gluconeogenesis (Fig. 6) in the liver, glucose absorption from food in the intestine and glucose uptake and utilization by peripheral tissues [START_REF] Saltiel | Insulin signalling and the regulation of glucose and lipid metabolism[END_REF]. (Adapted from [START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF]).

Central Regulation of Glucose Homeostasis

Glucose is the major source of energy for the brain and can enter the CNS via specific transporters like glucose transporter (GLUT) 1 in the BBB and is regulated based on the energy demand of the brain. GLUT1 is expressed throughout the CNS by astrocytes and endothelial cells of the BBB as well as in tanycytes. Circulating glucose is then taken up by perivascular astrocytes either via GLUT2 or passively through gap junctions and used for glycolysis or glycogenesis or could also represent a signal to inform glucose-sensing neurons about energy availability. Interestingly, several hormones have been reported to regulate glucose uptake in astrocytes, such as an increasing uptake via leptin and insulin-dependent pathway and a ghrelin-induced decrease in glucose uptake [START_REF] García-Cáceres | Astrocytic Insulin Signaling Couples Brain Glucose Uptake with Nutrient Availability[END_REF][START_REF] Freire-Regatillo | Non-neuronal cells in the hypothalamic adaptation to metabolic signals[END_REF]. Alternatively, glucose can alternatively be metabolized to lactate or derived from glycogen metabolism (Fig. 6). Lactate can then be secreted into the extracellular space for neurons to use as energy [START_REF] Freire-Regatillo | Non-neuronal cells in the hypothalamic adaptation to metabolic signals[END_REF]. Some studies suggest that lactate provided by astrocytes is the primary and preferred source of energy for neurons and seems to be neuroprotective in cases of hypoglycemia [START_REF] Wyss | In Vivo Evidence for Lactate as a Neuronal Energy Source[END_REF]. Furthermore, astrocytes store glucose in form of glycogen, which is converted to lactate via glycogenolysis for situations of shortage of nutrients or increased neuronal activity (Fig. 6). Interestingly, several hormones have been reported in increasing glycogen production in astrocytes, like insulin, insulin-like growth factor (IGF)-1 and leptin, while ghrelin seems to induce glycogenolysis in neurons of the hypothalamus [START_REF] Fuente-Martín | Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes[END_REF][START_REF] Freire-Regatillo | Non-neuronal cells in the hypothalamic adaptation to metabolic signals[END_REF]. In cases of prolonged fasting or when glycogen stores are exhausted, astrocytes can take up FAs as alternative source of energy, which undergo βoxidation in mitochondria FAs into ketone bodies like β-hydroxybutyrate.

These are either used themselves or released for neurons or other glia cells to use as energy.

Moreover, special glia cells lining the basal wall of the 3V in the hypothalamus, which are called tanycytes, are in close contact with capillaries and have been reported to sense and metabolize glucose and express GLUT2 [START_REF] Elizondo-Vega | The role of tanycytes in hypothalamic glucosensing[END_REF][START_REF] Barahona | Glial hypothalamic inhibition of GLUT2 expression alters satiety, impacting eating behavior[END_REF]. Interestingly, other glucose transporters like GLUT1, 3 and 5 have been found on astrocytes, neurons and microglia, respectively [START_REF] Pomytkin | Insulin receptor in the brain: Mechanisms of activation and the role in the CNS pathology and treatment[END_REF].

Central glucose sensing is attributed to two types of neurons. Firstly, there are neurons that are excited when glucose levels increase in the extracellular fluid and are mostly found in hypothalamic nuclei like VMH, Arc and PVN.

Secondly, neurons that are inhibited by declining extracellular levels, are located in the LH, ARC and the PVN. Interestingly, the dorsal vagal complex consisting of the NTS, area postrema and the dorsal motor nucleus of the vagus contain both type of glucose-sensing neurons [START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF].

Pancreatic Regulation of Glucose Homeostasis

The pancreas is a complex organ that is subdivided into four continuous parts, a hook-shaped head around the superior mesenteric vein, a neck, body and tail (Fig. 7). It is composed of a system of ducts connecting exocrine glands that regulate digestion by secreting digestive enzymes via a duct, which is fused at the distal end with the common bile duct, into the duodenum (Fig. 7, lower panel). Additionally, it contains endocrine glands called islets of Langerhans, which are involved in controlling nutrient metabolism such as glucose, protein and fat metabolism by secreting hormones into the circulation via the portal vein [START_REF] Cade | The pancreas[END_REF]. Around 1-2 million ovoid-shaped islets are interspersed throughout the pancreas between acini of a healthy human. The pancreas is located in between the duodenum underneath the stomach and connected via the vena cava to the liver and via ducts to the intestine as well as to lymphatic vessels (Fig. 7) [START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF][START_REF] Cade | The pancreas[END_REF]. It is regulated in part by the brain through dense innervations by sympathetic fibres and parasympathetic fibers as well as neurons expressing neuropeptides, which stimulate the release of both amines and peptides. While sympathetic fibers signal through the splanchnic nerves, the parasympathetic fibers transmit information via the vagus nerve to the brain.

The pancreas is subdivided into two functionally distinct glands, the exocrine pancreatic gland and the endocrine pancreatic gland.

The exocrine pancreas represents 99% of the pancreas and constitutes a large ductal system interconnecting a large amount of acini that form lobules of 1-10 mm size and connected by interlobular pancreatic duct. One acinus consists of around 40 pyramid-shaped acinar cells, collage, fibroblast and a tissue surround the acinus including capillaries. Acinar cells contain zymogen granules containing proenzymes, necessary for digestion such as trypsin, chymotrypsin, lipase, amylase and elastase [START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF]. (Adapted from [START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF]).

The endocrine pancreas consists mainly of islets of Langerhans scattered across the pancreas and represents 1% of the whole pancreas. The human pancreatic islets range in size between 50 to 250 µm and are made up of 30% glucagon-producing alpha cells; ~60% insulin-producing Beta cells; 10% somatostatin-producing Delta cells and 1% pancreatic polypeptideproducing PP cells (also known as F cells) [START_REF] Cabrera | The unique cytoarchitecture of human pancreatic islets has implications for islet cell function[END_REF][START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF]. Furthermore, the islets of Langerhans contain ghrelin-producing epsilon cells, which are primarily found during gestational development and can give rise to alpha cells and PP cells, but rarely to beta cells in the adult pancreatic islets [START_REF] Arnes | Ghrelin Expression in the Mouse Pancreas Defines a Unique Multipotent Progenitor Population[END_REF]. The pancreas is well irrigated as it contains 10% of the pancreatic blood supply, which allows for the direct access of hormones to blood vessels, which drains into the hepatic portal vein before entering the systemic circulation. Secretion of hormones is tightly regulated through humoral paracrine and neural communication, which is facilitated by an innervation of islets by both sympathetic and parasympathetic nerve fibers.

Furthemore, those innervations control not only blood flow of surrounding vessels but also increase cholinergic and adrenergic stimulation to increase or inhibit insulin secretion [START_REF] Cade | The pancreas[END_REF].

The pancreas is continuously secreting small amount of insulin, which passes the liver before acting on other organs and tissue, and is secreting a larger amount upon carbohydrate stimulation after a meal [START_REF] Katsarou | Type 1 diabetes mellitus[END_REF] 3.2.1. Glucagon Glucagon, a hormone derived from proglucagon precursor by enzymatic cleavage by PC2, is composed of 29 amino acids, produced, and secreted from α-cells of the pancreatic islets in response to declining blood glucose levels. This happens during fasting state as well as during aerobic exercise, but has also been shown to be induced by increased levels of amino acids and the intestinal peptides oxyntomodulin and gastric inhibitory polypeptide (GIP).

Interestingly, oxyntomodulin, like another peptide that has an inhibitory effect on glucagon, such as the intestinal incretin GLP-1, are likewise derived from the proglucagon peptide in the intestine through alternative cleavage by PC1/3 among other enzymes (references in [START_REF] Wewer Albrechtsen | The biology of glucagon and the consequences of hyperglucagonemia[END_REF].

Furthermore, glucagon secretion is also regulated through paracrine actions (intra-islet factors), such as somatostatin, which inhibits glucagon.

It has been shown to not only be a powerful stimulator of insulin secretion, but is highly implicated in glucose metabolism regulation through regulating hepatic glucose production. In a fasting state, when glucose concentration in the blood is low, glucagon stimulates the hepatic production of glucose, termed gluconeogenesis and the biochemical breakdown of glycogen to yield glucose to raise blood glucose levels and keep them stable, and thereby regulates blood glucose homeostasis [START_REF] Wewer Albrechtsen | The biology of glucagon and the consequences of hyperglucagonemia[END_REF]. The subsequent rise in blood glucose levels, in addition to a stimulatory effect of glucagon on insulin secretion, maintains the balance of euglycemia in healthy individuals. It regulates blood glucose homeostasis, by increasing glucose production, termed gluconeogenesis in the liver, as well as glycogenolysis, the biochemical breakdown of glycogen to yield glucose in a fasting state (Albrechtsen et al 2016).

Somatostatin

Somatostatin is a peptide hormone and not only produced in pancreatic δcells but also in neurons of the hypothalamus and other endocrine tissues like the GIT. It inhibits both a -and β-cell activity by binding to one of five inhibitory GPCRs called somatostatin receptors 1-5 and thus reduces the synthesis and release of insulin, glucagon and pancreatic polypeptide (PP) through paracrine action. In addition, somatostatin acts as a hormone by acting on the anterior pituitary and GIT to inhibit growth hormone and GIT hormone activity and decrease gut motility, gastric acid, gastrin secretion and also pancreatic exocrine secretion [START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF][START_REF] Cade | The pancreas[END_REF]. Somatostatin is secreted by macronutrients like glucose, amino acids, and fat-derived ketone bodies, but also by hormones like CCK, gastrin, secretin and insulin in hyperglycemic states to prevent hypoglycemia as well as by glucagon to regulate its own secretion [START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF].

PP Peptide

PP, a hormone produced and secreted by pancreatic PP or F cells in fasting and hypoglycemic conditions or after protein meals, it has an inhibitory action on the secretion of insulin and somatostatin. It also reduces gallbladder contractility, gastrointestinal motility, gastrin acid and pancreatic enzyme secretion [START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF][START_REF] Cade | The pancreas[END_REF].

Insulin

Insulin is an anabolic hormone produced and secreted in a constant fashion and increased in response to elevated plasma glucose levels by pancreatic β-cells. It is stored as hexameric insulin/Zn2+ crystals in secretory granules in β cells (Fig. 8) that fuse with the plasma membrane upon glucose stimulation [START_REF] Plum | The role of insulin receptor signaling in the brain[END_REF][START_REF] Pomytkin | Insulin receptor in the brain: Mechanisms of activation and the role in the CNS pathology and treatment[END_REF]. Insulin is a very important hormone for the regulation of carbohydrate, protein and lipid metabolism. Postprandially, insulin secretion happens in two phases. During the first phase, which lasts about 10 min, the preformed insulin stored in vesicles is released by stimulation of ATP-sensitive K + (K ATP ) channels upon glucose absorption that induces changes in the ratio of ATP/ADP and cell depolarization activating Ca 2+ channels. The entry of calcium then induces exocytosis of insulin granules. The second phase is much longer, can take up to an hour because insulin is newly synthetized, and released along with some remaining preformed insulin [START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF]. Interestingly, other nutrients like amino acids and FFAs as well as hormones and neurotransmitters are also able to trigger or potentiate insulin release but most likely through different mechanisms that lead to changes in the electrical activity of β-cells. The latter are glucose dependent and involve the activation of the parasympathetic nervous system. For example at the level of oral taste buds, that signal glucose to the brainstem; or through vagal afferent neurons connected to the portal vein of the liver that signal to the brainstem and hypothalamus;

and by direct glucose-sensing by neurons in the hypothalamus and respond by regulating β-cell activity [START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF].

Insulin Receptor Expression

Insulin can bind to IR, of which two isoforms are known until now, known as the short isoform IR-A and the long isoform IR-B. They are expressed either in cells of the brain or in adult peripheral tissues, respectively. Interestingly, neurons express exclusively IR-A, which binds, in addition to high affinity binding to insulin, IGF-2 that can activate it even at low concentrations [START_REF] Pomytkin | Insulin receptor in the brain: Mechanisms of activation and the role in the CNS pathology and treatment[END_REF]. Astrocytes however seem to express both isoforms of IR. IR is expressed in several regions of the brain and the highest expression is found in the olfactory bulb, the hypothalamus, cerebral cortex, cerebellum and hippocampus [START_REF] Plum | The role of insulin receptor signaling in the brain[END_REF][START_REF] Pomytkin | Insulin receptor in the brain: Mechanisms of activation and the role in the CNS pathology and treatment[END_REF].

Interestingly, IGF-2, which can bind also other receptors IGF-1R and IGF-2R, is also widely expressed throughout the brain like the hippocampus, is upregulated in situations of cellular stress such as acute hypoxia, chronic stress, cerebral ischemia and exposure to toxicity. IGF-2 seems to confer neuroprotection in these stressful scenarios [START_REF] Plum | The role of insulin receptor signaling in the brain[END_REF][START_REF] Pomytkin | Insulin receptor in the brain: Mechanisms of activation and the role in the CNS pathology and treatment[END_REF].

IRs are heterotetramers consisting of two a-subunits and two β-subunits, which are linked via disulfide bonds. While the extracellular a-subunits are responsible for ligand binding, the intracellular part of the transmembrane domain of the β-subunits are the sites of tyrosine kinase activity.

Additionally, it is also expressed in axons and especially in nodes of Ranvier in sensory neurons in dorsal root ganglia (DRG) [START_REF] Feldman | Diabetic neuropathy[END_REF].

Peripheral Insulin Action

It is a major regulator of glucose homeostasis and metabolism through a multitude of effects. Firstly, when plasma glucose levels rise postprandially, insulin is secreted from pancreatic β cell granules into the circulation, enters the liver via the portal vein in a pulsatile manner and is cleared there by 50%

via degradation, before it gets to the heart and is distributed to the periphery via arterial circulation (Fig. 8B andC) [START_REF] Tokarz | The cell biology of systemic insulin function[END_REF].

Insulin then can pass through the liver a second time via arterial circulation to regulate metabolic function. In the liver, the interaction of insulin with the IR activates the insulin receptor tyrosine kinase (IRTK) which induces the phosphorylation of IRS 1 or 2 that act through Akt to activate glycogen synthase (GS) to convert glucose to the storage form glycogen (Fig. 9).

Likewise, the increased intracellular amount of glucose inhibits glucogenolysis and is processed via glycolysis and used for de novo lipogenesis (DNL). This process is facilitated by sterol regulatory element-binding protein (SREBP1), whose expression is upregulated through insulin signaling (Fig. 9). Furthermore, Akt phosphorylation inhibits the transcription factor called factor Forkhead box O1 (FOXO1) and thereby downregulates the transcription of enzymes needed for gluconeogenesis [START_REF] Samuel | The pathogenesis of insulin resistance: Integrating signaling pathways and substrate flux[END_REF]. In addition to that, the liver takes up plasma FAs, which are absorbed after food intake or from chylomicron remnants that are taken up from the circulation and uses them for lipogenesis (Fig. 8C).

The microvasculature and arterial circulation transmits insulin to muscle cells, AT and the brain (Fig. 8C). In skeletal muscles and AT, activation of insulin signaling leads to a translocation of vesicle-stored glucose transporters (GLUT:

GLUT4 in AT and muscles; GLUT2 in liver, kidney, intestine, pancreas and neuronal and glial cells of the CNS [START_REF] Thorens | GLUT2, glucose sensing and glucose homeostasis[END_REF]) via the canonical Insulin signaling pathway to PI3K and Akt and downstream factors, to the plasma membrane to enable glucose uptake (Fig. 89) [START_REF] Samuel | The pathogenesis of insulin resistance: Integrating signaling pathways and substrate flux[END_REF][START_REF] Tokarz | The cell biology of systemic insulin function[END_REF]. Additionally, insulin signaling also activates GS to promote glycogen synthesis. However, glucose is not the only energy source of skeletal muscle as it also takes up FAs for oxidation.

AT is the primary location for energy storage in form of TG. Similar to skeletal muscles, insulin action in AT leads to increased glucose uptake via GLUT4 and insulin signaling via IRS and Akt inhibit lipolysis. However, glucose uptake and utilization is lower in AT then in muscles or brain and is converted to glyceride as a form of storage of excess glucose. Once insulin exerted its action in the target tissues, it is slowly internalized and degraded in the lysosome.

Finally, potentially remaining insulin is then degraded by the kidney within 30 min of release by the pancreas (Fig. 8) [START_REF] Tokarz | The cell biology of systemic insulin function[END_REF].

Insulin can also interact with endothelial cells of the vasculature to induce vasodilation to facilitate better substrate distribution via IR signaling which phosphorylates IRS2 and in turn activates Akt, which phosphorylates endothelial nitric oxide (NO) synthase (eNOS) to produce NO. NO then acts on the smooth muscle cell layer surrounding the blood vessels to induce vasorelaxation via cyclic guanosine monophosphate [START_REF] Tokarz | The cell biology of systemic insulin function[END_REF]. (Adapted from [START_REF] Samuel | The pathogenesis of insulin resistance: Integrating signaling pathways and substrate flux[END_REF]).

Central Insulin Action

Insulin exerts its effect not only in the periphery but has also important functions in the brain, such as in regulating glucose metabolism, food intake, sympathetic activity, neuroprotection and synaptic plasticity, reproductive endocrinology as well as hepatic gluconeogenesis inhibition [START_REF] Plum | The role of insulin receptor signaling in the brain[END_REF][START_REF] Pomytkin | Insulin receptor in the brain: Mechanisms of activation and the role in the CNS pathology and treatment[END_REF].

It uses receptor-mediated transport to transfer across the BBB, which is altered under certain conditions. For example, while fasting, obesity aging and certain medication decrease the transport of insulin, it is increased in some models of diabetes mellitus and in the neonatal period [START_REF] Plum | The role of insulin receptor signaling in the brain[END_REF].

Central insulin administration inhibits food intake, reduces of BW, while inhibition of insulin signaling increases food intake and BW and results in insulin resistance. Furthermore, ICV infusion of insulin decreases expression of orexigenic neuropeptide NPY in the ARC, upregulates a-MSH and consequently increases the anorexigenic expression of CRH in the PVN [START_REF] Plum | The role of insulin receptor signaling in the brain[END_REF].

Interestingly, insulin shares a signaling pathway with leptin via PI3K signaling and both activate hypothalamic K ATP channels in this way to hyperpolarize and inhibit glucose-sensitive neurons and thereby modulate glucose metabolism and potentially food consumption and BW (Fig. 10) [START_REF] Plum | The role of insulin receptor signaling in the brain[END_REF]. Insulin was further implicated in the modulation of reward pathways as it was shown to directly activate neurons of the mesolimbic dopamine-mediated pathway and might thus affect motivation to eat or its hedonic attributes [START_REF] Sipols | Insulin and raclopride combine to decrease short-term intake of sucrose solutions[END_REF]. (Adapted from [START_REF] Morton | Central nervous system control of food intake and body weight[END_REF]).

For example, it was shown that ICV infusion of insulin into the 3V suppressed hepatic glucose production, while central administration of insulin signaling blockers in presence of insulin blocked this insulin effect on hepatic glucose production [START_REF] Obici | Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats[END_REF][START_REF] Obici | Hypothalamic insulin signaling is required for inhibition of glucose production[END_REF].

Similarly, overexpression of actors of the insulin-signaling pathway like IRS2 and Akt in the hypothalamus promoted glucose-decreasing effects in diabetic rats [START_REF] Gelling | Insulin action in the brain contributes to glucose lowering during insulin treatment of diabetes[END_REF]. Interestingly, IR decrease in the hypothalamus (mostly observed in the ARC) was associated with a rapid onset hyperphagia and increase in fat mass [START_REF] Obici | Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats[END_REF]. The central effects of insulin seem to be mediated through an K ATP sensitive channel in hypothalamic neurons as well as via vagal efferent fibers, which constitute the connection of the brain-liver axis [START_REF] Spanswick | Insulin activates ATP-sensitive K+ channels in hypothalamic neurons of lean, but not obese rats[END_REF][START_REF] Roh | Emerging role of the brain in the homeostatic regulation of energy and glucose metabolism[END_REF]. Moreover, ICV administration of insulin leads to an increase in hepatic IL-6 expression, which in turn is associated with activation of STAT3 signaling. This results in inhibition of gluconeogenic gene expression via FOXO1 blocking by STAT3 in the liver.

The IR is expressed more specifically on MCH neurons in the LH of the hypothalamus as well as on NPY/AgRP-and POMC-expressing neurons of the ARC [START_REF] Obici | Hypothalamic insulin signaling is required for inhibition of glucose production[END_REF][START_REF] Hausen | Insulin-Dependent Activation of MCH Neurons Impairs Locomotor Activity and Insulin Sensitivity in Obesity[END_REF]. Previous studies have

shown that impairment of IR or IRS activity in the hypothalamus through different methods can lead to insulin resistance and glucose intolerance [START_REF] Withers | Disruption of IRS-2 causes type 2 diabetes in mice[END_REF][START_REF] Obici | Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats[END_REF][START_REF] Kubota | Insulin receptor substrate 2 plays a crucial role in β cells and the hypothalamus[END_REF][START_REF] Lin | Dysregulation of insulin receptor substrate 2 in β cells and brain causes obesity and diabetes[END_REF][START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF].

Gut Hormones: Incretins

Incretins are intestinal-derived humoral or neural factors capable of potentiating insulin secretion from β-cells after oral food ingestion. Two wellknown incretins are GLP-1 and GIP. Both GLP-1 and GIP are released by enteroendocrine cells in the small intestine within minutes upon food ingestion and increase glucose-dependent insulin secretion [START_REF] Nauck | Additive insulinotropic effects of exogenous synthetic human gastric inhibitory polypeptide and glucagon-like peptide-1-(7-36) amide infused at nearphysiological insulinotropic hormone and glucose concentrations[END_REF].

Incretins are responsible for about 50-70% of insulin secretion after glucosecontaining food consumption [START_REF] Baggio | Biology of Incretins: GLP-1 and GIP[END_REF].

GLP-1 is another hormone involved in glucose homeostasis and is produced from the proglucagon gene by alternative posttranslational processing and secreted by L-cells in the GIT in a postprandial manner but also in neurons of the NTS. It is both decreasing glucagon secretion and stimulating the second phase of insulin secretion as well as enhancing β-cell growth and survival [START_REF] Faulconbridge | Regulation of energy balance and body weight by the brain: a distributed system prone to disruption[END_REF][START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF]. Furthermore it was shown to suppress food intake and delay gastric emptying [START_REF] Faulconbridge | Regulation of energy balance and body weight by the brain: a distributed system prone to disruption[END_REF][START_REF] Pais | RANTES (CCL5) reduces glucose-dependent secretion of glucagon-like peptides 1 and 2 and impairs glucose-induced insulin secretion in mice[END_REF]. GLP-1 signal through the GPCR GLP-1R to exert it's action, which is expressed throughout the brain including the hypothalamus and brainstem, and on vagal afferent neurons in the GIT, portal vein, liver and in pancreatic islet cells [START_REF] Baggio | Biology of Incretins: GLP-1 and GIP[END_REF][START_REF] Faulconbridge | Regulation of energy balance and body weight by the brain: a distributed system prone to disruption[END_REF].

GIP, a 42 amino acid peptide, is produced in cells of the stomach and K cells of the duodenum, but was found to be expressed in the CNS, too [START_REF] Drucker | The biology of incretin hormones[END_REF]. In addition to that, GIP acts on AT and bones to promote energy storage and bone formation, respectively [START_REF] Baggio | Biology of Incretins: GLP-1 and GIP[END_REF]. GIP is secreted upon glucose or fat ingestion and like GLP-1 acts as an incretin to potentiate glucose-stimulated insulin secretion. GIP signals through the GIP receptor, which is also a GPCR and expressed in the pancreas, stomach, small intestine, AT and some parts of the brain among other tissues.

Interestingly, GIP is also implicated in AT function and the control of lipid metabolism by stimulating FA synthesis, re-esterification and insulin-induced incorporation into TG as well as decreasing lipolysis upon glucagon stimulation and thus plays a role in the development of obesity. Both incretins are degraded and thus regulated by dipeptidyl peptidase 4 (DPP4) [START_REF] Baggio | Biology of Incretins: GLP-1 and GIP[END_REF]. Thus, both present attractive potential therapeutical targets for diabetes mellitus.

Diabetes Mellitus (DM)

DM is an umbrella term for metabolic disorders that are united by a common As DM is a complex and heterogeneous group of metabolic disorders, many factors play into the etiology of the diseases such as genetic predisposition, epigenetic processes, auto-immunity, inflammation and environmental factors. Furthermore, other factors such as certain medication or chemicals and virus infections can induce or exacerbate diabetes and its symptoms [START_REF] Forrest | HIGH FREQUENCY OF DIABETES MELLITUS IN YOUNG ADULTS WITH CONGENITAL RUBELLA[END_REF][START_REF] Karjalainen | Relationship between serum insulin autoantibodies, islet cell antibodies and Coxsackie-B4 and mumps virus-specific antibodies at the clinical manifestation of type 1 (insulin-dependent) diabetes[END_REF][START_REF] Pandit | Drug-induced disorders of glucose tolerance[END_REF][START_REF] Esposti | Inhibition of mitochondrial complex I may account for IDDM induced by intoxication with the rodenticide vacor[END_REF].

Although many advances were made in the understanding of diabetes, a lot of knowledge is still missing to decipher the exact mechanism and develop effective cures or treatment options (International Diabetes Federation, 2017).

Independent of type, apart from hyperglycemia, DM is accompanied by symptoms such as excessive urine production, compensatory thirst and fluid intake, blurred vision as well as lack of energy, tiredness, unexplained weight loss and abnormal modifications in energy metabolism including carbohydrate, lipid and protein metabolism. The latter is most likely due to the anabolic actions of insulin action, which are impaired in DM and thus can't induce the adequate response in metabolic target tissues [START_REF] Lin | Current views on type 2 diabetes[END_REF][START_REF] Kharroubi | Diabetes mellitus: The epidemic of the century[END_REF]International Diabetes Federation, 2017).

The T1DM, previously known as juvenile diabetes, but with the increasing prevalence of T1DM with adult onset, this term is not justified anymore, is the least frequent form of DM with a prevalence of around 10% worldwide.

However, compared to T2DM the onset of disease is in younger patients.

While it is a chronic autoimmune form of the disease, causing the production of autoantibodies that destroy β-cells of the endocrine pancreas and result in insulin deficiency and eventually in hyperglycemia, a subtype with less than 10% of T1DM cases shows no signs of autoimmunity and the onset seems rather idiopathic [START_REF] Paschou | On type 1 diabetes mellitus pathogenesis[END_REF]. Several factors such as genetic, environmental and immunological factors can play into the pathogenesis of the disease. The major genetic alleles that are responsible for 15% of the genetic predisposition of T1DM are with 40% the gene region for major histocompatibility complex (MHC), also called human leucocyte antigen, polymorphisms in the insulin gene (ins-VNTR, IDDM 2) and cytotoxic T lymphocyte-associated antigen-4 gene (CTLA-4) [START_REF] Paschou | On type 1 diabetes mellitus pathogenesis[END_REF].

The environmental factors that represent a trigger for pathogenesis of T1DM include viruses, toxins and nutrients.

The autoantibodies produced by lymphocytes, which most likely escape the selection process of T cells and B cells during their production and maturation in thymus and bone marrow, respectively, occur early in the pathogenesis of patients with T1DM, when they are still asymptomatic and euglycemic (normal concentration of blood glucose). The most common autoantibodies associated with T1DM pathogenesis are antibodies targeting insulin, 65kDa glutamic acid decarboxylase/glutamate decarboxylase (GAD65), insulinoma-associated protein 2 (IA-2) and zinc transporter 8 (ZNT8) and can serve as biomarkers for diagnosis of the development of T1DM [START_REF] Katsarou | Type 1 diabetes mellitus[END_REF]. Interestingly, some autoantibodies like GAD65 and for insulin usually appear earlier, while the others tend to be detected later on during disease progression. They are used to separate the pathogenesis of the disorder into three different stages (Fig. 12). (Adapted from [START_REF] Katsarou | Type 1 diabetes mellitus[END_REF]).

This form of diabetes that is predominantly found in children and adolescents, is characterized by autoantibodies, β-cell destruction and an absence of insulin secretion [START_REF] Kharroubi | Diabetes mellitus: The epidemic of the century[END_REF]. Unfortunately, the mechanism leading to β-cell destruction and disease initiation as well as the role of the autoantibodies is not deciphered yet.

However, the theory suggests that the first stage of T1DM is characterized by a detection of serum autoantibodies, most commonly in early stages they are targeting GAD65 and insulin. However, patients in stage 1 are presymptomatic and have still normal glucose levels. Autoantibodies are often found months or years before β-cell damage occurs in stage 1. It was suggested that the cognate interaction between T and B cells might induce the production of autoantibodies against islet cell antigens, that might lead to β-cell damage (Fig. 13) [START_REF] Wong | How does β-cell tolerance contribute to the protective effects of diabetes following induced mixed chimerism in autoimmune diabetes?[END_REF]. Interestingly, certain autoantibody occurrence is associated with patients having certain haplotypes in the gene for human leukocyte antigen. The progression to stage 2 is characterized by a progressive destruction of pancreatic insulin-secreting β-cells that leads to hyperglycemia, although patients still remain asymptomatic. It is assumed that β-cell destruction is mediated through immune cell infiltration and action of T cells and B cells that induce an inflammatory reaction that can result in insulitis or pre-insulitis (Fig. 13) [START_REF] Kharroubi | Diabetes mellitus: The epidemic of the century[END_REF][START_REF] Katsarou | Type 1 diabetes mellitus[END_REF]. Recent evidence suggests that T cells preferably recognize β-cell peptides with post-translational modifications which are believed to be caused by cellular stress and thus lead to loss of tolerance to β-cell autoantigens [START_REF] Yang | CD4+ T cells recognize diverse epitopes within GAD65: Implications for repertoire development and diabetes monitoring[END_REF][START_REF] Van Lummel | Posttranslational modification of HLA-DQ binding islet autoantigens in type 1 diabetes[END_REF]. Interestingly, nonobese diabetic (NOD) mouse models of diabetes show no autoantibody production, which might be due to differences in the immune system and pathogenesis compared to humans. However, NOD mice display inflammation in and around the islets in the pancreas, known as insulitis and peri-insulitis, respectively. This occurs before mice present themselves with hyperglycemia and is associated with an infiltration of immune cells into pancreatic islets. Furthermore, it was reported that β-cells of NOD mice also show perturbation in ER UPR that were linked to β-cell destruction and T1DM phenotype [START_REF] Engin | Restoration of the unfolded protein response in pancreatic β cells protects mice against type 1 diabetes[END_REF][START_REF] Magnuson | Population dynamics of islet-infiltrating cells in autoimmune diabetes[END_REF]. T1DM is an autoimmune-mediated disease that is initiated upon antigenpresentation of DC and activated B cells that interact with and activate CD4+ and CD8+ T cells. Furthermore, contact with β-cell autoantigens drives the production of autoantibodies against islet peptides. Activated T cells can infiltrate the islets and lead to loss of β-cells due to damage and destruction. (Adapted from [START_REF] Katsarou | Type 1 diabetes mellitus[END_REF]).

Upon diagnosis, careful blood-glucose monitoring is required in addition to subcutaneous administration of insulin with the aim to achieve close to normal physiological glycemia, while avoiding hyperglycemia, hypoglycemia and ketoacidosis. Ketoacidosis occurs in some diabetic patients as a metabolic decompensation, when the insulin deficit leads to an increase in serum glycogen levels and a decrease of glucose utilization by metabolic tissues and once the liver glycogen stores are depleted, the body turns to ketones as alternative fuel derived from fat [START_REF] Fedorovich | Ketogenic diet versus ketoacidosis: what determines the influence of ketone bodies on neurons?[END_REF]. Ketone bodies produced by lipid degradation accumulate in the blood as ketone production is faster than its utilization and eventually overpowers the kidneys buffer capacity of the pH leading to metabolic acidosis, which in turn can lead to neuronal damage and coma or even death, if not treated [START_REF] Fedorovich | Ketogenic diet versus ketoacidosis: what determines the influence of ketone bodies on neurons?[END_REF]. Rigorous blood-glucose was shown to reduce HbA1c levels and reduce both microand microvascular complications in T1DM patients [START_REF] Nathan | The diabetes control and complications trial/epidemiology of diabetes interventions and complications study at 30 years: Overview[END_REF].

Hypoglycemia, like hyperglycemia can have a negative impact on health, as frequent hypoglycemia after insulin injections can result in a shortage of glucose by the CNS and peripheral nervous system (PNS) and neuronal cell death [START_REF] Auer | The distribution of hypoglycemic brain damage[END_REF][START_REF] Mohseni | Neurologic damage in hypoglycemia[END_REF][START_REF] Auer | Hypoglycemic brain damage', in Acute Neuronal Injury: The Role of Excitotoxic Programmed Cell Death Mechanisms: Second Edition[END_REF]. However, it also leads to fear and anxiety, which in turn affects glucose regulation and reduces the quality of life of not only the patient but also caregivers and family of the patient [START_REF] Katsarou | Type 1 diabetes mellitus[END_REF].

Type 2 Diabetes Mellitus (T2DM)

T2DM is the most common form that represents more than 90% of all diabetes cases (International Diabetes Federation, 2017) and occurs mostly in adults. In fact, incidence of T2DM increases with age and with the onset of hyperglycemia. Because it is closely linked with obesity, which is one of the most common risk factors leading to T2DM, I will be focusing on T2DM in this project. Along with obesity is has become a major global public health issue [START_REF] Defronzo | Type 2 diabetes mellitus[END_REF]. Other major risk factors for developing diabetes are age, family history, unhealthy lifestyle like poor diet and physical inactivity, smoking, increased adiposity and a history of GDM or IFG or IGT, which is indicative of a prediabetic state that, if not reversed through medication or lifestyle changes can progress into T2DM. T2DM occurs slightly more frequent in males than females as well as adults compared to children and adolescent. However, with the increase in obese cases and unhealthy sedentary lifestyle changes and increasingly poor diet, among younger adults and children, the incidence of T2DM also increases in this population (International Diabetes Federation, 2017). Similar to T1DM it is characterized by hyperglycemia, a progressive deficiency in insulin secretion of β-cells but also insulin resistance, which occurs in metabolic tissues such as skeletal muscle, liver and AT and worsens the hyperglycemia [START_REF] Defronzo | Type 2 diabetes mellitus[END_REF]. Due to the ineffectiveness of insulin, the body tries to compensate the rising glucose levels with a higher insulin production and secretion (hyperinsulinemia), but over times this overpowers the pancreas and becomes inadequate in light of increasing deficiency of β-cells and peripheral insulin resistance (International Diabetes Federation, 2017).

Different Stages of T2DM

Diabetes pathogenesis and development can be subdivided into different stages as proposed by [START_REF] Weir | Five of stages of evolving β-cell dysfunction during progression to diabetes[END_REF][START_REF] Weir | Five of stages of evolving β-cell dysfunction during progression to diabetes[END_REF][START_REF] Weir | Five of stages of evolving β-cell dysfunction during progression to diabetes[END_REF], or simply into prediabetes and diabetes.

The latter distinguishes diabetes from a prediabetic state, which is characterized by increased or impaired IFG, IGT (Fig. 11) or increased glycated hemoglobin A1c (HbA1c) levels (5.7-6.4%), which are increased or impaired compared to normal individuals but below clinical diagnostic values, and presents a high risk of progression to diabetes (Abdul-Ghani, [START_REF] Abdul-Ghani | Contributions of β-cell dysfunction and insulin resistance to the pathogenesis of impaired glucose tolerance and impaired fasting glucose[END_REF][START_REF] Defronzo | Type 2 diabetes mellitus[END_REF]. While, IGF is characterized by hepatic insulin resistance and impaired early or first-phase insulin secretion, people with IGT have rather muscular insulin resistance and impaired postprandial late or second-phase insulin secretion [START_REF] Abdul-Ghani | Contributions of β-cell dysfunction and insulin resistance to the pathogenesis of impaired glucose tolerance and impaired fasting glucose[END_REF][START_REF] Defronzo | Type 2 diabetes mellitus[END_REF].

The first sign of prediabetes tends to be a decline in β-cell function, which can begin as early as 12 years before diagnosis of diabetes and gets worse throughout the progression of disease and in combination with hyperglycemia and the associated glucose toxicity (Fig. 14) [START_REF] Fonseca | Defining and characterizing the progression of type 2 diabetes[END_REF].

This might be due to a higher intake in carbohydrates and fats that lead to higher fat accumulation and increased glucose and FFA levels that puts pressure on β-cells, muscles and liver, that leads to local insulin resistance, while inducing increased glucose production in liver [START_REF] Fonseca | Defining and characterizing the progression of type 2 diabetes[END_REF]. At the same time, β-cells try to compensate for hyperglycemia with increase in cell mass (hypertrophy) and insulin secretion, which leads to further deterioration of β-cells over time and causes progression to T2DM. Interestingly, β-cells dysfunction in pancreatic islets leads to apoptosis of β-cells.

The initial decline in β-cell function is presented as insulin resistance, which increases over time. However, in the beginning, β-cells respond to that with an increase in insulin production and secretion (hyperinsulinemia) to compensate for insulin resistance to keep blood glucose levels normal (Fig. 14).

There are two adaptations that islets adopt in the face of insulin resistance:

They adapt by increasing insulin secretion for each β-cell and/or increase in βcell mass to increase constant output of insulin [START_REF] Fonseca | Defining and characterizing the progression of type 2 diabetes[END_REF]. This is often the case in obese patients, that maintain normal glycemia but present with increased β-cell mass as well as insulin secretion, that does not decrease to baseline levels between meals [START_REF] Fonseca | Defining and characterizing the progression of type 2 diabetes[END_REF].

Interestingly, some obese individuals with prediabetes never progress to diabetes, as their β-cells are able continue to function and maintain normal glucose control while compensating for the insulin resistance with insulin secretion [START_REF] Fonseca | Defining and characterizing the progression of type 2 diabetes[END_REF].

Pathophysiology

The hallmarks of T2DM are hyperglycemia, β-cell loss and failure to secrete insulin as well as insulin resistance and high levels of blood FFAs. Insulin resistance is due to an inefficiency or defective and delayed insulin action or response. In other words, the target cells that express the IR will no longer respond to insulin, because the signal transduction is impaired and thus the target cells fails to take up glucose [START_REF] Lin | Current views on type 2 diabetes[END_REF].

There are several theories about the mechanisms behind these characteristics in T2DM, including inflammation, lipotoxicity, glucotoxicity, mitochondrial dysfunction and ER stress (Fig. 15). One of the proposed mechanisms is local inflammation-induced β-cell dysfunction. Similar to obese patients, a key aspect in the inflammation leading to insulin resistance is infiltration of AT by pro-inflammatory macrophages and T cells (Lumeng and Saltiel, 2011). Pro-inflammatory cytokines and chemokines have been implicated in insulin resistance, because the downstream pathways activated by them such as IKKβ, JNK1 and p38 MAPK can contribute to insulin resistance by either blocking IRS proteins through inducing the production of SOCSs or through phosphorylation of IRS proteins (Fig. 15) [START_REF] Defronzo | Type 2 diabetes mellitus[END_REF]. Furthermore, pharmacological and genetic models of NF-κB pathway invalidation as well as blocking TNF activity lead to an amelioration of insulin sensitivity in mice [START_REF] Uysal | Functional analysis of tumor necrosis factor (TNF) receptors in TNF-α-mediated insulin resistance in genetic obesity[END_REF][START_REF] Arkan | IKK-β links inflammation to obesity-induced insulin resistance[END_REF].

Moreover, increased FAs can activate TLR4, which induces IKK and JNK activity to stimulate the biosynthesis of the sphingolipid ceramide and thereby lead to a phosphorylation of IRS serine residues and inhibit IRS tyrosine phosphorylation and ultimately contribute to impaired insulin sensitivity and signaling (Fig. 1516) [START_REF] Shi | TLR4 links innate immunity and fatty acid-induced insulin resistance[END_REF][START_REF] Chavez | A ceramide-centric view of insulin resistance[END_REF]. This is supported by data showing that TLR4 deficient mice are resistant to DIO and insulin resistance [START_REF] Tsukumo | Loss-of-function mutation in toll-like receptor 4 prevents dietinduced obesity and insulin resistance[END_REF].

Another theory is based on hyperlipidemia induced lipotoxicity [START_REF] Cnop | Fatty acids and glucolipotoxicity in the pathogenesis of Type 2 diabetes[END_REF][START_REF] Giacca | Lipid-induced pancreatic β-cell dysfunction: focus on in vivo studies[END_REF][START_REF] Xiao | Sodium phenylbutyrate, a drug with known capacity to reduce endoplasmic reticulum stress, partially alleviates lipid-induced insulin resistance and β-cell dysfunction in humans[END_REF].

Several tissues, such as muscle, liver and others reflect the pathogenesis and metabolic abnormalities seen in T2DM. According to one of the hypotheses, adiposity and associated lipotoxicity is crucial for the development of insulin resistance and T2DM. Obesity or adiposity is closely linked with the occurrence of T2DM and is a major risk for T2DM. The increased adiposity is reflected by an excessive storage of fat in visceral AT leading to metabolic abnormalities.

When the capacity of AT storages is surpassed, lipids accumulate in ectopic sites, such as muscle, liver and heart among others, which contributes to insulin resistance as well as other metabolic comorbidities such as hepatic steatosis or cardiovascular disorders. Studies report that ectopic lipid accumulation in muscles precedes hyperglycemia and liver insulin resistance and leads to increased fat oxidation, citrate production and induces alterations in insulin-stimulated glucose metabolism such as glucose transport impairment (Fig. 16) [START_REF] Defronzo | Type 2 diabetes mellitus[END_REF][START_REF] Samuel | The pathogenesis of insulin resistance: Integrating signaling pathways and substrate flux[END_REF].

This can be due to abnormalities in mitochondrial oxidative and phosphorylation activity as offspring of T2DM in a study that were healthy, young and lean, but presented with insulin-resistance, had reduced muscle mitochondrial oxidative-phosphorylation activity as well as reduced mitochondrial density [START_REF] Petersen | Impaired Mitochondrial Activity in the Insulin-Resistant Offspring of Patients with Type 2 Diabetes[END_REF][START_REF] Befroy | Impaired mitochondrial substrate oxidation in muscle of insulinresistant offspring of type 2 diabetic patients[END_REF]. Previous studies showed that lipid infusion in rodents and healthy humans, as well as HFD feeding in rodents induced increased muscle diacylglycerol (DAG) content. This in turn activated PKCθ, which inhibits insulin signaling by preventing IRS-1 phosphorylation by IRTK and resulted in muscle insulin resistance [START_REF] Dresner | Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity[END_REF][START_REF] Yu | Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle[END_REF][START_REF] Szendroedi | Role of diacylglycerol activation of PKCθ in lipid-induced muscle insulin resistance in humans[END_REF]. Further support for the important role of muscle insulin resistance in the pathophysiology of T2DM and other metabolic disorders was provided by studies looking at transgenic mice with muscle-specific deletions of IRTK or GLUT4, which results in selective insulin resistance in muscles (Fig. 16). Those mouse models developed diabetes-like phenotypes with secondary glucose toxicity induced impairment in glucose uptake in AT and hepatic glucose production [START_REF] Kim | Glucose toxicity and the development of diabetes in mice with musclespecific inactivation of glut4[END_REF] as well as hepatic steatosis and increased adiposity due to decreased uptake of glucose by muscles and redistribution of glucose to other tissues [START_REF] Kim | Redistribution of substrates to adipose tissue promotes obesity in mice with selective insulin resistance in muscle[END_REF][START_REF] Petersen | The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome[END_REF]. In addition, it raises levels of acetyl-CoA, which drive gluconeogenesis through activation of PC activity and glycerol and ultimately increase hepatic glucose production. This induces hyperglycemia. Hepatic increase in DAG activates PKCε, which inhibits insulin signaling via IRS serine phosphorylation and decrease in glycogen synthesis. (Adapted from [START_REF] Samuel | The pathogenesis of insulin resistance: Integrating signaling pathways and substrate flux[END_REF]).

A recent study by [START_REF] Perry | Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes[END_REF] described another interesting mechanism involving the interaction between WAT and liver to induce hyperglycemia in an attempt to explain how hyperglycemia comes about in T2DM.

Using an in vivo metabolomics approach, they found that WAT lipolysis increases hepatic acetyl CoA concentration by increasing the flux of pyruvate carboxylase (Fig. 16). They thus show that hepatic acetyl CoA is a key regulator mediating insulin effects in the liver, and that insulin can suppress hepatic glucose production via inhibiting lipolysis in WAT and is responsible for mediating the inflammation-induced insulin resistance in the liver of T2DM patients [START_REF] Perry | Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes[END_REF]. In other words, in T2DM insulin loses its ability to inhibit lipolysis in WAT, which mediates hepatic glucose production and thereby leads to hyperglycemia. Interestingly, they found that WAT lipolysis was induced by local IL-6 increase in WAT macrophages in HFD fed rats, which was suggested to be mediated by WAT comparative gene identification-58 protein (CGI-58) [START_REF] Perry | Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes[END_REF].

The other suggested mechanism trying to explain the defects observed in T2DM and associated complications are based on hyperglycemia-induced glucotoxicity [START_REF] Dubois | Glucotoxicity inhibits late steps of insulin exocytosis[END_REF][START_REF] Wallace | Metabolomic analysis of pancreatic beta cells following exposure to high glucose[END_REF][START_REF] Luo | Hyperglycemic Stress and Carbon Stress in Diabetic Glucotoxicity[END_REF]. Chronically elevated glucose levels are toxic and increase the activation of certain pathways, which lead to toxicity and the dysfunction or destruction of different cells.

Under normal physiological conditions, glucose is usually processed by cells via glycolysis and the TCA cycle, however under pathological elevated glucose conditions, pathways with low affinity for glucose, which are usually dormant, are activated. The pathways activated or upregulated by hyperglycemic stress, also known as glucotoxicity, and carbon stress include the polyol pathway, the PKC activation pathway, the hexosamine pathway, the advanced glycation end products (AGE) pathway, and the enediol pathway, all of which ultimately end in oxidative stress (Fig. 17) [START_REF] Bensellam | The molecular mechanisms of pancreatic β-cell glucotoxicity: Recent findings and future research directions[END_REF][START_REF] Luo | Hyperglycemic Stress and Carbon Stress in Diabetic Glucotoxicity[END_REF].

Several in vitro experiments showed that β-cell cultures exposed to chronic high glucose medium presented with defects such as decreased levels of insulin mRNA, insulin content and insulin release, which were reversed in a time-dependent fashion when culture medium was switched back to low glucose-containing medium [START_REF] Robertson | Preservation of insulin mRNA levels and insulin secretion in HIT cells by avoidance of chronic exposure to high glucose concentrations[END_REF][START_REF] Gleason | Determinants of glucose toxicity and its reversibility in the pancreatic islet β-cell line, HIT-T15[END_REF]. It was shown that glucotoxicity seems to act through oxidative stress, because when β-cell lines were again cultured in high glucose-containing medium but supplemented with antioxidants, β-cells were protected from apoptosis and other toxic effects of chronic high glucose exposure [START_REF] Kaneto | Reducing sugars trigger oxidative modification and apoptosis in pancreatic β -cells by provoking oxidative stress through the glycation reaction[END_REF][START_REF] Poitout | Chronic exposure of βTC-6 cells to supraphysiologic concentrations of glucose decreases binding of the RIPE3b1 insulin gene transcription activator[END_REF]. In fact, due to a very low amount of antioxidant enzymes, β-cells appear to be more sensitive to oxidative stress compared to other tissues [START_REF] Grankvist | CuZn-superoxide dismutase, Mnsuperoxide dismutase, catalase and glutathione peroxidase in pancreatic islets and other tissues in the mouse[END_REF][START_REF] Tiedge | Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells[END_REF]. Similarly, the overexpression of antioxidant enzyme, glutathione peroxidase, that catabolizes hydrogen peroxide and lipid peroxides to oxygen and water, protected β-cells from ribose-induced damage [START_REF] Tanaka | A role for glutathione peroxidase in protecting pancreatic cells against oxidative stress in a model of glucose toxicity[END_REF]. Later, studies confirmed these results in in vivo studies using Zucker diabetic fatty (ZDF) rats and db/db mice, which harbor a mutation in the leptin receptor gene and develop both obesity and diabetes. These rodent models were treated with antioxidants, which protected them from elevated ROS and hyperglycemia, compared to placebo-treated mice that had elevated circulating levels of oxidative stress markers [START_REF] Kaneto | Beneficial effects of antioxidants in diabetes: Possible protection of pancreatic β-cells against glucose toxicity[END_REF][START_REF] Tanaka | Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants[END_REF].

Glucotoxicity leads to β-cells becoming sensitized to the increased presence of hyperglycemia, which depletes β-cells of their insulin secretory granules thereby decreasing the secretable amount of insulin in response to glucose [START_REF] Fonseca | Defining and characterizing the progression of type 2 diabetes[END_REF]. It is believed that loss of β-cell can be attributed to apoptosis and dysregulated autophagy [START_REF] Marchetti | The endoplasmic reticulum in pancreatic beta cells of type 2 diabetes patients[END_REF][START_REF] Marchetti | Autophagy and the pancreatic beta-cell in human type 2 diabetes[END_REF].

β-cell dysfunction is associated with ER stress that might be caused by elevated glucose concentrations [START_REF] Marchetti | The endoplasmic reticulum in pancreatic beta cells of type 2 diabetes patients[END_REF] and lipotoxicity [START_REF] Cnop | Fatty acids and glucolipotoxicity in the pathogenesis of Type 2 diabetes[END_REF][START_REF] Giacca | Lipid-induced pancreatic β-cell dysfunction: focus on in vivo studies[END_REF][START_REF] Xiao | Sodium phenylbutyrate, a drug with known capacity to reduce endoplasmic reticulum stress, partially alleviates lipid-induced insulin resistance and β-cell dysfunction in humans[END_REF]. Cultures of isolated islets from diabetes patients showed impaired secretion of insulin in response to glucose as well as a higher threshold for glucose levels to induce insulin secretion. Interestingly, other studies confirmed these results when they showed that prolonged exposure of high levels of glucose of FFAs, especially palmitate, induced similar disturbances in insulin secretion in islets of nondiabetic persons [START_REF] Igoillo-Esteve | Palmitate induces a pro-inflammatory response in human pancreatic islets that mimics CCL2 expression by beta cells in type 2 diabetes[END_REF][START_REF] Xiao | Sodium phenylbutyrate, a drug with known capacity to reduce endoplasmic reticulum stress, partially alleviates lipid-induced insulin resistance and β-cell dysfunction in humans[END_REF]. This shows evidence that lipotoxicity and toxicity of high glucose play important parts in the development of islet dysfunction and T2DM.

Other studies found strong associations between excessive consumption of sugar and fat, whereas others found relations between fat intake and T2DM and some did not find any association [START_REF] Peterson | Sucrose in the diet of diabetic patients -just another carbohydrate?[END_REF] Diabetes can often lead to the development of several complications or comorbidities, affecting multiple organs and tissues, such as eyes, kidnes, heart and nerves. They can be subdivided into macrovascular complications such as, coronary disease, stroke and neuropathy [START_REF] Katsarou | Type 1 diabetes mellitus[END_REF] and microvascular complications like retinopathy [START_REF] Harris | Onset of NIDDM occurs at least 4-7 yr before clinical diagnosis[END_REF] and nephropathy, among others. The risk of developing complications increases with earlier age of onset, delayed diagnosis and the length of duration of T2DM and T1DM but also with certain genes and mutations [START_REF] Kharroubi | Diabetes mellitus: The epidemic of the century[END_REF]. Other severe complications include blindness, renal failure and slow wound healing [START_REF] Lin | Current views on type 2 diabetes[END_REF]. These complications are strongly associated with hyperglycemia and are more likely to occur if hyperglycemia is not tightly controlled [START_REF] Stratton | Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study[END_REF].

One of the most common comorbidity of diabetes is diabetic neuropathy (DN). The rate of incidence of this type of neuropathy among diabetic patients varies and can range between 50% and 90%.

Neuropathic Pain

Neuropathy commonly refers to diseases, injury or dysfunction of nerves and can affect either the CNS such as cranial nerves; the ANS, namely nerves belonging to the involuntary nervous system. Then there is focal neuropathy, which refers to neuropathy restricted to one body part or nerve or group of nerves and peripheral neuropathy, which affects nerves of the peripheral nervous system innervating the extremities.

The International Association for the Study of Pain (IASP) has defined neuropathic pain as "pain caused by a lesion or disease of the somatosensory nervous system" [START_REF] Jensen | A new definition of neuropathic pain[END_REF]; International Association for the Study of Pain, 2017).

The somatosensory system is a system of interconnected neurons of the periphery and CNS and in close contact with the motor system to receive and respond to different stimuli. The somatosensory system can perceive different forms of stimuli, apart from the 5 senses like olfaction, gustatory, vision, hearing and touch, it is also responsible for the perception of pain, temperature, movement, vibration and the position of the body or body parts, termed proprioception. To be able to perceive stimuli, somatosensory nerves or DRG are connected to peripheral tissues like skin, muscles, joints and facia and contain different receptors at their nerve endings that detect specific stimuli and transduce this information into electrical stimulation that is transmitted to and through the dorsal horn of the spinal cord and to the brain.

In the area of the face, the stimulus is detected by nerves called trigeminal nerve ganglia and transmitted directly to the brain. There are different fibers and different receptors that are specific for certain stimuli. For example, there are thermoreceptor containing nerves responsible for detection of different temperature ranges; mechanoreceptors that detect mechanical stimuli such as pressure; chemoreceptors that perceive different molecules such as CO 2 , O 2 or pH; prurireceptors that detect itchy sensations and nociceptors that can perceive noxious stimuli like pain [START_REF] Colloca | Neuropathic pain [Review][END_REF].

Pain is a protective mechanism of the organism in order to protect from potential or further damage or injury and encourages a behavior in aid of wound healing and escaping the dangerous situation that could possible inflict injury.

Nociceptive pain is a protective mechanism that is transmitted through nociceptors, also known as sensory nerve fibers, which are activated by painful stimuli in order to protect us from potential or further tissue injury [START_REF] Basbaum | Cellular and Molecular Mechanisms of Pain[END_REF][START_REF] Fernandes | Cellular and molecular mechanisms driving neuropathic pain: recent advancements and challenges[END_REF]. Tissue damage or injury of peripheral nerves, DRG or parts of the CNS can cause hypersensitivity and ultimately neuropathic pain. Nociceptors can be subdivided into two major classes, the first being myelinated Aδ afferent sensory nerve fibers with a medium diameter, transduce acute, well-localized fast pain. This class includes also Aβ afferent fibers that have a fast conducting speed due to their large diameter and myelination and are activated by mechanical stimuli, such as light touch. The second class of afferent nociceptor fibers consist of C-fibers, small diameter and unmyelinated fibers that respond to poorly localized and slow pain [START_REF] Basbaum | Cellular and Molecular Mechanisms of Pain[END_REF]. Aδ-fibers respond to both mechanical and chemical stimuli, but dependent on the subtype they can also be activated by lower or higher temperature thresholds. Similarly, Cfibers are also heterogeneous and polymodal, and can be further subdivided into subgroups, for example into peptidergic C-fibers, which release neurotransmitters and non-peptidergic C-fibers that express different receptors including GPCRs. There are many more subpopulations of C-fibers, and depending on the population, they convey either heat and mechanical stimuli or chemical stimuli such as itch-producing pruritogens, whereas others mediate non-noxious stimuli like "pleasant touch" or cool temperatures. But they do not respond to heat and mechanical stimuli and thus are not regarded as nociceptors [START_REF] Basbaum | Cellular and Molecular Mechanisms of Pain[END_REF].

Different channels and receptors are present on free nerve endings of different fibers adding to the complexity and providing another potential classification method. These channels include the transient receptor potential cation channel subfamily V Member 1 (TRPV1), which is sensitive to heat, the transient receptor potential cation channel subfamily M member 8 (TRPM8) channel that conveys cold stimuli, acid-sensing ion channels (ASICs), and channels that respond to chemical irritants like the transient receptor potential cation channel subfamily A member 1 (TRPA1) [START_REF] Basbaum | Cellular and Molecular Mechanisms of Pain[END_REF].

The most common cause of neuropathy worldwide is DM and increases with poor intervention and blood glucose control [START_REF] Said | Diabetic neuropathy-a review[END_REF].

Diabetic Neuropathy (DN)

DN is a heterogeneous and complex disorder, which can be a result of nerve injury or damage, secondary to stroke or autoimmune disorders but often occurs as a comorbidity of metabolic disorders such as diabetes. In fact, diabetes is the most common cause of neuropathy worldwide, with an estimated 60% of diabetic patients that will develop peripheral DN and more than $10 billion annual costs of DN alone in the United States [START_REF] O'brien | Mouse models of diabetic neuropathy[END_REF][START_REF] Feldman | Diabetic neuropathy[END_REF]. While, Type 1 and T2DM patients have similar symptoms, they seem to be caused by different pathophysiological mechanisms.

Furthermore, the incidence of neuropathy is higher in patients of T2DM than T1DM , whereas the prevalence is similar and increases with duration of disease and age [START_REF] Feldman | Diabetic neuropathy[END_REF]. Components of metabolic syndrome like obesity, hypertriglyceridemia, hyperglycemia, hypertension, dyslipidemia and prediabetes seem to increase the risk of developing DN and might play a role in the pathogenesis [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF].

Unfortunately, apart from tight control of glycemia, there seems to be no effective treatment for DN. While glucose control works well for T1DM patients to prevent neuropathy complications, it is not as successful for T2DM patients [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF][START_REF] O'brien | Mouse models of diabetic neuropathy[END_REF]. Paradoxically, glucose control can reduce the risk for neuropathies, but the treatment for diabetes and hyperglycemia can also cause neuropathies [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF]. With the progression of neuropathy and implication of more nerve fibers, it will result in hyperalgesia and ultimately the complete loss of sensation [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF]. Furthermore, DPN is associated with impaired wound healing and together with the loss of sensation, it can lead to ulcers and the common comorbidity known as "diabetic foot" that might result in amputation [START_REF] O'brien | Mouse models of diabetic neuropathy[END_REF]. In fact, diabetes is the major cause for lower extremity amputations, which is in turn associated with a 30% risk of death in diabetic patients with neuropathy [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF][START_REF] O'brien | Mouse models of diabetic neuropathy[END_REF]. The most common neuropathy seen in diabetes is diabetic polyneuropathy or length-dependent diabetic polyneuropathy (DPN), defined as the symmetric loss of nerve function of long-distant nerves innervating the outermost limbs such as toes of the feet and fingers of the hands and progressing from distal to proximal parts of the extremities (Fig. 18a). This dysfunction or degeneration of peripheral nerves leads to symptoms such as weakness, tingling, pain and eventual loss of sensation [START_REF] O'brien | Mouse models of diabetic neuropathy[END_REF]. Symptoms depend largely on which nerves and nerve fibers are affected and is not always accompanied by pain. Clinical manifestations of DN can include numbness, tingling, weakness, that spreads in a symmetrical fashion from distal to proximal parts of the body according to the nerve dysfunction pattern as described above, and is often referred to as " stocking and glove distribution" [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF]. The pattern of symptoms can be described by the underlying pathological pattern that usually starts with the degeneration of the small unmyelinated C-fibers, which have a small diameter and thus belong to the slow conducting nerve fibers and are responsible for transduction of temperature and pain. This first step leads to symptoms of hyperalgesia and allodynia. Whereas hyperalgesia is referred to increased sensitivity to painful stimuli, allodynia is when nonpainful stimuli is perceived as painful [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF]. DPN then progresses involving the larger diameter and myelinated nerve fibers leading to loss of ankle reflexes, sensory ataxia and decreased proprioception, which is the awareness of body position and sense of movement [START_REF] Tesfaye | Diabetic neuropathies: Update on definitions, diagnostic criteria, estimation of severity, and treatments[END_REF].

These negative impacts of this disabling disease can lead to secondary effects, such as injuries from falls, due to balance problems, affect patients negatively both physically and mentally, and reduce quality of life [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF].

Loss of C-fibers along with their sensation, which paradoxically can lead to development of painful neuropathy with painful sensation despite the loss of sensation. This is likely due to a different mechanism of detecting different sensory and noxious painful stimuli.

Different fibers exist, that are important in sensory sensation including myelinated A-fibers, and unmyelinated C-fibers.

Once, polyneuropathy is established, it is irreversible and at best remains stable or worsens slowly over time with treatment of diabetes [START_REF] Said | Diabetic neuropathy-a review[END_REF].

Pathophysiological Mechanisms

Despite a great effort in research, the underlying mechanism is still unclear.

However, it was identified that several factors may play a major role in the development of DN like hyperglycemia, visceral adiposity and associated dyslipidemia, impaired insulin signaling and sensitivity of neurons and chronic inflammation associated with obesity [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF]. Apart from those, other factors such as macro-and microvascular dysfunction in nerve and endoneurial perfusion as well as hypertension are potential contributors to DN.

Excess and prolonged hyperglycemia can saturate the traditional metabolic pathways and increase the processing of glucose through alternative pathways that can result in metabolites, which if in abundance, can create cellular damage. These pathways are the polyol-, the hexosamine pathway, and the overload of mitochondrial oxidative phosphorylation that leads to the production of ROS (Fig. 19). Excess of glucose can also result in the increased generation of AGEs, which form reactive attachments to proteins, lipids and nucleic acids and thereby impair their function and cellular maintenance.

Dyslipidemia is a very common comorbidity of T2DM that could lead to DN via several mechanisms. Increased FFAs can either cause direct damage to glia cells like Schwann cells or induce systemic inflammation by stimulating the secretion of cytokines in AT and immune cells. Moreover, the modification of lipoproteins like low-density lipoproteins (LDL) via either oxidation or glycation can trigger signaling cascades through several receptors such as oxLDL receptor Lectin-like oxidized LDL receptor 1 (LOX1), TLR4 and the receptor for advanced glycation end products (RAGE) to induce oxidative stress and inflammatory processes [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF]. Similarly, the oxidation of cholesterol by oxysterols might induce apoptosis in neurons and thereby potentially contribute to DN [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF].

While insulin signaling has been implicated as a neurotrophic factor, impaired insulin signaling such as IR or insulin deficiency has been suggested to play a contributing role to DN through inhibition of the PI3K/Akt pathway in neurons and thereby disrupting mitochondrial function and inducing oxidative stress (Fig. 19) [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF].

Most of these pathways end up inducing oxidative stress through mitochondrial dysfunction, ER stress or inflammatory pathways that result in cellular and DNA damage as well as apoptosis [START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF].

Chemokines

Chemokines were first discovered in 1987, when the cytokine called IL-8 was identified and demonstrated chemotactic ability for neutrophils. It was later called CXCL8 and considered a chemokine in 1992 [START_REF] Yoshimura | Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines[END_REF][START_REF] Rostène | Chemokines: a new class of neuromodulator?[END_REF]. Chemokines represent since a subgroup within the large family of cytokines as cytokines with chemotactic ability and have grown in number as more than 50 chemokines and around 23 chemokine receptors have been identified since [START_REF] Xue | The chemokine system and its role in obesity[END_REF]. They are small heparin-binding proteins ranging from 8 to 14 kDa with up to four conserved cysteine residues that are linked by disulfide bonds, giving rise to the characteristic structure called the "chemokine fold" [START_REF] Bajetto | Characterization of chemokines and their receptors in the central nervous system: physiopathological implications[END_REF][START_REF] Legler | Chemokines: Chemistry, Biochemistry and Biological Function[END_REF]. Chemokines are structurally classified into four subgroups based on the number and position of their first N-terminal cysteine residues: the α-chemokines or CXC group, whose cysteine group is separated by one amino acid, β-chemokines or CC group with adjacent cysteines, γchemokines or C group and the δ-chemokines or CX3C group [START_REF] Bajetto | Characterization of chemokines and their receptors in the central nervous system: physiopathological implications[END_REF][START_REF] Rostène | Chemokines: a new class of neuromodulator?[END_REF].

The α-chemokines and β-chemokines are the largest group of chemokines and are similar in that they both contain four cysteine groups, with the former having an amino acid separating the first cysteine residues, while the latter has two adjacent cysteines. Members of the CXC group can chemoattract neutrophils, NK cells and B and T lymphocytes, while the CC group attracts basophils, eosinophils, DCs, macrophages, monocytes, NK cells and T lymphocytes, but not or only barely neutrophils [START_REF] Mantovani | The chemokine system: redundancy for robust outputs[END_REF][START_REF] Rostène | Chemokines: a new class of neuromodulator?[END_REF].

Unlike the other groups γ-chemokines possess only two of the four conserved cysteine residues and one disulfide bond. They consist only of two members, called XCL1 or lymphotactin-α and XCL2 or lymphotactin-β and attract T lymphocytes [START_REF] Bajetto | Characterization of chemokines and their receptors in the central nervous system: physiopathological implications[END_REF][START_REF] Rostène | Chemokines: a new class of neuromodulator?[END_REF][START_REF] Legler | Chemokines: Chemistry, Biochemistry and Biological Function[END_REF]. These two chemokines bind, according to current knowledge, specifically to XCR1 receptor.

The δ or CX3C group is at present represented by only one chemokine called fractalkine or CX3CL1 and has a characteristic structure, whose N-terminal cysteines are separated by three variable amino acids, in addition to a transmembrane region and a mucin-like domain. It exists in both a secreted soluble form, that is chemoattractive for T lymphocytes, NK cells and monocytes but not neutrophils, and as a membrane-anchored state on activated primary endothelial cells, where it facilitates adhesion of leukocytes [START_REF] Bazan | A new class of membrane-bound chemokine with a CX3C motif[END_REF][START_REF] Mantovani | The chemokine system: redundancy for robust outputs[END_REF][START_REF] Rostène | Chemokines: a new class of neuromodulator?[END_REF].

Structure

Chemokines can be active as monomers as well as homodimers, heterodimers or other forms of higher-order aggregates which can include either one or more chemokine types but also be more complicated by interaction with other molecules such as glycosaminoglycans (GAG) and thereby adding to the complexity of the system of chemokine interaction [START_REF] Miller | Chemokines from a Structural Perspective[END_REF][START_REF] Hughes | A guide to chemokines and their receptors[END_REF].

Monomeric forms of chemokines are fully active, so dimeric or other forms of oligomerization, often supported by heparin sulfate or other sulfated sugars, could elicit different responses within the same receptor as shown for some chemokines. Furthermore, oligomerization especially with other chemokines might enable synergistic effects that have other or more enhanced effects, such as in case of inflammation or infection when many different chemokines are secreted simultaneously [START_REF] Legler | Chemokines: Chemistry, Biochemistry and Biological Function[END_REF]. Despite differences in their sequence identity, all chemokines have a common secondary or tertiary structure, which is given by their cysteine residues and disulfide bonds, and share the resulting chemokine fold [START_REF] Bachelerie | International Union of Pharmacology. LXXXIX. Update on the Extended Family of Chemokine Receptors and Introducing a New Nomenclature for Atypical Chemokine Receptors[END_REF]. A chemokine monomer is usually composed of a flexible but unstructured N-terminus followed by one or more cysteine residues, continued by a long rigid loop and ending in a central three antiparallel-stranded β-sheet, which contains cysteines that are connected to the N-terminal through disulfide bridges. The construct ends with a helical Cterminus overlying the sheets and a short unfolded end (Fig. 20) [START_REF] Bachelerie | International Union of Pharmacology. LXXXIX. Update on the Extended Family of Chemokine Receptors and Introducing a New Nomenclature for Atypical Chemokine Receptors[END_REF][START_REF] Goazigo | Current status of chemokines in the adult CNS[END_REF][START_REF] Legler | Chemokines: Chemistry, Biochemistry and Biological Function[END_REF][START_REF] Hughes | A guide to chemokines and their receptors[END_REF].

The functionally most relevant domains for chemokines seem to be the Nterminus including the rigid loop, which are important for receptor binding, and GAG binding domains within the β-sheets or in the C-terminus [START_REF] Clark-Lewis | Structure-activity relationships of chemokines[END_REF][START_REF] Rossi | The Biology of Chemokines and their Receptors[END_REF][START_REF] Rostène | Chemokines and chemokine receptors: New actors in neuroendocrine regulations[END_REF].

Chemokine Receptors

Chemokines bind and activate heptahelical GPCRs with 7TM domains and thereby mediate their hallmark function to chemoattract leukocytes and polymorphonuclear neutrophils (PMN) [START_REF] Baggiolini | Chemokines and leukocyte traffic[END_REF][START_REF] Moser | Chemokines: multiple levels of leukocyte migration control☆[END_REF].

These receptors are funtionally coupled to heterotrimeric and pertussis-toxin sensitive Gαi proteins [START_REF] Hughes | A guide to chemokines and their receptors[END_REF].

Despite their redundancy in their interaction with promiscuous receptors, chemokines usually associate with receptors of the same class and thus the receptors match the nomenclature of chemokines they bind, followed by the letter R designating "receptor" and a number [START_REF] Mantovani | The chemokine system: redundancy for robust outputs[END_REF][START_REF] Bajetto | Characterization of chemokines and their receptors in the central nervous system: physiopathological implications[END_REF]. Only two monogamous chemokine-receptor interaction exist so far: CXCL12/ stromal cell-derived factor 1 (SDF-1) with CXCR4 and CX3CL1/fraktalkine with CX3CR1 [START_REF] Bajetto | Characterization of chemokines and their receptors in the central nervous system: physiopathological implications[END_REF].

So far five CXC (CXCR1-5), eight CC (CCR1-8), one of CX3C (CX3CR1) and C (XCR1) receptors have been described [START_REF] Mantovani | The chemokine system: redundancy for robust outputs[END_REF][START_REF] Rostène | Chemokines and chemokine receptors: New actors in neuroendocrine regulations[END_REF].

Most chemokine receptors share the following common features: a relatively short, extracellular N-terminus connected with four extracellular loops, that each contain a cysteine residue, in addition to a conserved sequence of amino acids (DRYLAIVHA) in the second intracellular loop, seven TM domains and ending in a C-terminus [START_REF] Rajagopalan | Structural basis of chemokine receptor function-a model for binding affinity and ligand selectivity[END_REF][START_REF] Goazigo | Current status of chemokines in the adult CNS[END_REF]. The C-terminus contains multiple serine and threonine residues that are targets of phosphorylation during receptor-ligand interaction.

Apart from the above-mentioned "conventional" chemokine receptors (cCKRs) chemokines are also able to bind so called atypical chemokine receptors (ACKRs), of which only 4 are known so far: ACKR1 or Duffy antigen receptor for chemokines (DARK), ACKR2 or D6 or chemokine-binding protein 2 (CCBP2), ACKR3 or CXCR7 and ACKR4 or CCRL1 [START_REF] Hughes | A guide to chemokines and their receptors[END_REF].

Although atypical chemokine receptors are similar in structure to cCKRs, they are not coupled to signaling pathways due to modifications in or lack of a part of the conserved region (DRYLAIVHA) in the second intracellular loop that is important for coupling with the G protein [START_REF] Zlotnik | The chemokine superfamily revisited[END_REF].

Thus, instead of exerting the characteristic effects of chemokines such as their chemotaxis for leukocytes, they have rather been reported in chemokine scavenging or as decoy/interceptor receptor and thereby keep chemokine levels low or preventing them to bind to other receptors [START_REF] Zlotnik | The chemokine superfamily revisited[END_REF].

Ligand-Receptor Interaction and Signaling

It was reported that chemokines interact with their receptors mainly with their N-terminus and a region within their exposed loop of the backbone that extends beyond the second cysteine [START_REF] Baggiolini | Chemokines and leukocyte traffic[END_REF].

In general, structure-function studies have suggested the two-step model for chemokine ligand-receptor binding and activation. Simplified, this model states that the first contact between chemokine and its receptor is established between the rigid loop of the ligand and the N-terminus of the cognate receptor, which acts as chemokine recognition site and determines the affinity and specificity of binding [START_REF] Kleist | New paradigms in chemokine receptor signal transduction: moving beyond the two-site model[END_REF][START_REF] Legler | Chemokines: Chemistry, Biochemistry and Biological Function[END_REF]. Subsequently, the N-terminus of the chemokine is inserted into the activation pocket of transmembrane helices of the receptor and induces a conformational change and activation of the latter [START_REF] Crump | Solution structure and basis for functional activity of stromal cellderived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1[END_REF][START_REF] Legler | Chemokines: Chemistry, Biochemistry and Biological Function[END_REF]. This then leads to the signal transduction via the heterotrimeric coupled G protein to exert its manifold actions via different secondary pathways.

Function

Chemokines exert their function through interaction with their receptors and target cells that express those receptors. Each of these interactions is redundant, because several chemokines can bind several chemokine receptors, and vice versa. The same is true for their target cells, although chemokines are diverse in which cells they attract, many chemokines can target similar immune cells, and one cell can express and secrete several chemokines [START_REF] Mantovani | The chemokine system: redundancy for robust outputs[END_REF].

Apart from the structural categorization, chemokines can be further discriminated based on their functional classification in "inflammatory" or inducible, "homeostatic" or constitutive, or in "dual-function" for a few chemokines [START_REF] Rajagopalan | Structural basis of chemokine receptor function-a model for binding affinity and ligand selectivity[END_REF]. Chemokines in the inflammatory category are principally induced and produced during inflammatory conditions and function, mainly in the recruitment of immune cells to target locations upon injury or infection [START_REF] Zlotnik | The chemokine superfamily revisited[END_REF]. For example, chemokines such as CCL3-5 or CCL2 can act on their respective receptors CCR1/CCR5 and CCR2, to regulate the cytolytic activity of NK cells as well as bactericidal protease release upon infection with a pathogen [START_REF] Maghazachi | Role of chemokines in the biology of natural killer cells[END_REF][START_REF] López-Cotarelo | Beyond Chemoattraction: Multifunctionality of Chemokine Receptors in Leukocytes[END_REF]. Interestingly, chemokines can be induced by pro-inflammatory cytokines such as IL-6, IL-1 and TNF-α [START_REF] Kalinkovich | Chemokines and chemokine receptors: Role in HIV infection[END_REF]. Different from that, homeostatic chemokines are rather expressed constitutively in different tissues and aid in immune cells circulation through tissues for immune surveillance and under physiological conditions. For example, CXCL12 acts through CXCR4 in a homeostatic context to regulate hematopoiesis [START_REF] Moser | Chemokines: multiple levels of leukocyte migration control☆[END_REF].

Chemokines in the dual-function category can be both homeostatic and inflammatory [START_REF] Mantovani | The chemokine system: redundancy for robust outputs[END_REF][START_REF] Zlotnik | The chemokine superfamily revisited[END_REF]. Dual-function chemokines can be act in both homeostatic and inflammatory conditions like CCL5 that is acting both as a chemoattractant to recruit macrophages to the site of inflammation for example in obesity, but also can play a role in a homeostatic context by controlling glucose uptake in T cells [START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF][START_REF] Chan | The chemokine CCL5 regulates glucose uptake and AMP kinase signaling in activated T cells to facilitate chemotaxis[END_REF].

The chemokine system is tightly regulated and destabilization of this system can have detrimental effects and result in potential pathological conditions such as development of allergies, autoimmunity, inflammatory diseases and chronic inflammatory conditions and cancer progression and metastasis [START_REF] Rajagopalan | Structural basis of chemokine receptor function-a model for binding affinity and ligand selectivity[END_REF][START_REF] Sahin | Functional role of chemokines in liver disease models[END_REF][START_REF] Roh | Chemokines and Chemokine Receptors in the Development of NAFLD[END_REF].

Chemokines are most famous for their functions in cell migration especially in the pathological context of infection, injury or inflammation, where they regulate leukocyte recruitment and their trafficking to the source of the inflammatory stimuli. In general, invading pathogens or other inflammatory stimuli lead to a local secretion of chemokines, forming a chemokine gradient. Circulating leukocytes can then migrate along this gradient to the source of the gradient and exert a localized and specific effect to contain the infection or inflammation, or promote wound healing and repair. In addition to that, many different cells and tissues throughout the body can express chemokines and their cognate receptor and depending on which cell or tissue they act on it can result in various other responses [START_REF] Stellato | Cutting Edge: Expression of the C-C Chemokine Receptor CCR3 in Human Airway Epithelial Cells[END_REF][START_REF] Cinamon | Follicular shuttling of marginal zone B cells facilitates antigen transport[END_REF][START_REF] Griffin | Chemokine expression and control of muscle cell migration during myogenesis[END_REF][START_REF] Beck | Functional Analysis of the Chemokine Receptor CCR3 on Airway Epithelial Cells[END_REF][START_REF] Kulkarni | Role of chemokine receptors and intestinal epithelial cells in the mucosal inflammation and tolerance[END_REF]. This includes but is not limited to proliferation, activation and differentiation, extracellular matrix remodeling [START_REF] Baggiolini | Chemokines and leukocyte traffic[END_REF][START_REF] Moser | Chemokines: multiple levels of leukocyte migration control☆[END_REF]. Additionally, some studies report a chemorepulsive effect (also called chemofugetaxis) in that some cells can move against the chemokine gradient [START_REF] Tharp | Neutrophil chemorepulsion in defined interleukin-8 gradients in vitro and in vivo[END_REF].

However, chemokines can act also under physiological conditions, such as chemokines belonging to the homeostatic functional group. They are constitutively expressed in different tissues and participate in normal immune surveillance, and cell adhesion, especially in the context of transcellular migration or diapedesis via the interaction with GAG [START_REF] Cinamon | Follicular shuttling of marginal zone B cells facilitates antigen transport[END_REF].

A few other studies have even reported direct microbicidal activity by some chemokines such as CXCL14, CXCL17 and CCL28 [START_REF] Poznansky | Active movement of T cells away from a chemokine[END_REF][START_REF] Poznansky | Thymocyte emigration is mediated by active movement away from stroma-derived factors[END_REF][START_REF] Hieshima | CCL28 Has Dual Roles in Mucosal Immunity as a Chemokine with Broad-Spectrum Antimicrobial Activity[END_REF][START_REF] Ogilvie | Eotaxin-3 is a natural antagonist for CCR2 and exerts a repulsive effect on human monocytes[END_REF][START_REF] Vianello | A CXCR4-dependent chemorepellent signal contributes to the emigration of mature single-positive CD4 cells from the fetal thymus[END_REF][START_REF] Tharp | Neutrophil chemorepulsion in defined interleukin-8 gradients in vitro and in vivo[END_REF][START_REF] Maerki | Potent and broad-spectrum antimicrobial activity of CXCL14 suggests an immediate role in skin infections[END_REF][START_REF] Burkhardt | CXCL17 Is a Mucosal Chemokine Elevated in Idiopathic Pulmonary Fibrosis That Exhibits Broad Antimicrobial Activity[END_REF]. Some chemokines and chemokine receptors are even indispensable for life, such as CXCL12 and the receptors CXCR4 and ACKR3 because they play crucial roles in the development of various organs and cells including the heart, brain, hematopoietic system and vascular system [START_REF] Nagasawa | Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1[END_REF][START_REF] Tachibana | The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract[END_REF][START_REF] Zou | Function of the chemokine receptor CXCR4 in heaematopolesis and in cerebellar development[END_REF][START_REF] Finke | CD4+CD3-cells induce Peyer's patch development: Role of α4β1 integrin activation by CXCR5[END_REF][START_REF] Sierro | Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7[END_REF][START_REF] Valentin | The Chemokine SDF1a Coordinates Tissue Migration through the Spatially Restricted Activation of Cxcr7 and Cxcr4b[END_REF][START_REF] Klein | Decoy receptor CXCR7 modulates adrenomedullin-mediated cardiac and lymphatic vascular development[END_REF].

Moreover, chemokines play important roles in angiogenesis [START_REF] Dimberg | Chemokines in angiogenesis[END_REF], tumor metastasis [START_REF] Müller | Involvement of chemokine receptors in breast cancer metastasis[END_REF] and thereby can contribute to cancer or in some cases have anti-tumor effects (Keeley, Mehrad andStrieter, 2010, 2011;[START_REF] Rezaeeyan | Role of chemokines in metastatic niche: new insights along with a diagnostic and prognostic approach[END_REF].

Besides their well-known role in the immune system, an accumulating body of research seems to increasingly suggest that chemokines might play an important role in the CNS [START_REF] Bajetto | Characterization of chemokines and their receptors in the central nervous system: physiopathological implications[END_REF][START_REF] Rostène | Neurochemokines: A menage a trois providing new insights on the functions of chemokines in the central nervous system[END_REF][START_REF] Goazigo | Current status of chemokines in the adult CNS[END_REF].

Chemokines Function in the CNS

Several different in vivo and in vitro studies have indicated that chemokines and their receptors are expressed in the CNS, not only in pathological conditions but also in a constitutive manner, which will be described more in the following sections.

For chemokine receptors studies reported the expression of all CXC type receptors, all CC type receptors apart from CCR6 and CCR7, and even CX3CR1 in the CNS. Moreover, many studies have described the expression of the respective chemokine ligands, too. It was reported that chemokines and chemokine receptors are expressed by nearly all cell types in the CNS, including different glia cells like astrocytes, oligodendrocytes, microglia but also neurons and endothelial cells (reviewed in [START_REF] Dorf | Astrocytes express functional chemokine receptors[END_REF]). Apart from constitutive expression chemokines have not only been found to play important roles in many inflammatory diseases of the periphery but many studies found their implication in diseases of the CNS, such as experimental autoimmune encephalomyelitis (EAE), Alzheimer's disease (AD), multiple sclerosis (MS), acquired immune deficiency syndrome dementia complex and many other inflammatory brain conditions. Other than that, chemokines were also found to be expressed in normal healthy brains. For example, studies have found that fetal human and murine cells express chemokine receptors CCR5, CCR3 and CXCR4 [START_REF] Klein | Chemokine Receptor Expression and Signaling in Macaque and Human Fetal Neurons and Astrocytes: Implications for the Neuropathogenesis of AIDS[END_REF][START_REF] Croitoru-Lamoury | Expression of chemokines and their receptors in human and simian astrocytes: Evidence for a central role of TNFα and IFNγ in CXCR4 and CCR5 modulation[END_REF].

Chemokine receptors (CXCR4/CCR5) have been found to be expressed in neurons and many other chemokine receptors also in glia cells such as microglia and astrocytes [START_REF] Dorf | Astrocytes express functional chemokine receptors[END_REF][START_REF] Banisadr | Neuroanatomical distribution of CXCR4 in adult rat brain and its localization in cholinergic and dopaminergic neurons[END_REF][START_REF] Westmoreland | Developmental expression patterns of CCR5 and CXCR4 in the rhesus macaque brain[END_REF].

Another three studies used immunofluorescence and confocal microscopy to detect chemokine expression in primary cultures of human embryonic neurons, microglia and astrocytes. They found functional CCR5 and CXCR4 expression and transcript synthesis in cell body and processes of embryonic neurons, while embryonic astrocytes and microglia expressed in addition to CCR5 and CXCR4 also CCR3. While microglia were the only cell that responded to eotaxin or CCL5, a CCR3 agonist, with modulated calcium response, neurons and astrocytes had increased intracellular Ca 2+ levels only upon stimulation with the respective agonist of CCR5 (CCL5 and MIP-1β) and CXCR4 (SDF-1α) [START_REF] Hegg | β-Chemokines and human immunodeficiency virus type-1 proteins evoke intracellular calcium increases in human microglia[END_REF][START_REF] Boutet | Cellular expression of functional chemokine receptor CCR5 and CXCR4 in human embryonic neurons[END_REF][START_REF] Boutet | Isolated human astrocytes are not susceptible to infection by M-and T-tropic HIV-1 strains despite functional expression of the chemokine receptors CCR5 and CXR4[END_REF]. Moreover, in 2005, Omari and colleagues found chemokine receptors CXCR1-3 expressed on oligodendrocytes in the adult human CNS [START_REF] Omari | CXC chemokine receptors on human oligodendrocytes: Implications for multiple sclerosis[END_REF]. Chemokines were not only expressed but also involved in brain development. For example, CXCR4 is not only a coreceptor for viral entry of HIV-1, but also was found to be involved in neuronal cell migration and patterning during cerebellar development [START_REF] Zou | Function of the chemokine receptor CXCR4 in heaematopolesis and in cerebellar development[END_REF][START_REF] Tran | HIV-1, chemokines and neurogenesis[END_REF].

Chemokine (BNDF and SDF-1) signaling through CXCR4 and CCL5 signaling was shown to be neuroprotective and promoting neuronal survival [START_REF] Bachis | Brain-derived neurotrophic factor inhibits human immunodeficiency virus-1/gp120-mediated cerebellar granule cell death by preventing gp120 internalization[END_REF][START_REF] Imitola | Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1 /CXC chemokine receptor 4 pathway[END_REF][START_REF] Bachis | Brain-derived neurotrophic factor is neuroprotective against human immunodeficiency virus-1 envelope proteins[END_REF][START_REF] Nosheny | Brain-derived neurotrophic factor as a prototype neuroprotective factor against HIV-1-associated neuronal degeneration[END_REF][START_REF] Kaul | HIV-1 coreceptors CCR5 and CXCR4 both mediate neuronal cell death but CCR5 paradoxically can also contribute to protection[END_REF].

Interestingly, chemokines have been also been implicated in the regulation of energy balance. For example, the chemokine CCL2 has been recently shown to mediate the LPS-induced anorexic effect in the hypothalamus by modulating the activity of MCH neurons and thereby leading to weight loss (Le Thuc et al., 2016).

Role of Chemokines in Obesity and Comorbidities

Chronic Inflammation is a common factor, and plays an important role in the etiology of several diseases such as obesity and associated comorbidities such as type 2 diabetes and neuropathic pain, metabolic syndrome, cardiovascular disease and non-alcoholic fatty liver disease (NAFLD) and liver steatosis [START_REF] Esser | Inflammation as a link between obesity, metabolic syndrome and type 2 diabetes[END_REF].

Although a multifactorial condition, obesity is often caused by environmental factors such as a diet rich in saturated fats and sugars in addition to a sedentary life-style. Although the exact mechanism behind the etiology of obesity is not quite clear, several studies have identified some characteristics of this pathology.

Obesity is a complex condition caused by interaction of different factors and is often associated with chronic low-grade inflammation and a secretion of pro-inflammatory cytokines and chemokines. The cytokines most often associated and studied in relation to obesity are IL-1β, IL-6 and TNF-α [START_REF] Hotamisligil | Adipose expression of tumor necrosis factor-α: Direct role in obesity-linked insulin resistance[END_REF][START_REF] Uysal | Protection from obesity-induced insulin resistance in mice lacking TNFα function[END_REF][START_REF] Spranger | Inflammatory cytokines and the risk to develop type 2 diabetes: Results of the prospective population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study[END_REF][START_REF] Kristiansen | Interleukin-6 and diabetes: The good, the bad, or the indifferent?[END_REF].

In addition to that, activation of pathways including JNK, IKKβ/NF-κB as well as mammalian target of rapamycin (mTOR)/S6 kinase, that were found to affect insulin action in both adipocytes and hepatocytes, seem to be inherent to obesity-associated inflammatory processes [START_REF] Ota | Chemokine systems link obesity to insulin resistance[END_REF].

Despite the fact that chronic inflammation in obesity is multifactorial and involves many chemokines, chemokines like CCL2, CCL5 and CCL3/MIP-1α among others, have been particularly implicated in obesity. For example, they are expressed in WAT of obese mice and with their chemotactic ability it is not a surprise that chemokines have been found to play a major role in macrophage recruitment and contribution to insulin resistance in obesity (H. Xu et al., 2003;[START_REF] Sartipy | Monocyte chemoattractant protein 1 in obesity and insulin resistance[END_REF][START_REF] Kanda | MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity[END_REF]. CCL2 and its receptor CCR2 in particular are highly increased in obese AT and the overexpression of CCL2 leads to an elevated increase in macrophage recruitment of AT macrophages and ultimately insulin resistance [START_REF] Kanda | MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity[END_REF]. In support of this, CCR2 genetic deficiency protected partially from glucose intolerance, and AT macrophage infiltration as well as inflammation, when mice were fed a HFD for an extended period of time [START_REF] Weisberg | CCR2 modulates inflammatory and metabolic effects of high-fat feeding[END_REF][START_REF] Lumeng | Obesity induces a phenotypic switch in adipose tissue macrophage polarization[END_REF][START_REF] Gutierrez | Aberrant accumulation of undifferentiated myeloid cells in the adipose tissue of CCR2-deficient mice delays improvements in insulin sensitivity[END_REF]. Contrary to that, deficiency of the chemokine CCL3 seemed to have no ameliorating effect on macrophage or T cell recruitment to AT in DIO mice [START_REF] Kennedy | Impact of macrophage inflammatory protein-1α deficiency on atherosclerotic lesion formation, hepatic steatosis, and adipose tissue expansion[END_REF]. However, the study demonstrated that CCL3-deficient bone marrow had an effect on plasma cholesterol, TG concentrations and reduced AT mass, plasma insulin and leptin levels, compared to bone marrow transplants from either C57BL/6 or CCL2 -/-mice. This indicates that the effects are specifically mediated by bone marrow-derived CCL3-deficient cells and that CCL3 has a potential role in lipid metabolism in mice fed a hyperlipidemic diet [START_REF] Kennedy | Impact of macrophage inflammatory protein-1α deficiency on atherosclerotic lesion formation, hepatic steatosis, and adipose tissue expansion[END_REF].

Several studies implicated a number of chemokines also in the pathogenesis of type 1 and 2 diabetes and showed their upregulation in human and murine pancreatic tissue [START_REF] Saurer | Differential expression of chemokines in normal pancreas and in chronic pancreatitis[END_REF][START_REF] Herder | Chemokines as risk factors for type 2 diabetes: Results from the MONICA/KORA Augsburg study, 1984-2002[END_REF][START_REF] Marselli | Gene expression of purified β-cell tissue obtained from human pancreas with laser capture microdissection[END_REF][START_REF] Planas | Gene expression profiles for the human pancreas and purified islets in Type 1 diabetes: New findings at clinical onset and in long-standing diabetes[END_REF][START_REF] Sarkar | Expression and regulation of chemokines in murine and human type 1 diabetes[END_REF][START_REF] Esser | Inflammation as a link between obesity, metabolic syndrome and type 2 diabetes[END_REF][START_REF] Moretto | The effects of calorie-matched high-fat diet consumption on spontaneous physical activity and development of obesity[END_REF].

CCL5/RANTES

First identified by Schall in 1988 the chemokine (C-C motif) ligand 5 or also known as Regulated upon Activation Normal T cells Expressed and Secreted (RANTES), CCL5 is encoded by a gene on the q-arm of chromosome 17 in humans (4 exons) [START_REF] Donlon | Localization of a human T-cell-specific gene, RANTES (D17S136E), to chromosome 17q11.2-q12[END_REF] and on 11 (3 exons) in mice. The gene product of human CCL5 was predicted to be 10 kDa in size, that upon cleavage results in an 8 kDa sized peptide, with 68 amino acid residues, among which are 4 cysteines. Schall and colleagues have not found any sites for N-linked glycosylation, nor a transmembrane domain in the positively charged, basic and mature protein [START_REF] Schall | A human T cell-specific molecule is a member of a new gene family[END_REF].

CCL5 belongs to the group of inflammatory β-chemokines [START_REF] Levy | The Unexpected Pleiotropic Activities of RANTES[END_REF] and exerts its effects through 7TM GPCRs including CCR1, CCR3 and CCR5 [START_REF] Raport | Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1β, and MIP-1α[END_REF] .

In addition, a few studies have found that apart from its cognate chemokine receptors CCL5 can bind also ACKRs such as CCBP2, DARK [START_REF] Horuk | The Duffy Antigen Receptor for Chemokines DARC/ACKR1[END_REF] as well as the orphan GPCR 75 (GPR75) [START_REF] Ignatov | RANTES stimulates Ca 2+ mobilization and inositol trisphosphate (IP 3 ) formation in cells transfected with G protein-coupled receptor 75[END_REF].

Characterisation of T cell-derived human CCL5 showed that although CCL5 was constitutively expressed at basal levels, after cellular activation, expression reaches high levels only 5 to 7 days after stimulation and decreases dramatically 6 to 24 hours after, before returning to basal levels within 3 days [START_REF] Schall | A human T cell-specific molecule is a member of a new gene family[END_REF]. Contrary to initial belief, CCL5 is inducible in many different cell types, including macrophages, T lymphocytes and human Th1 clones but only minimally by Th2 cells [START_REF] Schrum | Synthesis of the CC-chemokines MIP-1alpha, MIP-1beta, and RANTES is associated with a type 1 immune response[END_REF]. Furthermore, it is secreted by fibroblasts, thrombin-stimulated platelets, endothelial cells, mesangial cells, TNF-α or IL-1β-stimulated renal tubular epithelial cells, macrophages, eosinophils, AT [START_REF] Skurk | Expression and secretion of RANTES (CCL5) in human adipocytes in response to immunological stimuli and hypoxia[END_REF].

CCL5 is a pleiotropic chemokine that exerts chemotactic activity for monocytes, CD4 T cells [START_REF] Schall | Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES[END_REF] and is also chemotactic and activating for eosinophils [START_REF] Alam | RANTES is a chemotactic and activating factor for human eosinophils[END_REF] in addition to modulating histamine release from basophils (degranulation) [START_REF] Alam | Interleukin-8 and RANTES inhibit basophil histamine release induced with monocyte chemotactic and activating factor/monocyte chemoattractant peptide-1 and histamine releasing factor[END_REF][START_REF] Kuna | RANTES, a monocyte and T lymphocyte chemotactic cytokine releases histamine from human basophils[END_REF].

Many studies reported CCL5 to be involved in the pathophysiology of many inflammatory diseases, or conditions characterized by a chronic inflammatory state such as, atherosclerosis [START_REF] Braunersreuther | Ccr5 but not Ccr1 deficiency reduces development of dietinduced atherosclerosis in mice[END_REF][START_REF] Jones | Chemokine receptor CCR5: from AIDS to atherosclerosis[END_REF], liver disease [START_REF] Pérez-Martínez | Maraviroc, a CCR5 antagonist, ameliorates the development of hepatic steatosis in a mouse model of non-alcoholic fatty liver disease (NAFLD)[END_REF][START_REF] Mohs | Functional role of CCL5/RANTES for HCC progression during chronic liver disease[END_REF], rheumatoid arthritis [START_REF] Rathanaswami | Expression of the cytokine RANTES in human rheumatoid synovial fibroblasts. Differential regulation of RANTES and interleukin-8 genes by inflammatory cytokines[END_REF] but also neurodegenerative diseases like AD [START_REF] Xia | Immunohistochemical study of the β-chemokine receptors CCR3 and CCR5 and their ligands in normal and Alzheimer's disease brains[END_REF], MS [START_REF] Trebst | CCR1+/CCR5+ mononuclear phagocytes accumulate in the central nervous system of patients with multiple sclerosis[END_REF][START_REF] Mori | RANTES correlates with inflammatory activity and synaptic excitability in multiple sclerosis[END_REF], EAE and cancer [START_REF] Conti | MCP-1 and RANTES are mediators of acute and chronic inflammation[END_REF][START_REF] Vaday | Expression of CCL5 (RANTES) and CCR5 in prostate cancer[END_REF][START_REF] Soria | The inflammatory chemokines CCL2 and CCL5 in breast cancer[END_REF][START_REF] Goazigo | Current status of chemokines in the adult CNS[END_REF][START_REF] Weitzenfeld | The chemokine system, and its CCR5 and CXCR4 receptors, as potential targets for personalized therapy in cancer[END_REF][START_REF] D'esposito | Adipose microenvironment promotes triple negative breast cancer cell invasiveness and dissemination by producing CCL5[END_REF].

Expression

It was found to be upregulated in psoriatic lesions and secreted in epidermal keratinocytes upon stimulation by a combination of TNF-α and interferon-γ (IFN-γ) and production is inhibited by an active vitamin D3 analogue [START_REF] Fukuoka | RANTES expression in psoriatic skin, and regulation of rantes and IL-8 production in cultured epidermal keratinocytes by active vitamin D3 (tacalcitol)[END_REF][START_REF] Warhurst | Interferon gamma induces differential upregulation of alpha and beta chemokine secretion in colonic epithelial cell lines[END_REF]. This indicates that CCL5 signaling is regulated downstream of TNF-α pathway in synergy with other cytokines. Interestingly, CCL5 is expressed also in adipocytes and murine AT stromal/vascular cells of mice and humans and CCL5 mRNA levels are negatively correlated with murine AT adiponectin [START_REF] Wu | T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity[END_REF].

Adiponectin-deficient mice on a HFD had upregulation of CCL5 mRNA levels in AT compared to WT mice [START_REF] Wu | T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity[END_REF].

Interestingly, expression of the mRNA of CCL5 was also found in small intestine, with highest expression in duodenum, jejunum and ileum of mice [START_REF] Shang | Expression of the Chemokine Binding Protein M3 Promotes Marked Changes in the Accumulation of Specific Leukocytes Subsets Within the Intestine[END_REF].

Apart from peripheral expression, CCL5 was found to be expressed in and secreted by astrocytes (and secreted by astrocytes) [START_REF] Chou | Expanded-Polyglutamine Huntingtin Protein Suppresses the Secretion and Production of a Chemokine (CCL5/RANTES) by Astrocytes[END_REF], microglia [START_REF] Hu | Inhibition of microglial cell RANTES production by IL-10 and TGF-β[END_REF][START_REF] Avdoshina | Morphine Induces the Release of CCL5 from Astrocytes: Potential Neuroprotective Mechanism Against the HIV Protein gp120[END_REF] and neurons in the CNS [START_REF] Lanfranco | Glial-and Neuronal-Specific Expression of CCL5 mRNA in the Rat Brain[END_REF]. Interestingly, CCL5 was expressed, and induced differentiation in an age-dependent manner, in human fetal astrocytes and seems to have an important role in the ontogenetic development of the CNS and PNS [START_REF] Bolin | Primary sensory neurons migrate in response to the chemokine RANTES[END_REF][START_REF] Bakhiet | RANTES promotes growth and survival of human first-trimester forebrain astrocytes[END_REF].

Additional support for a central role of CCL5 was provided in a murine Huntington's disease (HD) model, in which CCL5 promoted cortical neurite outgrowth and neuronal activity. They also confirmed previous results showing that astrocytes express and secrete CCL5, which is impaired in HD [START_REF] Chou | Expanded-Polyglutamine Huntingtin Protein Suppresses the Secretion and Production of a Chemokine (CCL5/RANTES) by Astrocytes[END_REF][START_REF] Park | Chemokines released from astrocytes promote chemokine receptor 5-mediated neuronal cell differentiation[END_REF]. Interestingly, HD is associated with metabolic impairment, such as insulin and glucose regulation impairments [START_REF] Hurlbert | Mice transgenic for an expanded CAG repeat in the Huntington's disease gene develop diabetes[END_REF][START_REF] Lalić | Glucose homeostasis in Huntington disease: Abnormalities in insulin sensitivity and early-phase insulin secretion[END_REF].

Researcher have also found that CCL5 among other chemokines is a downstream factor of hepatocyte growth factor in promoting axon extension [START_REF] Bhardwaj | Chemokines induce axon outgrowth downstream of Hepatocyte Growth Factor and TCF/β-catenin signaling[END_REF].

CCL5 is not only expressed in embryonic DRG cells, Schwann cells and spinal cord, but was also found to elicit neural migration in DRG sensory neurons, that differentiate into NPY and CGRP expressing cells, which are associated with the phenotype of thermoceptive and nociceptive DRG neurons, that transmit pain and temperature sensation [START_REF] Bolin | Primary sensory neurons migrate in response to the chemokine RANTES[END_REF]. Furthermore, CCL5 elicits a transient Ca 2+ mobilization in embryonic DRG cells through coupling to a GPCR [START_REF] Bolin | Primary sensory neurons migrate in response to the chemokine RANTES[END_REF].

Receptor Interaction and Synergistic Effects With Other

Chemokines

Induced by TNF-α, IL-1β and IFN-γ and when TNF-α or IL-1β receptor are inhibited, CCL5 induction is inhibited [START_REF] Pattison | The RANTES Chemokine[END_REF][START_REF] Fukuoka | RANTES expression in psoriatic skin, and regulation of rantes and IL-8 production in cultured epidermal keratinocytes by active vitamin D3 (tacalcitol)[END_REF]. Additionally, corticosteroids are able to inhibit the transcription of CCL5 in fibroblasts [START_REF] Pattison | The RANTES Chemokine[END_REF]. Furthermore, IL-4 and IL-13 inhibit CCL5 expression and secretion in endothelial cells [START_REF] Marfaing-Koka | Regulation of the production of the RANTES chemokine by endothelial cells. Synergistic induction by IFN-gamma plus TNF-alpha and inhibition by IL-4 and IL-13[END_REF]. The ability of microglia cells to produce CCL5 can be inhibited by IL-10 and TGF-β, which shows that also other cytokines can regulate CCL5 at least in microglia cells [START_REF] Hu | Inhibition of microglial cell RANTES production by IL-10 and TGF-β[END_REF]. This study found also that chemokine binding protein M3, which interacts with many members of all chemokine families and blocks their activity, binds with high affinity to CCL5 among others. CCL5 expression and secretion was inducible in a time-dependent manner by opiates, and morphine in astrocytes and LPS seemed to induce an increased production of CCL5 in microglia cells in vitro [START_REF] Avdoshina | Morphine Induces the Release of CCL5 from Astrocytes: Potential Neuroprotective Mechanism Against the HIV Protein gp120[END_REF].

Furthermore, CCL5 has been shown to act as a substrate for DPP4, which appears to cleave the N-terminal serine and proline residues in CCL5. It might thereby modulate its activity, by affecting its binding affinity to chemokine receptors or by potentially decreasing the bio-availability of CCL5 [START_REF] Schols | CD26-processed RANTES(3-68), but not intact RANTES, has potent anti-HIV-1 activity[END_REF][START_REF] Oravecz | Regulation of the Receptor Specificity and Function of the Chemokine RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted) by Dipeptidyl Peptidase IV (CD26)-mediated Cleavage[END_REF][START_REF] Liu | The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis[END_REF].

In support of its potential role in neuromodulation and neurotransmission studies have found that increases in Intracellular Ca 2+ induced by CCL5 have been also demonstrated in spinal cord synaptosomes. Additionally, studies found CCL5 to be involved in the control of glutamate release, with opposing effects depending on which receptor is activated [START_REF] Meucci | Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity[END_REF][START_REF] Klein | Chemokine Receptor Expression and Signaling in Macaque and Human Fetal Neurons and Astrocytes: Implications for the Neuropathogenesis of AIDS[END_REF][START_REF] Musante | RANTES Modulates the Release of Glutamate in Human Neocortex[END_REF][START_REF] Di Prisco | RANTES-mediated control of excitatory amino acid release in mouse spinal cord[END_REF].

Studies indicate that CCL5 might regulate glucose uptake and 5' adenosine monophosphate-activated protein kinase (AMPK)-a signaling in T cells through activation of the mTOR pathway in an attempt to provide enough energy for chemotaxis [START_REF] Chan | The chemokine CCL5 regulates glucose uptake and AMP kinase signaling in activated T cells to facilitate chemotaxis[END_REF]. Interestingly, a recent study implicated CCL5-CCR5 signaling in the hypothalamic regulation of insulin signaling [START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF]. In a study, administration of CCL5 (50ng/ml) induced rapid calcium influx from the extracellular environment through CCR3 activation in human fetal microglia cultures [START_REF] Hegg | β-Chemokines and human immunodeficiency virus type-1 proteins evoke intracellular calcium increases in human microglia[END_REF] and potentially through CCR5, as it is expressed in microglia, too [START_REF] He | CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia[END_REF][START_REF] Albright | Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates[END_REF].

Apart from identifying CCR5 expression specifically in neurons of the ARC and CCL5 immunoreactivity in both ARC and VMH of the hypothalamus, they found increased phosphorylated AMPKa (pAMPKa) immunoreactivity in the ARC of CCR5 -/-mice compared to control mice. Furthermore, whereas pAMPKa was increased in fasted conditions in WT mice, CCR5 and CCL5 deficient mice showed elevated pAMPKa in both fed and fasted conditions [START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF]. Interestingly, qPCR analysis of hypothalamic neuropeptide expression indicated an upregulation of both orexigenic (NPY and AgRP) and anorexigenic (POMC) neuropeptides in KO mice for CCL5 and its receptors compared to WT controls fed a regular diet. In agreement with this, primary hypothalamic neuron cultures identified CCR5 expression in POMC + neurons.

These results suggest a potential role of CCL5 signaling in the regulation of energy balance and feeding regulation as proposed by the authors.

According to the authors, both, CCL5 and CCR5 deficient mice revealed an impaired insulin response, which was supported by downregulation of genes associated with energy and insulin signaling regulation in KO mice.

CCL5 seems to have different ways of eliciting its function and through different pathways, depending on its concentration and state. Apart from chemotaxis, it was found to activate different types of cells including T cells, monocytes and neutrophils [START_REF] Appay | Aggregation of RANTES is responsible for its inflammatory properties. Characterization of nonaggregating, noninflammatory rantes mutants[END_REF][START_REF] Appay | RANTES activates antigen-specific cytotoxic T lymphocytes in a mitogen-like manner through cell surface aggregation[END_REF]. CCL5 is capable of aggregation, and depending on its oligomerized forms as a dimeric, trimeric or a monomer it can have different effects. For example, it was reported that activation of immune cells requires CCL5 to be in aggregated form, as nonaggregating variants had less or no effect on leukocyte activation [START_REF] Appay | Aggregation of RANTES is responsible for its inflammatory properties. Characterization of nonaggregating, noninflammatory rantes mutants[END_REF]. Cell-surface GAG seem to have a promoting effect on the oligomerization ability of CCL5 and other chemokines and thus are important for leukocyte activation, while soluble GAG seem to have a more inhibiting effect on chemokine action [START_REF] Hoogewerf | Glycosaminoglycans mediate cell surface oligomerization of chemokines[END_REF][START_REF] Kuschen | Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses[END_REF].

Similarly, the concentration of chemokines has an impact on their function. At high micromolar concentrations, CCL5 can activate leukocytes through a GPCR independent manner (protein tyrosine kinase-mediated pathway). The activation of this pathway is believed to elicit cell activation and cytokine release or cell proliferation via induction of a prolonged calcium influx, hyperphosphorylation [START_REF] Appay | Aggregation of RANTES is responsible for its inflammatory properties. Characterization of nonaggregating, noninflammatory rantes mutants[END_REF][START_REF] Appay | RANTES activates antigen-specific cytotoxic T lymphocytes in a mitogen-like manner through cell surface aggregation[END_REF][START_REF] Bacon | RANTES induces tyrosine kinase activity of stably complexed p125FAK and ZAP-70 in human T cells[END_REF]. In contrast to this, CCL5 seems to act in a manner characteristic for chemokines at nanomolar concentrations to induce chemotaxis and receptor polarization by activating GPCRs and generating a transient calcium influx [START_REF] Nieto | Polarization of Chemokine Receptors to the Leading Edge during Lymphocyte Chemotaxis[END_REF].

Receptors of CCL5

C-C Motif Chemokine Receptor 1 (CCR1)

CCR1 is a typical 7TM GPCR and expressed on immune cells like monocytes, memory T cells, basophils and DCs [START_REF] Neote | Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor[END_REF][START_REF] Bachelerie | International Union of Pharmacology. LXXXIX. Update on the Extended Family of Chemokine Receptors and Introducing a New Nomenclature for Atypical Chemokine Receptors[END_REF].

In addition, it seems to be also expressed on intestinal eosinophils and macrophages [START_REF] Shang | Expression of the Chemokine Binding Protein M3 Promotes Marked Changes in the Accumulation of Specific Leukocytes Subsets Within the Intestine[END_REF], lung airway smooth muscle cells, murine neutrophils, endothelial cells and different myeloma cells [START_REF] Horuk | Chemokine receptors[END_REF].

Moreover, it seems to be expressed in cells of the CNS such as astrocytes and hippocampal neurons, in intestinal cell lines, GLP-1 secreting enteroendocrine L-cells and other cells of murine small intestine [START_REF] Meucci | Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity[END_REF][START_REF] Dorf | Astrocytes express functional chemokine receptors[END_REF][START_REF] Pais | RANTES (CCL5) reduces glucose-dependent secretion of glucagon-like peptides 1 and 2 and impairs glucose-induced insulin secretion in mice[END_REF].

CCR1 is like many chemokine receptors promiscuous because it can bind to several chemokines including CCL3, CCL5 and with lower affinity to CCL6, CCL9, CCL15 and CCL23 but does not seem to have a selective ligand so far [START_REF] Neote | Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor[END_REF][START_REF] Bachelerie | International Union of Pharmacology. LXXXIX. Update on the Extended Family of Chemokine Receptors and Introducing a New Nomenclature for Atypical Chemokine Receptors[END_REF]. Surprisingly, it was reported, that although CCL5 binds with lower affinity to CCR1 compared to other ligands, its ability to induce a calcium signal was much more efficient than chemokines that bind with higher affinity to the receptor (CCL2 and human CCL4) [START_REF] Neote | Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor[END_REF]. CCR1 signaling follows the classical chemokine pathway and induces inhibition of adenylyl cyclase and activation of phospholipase C (PLC), PKC, calcium flux and phospholipase A2 via G proteins belonging to the Gi/G0 class [START_REF] Bachelerie | International Union of Pharmacology. LXXXIX. Update on the Extended Family of Chemokine Receptors and Introducing a New Nomenclature for Atypical Chemokine Receptors[END_REF].

CCR1 invalidation studies implicated the receptor in the pathophysiology of MS and organ transplant rejection, protection from secondary pulmonary inflammation and injury in an acute pancreatitis model [START_REF] Gerard | Targeted disruption of the beta-chemokine receptor CCR1 protects against pancreatitis-associated lung injury[END_REF].

C-C Motif Chemokine Receptor 3 (CCR3)

Belonging to the CC group of chemokine receptors, CCR3 is expressed on intestinal eosinophils and contrary to the expression of CCR5, it is highly expressed in CD4 + Th2 cells but undetected in Th1 cells [START_REF] Loetscher | CCR5 is characteristic of Th1 lymphocytes [6[END_REF][START_REF] Shang | Expression of the Chemokine Binding Protein M3 Promotes Marked Changes in the Accumulation of Specific Leukocytes Subsets Within the Intestine[END_REF]. Furthermore, expression was found in the CNS, more specifically in astrocytes [START_REF] Dorf | Astrocytes express functional chemokine receptors[END_REF] and microglia [START_REF] He | CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia[END_REF][START_REF] Ghorpade | Role of the beta-chemokine receptors CCR3 and CCR5 in human immunodeficiency virus type 1 infection of monocytes and microglia[END_REF][START_REF] Hegg | β-Chemokines and human immunodeficiency virus type-1 proteins evoke intracellular calcium increases in human microglia[END_REF] and subpopulations of neurons [START_REF] Klein | Chemokine Receptor Expression and Signaling in Macaque and Human Fetal Neurons and Astrocytes: Implications for the Neuropathogenesis of AIDS[END_REF].

CCR3 has several ligands including the selective agonist eotaxin (CCL11), but can be also activated by CCL5 and CCL7, CCL8, CCL13, CCL15, CCL24, CCL26 and CCL28 [START_REF] Heath | Chemokine receptor usage by human eosinophils: The importance of CCR3 demonstrated using an antagonistic monoclonal antibody[END_REF][START_REF] Albright | Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates[END_REF][START_REF] Alexander | Guide to Receptors and Channels (GRAC), 5th edition[END_REF] A study reported that signaling through CCR3 can be induced by binding of its ligands CCL5 (or CCL11) and evokes a rapid influx of extracellular calcium in 26% of microglial cells [START_REF] Hegg | β-Chemokines and human immunodeficiency virus type-1 proteins evoke intracellular calcium increases in human microglia[END_REF].

G Protein-coupled Receptor 75 (GPR75)

First identified by [START_REF] Tarttelin | Cloning and characterization of a novel orphan G-proteincoupled receptor localized to human chromosome 2p16[END_REF] as a novel human orphan GPCR, GPR75 is a 540 amino-acid sequence protein that belongs to the Gqα family of G proteins. It was reported to be mainly expressed in the CNS, the retina and vascular endothelial cells, but not in immune organs contrary to chemokine receptors [START_REF] Tarttelin | Cloning and characterization of a novel orphan G-proteincoupled receptor localized to human chromosome 2p16[END_REF][START_REF] Sauer | Evaluation of the G protein coupled receptor-75 (GPR75) in age related macular degeneration[END_REF][START_REF] Garcia | 20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension[END_REF]. So far, only few ligands have been identified that bind GPR75, namely, the eicosanoid 20-hydroxyeicosatetraenoic acid, CCL3 and CCL5 [START_REF] Ignatov | RANTES stimulates Ca 2+ mobilization and inositol trisphosphate (IP 3 ) formation in cells transfected with G protein-coupled receptor 75[END_REF][START_REF] Garcia | 20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension[END_REF]. It is quite surprising that CCL5 is an agonist for GPR75, seeing as it shares only 12-16% amino acid identity with chemokine receptors. The highest homology of GPR75 was found with neuropeptide Y receptor. Apart from the basic 7TM structure, GPR75 differs from chemokine receptors in that it has a longer C-terminal tail, a lack of the characteristic "aspartate-arginine-tyrosine" motif in the transmembrane region and signaling is unimpaired by pertussis toxin [START_REF] Ignatov | RANTES stimulates Ca 2+ mobilization and inositol trisphosphate (IP 3 ) formation in cells transfected with G protein-coupled receptor 75[END_REF][START_REF] Liu | The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis[END_REF][START_REF] Garcia | 20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension[END_REF].

It has been reported, that CCL5 signaling through endogenously expressed GPR75 in hippocampal cell lines protected Aβ-induced neuron death by activating signaling pathway PLC/PI3K/Akt/MAPK (Fig. 21) [START_REF] Ignatov | RANTES stimulates Ca 2+ mobilization and inositol trisphosphate (IP 3 ) formation in cells transfected with G protein-coupled receptor 75[END_REF]. A recent study has further demonstrated the constitutive expression of GPR75 in islets of murine and human pancreas, where it was involved in CCL5 mediated calcium modulation and related insulin secretion [START_REF] Liu | The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis[END_REF].

Duffy Antigen Receptor For Chemokines (DARC)

Apart from the most common receptors CCL5 binds to, it was shown that it can also bind to a promiscuous and atypical and non-signaling chemokine receptor, called DARC [START_REF] Horuk | The Duffy Antigen Receptor for Chemokines DARC/ACKR1[END_REF]. DARC consists of 338 amino acids and with its 7TM domains, it belongs to the GPCR family. However, it has only less than 20% amino acid identity with chemokine receptors belonging to the CXC or CC family. Apart from erythrocytes and some B and T cells, the receptor is also found in many tissues like heart, lung, thymus, spleen, kidney and pancreas, which might be due to its expression in endothelial cells lining post-capillary venules. It is postulated that expression of DARC on erythrocytes might attenuate the inflammatory chemokine activity in the blood by internalization of the bound ligand, or similar to binding to GAG it might keep them cleared from circulation. Furthermore, it was found that chemokine binding to DARC on erythrocytes protects from malaria-causing parasite Plasmodium vivax by blocking their binding to and infection of erythrocytes [START_REF] Chitnis | The domain on the Duffy blood group antigen for binding Plasmodium vivax and P. knowlesi malarial parasites to erythrocytes[END_REF]. Endothelial cell expression of DARC might further provide a path for attachment to establish a chemokine gradient, attract leukocytes and potentially induce diapedesis or transport chemokines across endothelial cells [START_REF] Horuk | The Duffy antigen receptor for chemokines: structural analysis and expression in the brain[END_REF][START_REF] Baggiolini | Human Chemokines: An Update[END_REF][START_REF] Horuk | The Duffy Antigen Receptor for Chemokines DARC/ACKR1[END_REF].

Interestingly, DARC is also expressed in the brain, more specifically in Purkinje cells of the cerebellum, which opens up the possibility of DARC having a role in modulating neuronal activity stimulated by chemokines. CCL5 appears to bind to DARC receptor on human and mouse erythrocytes with a high affinity (K D ) of 4.8 nM and 8.3 nM respectively [START_REF] Horuk | The Duffy antigen receptor for chemokines: structural analysis and expression in the brain[END_REF]. 5.2.2.5. Chemokine-binding Protein 2 (CCBP2) CCBP2, also called ACKR2 or D6 is an atypical 7TM chemokine receptor and different from other chemokine receptors, it does not transduce ligand binding into an intracellular signal. This is most likely due to changes in the characteristic chemokine motif "DRYLAIV" to "DKYLEIV" and the lack of coupling to G proteins [START_REF] Nibbs | Cloning and characterization of a novel promiscuous human βchemokine receptor D6[END_REF][START_REF] Hughes | A guide to chemokines and their receptors[END_REF] Therefore it is often referred to as decoy receptor that ia able to bind promiscuously to most members of the inflammatory β-chemokine family, which has 12 identified ligands so far [START_REF] Bonecchi | Differential Recognition and Scavenging of Native and Truncated Macrophage-Derived Chemokine (Macrophage-Derived Chemokine/CC Chemokine Ligand 22) by the D6 Decoy Receptor[END_REF]. Several tissues express D6 receptor including skin, gut and lung tissues but mostly lymphatic endothelial cells [START_REF] Nibbs | The β-chemokine receptor D6 is expressed by lymphatic endothelium and a subset of vascular tumors[END_REF].

Similar to DARK, it seems to act on chemokines by internalizing them through a β-arrestin dependent way and degrading them upon binding and thereby potentially playing a role in the homeostasis of circulating chemokine levels and their clearance from inflamed tissues [START_REF] Nibbs | Chemokines on the move: Control by the chemokine "interceptors" Duffy blood group antigen and D6[END_REF][START_REF] Galliera | β-arrestin-dependent constitutive internalization of the human chemokine decoy receptor D6[END_REF].

C-C Motif Chemokine Receptor 5 (CCR5)

CCR5 is like the other above mentioned chemokine receptors a 7TM GPCR (Fig. 22) and well known for its role in the immune system but also as primary co-receptor facilitating the cell entry for HIV [START_REF] Sorce | The chemokine receptor CCR5 in the central nervous system[END_REF].

Most studies report that apart from CCL5, CCR5 binds to other chemokines such as macrophage inflammatory proteins (MIP-1α or CCL3) and MIP-1β (CCL4) with high affinity [START_REF] Raport | Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1β, and MIP-1α[END_REF][START_REF] Blanpain | CCR5 binds multiple CC-chemokines: MCP-3 acts as a natural antagonist[END_REF][START_REF] Waller | Chemotherapy of infections[END_REF].

However, it was reported that it seems to bind also MCP-2 (CCL8), eotaxin (CCL11), HCC-1 (CCL14a), HCC-4 (CCL16) and that CCL7 (MCP-3) can act as an antagonist on CCR5 [START_REF] Blanpain | CCR5 binds multiple CC-chemokines: MCP-3 acts as a natural antagonist[END_REF][START_REF] Alexander | Guide to Receptors and Channels (GRAC), 5th edition[END_REF].

It is expressed on many immune cells such as CD4 + Th1 cells (high expression) and almost undetectable in Th2 cells [START_REF] Loetscher | CCR5 is characteristic of Th1 lymphocytes [6[END_REF], but is found on macrophages and DCs [START_REF] Waller | Chemotherapy of infections[END_REF]. CCR5 mRNA expression was also found on intestinal T cells and macrophages [START_REF] Shang | Expression of the Chemokine Binding Protein M3 Promotes Marked Changes in the Accumulation of Specific Leukocytes Subsets Within the Intestine[END_REF].

Interestingly, several studies have reported the constitutive expression of CCR5 in the CNS [START_REF] Sorce | The chemokine receptor CCR5 in the central nervous system[END_REF]. For example, CCR5 expression was found on microglia [START_REF] He | CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia[END_REF][START_REF] Ghorpade | Role of the beta-chemokine receptors CCR3 and CCR5 in human immunodeficiency virus type 1 infection of monocytes and microglia[END_REF][START_REF] Albright | Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates[END_REF][START_REF] Waller | Chemotherapy of infections[END_REF], astrocytes and neurons in the CNS [START_REF] Meucci | Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity[END_REF][START_REF] Klein | Chemokine Receptor Expression and Signaling in Macaque and Human Fetal Neurons and Astrocytes: Implications for the Neuropathogenesis of AIDS[END_REF][START_REF] Avdoshina | Neurotrophins modulate the expression of chemokine receptors in the brain[END_REF]. Moreover, when microglia were stimulated with CCL3 and CCL5, it lead to a response in intracellular calcium levels through either CCR3 or CCR5, which has higher expression levels in microglia than CCR3 [START_REF] Albright | Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates[END_REF]. Heterozygous mice for BNDF have increased expression levels of CCR5 mRNA in the hippocampus, striatum and cerebral cortex. This means that BDNF seems to be able to negatively regulate and reduce CCR5 expression in the brain and that CCR5 is indeed expressed in parts of the brain [START_REF] Ahmed | Brain-derived neurotrophic factor modulates expression of chemokine receptors in the brain[END_REF]. Another study reinforces the idea that CCL5 through CCR5 signaling is involved in brain development as they found CCR5 expression and production in human embryonic neurons, located inside the neuronal cell body and processes, and upon stimulation evoked an increased intracellular Ca 2+ response [START_REF] Boutet | Cellular expression of functional chemokine receptor CCR5 and CXCR4 in human embryonic neurons[END_REF]. CCR5 was localized in neuronal cell body and processes, especially in Purkinje cells of the cerebellum and pyramidal neurons of the hippocampus, as well as subpopulations of striatal and cortical neurons [START_REF] Avdoshina | Neurotrophins modulate the expression of chemokine receptors in the brain[END_REF]. Other findings confirmed and added to these results by showing CCR5 expression in subpopulations of cortical neurons of human and macaque brain, in fetal neurons and astrocyte culture cells [START_REF] Klein | Chemokine Receptor Expression and Signaling in Macaque and Human Fetal Neurons and Astrocytes: Implications for the Neuropathogenesis of AIDS[END_REF][START_REF] Westmoreland | Developmental expression patterns of CCR5 and CXCR4 in the rhesus macaque brain[END_REF].

Although CCL5-CCR5 signaling seems to play an important role in development, it is intriguing that KO models or humans with a deletion in the gene for CCR5 do not have any particular or obvious phenotype, apart from partial impairments in immune function in mice. Homozygous CCR5 -/-in humans even established protection from HIV-1 infection and progression. But it was reported that homozygous individuals also are more prone to other diseases, such as listeria infection or West Nile virus and with more serious outcomes than people without the mutation [START_REF] Alkhatib | The biology of CCR5 and CXCR4.', Current opinion in HIV and AIDS[END_REF][START_REF] Sorce | The chemokine receptor CCR5 in the central nervous system[END_REF]. In this study we were particularly interested in CCL5 and CCR5 singling and will thus focus more in detail on this chemokine and receptor.

Signaling

GPCRs such as CCR5 mediate their effects through heterotrimeric G proteins.

It has been reported that CCR5 can signal through all G protein subunits such as Ga i and βγ dimers to induce several different effectors (Fig. 23). CCR5

activation by its ligands leads to signal transduction via three members of the MAPK family, namely ERK1/2, p38 and stress-activated protein kinase/JNK.

However, CCR5 can also signal through adenylyl cyclase or PLCβ leading to intracellular calcium influx and formation of inositol-1,4,5-trisphosphate (IP 3 ) (Fig. 23) [START_REF] Oppermann | Chemokine receptor CCR5: Insights into structure, function, and regulation[END_REF][START_REF] Wu | Chemokine Coreceptor Signaling in HIV-1 Infection and Pathogenesis[END_REF]. Firstly, signal transduction via the Ga i subunit usually results in adenylyl cyclase inhibition, followed by a decline in intracellular cAMP levels. Secondly, signaling through the G qa subunit of the G protein can activate PLC leading to a transient increase in intracellular calcium, via DAG and IP 3 production followed by increased PKC activity (Fig. 23)(Réaux-Le [START_REF] Goazigo | Current status of chemokines in the adult CNS[END_REF]. Signaling by interaction with

Regulation

Chemokine receptors like CCR5 can be regulated by different mechanisms, including endocytosis, palmitoylation, arrestin interaction, receptor oligomerization and phosphorylation, which modulate receptor function and cell surface expression (reviewed in [START_REF] Oppermann | Chemokine receptor CCR5: Insights into structure, function, and regulation[END_REF]).

β-Arrestins are known for their role as GPCR signaling regulators, which can be involved in either initiation or termination of cellular responses induced by ligand binding to C-terminal serine phosphorylation sites. They are involved in receptor desensitization, clathrin-mediated receptor internalization, but also in signal transduction and scaffolding protein that can mediate a second signaling response through the recruitment of different kinases such as MAPK, src family tyrosine kinases, ERK1/2 or JNK3 [START_REF] Oppermann | Chemokine receptor CCR5: Insights into structure, function, and regulation[END_REF][START_REF] Lopalco | Natural anti-CCR5 antibodies in HIV-infection and -exposure[END_REF]. receptors accumulate in perinuclear endosomes, where they can be recycled and sent back to the membrane in a dephosphorylated state, or be degraded [START_REF] Oppermann | Chemokine receptor CCR5: Insights into structure, function, and regulation[END_REF].

Furthermore, phosphorylation of C-terminal serine residues of chemokine receptors is performed by GPCR kinases (GRK) or PKC, which belong to the family of serine threonine protein kinases and mediate ligand-induced receptor phosphorylation and homologous desensitization. The latter describes the refractory period of an activated receptor unable to get stimulated again for a short period of time [START_REF] Oppermann | Chemokine receptor CCR5: Insights into structure, function, and regulation[END_REF]. In addition to that, CCR5 is capable of oligomerization into homo-or heterooligomers, which can be modulated by its agonists and alter signaling properties. This only adds to the already complex promiscuous and redundant chemokine system, whose exact mechanisms of signaling and regulation have not been sufficiently defined yet.

Role of CCL5/CCR5 in Peripheral Inflammation in Obesity

As mentioned before, CCL5 and its receptors, have a crucial role in the immune system including leukocyte trafficking and attraction to sites of injury, infection and inflammation, adhesion among others. Obesity is a condition defined by excessive accumulative of body fat in AT and its expansion, which is associated with lipotoxicity and secretion of cytokines and chemokines. This in turn leads to infiltration of AT and other organs by macrophages, monocytes and other immune cells, which further the inflammatory state and lead to chronic and systemic inflammation, hyperglycemia, impaired glucose metabolism and ultimately to dysfunction of pancreatic β-cells and insulin resistance among other comorbidities.

CCL5 has been repeatedly reported in studies that provide evidence for its role in obesity. An important correlation between obesity and CCL5 signaling was made when studies reported increased serum levels of CCL5 in obese humans and mice [START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF][START_REF] Abubaker | DNAJB3/HSP-40 Cochaperone Is Downregulated in Obese Humans and Is Restored by Physical Exercise[END_REF][START_REF] Baturcam | Physical exercise reduces the expression of RANTES and its CCR5 receptor in the adipose tissue of obese humans[END_REF][START_REF] Montecucco | Gastric bypass in morbid obese patients is associated with reduction in adipose tissue inflammation via N-oleoylethanolamide (OEA)-mediated pathways[END_REF][START_REF] Hu | High-fat diet-induced adipokine and cytokine alterations promote the progression of prostate cancer in vivo and in vitro[END_REF].

In contrast to this, Hu et al have found levels of serum levels of CCL5 to be decreased in HFD condition compared to control diet condition in a mouse model of transgenic adenocarcinoma of mouse prostate, that mimics prostate cancer progression associated with obesity [START_REF] Hu | High-fat diet-induced adipokine and cytokine alterations promote the progression of prostate cancer in vivo and in vitro[END_REF].

In collaboration with our team, Dumas and colleagues performed a study in which they followed up obese patients before and after they underwent bariatric surgery. It was demonstrated that among a panel of cytokines and chemokines CCL5 was increased in obese human patients compared to healthy control patients and seemed to correlate with the weight of the patients, because three months after Roux-en-Y gastric bypass, CCL5 levels dropped and so did the obese patients weight [START_REF] Dalmas | Variations in circulating inflammatory factors are related to changes in calorie and carbohydrate intakes early in the course of surgery-induced weight reduction1-3[END_REF]. These results have been confirmed a few years later, when Montecucco and colleagues observed elevated circulating and SC AT CCL5 levels in morbid obese patients with higher insulin levels compared to control patients and one year after gastric bypass surgery CCL5 levels dropped in circulation and SC AT [START_REF] Montecucco | Gastric bypass in morbid obese patients is associated with reduction in adipose tissue inflammation via N-oleoylethanolamide (OEA)-mediated pathways[END_REF]. Other studies have found increased CCR5 expression in obese mice and human patients [START_REF] Wu | T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity[END_REF][START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF].

Studies indicate the constitutive expression of CCL5-binding chemokine receptors including CCR1 and CCR5 on the cell-surface of human pre-adipocytes and mature adipocytes. Furthermore, CCR5 expression was found to be increased upon differentiation of pre-adipocytes into mature adipocytes, while CCR1 levels remained constant [START_REF] Gerhardt | Chemokines control fat accumulation and leptin secretion by cultured human adipocytes[END_REF]. The study further showed that apart from chemokines CCL2 and IL-8 also CCL3/MIP-1α (CCR5 ligand) were produced in pre-adipocytes and decreased expression in mature adipocytes, but could get upregulated through stimulation with TNF-α. Interestingly, stimulation of adipocytes by the above mentioned chemokines also induced TNF-α, which shows a reciprocal regulation between chemokines and cytokines [START_REF] Gerhardt | Chemokines control fat accumulation and leptin secretion by cultured human adipocytes[END_REF].

Intriguingly, the study found that mature adipocytes had a great reduction in the quantity of TG and PPARβ expression and tyrosine phosphorylation of proteins (one of the pathways activated by chemokine receptor stimulation)

upon stimulation with the above-mentioned chemokines. In addition, long term stimulation of adipocytes with CCL3 significantly increased leptin concentrations starting after 8 days, whereas acute stimulation with chemokines lead to an increasing effect on leptin concentrations in the medium by all above chemokines after 6 hours, which disappeared for IL-8 but were still increased for CCL3 and CCL2 24 hours post-stimulation. The increase in leptin secretion seemed to be induced by post-transcriptional processes [START_REF] Gerhardt | Chemokines control fat accumulation and leptin secretion by cultured human adipocytes[END_REF]. It would be interesting to repeat the study with a bigger panel of chemokines in particular including CCL5.

Other studies that have linked CCL5/CCR5 to obesity and AT inflammation showed that CCL5 and CCR5 are upregulated in WAT of obese humans [START_REF] Wu | T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity[END_REF][START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF], which was confirmed by other studies in both genetic and DIO mouse models [START_REF] Huber | CC chemokine and CC chemokine receptor profiles in visceral and subcutaneous adipose tissue are altered in human obesity[END_REF][START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF].

In addition, gene expression studies have found an increase in several chemokines such as CCL2, CCL3 and CCL5 among others and chemokine receptors including CCR1-3 and CCR5 in not only visceral AT, but also in SC AT of obese and morbidly obese patients [START_REF] Huber | CC chemokine and CC chemokine receptor profiles in visceral and subcutaneous adipose tissue are altered in human obesity[END_REF][START_REF] Duffaut | Interplay between human adipocytes and T lymphocytes in obesity: CCL20 as an adipochemokine and T lymphocytes as lipogenic modulators[END_REF][START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF][START_REF] Montecucco | Gastric bypass in morbid obese patients is associated with reduction in adipose tissue inflammation via N-oleoylethanolamide (OEA)-mediated pathways[END_REF]. CCR1 was found to be the most expressed among them in monocytes, AT macrophages and visceral WAT [START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF].

Interestingly, one of the studies found that CCL5 was secreted at higher levels in visceral AT compared to SC AT. Furthermore, the expression of CCL5 was in particular high in WAT macrophages and correlated positively with macrophage marker expression such as CD11b and CD3 and also with TNF and IL-6 expression levels, that are markers associated with M1 macrophages, but not with M2 macrophage markers (CD206 or AMAC-1) [START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF]. Additionally, CCL5 seems to trigger adhesion of blood monocytes to AT endothelial cells and their transmigration through endothelial cells in human WAT [START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF]. CCL5 also protected AT macrophages from apoptosis, induced by free cholesterol accumulation, via activating Akt/Erk pathways. Interestingly, M2 macrophages were more sensitive to protection from free cholesterol-induced apoptosis by CCL5 than M1 macrophages, while in vitro M1 macrophages were found to secrete higher levels of CCL5 compared to M2 macrophages [START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF].

Kitade and colleagues further show not only that both CCR5 and its ligands (CCL3, 4 and 5) are highly elevated in WAT of both genetic and DIO obese mouse models, but also an upregulation of CCR5, specifically on AT macrophages in the DIO model. Interestingly, they also found a phenotypic shift of macrophages from the "classically activated" pro-inflammatory M1 polarization state to a "alternatively activated" non-inflammatory activation state that usually predominates in lean mice compared to the M1 state of obese mice. M1 AT macrophages are associated with increased proinflammatory cytokine expression and secretion like TNF-a and IL-6. CCR5

deficiency and the shift to M2 macrophages resulted in an attenuated inflammatory response and improval of impaired glucose tolerance and insulin sensitivity in DIO [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF].

When characterizing the metabolic state of the KO animals versus WT mice, they found no difference in BW or food intake, but an improvement of diabetes and liver steatosis associated with obesity. Furthermore, CCR5 -/- presented themselves with a marked reduction in infiltrated macrophages, that expressed less TNF-a and iNOS, and reduced serum levels of TG and TNFa in HFD condition and lower FFAs in normal diet condition [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF].

Adiponectin-deficient mice on a HFD had upregulation of CCL5 mRNA levels in AT compared to WT mice [START_REF] Wu | T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity[END_REF]. Similarly, CCL5 levels were lowered in WAT of a mouse model deficient for phosphatidylethanolamine Nmethyltransferase (molecule involved in adipocyte differentiation) and resistant to DIO [START_REF] Gao | Decreased lipogenesis in white adipose tissue contributes to the resistance to high fat diet-induced obesity in phosphatidylethanolamine N-methyltransferasedeficient mice[END_REF].

However, although some studies report beneficial findings of CCR5 signaling in obesity, there is some controversy as other studies report conflicting results [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF][START_REF] Kennedy | Loss of CCR5 results in glucose intolerance in diet-induced obese mice[END_REF][START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF].

For instance, Kennedy and colleagues presented quite contrasting results. In fact, the only results that both studies seem to have in common is a similar diet and the genotype of the mice and the result showing a reduction in CD11c + M1-macrophages in WAT of CCR5 -/-DIO mice [START_REF] Kennedy | Loss of CCR5 results in glucose intolerance in diet-induced obese mice[END_REF].

Different from the study of Kitade and colleagues, CCR5 did not seem to play a major but rather minor role in immune cell infiltration and inflammation. They demonstrated that CCR5 deficient mice had even an increase in T cells infiltration in WAT compared to WT mice fed a HFD and no reduction in macrophages as reported by Kitade et al. [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF][START_REF] Kennedy | Loss of CCR5 results in glucose intolerance in diet-induced obese mice[END_REF]. Another contrasting result they found was that CCR5 -/-mice became more glucose intolerant with reduced insulin-induced phosphorylated Akt and IRβ (in AT and muscle) compared to WT mice, but had equal levels of adiposity, blood glucose and plasma insulin [START_REF] Kennedy | Loss of CCR5 results in glucose intolerance in diet-induced obese mice[END_REF].

Surprisingly, in 2018 a study by Kim et al in 2018 implicated CCL5 in lipid metabolism. They found that CCL5 among other chemokines was produced in human SC fat upon UV exposure and downregulated several lipogenic enzymes via CCR5, which led to impaired TG synthesis (E. J. Kim et al., 2018).

Although some of the above mentioned studies, have not found any effect on food intake in CCR5 -/-mice on SD or HFD, there are some that report an effect of CCL5 on modulation of food intake, suggesting a neuroendocrine role in the brain, as well as changes in body temperature and water intake (Tavares andMiñano, 2000, 2004). All these processes are regulated by the brain and provide evidence in support of a central role for CCL5.

Intriguingly, CCL5 was found to be a potent pyrogen and inducing a dosedependent fever in rats, when injected into the preoptic area of the anterior hypothalamic containing thermosensitive-cells [START_REF] Tavares | RANTES: A new prostaglandin dependent endogenous pyrogen in the rat[END_REF].

This response seemed to be mediated specifically through CCR5 signaling, when activated by injected CCL5 and not other ligands like CCL4 [START_REF] Tavares | Differential sensitivities of pyrogenic chemokine fevers to CC chemokine receptor 5 antibodies[END_REF].

It was previously discovered that cytokines like TNF-a (Plata-Salamán, Oomura and [START_REF] Plata-Salamán | Tumor necrosis factor and interleukin-1β: suppression of food intake by direct action in the central nervous system[END_REF][START_REF] Schreyer | Obesity and diabetes in TNF-α receptordeficient mice[END_REF][START_REF] Romanatto | TNF-α acts in the hypothalamus inhibiting food intake and increasing the respiratory quotient-Effects on leptin and insulin signaling pathways[END_REF], IL-1β (Plata-Salamán, 1991), and IFN (Plata-Salaman, 1992) can induce, similar to bacterial endotoxins (Plata-Salamán and Borkoski, 1993), a reduction in food intake and play an important role in inflammation in DIO. Likewise, a few studies show that chemokines might have a modulatory role on neural networks that regulate food intake as well [START_REF] Plata-Salaman | Interferons and central regulation of feeding[END_REF]Borkoski, 1993, 1994;Le Thuc et al., 2016). As already mentioned above, cytokines like TNF-a, IFN-γ and IL-1β have been shown to induce the production of chemokines.

Thus, it makes sense that chemokines could mediate the effects of cytokines on feeding behavior and be a modulator bridging the gap between cytokine and neuronal communication. Interestingly, CCL5 has been shown to modulate a short-term reduction in food intake, 2 hours after intracerebral infusion into the 3V at 20ng/rat but not at higher concentrations (Plata-Salaman and Borkoski, 1994).

Another study was looking at the effects of chemokines in the context of HIVinduced wasting syndrome and performed ICV injections of HIV120IIIB or various chemokines including CCL5, or a combination of HIV120IIIB and either one of the chemokines. They found that HIV120IIIB induced a reduction in food and water intake over the course of 5 days with daily injections, as well as a reduction in BW after the first day. Although they found no effect on food or water intake nor BW for CCL5 alone, CCL5 injection in combination with HIV120IIIB seemed to from the effect of HIV120IIIB on all three factors [START_REF] Guzmán | RANTES, MDC and SDF-1α, prevent the HIVgp120-induced food and water intake decrease in rats[END_REF]. The difference in effect of CCL5 on food intake might be due to differences in time of measurement or different concentration of injections.

The other study found only an effect of CCL5 after 2 hours but not after, whereas in this study the food intake was measured only every 24h for 5 days.

Interestingly, CCL5 seemed to not only to protect form HIV120IIIB-induced motor impairment, but even improved the rat's ability on the rota rod [START_REF] Guzmán | RANTES, MDC and SDF-1α, prevent the HIVgp120-induced food and water intake decrease in rats[END_REF].

Another interesting study, performed an oral lipid tolerance test in 100 healthy subjects and found that while other chemokines' blood concentrations like CCL2 and IP-10 (CXCL10) were decreased, CCL5 levels increased after TG administration [START_REF] Schmid | Pro-inflammatory chemokines CCL2, chemerin, IP-10 and RANTES in human serum during an oral lipid tolerance test[END_REF].

Ueba et al found in a cross-sectional study of human patients, that elevated serum CCL5 levels were positively associated with metabolic syndrome and correlated with hypertension and glucose intolerance. Furthermore, age and IL-6 were one of the most significant predictors of CCL5 levels [START_REF] Ueba | Elevated RANTES Level Is Associated With Metabolic Syndrome and Correlated With Activated Platelets Associated Markers in Healthy Younger Men[END_REF]. Another study investigating sex differences between middle-aged female and male mice, points to an interesting increase in CCL5 levels in AT, accompanied by a higher AT mass and higher levels of pro-inflammatory T cells of middle-aged female mice as opposed to males [START_REF] Ahnstedt | Sex differences in adipose tissue CD8+ T cells and regulatory T cells in middle-aged mice[END_REF].

Urinary CCL5 was increased in both hypertensive obese and obese children compared to control patients [START_REF] Hacıhamdioğlu | Elevated urinary t helper 1 chemokine levels in newly diagnosed hypertensive obese children[END_REF]. Interestingly, this study found that anti-hypertensive treatment reduced levels of CCL5, but also TG, LDL cholesterol levels and improved insulin sensitivity among other effects.

Another interesting study, confirmed previous findings of CCL5 being increased in non-diabetic obese patients, along with TG and leptin and a reduction in resistin in serum, along with TNF-a and IL-6 in AT [START_REF] Baturcam | Physical exercise reduces the expression of RANTES and its CCR5 receptor in the adipose tissue of obese humans[END_REF]. However, apart from CCL5 and IP-10 no other elevated inflammatory markers among cytokines and chemokines were found. Circulating CCL5 seemed to correlate negatively with IL-1ra and positively with IP-10 ( et al., 2014). Surprisingly, while CCR5 mRNA expression was also upregulated in AT in obese compared to lean patients, peripheral blood mononuclear cells demonstrated a lower expression of CCR5.

Baturcam
Additionally, this team investigated the effects of physical exercise intervention for 3 months on CCL5 and other factors in the circulation in a previous study and on CCL5 and CCR5 levels in AT in this study. Interestingly, while they found physical intervention to decrease circulating inflammatory markers like TNF-a and IL-6, no difference was found in CCL5 levels [START_REF] Abubaker | DNAJB3/HSP-40 Cochaperone Is Downregulated in Obese Humans and Is Restored by Physical Exercise[END_REF]. However, both CCL5 and CCR5 levels decreased after physical exercise in AT of obese patients, along with other pro-inflammatory factors [START_REF] Baturcam | Physical exercise reduces the expression of RANTES and its CCR5 receptor in the adipose tissue of obese humans[END_REF]. Contrasting these result another study looked at the effects of either caloric restriction or exercise while remaining on a HFD on metabolic and inflammatory parameters. They study reported that after a HFD for 16 weeks, swimming exercise and caloric restriction had different results and showed that, although both interventions improved BW and AT weight in addition to circulating values of TNF-a and IL-1β, exercise surprisingly increased BAT weight and serum IL-6 as well as CCL5 levels. Contrary to other studies, HFD-fed mice without any intervention had no significant difference in CCL5 levels compared to control diet fed mice and had lower levels than mice in the exercise group [START_REF] Wasinski | Exercise and Caloric Restriction Alter the Immune System of Mice Submitted to a High-Fat Diet[END_REF]. The increase in CCL5 and IL-6 levels was accompanied by an increase in NK cell numbers in AT in exercise conditions, while HFD condition without intervention had increased expression of CD4 + and CD8 + cells that was reduced in intervention groups with higher decline in the caloric restriction group for the latter [START_REF] Wasinski | Exercise and Caloric Restriction Alter the Immune System of Mice Submitted to a High-Fat Diet[END_REF].

Another interesting study looked at the effects of different dietary protein sources in a HFD in obese nondiabetic patients on postprandial inflammatory markers and found an interesting but different effect of proteins on CCL2 and CCL5 blood values. While CCL2 levels decreased post-prandially, after all four protein diets supplemented with either cod protein, whey protein, gluten or casein, CCL5 levels increased for all diets. Interestingly, the smallest overall increase in CCL5 levels resulted after a diet supplemented with whey protein, while the same diet induced the smallest decrease in CCL2 levels (Holmer- [START_REF] Jensen | A new definition of neuropathic pain[END_REF]. The study further established a positive correlation between CCL2 levels and postprandial insulin, and a negative correlation between CCL5 levels and postprandial insulin. Intriguingly, whey protein has been associated with reductions in postprandial lipedema [START_REF] Mortensen | Differential effects of protein quality on postprandial lipemia in response to a fat-rich meal in type 2 diabetes: Comparison of whey, casein, gluten, and cod protein[END_REF][START_REF] Pal | Acute effects of whey protein isolate on cardiovascular risk factors in overweight, post-menopausal women[END_REF] in other studies, which has not been confirmed by the above-mentioned study.

Role of CCL5/CCR5 Signaling in Insulin Signaling and Diabetes

There is quite some controversy of results implicating both CCL2/CCR2 and CCL5/CCR5 signaling in obesity-associated T2DM. A great amount of studies suggested that inflammation in obese mice represents the missing link between development of obesity and T2DM.

For instance, a study in male mice fed either a standard or HFD, or either of both supplemented with a dual antagonist of both CCR2 and CCR5, found that the antagonist protected mice fed a HFD not only from BW gain and increase of adipocyte size of WAT, but also improved insulin resistance and glucose tolerance [START_REF] Huh | Dual CCR2/5 Antagonist Attenuates Obesity-Induced Insulin Resistance by Regulating Macrophage Recruitment and M1/M2 Status[END_REF]. This was accompanied by reduction in ATM and CD8 + T cell infiltration and pro-inflammatory cytokines and shifted macrophage polarization towards M2 macrophages within WAT of HFD-fed mice with antagonist [START_REF] Huh | Dual CCR2/5 Antagonist Attenuates Obesity-Induced Insulin Resistance by Regulating Macrophage Recruitment and M1/M2 Status[END_REF]. Although impressive in showing the implication of both chemokine receptors in the etiology of obesity and diabetes, this study cannot distinguish between the distinct roles of both chemokines and receptors in the pathological processes. Fortunately, other studies helped to shed more light into this issue.

For example, a study showed that CCR5 and its ligands were upregulated in the adipocyte and macrophage fraction in WAT of obese mice and serum CCL5 is increased in obese humans and mice. Additionally, Kitade and colleagues showed that CCR5 -/-mice have a better glucose tolerance and insulin sensitivity including a lower plasma insulin concentration in fed state compared to HFD fed controls. Hence, CCR5 deficiency seems to protect from insulin resistance and diabetes associated with HFD. Moreover, bone transplanted mice from CCR5 -/-on HFD were equally protected from hyperinsulinemia and glucose intolerance [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF].

Contrasting these results, Kennedy et al. found that HFD feeding of CCR5 -/- resulted in greater, systemic impairment of glucose tolerance and reduced insulin-induced phosphorylation of Akt and IRβ in AT and muscle than WT mice [START_REF] Kennedy | Loss of CCR5 results in glucose intolerance in diet-induced obese mice[END_REF].

Interestingly, in a diabetes type 1 model, different chemokines including CCL5

were induced by separate or a combined administration of the cytokines IL-1β, TNF-a and IFN-γ in islet cells of human pancreata donors. CCL5 had a more than 30-fold upregulation after treatment of islet cells with the cytokine cocktail. Notably, while those cytokines usually signal through the NF-κB signaling pathway as well as through engagement with pro-inflammatory iNOS and immunomodulatory factors, the upregulation of chemokine mRNA observed in this study in human islet cells was not due to NF-κB signaling but dependent on iNOS signaling [START_REF] Sarkar | Expression and regulation of chemokines in murine and human type 1 diabetes[END_REF]. Immunohistochemical and RT-PCR analysis revealed that CCL5 was not expressed in pancreata of healthy donors or mice, but, while not being the most abundant chemokine, it was expressed in islet and α-cells of pancreas samples of different models of type 1 diabetes in mice and diabetic human patients [START_REF] Sarkar | Expression and regulation of chemokines in murine and human type 1 diabetes[END_REF].

Further evidence implicating CCL5 in T2DM was provided by a study showing higher CCL5 serum levels in type 2 diabetic patients and further identified strong correlation between CCL5 levels and insulin resistance as determined by the Ohkura index as well as 1,5-anhydro-D-glucitol serum levels [START_REF] Dworacka | Increased circulating RANTES in type 2 diabetes[END_REF]. The latter appears to be an indicator of metabolic control and reflect short-term hyperglycaemic episodes.

The potential involvement of CCL5 signaling through CCR5 was further demonstrated by a study showing CCL5 expression and production by β cells of pancreatic islets in NOD and control mice and high levels of CCR5 by most peripheral T cells and B cells [START_REF] Carvalho-Pinto | Leukocyte attraction through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice[END_REF]. NOD mice are often used as mouse model for diabetes research as they spontaneously develop type 1 diabetes, which shares many features with human diabetes.

Neutralizing CCR5 via antibodies in the NOD mouse model of diabetes, did not amend insulitis development but prevented from progression of insulitis to β-cell destruction and diabetes [START_REF] Carvalho-Pinto | Leukocyte attraction through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice[END_REF].

Supporting this, the islet-protecting role of CCR5 was also shown in islet allografts into CCR5 deficient mice and control mice that developed chemically induced diabetes. Islet allografts survival in CCR5 -/-recipients was much more prolonged compared to CCR5 +/+ mice [START_REF] Abdi | The role of CC chemokine receptor 5 (CCR5) in islet allograft rejection[END_REF]. It appears that cytokine-induced CCL5 production in islet cells leads to chemotaxis of lymphocytes and their activation via CCR5 signaling. This leads to Th1 cell polarization and destruction of islet cells [START_REF] Carvalho-Pinto | Leukocyte attraction through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice[END_REF]. It seems that the cytokine IFN-γ might be responsible for the progression of insulitis to diabetes, as its expression is associated with lymphocyte infiltration of islets. However, the NOD mouse model of diabetes indicates that CCR5 is less responsible for lymphocyte invasion of islets, but more for their invasiveness and deleterious actions. Other chemokines also secreted by pancreatic islet cells seem to be responsible for the initial stages of T cell infiltration leading to insulitis [START_REF] Frigerio | β cells are responsible for CXCR3-mediated t-cell infiltration in insulitis[END_REF][START_REF] Carvalho-Pinto | Leukocyte attraction through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice[END_REF].

These results were confirmed and extended by another study implicating the chemokines CCL3 and 4, in particular the CCL3 to CCL4 ratio in the development of diabetes and the progression of insulitis to destructive diabetes [START_REF] Cameron | Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes[END_REF]. Interestingly, these chemokines can act like CCL5 through CCR5. Moreover, IL-4 administration to NOD mice was shown to protect from diabetes through promoting a Th2-like cytokine environment by diminishing pancreatic CCL3 expression, while increasing CCL4 expression.

Likewise, IL-4 treatment downregulated CCR5 expression, but not other chemokine receptors like CCR1, 2 , 3 and others, in splenic T cells and islets [START_REF] Cameron | Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes[END_REF]. In fact, this is interesting in particular in the context that CCR5 expression is associated with Th1-type immune responses, whose activation in turn is believed to mediate β-cell destruction [START_REF] Bonecchi | Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s[END_REF][START_REF] Loetscher | CCR5 is characteristic of Th1 lymphocytes [6[END_REF][START_REF] Cameron | Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes[END_REF]. Recent studies have described the constitutive expression of CCL5 in pancreatic islets of both mice and humans [START_REF] Liu | The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis[END_REF] and expression of CCL5 in islets of NOD mice and human patients with type 1 diabetes [START_REF] Bouma | Evidence for an enhanced adhesion of DC to fibronectin and a role of CCL19 and CCL21 in the accumulation of DC around the pre-diabetic islets in NOD mice[END_REF][START_REF] Sarkar | Expression and regulation of chemokines in murine and human type 1 diabetes[END_REF].

Data in support of the assumption that CCL5 has a critical role in diabetes have shown that plasma CCL5 is increased in human type 1 and type 2 diabetes [START_REF] Nomura | Significance of chemokines and activated platelets in patients with diabetes[END_REF][START_REF] Pfleger | Relation of circulating concentrations of chemokine receptor CCR5 ligands to C-peptide, proinsulin and HbA1c and disease progression in type 1 diabetes[END_REF][START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF].

In addition to that, patients with T2DM have reduced levels of GLP-1 [START_REF] Nauck | Reduced incretin effect in type 2 (non-insulin-dependent) diabetes[END_REF][START_REF] Toft-Nielsen | Determinants of the impaired secretion of glucagon-like peptide-1 in type 2 diabetic patients[END_REF], which is an incretin that amplifes insulin secretion among other effects [START_REF] Drucker | The biology of incretin hormones[END_REF][START_REF] Campbell | Pharmacology, physiology, and mechanisms of incretin hormone action[END_REF].

Recently, CCR1, a receptor for CCL5 was found to be expressed in enteroendocrine and GLP-1 positive cells in murine small intestine and in a human intestinal enteroendocrine cell line [START_REF] Pais | RANTES (CCL5) reduces glucose-dependent secretion of glucagon-like peptides 1 and 2 and impairs glucose-induced insulin secretion in mice[END_REF]. In addition to that, CCL5 inhibited glucose stimulated GLP-1 and GLP-2 secretion both in vitro and in vivo, while the effect was prevented with Met-RANTES, an antagonist for CCL5 receptors. The inhibitory effect of CCL5 on GLP-1 secretion appeared to be mediated through a decrease in intracellular cAMP and PKA activity [START_REF] Pais | RANTES (CCL5) reduces glucose-dependent secretion of glucagon-like peptides 1 and 2 and impairs glucose-induced insulin secretion in mice[END_REF]. The study further showed that CCL5 diminished transport activity of rheogenic sodium-coupled glucose transporter, which seems to mediate glucose-dependent GLP-1 secretion by enteroendocrine cells [START_REF] Pais | RANTES (CCL5) reduces glucose-dependent secretion of glucagon-like peptides 1 and 2 and impairs glucose-induced insulin secretion in mice[END_REF].

Further evidence was provided by a study demonstrating the constitutive expression and production of CCL5 by glucagon expressing pancreatic cells in mice, and in both human glucagon producing a and insulin producing β cells [START_REF] Liu | The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis[END_REF].

Recently, it was shown that CCL5 can bind and signal through a newly discovered orphan GPCR called GPR75 [START_REF] Ignatov | RANTES stimulates Ca 2+ mobilization and inositol trisphosphate (IP 3 ) formation in cells transfected with G protein-coupled receptor 75[END_REF]. While the above mentioned study confirmed the expression of CCR1 in nondiabetic human islets, and CCR3 and CCR5 in healthy mouse pancreatic islets, they also demonstrated the expression of GPR75 in a and β cells of both mice and human islets and at higher levels than the other receptors of CCL5 [START_REF] Liu | The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis[END_REF]. The study further demonstrates an alternative physiological role of CCL5 in pancreatic β cell function. They showed that CCL5 seems to stimulate insulin secretion in mouse and human β cells through GPR75 activation, which in turn appears to act by coupling to Gq proteins to induce an extracellular calcium influx and PLC activation [START_REF] Liu | The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis[END_REF]. Lastly, Liu and colleagues have found that in vivo peripheral co-administration of CCL5 during a glucose tolerance test (GTT) and insulin tolerance test (ITT), improved glucose tolerance and increased insulin secretion in both lean and insulin-resistant ob/ob mice, but did not alter insulin sensitivity [START_REF] Liu | The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis[END_REF].

Interestingly, insulin was reported to have anti-inflammatory effects. Insulin infusion of obese patients suffering from type 2 diabetes for 4 hours significantly reduced plasma concentrations of chemokines including CCL3 and CCL5 as well as the expression of CCL4 and CCL5 and chemokine receptors such as CCR2 and CCR5 in mononuclear cells [START_REF] Ghanim | Suppressive effect of insulin infusion on chemokines and chemokine receptors[END_REF]. Should CCL5 signaling really be involved in insulin secretion as suggested in the data presented so far, it would be tempting to assume that the suppressive effect of insulin on chemokines could be part of a regulatory feedback loop.

In a Finnish diabetes prevention study, Herder et al. found a positive correlation between elevated CCL5 levels and the development of T2DM. It seems that CCL5 concentrations were indicative of high-risk patients that are more likely to become diabetic (Christian Herder et al., 2006). These results

were confirmed in a second population-based study, in which systemic CCL5 levels were higher and correlated in both patients with impaired glucose and T2DM [START_REF] Herder | Association of systemic chemokine concentrations with impaired glucose tolerance and type 2 diabetes: Results from the cooperative health research in the region of augsburg survey S4 (KORA S4)[END_REF].

Important evidence for a role of CCL5-CCR5 signaling in hypothalamic insulin regulation was shown by a recent study, which not only confirmed CCR5 expression in the ARC of the hypothalamus, most likely in POMC-expressing neurons as shown by immunostaining of primary hypothalamic neurons, but also that CCR5 seems to associate with IR in the hypothalamus. Furthermore, they established that CCR5 and CCL5 deficient mice or mice treated with Met CCL5, an antagonist for CCR5, are insulin resistant and glucose intolerant on a normal diet [START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF]. On the cellular level, this was likely due to modification in insulin signaling pathways in the hypothalamus, such as raised AMPKa and phosphorylation of IRS-1, which is indicative of a reduced activation of insulin signaling and CCL5 can induce GLUT4 translocation in hypothalamic neurons [START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF].

Role of CCL5/CCR5 Signaling in Neuropathic Pain

Some opiate abusers tend to also be infected with HIV and have higher probability of acquiring neurological complications such as ADC reviewed in [START_REF] Anthony | The effects of illicit drugs on the HIV infected brain[END_REF]. It seems that the neuronal degeneration caused by HIV is modulated by opiates. HIV-1 infection is facilitated by chemokine coreceptors such as CCR5 and CXCR4 and thus it seems possible that opiates might act through chemokine receptors to modulate the effects on neurons, especially since CCL5 was shown to prevent neurotoxic effect of a HIV envelope glycoprotein called gp120IIIB. This neuroprotective effect was blocked in CCL5-immunoneutralized cerebellar cultures of neurons and glia cells that subsequently were treated with morphine and gp120 [START_REF] Avdoshina | Morphine Induces the Release of CCL5 from Astrocytes: Potential Neuroprotective Mechanism Against the HIV Protein gp120[END_REF]. This study seems to indicate that morphine might have a neuroprotective effect against gp120Bal toxicity, which is mediated by CCL5 released by astrocytes.

Interestingly, a study found that morphine induced CCL5 secretion from astrocytes in primary cultures in a time-dependent manner [START_REF] Avdoshina | Morphine Induces the Release of CCL5 from Astrocytes: Potential Neuroprotective Mechanism Against the HIV Protein gp120[END_REF]. Furthermore, CCR5 expression and secretion was increased in the immune system and astrocytes by morphine and a µ-opioid receptor agonist treatment [START_REF] Suzuki | Interactions of opioid and chemokine receptors: Oligomerization of mu, kappa, and delta with CCR5 on immune cells[END_REF][START_REF] Steele | μ-Opioid modulation of HIV-1 coreceptor expression and HIV-1 replication[END_REF].

Lack of CCL5 in a chronic neuropathic pain model of a partial sciatic nerve ligation (PNL) model was investigated using CCL5 -/-mice [START_REF] Liou | Absence of C-C motif chemokine ligand 5 in mice leads to decreased local macrophage recruitment and behavioral hypersensitivity in a murine neuropathic pain model[END_REF].

They found less hypersensitive behaviour in CCL5 -/-mice compared to controls, which was accompanied by diminished macrophage infiltration of damaged nerves and pro-inflammatory cytokines while anti-inflammatory proteins such as IL-4 and IL-10 were increased in CCL5 deficient mice but not in controls. Interestingly, significant differences in levels of opioid peptides (ekephalins, dynorphins and β-endorphins) have been found between KO and control mice [START_REF] Liou | Absence of C-C motif chemokine ligand 5 in mice leads to decreased local macrophage recruitment and behavioral hypersensitivity in a murine neuropathic pain model[END_REF]. These results are among the first to implicate CCL5 and CCR5 in regulating pathways of hyperalgesia in neuropathic pain associated with inflammation and authors suggest that decreased hyperalgesia might be due to a decreased macrophage infiltration and activity in the injured nerve. CCR5 expression was found to be increased in liver tissue of obese mice and humans that are associated with hepatic steatosis and NASH [START_REF] Wu | T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity[END_REF][START_REF] Huber | CC chemokine and CC chemokine receptor profiles in visceral and subcutaneous adipose tissue are altered in human obesity[END_REF][START_REF] Bertola | Hepatic expression patterns of inflammatory and immune response genes associated with obesity and nash in morbidly obese patients[END_REF][START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF]. However, apart from a decreased inflammatory phenotype in WAT and decreased diabetic phenotype on HFD, CCR5 -/-mice had also reduced liver steatosis, hepatic TG and lipogenic mRNA expression, after 15 weeks on a HFD diet [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF]. Interestingly, bone marrow transplant from CCR5 -/-to WT mice could ameliorate also the DIO-induced liver steatosis, which seems to be mediated by myeloid specific CCR5 signaling. [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF]. HIV preferentially uses CXCR4 chemokine receptor for entry. However, dual tropism for some strains of HIV have been also observed, meaning they can use either of the receptors for viral entry [START_REF] Waller | Chemotherapy of infections[END_REF]. HIV tropism for CCR5-expressing cells happens usually early during infection and can transit tropism to CXCR4 or can even develop a dual tropism for both Chemokineexpressing cells later on during infection.

Surprisingly, some studies report that although CCR5 facilitates HIV-1 infection, its chemokine ligands CCL5, MIP-1α and MIP-1β seemed in contrary to act as suppressive factors for HIV infection by blocking viral infection of cells, or by delaying of disease progression [START_REF] Cocchi | Identification of RANTES, MIP-1α, and MIP-1β as the major HIVsuppressive factors produced by CD8+ T cells[END_REF]. However, these results seem to be controversial as other studies have either confirmed [START_REF] Zanussi | Serum levels of RANTES and MIP-1α in HIV-positive long-term survivors and progressor patients [1[END_REF][START_REF] Mackewicz | Do β-chemokines have clinical relevance in HIV infection?[END_REF][START_REF] Berger | CHEMOKINE RECEPTORS AS HIV-1 CORECEPTORS: Roles in Viral Entry, Tropism, and Disease[END_REF][START_REF] Polo | Longitudinal analysis of serum chemokine levels in the course of HIV-1 infection[END_REF][START_REF] Levy | The search for the CD8+ cell anti-HIV factor (CAF)[END_REF] or contradicted these data and even found that these ligands rather increase HIV infection [START_REF] Schmidtmayerova | Chemokines and HIV replication[END_REF][START_REF] Kelly | Cutting Edge: Dichotomous Effects of {beta}-Chemokines on HIV Replication in Monocytes and Monocyte-Derived Macrophages[END_REF][START_REF] Kinter | CC-chemokines enhance the replication of T-tropic strains of HIV-1 in CD4(+) T cells: role of signal transduction[END_REF][START_REF] Ye | Lack of good correlation of serum CC-chemokine levels with human immunodeficiency virus-1 disease stage and response to treatment[END_REF].

It is not surprising that mutations in CCR5 genes have been observed in the human populations that have a resistance for HIV-1 infection and delays AIDS progression of infected patients.

A polymorphism found in the gene for CCR5 is a 32 base pair deletion in the coding domain, leading to a frame shift mutation (CCR5Δ32), that results in a truncated protein that no longer is expressed at the cell surface and thus prevents HIV-1 infection by M-tropic or dual tropic strains [START_REF] Samson | Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene[END_REF]. Whereas rare individuals homozygous for the CCR5Δ32 allele seem to be resistant to M-tropic strains of HIV-1 despite repeated exposure, people who are heterozygous seem to have a partial protection from HIV-1 infection, slower disease progression and better survival compared to patients with a normal CCR5 allele [START_REF] Mellors | Prognosis in HIV-1 infection predicted by the quantity of virus in plasma[END_REF]. A cohort frequency of HIV-infected

Caucasian patients presented with a 32% lower frequency of heterozygous and no homozygous alleles than the general population [START_REF] Mellors | Prognosis in HIV-1 infection predicted by the quantity of virus in plasma[END_REF][START_REF] Samson | Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene[END_REF]. People being heterozygous for the polymorphous allele are quite common compared to its homozygous allele frequency and was reported to be ~20% in some populations [START_REF] Landau | Homozygous defect in {HIV-1} coreceptor accounts for resistance of some multiply-exposed individuals to {HIV-1} infection[END_REF]. With the highest prevalence of the allele in European people and a frequency of 1-3% in the Caucasian population, homozygous CCR5Δ32 allele was not found so far in the population of African or Asian origin [START_REF] Huang | The role of a mutant CCR5 allele in HIV-1 transmission and disease progression[END_REF][START_REF] Samson | Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene[END_REF].

In a famous case known as "Berlin patient", a patient called Timothy Ray Brown was HIV positive, before developing acute myeloid leukemia and ultimately undergoing hematopoietic stem cell transplantation from a donor homozygous for CCR5 the CCR5Δ32 mutation. Despite the discontinuation of antiretroviral treatment, the patient's viral load remained undetectable for 20 months in both lymphocytes and serum after the transplant [START_REF] Hütter | Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation[END_REF]. The patient still remains HIV-free, while a second patient, who similar to Timothy was HIV positive and undergoing alleiogenic hematopoietic stem cell transplant for Hodgin's lymphoma from a CCR5-deficient donor. Similar to the first patient to undergo this procedure also this patient virus free in the most recent follow-up 18 months after stopping his antiretroviral medication [START_REF] Gupta | HIV-1 remission following CCR5Δ32/Δ32 haematopoietic stem-cell transplantation[END_REF]. These successful treatments, including with the presented studies show how CCR5 and its ligands could represent a potential therapeutic target for HIV treatment. Indeed one drug named maraviroc (Pfitzer) that acts as a CCR5 antagonist, was FDA approved in 2007, whereas some other candidates are still in different stages of clinical trials ( [START_REF] Melica | Maraviroc-containing regimen suppresses HIV replication in the cerebrospinal fluid of patients with neurological symptoms[END_REF][START_REF] Tiraboschi | Maraviroc concentrations in cerebrospinal fluid in HIV-infected patients[END_REF][START_REF] Sorce | The chemokine receptor CCR5 in the central nervous system[END_REF].

Interestingly, although the presence of CCR5 and CXCR4 was found to facilitate viral entry to Immune cells in the periphery, a study found that astrocytes and microglia express both co-receptors CCR5 and CXCR4, and are equally susceptible to viral HIV-1 infection. Since chemokine co-receptors are expressed in the CNS, it makes sense that HIV-1 infection affects also the CNS and causes several neuropathological and behavioral disorders [START_REF] Liu | CD4-Independent Infection of Astrocytes by Human Immunodeficiency Virus Type 1: Requirement for the Human Mannose Receptor[END_REF].

Objectives

In summary, the here presented data show that many of the conditions mentioned above such as AD, PD, Huntington's disease, HIV-associated metabolic changes and T2DM have an inflammatory component in common, as well as energy metabolism and glucose impairment with insulin resistance. Cytokines and chemokines are very important in the regulation of immune system responses in many conditions such as the ones mentioned above and have been implicated manifold in functions of the CNS and in the periphery.

A certain chemokine, CCL5 seems to share a special connection, as CCL5 signaling through its cognate receptor CCR5 is not only Implicated in all diseases mentioned above in particular in obesity and diabetes, but also seems to be expressed in the CNS and was suggested to have a neuromodulatory function and effects on insulin regulation. Moreover, CCL5

and its receptor represent potential and interesting therapeutic candidates for various conditions.

However, the mechanisms behind CCL5-CCR5 signaling in the central regulation of energy metabolism, insulin regulation and glucose homeostasis remain unclear and need further investigation.

For this reason, the present study is divided into two parts that focus on:

1. Developing a mouse model of DIO to test the effect of different lipid nature and ω6/ω3 ratio on the development of DIO and associated comorbidities such as diabetes and neuropathic pain.

Experimental Strategy

In the first part of this study, our aim was to establish a mouse model of DIO by testing different HFDs with different lipid nature and ω6/ω3 ratio on the development of DIO and associated comorbidities. We used 4 weeks old weight-matched male mice, which were were given either a SD, a HFD-B with 40% of animal-derived lipids based on butter, HFD-C with 20% vegetal lipids (ω6/ω3=2.3), HFD-SC with 20% of vegetal lipids (ω6/ω3=7.3) or HFD-SN with 20% vegetal lipids (ω6/ω3=17.3) (Table 1) and measured their development of obesity and monitored glucose metabolism as well as inflammatory markers in the hypothalamus after 12 weeks of experimental protocol (Fig. 24). In the second part of our project, our aim was to study the effect of CCL5-CCR5 signaling in the regulation of energy balance in the context of dietinduced obesity and its associated comorbidities such as diabetes and neuropathic pain.

STANDARD (SD

To study the role of CCL5 and its receptor CCR5 in the regulation of energy balance and in DIO, we decided to use a loss of function approach by employing a mouse model of DIO as depicted in figure 25 with mice deficient for CCL5 and CCR5 as compared to WT littermates. We decided to use the most obesogenic diet identified in the first part of our experiment (HFD-B which will be called HFD in the second part of the study) to assess the effects of CCL5 and CCR5 deficiency in KO mice in a HFD context. We assessed the development of obesity and T2DM as well as neuropathic pain by measuring food intake, body weight and other metabolic parameters at 8 and 16 weeks of diet as representative time points of obesity installation and maintenance.

At the end of the experiment, we sacrificed mice and took different blood and tissue samples to be able to assess the modulation of peptides, and inflammatory markers. Furthemore, to assess the role of CCL5/CCL5 signaling in a physiological context in the regulation of energy balance we decided to use an immunohistochemical/in situ hybridization approach to verify the expression of CCL5 and CCR5 in hypothalamic nuclei. In addition to that, we employed stereotaxic surgery to implant cannula and repeatedly inject CCL5 and its antagonist Met CCL5 ICV to assess its role on BW and food intake regulation in a physiological context (Fig. 26 and 27 in Materials & Methods).

Materials and Methods

Animal Procedures

8-week-old C57Bl/6J male mice (20-25g) used in this study were from Janvier Labs, France. CCL5 -/-(CCL5 KO; B6.129P2-Ccl5 tm1Hso /J) and CCR5 -/-(CCR5 KO;

B6.129P2-Ccr5 tm1Kuz /J) mice were originally from the Jackson Laboratory, USA.

Animals were housed in an SPF facility in medium-size (up to 5 animals/cage) or large cages (up to 10 animals/cage) filled with wooden bedding, one plastic house and nesting material for enrichment. All CCL5-WT/CCR5-WT and CCL5-KO/CCR5-KO male mice used in this study were littermates born to heterozygous females. Pups were weaned at 28 days of age, identified by ear tags, and genotyped by PCR as described by Jackson Laboratory.

Pups from various litters were then randomly grouped according to their genotype in cages and had ad libitum access to water and standard chow (SD; 3 395 kcal/kg of which 25.2% are from proteins, 13.5% fat and 61.3% from carbohydrates; Safe #A03) (Table 1).

Animals were housed in a temperature (21-23°C) and hygrometry (70-80%)controlled room with a 12-h light/12-h inversed dark cycle (lights on at 8 pm).

All the described diet experiments were performed on male animals and started at 4 or 12 weeks of age.

For experiments involving treatment, animals from the same genotype were randomly assigned to experimental groups.

All of the protocols were carried out in accordance with French standard ethical guidelines for laboratory animals and with approval of the Animal Care Committee (Nice-French Riviera, registered number 04042.02).

Blood and Tissue Collection

At the end of the experimental protocol, mice were anesthetized via inhalation of 3% isofluarane and blood was withdrawn from retro-orbital sinuses using heparinized Pasteur pipettes (heparin 50 mg/mL, Sigma) from animals in non-fasting or fasting conditions as stated. Blood samples were collected in 1.1mL Z-Gel Micro tubes (Sarstedt) and centrifuges for 10 min at 4°C, at 10000 rpm. The supernatant was recovered in Eppendorf tubes and stored at -80°C. In nonfasting conditions, animals were fed ad libitum and in fasting conditions, animals were fasted overnight for 6 or 16 hr. Furthermore, brain, dorsal root ganglia, spinal cord and AT were collected, frozen on dry ice and stored at -80°C until further processing. For GTT serum was collected from tubes (Sarstedt, France) after centrifugation at 3000 rpm for 10 min.

Diets

Mice were food deprived 1h-2h prior the onset of the dark period to synchronize groups. For the first part of our study, mice were administered one of four HFDs (custom diets by Safe diets, France) or SD according to table 1

Glucose Tolerance Test (GTT)/Insulin Tolerance Test (ITT)

To measure the capacity to regulate glucose metabolism and insulin sensitivity a GTT and ITT were performed according to the IMPReSS guidelines (International Mouse Phenotyping Resource of Standardized Screens) and in agreement with [START_REF] Heikkinen | Evaluation of Glucose Homeostasis[END_REF] and [START_REF] Ayala | Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice[END_REF]. To further, characterize glucose metabolism and the capacity of HFD-fed mice to secrete insulin we measured glucose-stimulated insulin secretion. This was done during a GTT as described above, but in addition to measuring blood glucose, blood samples of ~20µL were collected from tail vein after incision at the tip of the tail at basal conditions "T0" and 15, 30 and 60min after glucose injection. Blood was collected using Microvette® CB 300 Z tubes (Sarstedt) and kept on ice until centrifuged was done at 4°C for 10 min at 3000 rpm after the test. Plasma supernatant was recuperated and kept at -80°C until ELISA for insulin testing was performed.

Microcomputed Tomography Analysis

AT quantification of anesthetized mice was carried out using a SkyScan-1178 X-ray micro-computed tomography analysis and performed as previously described [START_REF] Beranger | Oxytocin reverses ovariectomy-induced osteopenia and body fat gain[END_REF].

Briefly, mice were scanned using the following parameters: 104 μm of voxel size, 49 kV, 0.5 mm thick aluminum filter, 0.9° of rotation step. Total AT volume was determined between the cervical vertebra 1 (C1) and the thoracic vertebra 13 (T13), whereas intra-abdominal and subcutaneous AT area was measured on one section at the lumbar 5 (L5) level. AT area and volume were analyzed using NRecon and CTAn software (Bruker microCT, Kontich, Belgium).

Adipose Tissue Histology

Tissues were collected, fixed for 24 hours in 4% paraformaldehyde, paraffinembedded, sliced into 5-μm thick sections using a microtome RM 2255 (Leica)

and dried overnight at 37°C. All sections were then deparaffinized in xylene, rehydrated through alcohol, and washed in phosphate-buffered saline.

Hematoxylin-eosin-stained sections were imaged with the Vectra Polaris slide scanner using 20X NA 0.45 objective and visualized with Phenochart software.

Automated analysis of adipocytes surface was performed using an in-house

ImageJ software macro (F. Brau, imaging and cytometry platform).

Plasma Triglyceride (TG) and Glycerol Contents

TG plasma contents and glycerol plasma contents were immediately measured with a serum TG kit (T2449; Sigma-Aldrich France), and free glycerol reagent (F6428; Sigma-Aldrich), according to the manufacturer's instructions and as described in [START_REF] Pisani | The K+ channel TASK1 modulates b-adrenergic response in brown adipose tissue through the mineralocorticoid receptor pathway[END_REF]. We used 50µl of plasma and 200µl of reagent.

RNA Isolation and Quantitative qPCR

Total RNA from frozen hypothalamus or AT was isolated using Fast Prep apparatus (Q-Biogene, France) as we previously described (Le Thuc et al., 2016). First-strand cDNAs were synthesized from 1-2 μg of total RNA with 200U

of SuperScript III reverse transcriptase (SuperScriptIII, Invitrogen, France) in the appropriate buffer in the presence of 25 ng/µl oligo-dT primers, 0. for normalization with the 2 ΔΔ CT method. Primers (detailed in Table 3) were purchased from Eurogentec (France). Forward and reverse primers were designed using Primer Express software. Genes were analyzed with a SYBR® Green I system.

-a-R AGGGCAATTACAGTCACGGC nM mCCL2-F CCAACTCTCACTGAAGCCAGC mCCL2-R CAGGCCCAGAAGCATGACA nM mCCL5-F ACACCACTCCCTGCTGCTTT mCCL5-R AAATACTCCTTGACGTGGGCA nM mIba1-F GGATTTGCAGGGAGGAAAA mIba1-R TGGGATCATCGAATTG nM mGFAP-F TCGACATCGCCACCTACAG mGFAP-R GTCTGTACAGGAATGGTGATGC nM mMCH-F GAAGGAGAGATTTTGACATGCTCA mMCH-R CCAGCAGGTATCAGACTTGCC nM mORX-F GCCGTCTCTACGAACTGTTGC mORX-R CGCTTTCCCAGAGTCAGGATA nM mPOMC-F AGTGCCAGGACCTCACCA mPOMC-R CAGCGAGAGGTCGAGTTTG nM mCART-F CGAGAAGAAGTACGGCCAAG mCART-R CTGGCCCCTTTCCTCACT nM mNPY-F CCGCTCTGCGACACTACAT mNPY-R TGTCTCAGGGCTGGATCTCT nM mAgRP-F CCCAGAGTTCCCAGGTCTAAGTCT mAgRP-R CACCTCCGCCAAAGCTTCT nM mIRS1-F CTATGCCAGCATCAGCTTCC mIRS1-R TTGCTGAGGTCATTTAGGTCTTC nM mPPARγ-F GGGGGTGATATGTTTGAACTTG mPPARγ-R GAAAGACAACGGACAAATCACC nM mGAPDH-F GAACATCATCCCTGCATCC mGAPDH-R CCAGTGAGCTTCCCGTTCA nM

Cytokines, Chemokines and Hormones Quantification

A V-Plex multiplex assay (Meso Scale Discovery, USA) and mouse ELISA kits for CCL5 (RayBiotech, France), leptin (AssayPro, France) were used to measure respectively the plasma levels of inflammatory mediator and leptin, according to the manufacturer's protocols. Plasma insulin concentrations were determined using the Insulin AlphaLisa detection kit (cat. number AL204C) from Perkin Elmer.

Immunohistochemical Analysis

Brains were harvested from mice perfused with 4% paraformaldehyde in phosphate buffered saline (PBS) and post-fixed in the same fixative overnight 4°C. Thirty µm thick brain coronal sections were cut on a vibratome (HM 650V, Microm, Germany), blocked for 1h with 3% normal goat serum in PBS containing 0.1% Triton X-100 and incubated with primary antibodies overnight at 4°C. Primary anti-rabbit antibodies were against Iba1 (1:500, CP290A, B, Biocare Medical, USA) and GFAP (1:300, Z0334, Dako, Danemark). Primary anti-rabbit antibodies were against Iba1 (1:500, CP290A, B, Biocare Medical, USA) and GFAP (1:300, Z0334, Dako, Danemark). The immunoreactivity of the receptors of CCL5, namely CCR1, CCR3 and CCR5 were tested with the following antibodies: anti-CCR1 Pa1-41062 (Invitrogen -Thermofisher Scientific,

France; tested with dilutions: 1/200, 1/3000 and 1/9000), anti-CCR1 (CAT#:

TA336235 -lot010509-03, OriGene, France, tested at 1/200, 1/500 and 1/1000), anti-CCR3 (ab36827, Abcam, France tested at 1/1000), anti-CCR5 (17833 lotK1317, Santa Cruz Biotech, USA tested at 1/300, 1/500, 1/1000, 1/3000 and 1/9000).

Adequate AlexaFluor conjugated secondary antibodies were used for immunofluorescence microscopy. Sections were mounted in Vectashield solution (H-1000, Vector Laboratories). Images were acquired with confocal laser-scanning microscope (TCS SP5, Leica Biosystems, Germany).

Single-molecule RNA In Situ Hybridization

RNA in situ hybridization experiments were performed using the RNAScope® technology, which has been previously described [START_REF] Wang | RNAscope[END_REF]. Paired double-Z oligonucleotide probes were designed against target RNA using custom software. The following probes were used:

-RNAScope® Probe-Mm-Ccl5, cat.no. 469601, NM_013653.3, 11pairs, nt 4-527.

-RNAScope® Probe-Mm-Ccr5-C3, cat.no. 438651-C3, NM_009917.5, 20pairs, nt 1337-2336.

-RNAScope® Probe-Mm-Aif1-C2, cat.no. 319141-C2, _NM_019467.2, 18 pairs, nt 31-866.

The RNAScope® Multiplex Fluo kit v2 (cat. no. 323100) (Advanced Cell Diagnostics, Newark, CA) was used according to the manufacturer's instructions. Perfused frozen mouse brains were cut into sections of 10 µm thickness and prepared according to manufacturer's recommendations.

Each sample was quality controlled for RNA integrity with a mix of probes specific to the housekeeping genes PPIB, POLR2A and UBC (RNAScope® 3plex Positive Control Probe -Mm, cat.no. 320881). Negative control background staining was evaluated using a probe specific to the bacterial dapB gene (RNAScope® 3-plex Negative Control Probe -Mm, cat.no.

320871

). Probes were visualized by conjugation to Opal™ flourescent dyes (Opal™ 570, 650 and 520) according to manufacturer's recommendations.

Fluorescent images were acquired using a confocal microscope (SP5) using a 40x objective.

Stereotaxic Cannula Guide Placement

ICV injections were performed via guide cannula (26G PED 4 mm, 4 mm length below pedestal PHYMEP, France) into the right lateral ventricle of male C57BL6/J mice purchased from Jackson Laboratory in France. To place guide

Composition of Solutions for ICV Infusion

The aCSF is composed of 100 mL of physiological stock (119.0 mM NaCl, 2. Recombinant murine RANTES/CCL5, a 7.8kDa protein in powdered form, was purchased from Peprotech, France and reconstituted and diluted in aCSF (as described above) to achieve a final concentration of 10 ng, 50 ng and 100 ng per mouse per day for the intial experiment and 0.15 µg/µL for the second experiment and stored frozen at -20°C until injection.

Met RANTES human trifluoroacetate salt stock (50µg) purchased from Bachem AG, Switzerland was diluted in aCSF to a final concentration of 0.15 µg/µL and stored frozen at -20°C until infusion [START_REF] Proudfoot | Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist[END_REF]. Met RANTES will be referred to as Met CCL5 in the manuscript.

Chronic ICV Infusions

After two weeks of recuperation and handling of mice, including the removal and reattachment of the dummy cap for the guide cannula and verifying that guide cannula is not blocked, mice were habituated for injections. Then 2 µL of artificial cerebrospinal fluid (aCSF) were infused at a rate of 0.5 µL/min via an internal injector (33 GA, 4 mm PED, with 1 mm projection, PHYMEP, France) connected to the syringe through a polyethylene tube (0.023"X.0.038"X0.0075", PHYMEP, France). Once mice started to recover from the initial weight loss from aCSF infusions, they were randomly assigned to groups and infused every second day with 2 µL of either aCSF, Met CCL5 (0.15 µg/µL) or CCL5 (0.15 µg/µL) and BW and food intake were measured before and 2h and 24h after infusion. For the second experiment, Hargreaves' thermal pain sensitivity, GTT, ITT and was tested at 3 and 4 weeks after initiation of injections as depicted in Fig. 27.

Hargreaves' Test of Thermal Pain Sensitivity

The thermal heat pain thresholds were measured with the plantar Hargreaves' radiant heat test (Analgesia Meter, Bioseb). Mice were placed individually in elevated observation chambers (8 x 8 cm) with translucent glass at the bottom, where they were acclimated for at least 20 min before measures. A radiant infrared heat source was then focused under the animal's hind paw and maintained until the mice retrieved its paw. Reaction scores, paw withdrawal thresholds (PWT), were the results of two measures on both hind paws, with a minimum of 5 min interval between measures.

Statistical Analysis.

Displayed values are means ± SD. The ROUT method (Robust regression and

Outlier removal) was used to identify outliers with a Q coefficient equal to 1%.

Comparisons between groups were carried out using a non-parametric Mann-Whitney test for single comparison and a Kruskal-Wallis test with Dunn's correction or two-way ANOVA for multiple comparisons and interaction. A Pvalue of ≤ 0.05 was considered statistically significant. All tests were performed using GraphPad Prism 7.02 and Microsoft Office Excel. Numbers of animals and cells are given in the legends.

Results

The Effects of Lipid Nature on the Development of Obesity

In the first part of the project, we focused on establishing a mouse model of We did not find any significant difference between HFDs with 20% lipids and different ω6/ω3 ratios. We also measured food intake every second day and found, that although HFD-fed mice, independent on source of lipids or ω6/ω3 ratio, consumed on average less grams of food per mice, that there was no significant difference between kcal intake inbetween diets, and it remained stable over time (not shown). It thus seems that a different ratio of ω6/ω3 does not seem to affect BW gain or food intake differently, especially seeing that HFD-B has a lower ω6/ω3 ratio with 8.0 compared to HFD-SN, which has lower amounts of lipids but a ratio of 17.3.

Our measurement of serum leptin concentrations at 12 weeks of diet confirmed the trend seen in BW gain, as HFD-B had the highest concentration of Leptin at 12 weeks of diet compared to SD diet (Fig. 28C). We did not observe any significant difference for any other HFDs with 20% of lipids, but the data suggests a tendency for an increase in leptin concentration according to the ω6/ω3 ratio, as we see the highest increase in leptin with HFD-SN, which has the highest ω6/ω3 ratio of 17.3 at 12 weeks of diet. This is followed by an increase in serum leptin levels in mice fed HFD-SC (ω6/ω3=7.3), which in turn is higher than HFD-C (ω6/ω3=2.3), that resembles the closest to the leptin concentrations of SD-fed mice (Fig. 28C).

Expression

Although, we have no perceived any differences in kcal intake between mice fed different diets, we still were interested in determining, whether the consumption of HFDs based on different lipid nature and ω6/ω3 ratio could distingly affect the expression of hypothalamic neuropeptides, which are implicated in the control of food intake and energy balance.

We thus performed qPCR to quantify the relative expression of hypothalamic neuropeptides and found that mice fed a HFD-B had significant downregulation of anorexigenic POMC mRNA, as well as an upregulation of orexigenic neuropeptides NPY and MCH mRNA in the hypothalamus but no differences in other neuropeptides tested (Fig 29A -F). Interestingly, we did not find any significant changes in neuropeptide expression between HFDs based 20% of lipids from a plant source and different ω6/ω3 ratio, indicating that ω6/ω3 ratio seems to have no effect on neuropeptide expression in the hypothalamus after 12 weeks of diet. Our results indicate that a HFD based on butter had an effect on neuropeptide expression. However, we cannot infer from the data obtained, whether the effect is due to higher lipid content in HFD-B as opposed to all other diets or due to the nature of lipids, which are retrieved from animal products.

The Effects of Lipid Nature on Glucose Homeostasis

Due to the close association between obesity and diabetes, we decided to test whether the weight gain observed in mice on a HFD is accompanied by dysfunctions in glucose metabolism. We performed an ipGTT and ITT at 12 weeks of diet and found that our results reflect the changes in BW we showed earlier (Fig. 28). Mice fed a HFD-B showed perturbations in glucose tolerance)

after 12 weeks of diet compared to SD-fed mice (Fig. 30A-C, D). When comparing diets based on vegetal sources of lipids with different ω6/ω3 ratio, we did not find any difference between diets. None of the HFDs containing 20% of lipids showed a measurable impairment in glucose tolerance irrespective of lipid sourse, when compaired to SD (Fig. 30A-B). In accordance with these results, we observed a slight increase, although not significant, of fasting blood glucose levels for HFD-B and HFD-C at 8 weeks of diet and a significant increase in fasted blood glucose levels of mice fed a HFD-B after 12 weeks of diet (Fig. 30D). Concurrently, when we performed an ITT at 12 weeks of diet, we found that mice on a HFD-B were significantly resistant from SD-fed mice, while mice fed one of the other vegetal lipid based diets, showed no impairment in insulin sensitivity (Fig. 30C).

The Effect of Lipid Nature on HFD-Induced Central Inflammation

After 12 weeks of HFD treatment, we collected the hypothalamus of all mice and quantified the mRNA expression of inflammatory markers relative to SDfed mice. Our data indicates that mice fed a HFD-B, based on animalderived lipids, had higher expression levels of inflammatory cytokines such as TNF-a and IL-6 as well as upregulaton of glial cell markers Iba1 and GFAP compared to the SD control group (Fig. 31A-G). Among the HFDs with vegetal-derived lipids we found increased upregulation of pro-inflammatory cytokines only in the hypothalamus of HFD-SN fed mice, indicating that a HFD with lower lipid content but unfavorable ω6/ω3 ratio of 17,3 equally is able to induce inflammation (Fig. 31A-G). In addition to that, HFD-SN induced also an upregulation of iba1 expression but not GFAP. Interestingly, HFD-SC induced an upregulation of both iba1 and GFAP expression without any increase in pro-inflammatory markers in the hypothalamus. Strikingly, only HFD-B induced hypothalamic upregulation of the chemokine CCL5 (Fig. 31D).

The Effects of Lipid Nature on Thermal Pain Sensitivity

Because T2DM is associated with many macro-and microvascular complications, one of which is neuropathic pain, we were interested to assess, whether HFDs with different nature of lipids have a different propensity to reduce the pain threshold called hyperalgesia. Thus, in collaboration with J.

Noël in the team of E. Lingueglia at IPMC, we performed a Hargreaves' thermal pain test after 12 weeks of diet and found that only HFD-B the most obesogenic diet that rendered mice diabetic also reduced the threshold for thermal pain sensitivity compared to mice fed a SD (Fig. 32). In summary, the first part of our study focused on testing four diets high in fat with different lipid nature and composition on the development of obesity and associated complications such as diabetes, neuroinflammation, alteration of hypothalamic neurons and neuropathic pain. We have identified that HFD-B seem to be the most obesogenic diet, that induces both hypothalamic inflammation as well as modulation of the expression of hypothamamic neuropeptides. In addition to that, it was the only diet that after 12 weeks of diet rendered mice diabetic and more sensitive to thermal pain. For these reasons, we focused on HFD-B in the second part of our experiment to investigate the effects of CCL5 and CCR5 in an obese context.

In the following section "HFD-B" will be referred to simply as "HFD".

Role of Chemokine CCL5 in the Development of Obesity

Previously, we have identified that a HFD and based on lipids obtained from an animal source such as butter is the most obesogenic diet and induces a rapid development of obesity, with both peripheral and central inflammation.

We have also shown that CCL5 is one of the inflammatory markers that is persistently increased in the serum of obese mice and as previously reported in obese human patients [START_REF] Dalmas | Variations in circulating inflammatory factors are related to changes in calorie and carbohydrate intakes early in the course of surgery-induced weight reduction1-3[END_REF]. This part of the study thus investigated the role chemokine CCL5 plays in the development and maintenance of obesity. To investigate this, we employed our previously developed mouse model of DIO and applied it to our KO model of the chemokine CCL5. In other words, to identify the effects of chemokine CCL5 on DIO, we gave either SD or HFD to either wildtype littermates (WT) or CCL5 KO mice (CCL5 -/-) and characterized several physiological and metabolic parameters. The following section of this manuscript will develop the results we have obtained.

Chemokine CCL5 Deficiency Reduces HFD-Induced BW Gain

and Food Intake.

To elucidate the role of CCL5 in DIO we compared age-matched WT and CCL5 -/-mice that were started on either a SD or a HFD at an adult age of ~3 months, maintained on the diet for 16 weeks and measured different physiological and metabolic parameters.

We have observed no difference in BW gain in CCL5 -/-mice compared to WT when fed a SD. However, as expected we have confirmed previous findings of our team and literature that when WT mice were fed a HFD, they rapidly gain weight and develop obesity with significant changes in BW starting from week three (Fig. 33A). Interestingly, when we compare CCL5 -/-with WT mice on a HFD, we see a significant reduction in the BW gain in CCL5 -/-mice starting from three weeks of HFD with a milder and delayed development of obesity (Fig. 33A). This finding is equally represented as area under the curve (AUC) with an average reduction of BW in CCL5 -/-as opposed to WT mice on a HFD with no difference in SD-fed mice in fig. 33B.

Interestingly, despite the difference in BW gain, we have not found any difference in serum leptin levels between HFD fed CCL5 -/-and WT mice (Fig. 33F), while the leptin level increases in the serum of HFD WT mice compared to SD mice in proportion to BW as described in literature [START_REF] Ahrén | Regulation of plasma leptin in mice: Influence of age, high-fat diet, and fasting[END_REF] Next, we wanted to find out what might cause the observed reduction in BW gain and focused on food intake. When we looked at food intake, we observed that CCL5 -/-mice eat on average less grams of food compared to WT mice independent on diet. Surprisingly, although CCL5 -/-mice on a SD do not differ in BW from their controls, they eat significantly less food (Fig 33C). As expected, both genotypes consumed on average significantly less grams of food compared to their controls on a SD (Fig. 33C).

However, there was no difference in the average kcal consumption between WT mice or CCL5 -/-mice independent of diet. However, although CCL5 -/- mice on a SD still showed a reduced kcal intake, it was no longer significantly different from HFD fed CCL5 -/-mice, who showed a significant reduction in average consumed kcal compared to their HFD-fed controls (Fig. 33D).

Interestingly, mice on a HFD drank less water than SD-fed mice, and CCL5 -/- mice on a SD drank significantly less water than WT controls.

Chemokine CCL5 Has a Neuromodulatory Effect on

Hypothalamic Neurons

Due to the difference observed in food intake between genotypes, and because we are also interested in the role of CCL5 in the regulation of energy balance within the CNS, we next used realtime qPCR to quantify and compare the relative mRNA expression of both anorexigenic and orexigenic neuropeptides in hypothalamic samples of CCL5 -/-and WT mice after 16 weeks of SD or HFD feeding. First, we wanted to know whether HFD could alter the neuropeptide expression within a genotype, so we normalized the mRNA expression of each HFD-fed group to their proper SD control group as you can see in Fig. 34. Then, because we wanted also to find out whether CCL5 deficiency has a neuromodulatroy effect in physiological conditions on a SD or in pathological conditions on a HFD, we also normalized the mRNA expression of the neuropeptides by the WT SD or HFD condition, respectively to compare between genotypes (Fig. 35).

We found that diet does not seem to change the expression of orexigenic neuropeptides of the hypothalamus in WT or CCL5 deficient mice after 16

weeks as there was no significant difference between HFD and SD fed mice (Fig. 34). Interestingly, there was a tendency for increased mRNA expression in orexigenic NPY in WT HFD mice compared to their SD fed controls (P=0.072) and a decrease in AgRP in CCL5 -/-mice on a HFD compared to their SD controls (P=0.077) (Fig. 34C andD). However, we found a significant increase in both HFD fed groups in the mRNA expression of anorexigenic neuropeptide CART relative to their SD controls (Fig 34B). When comparing genotypes within the same diet, we have found a slight but significant increase in MCH and a 2-fold increase in ORX expression in SD-fed CCL5 -/-mice compared to their WT controls (Fig. 35E andF).

Interestingly, when comparing between HFD-fed mice, we found a significant decline in orexigenic NPY mRNA expression in the hypothalami of CCL5 -/- relative to WT mice but no difference in any of the other neuropeptides (Fig. 35C).

Chemokine CCL5 Deficiency Improves Glucose Metabolism in

Obese Mice

Because obesity is associated with many other comorbidities, of which a common one is diabetes, we wanted to further investigate if CCL5 deficiency is not only reducing weight gain but also might impact the impaired glucose metabolism seen in obese mice and humans as mentioned earlier. We performed an IP injected GTT at 8 and 16 weeks of diet and found that WT mice on a HFD showed signs of diabetes already at 8 weeks of diet compared to WT mice on SD (Fig. 36A andC-D). Diabetes in mice has been previously defined by basal blood glucose levels of 150mg/dL, which was the case for the HFD-fed WT mice at 8 weeks and 16 weeks of diet after both 16h and 6h of fasting [START_REF] Sullivan | Mouse models of diabetic neuropathy[END_REF](Fig. 36D-E andG-H). The GTT of HFD-fed WT mice showed an impaired glucose-stimulated response at both 8 and 16

weeks of diet compared to SD fed control mice (Fig. 36A andB). While there seems to be no difference in glucose metabolism between CCL5 -/-and WT mice on a SD diet, CCL5 -/-mice on a HFD are significantly more tolerant to glucose with lower fasting blood glucose levels (Fig. 36D) compared to HFDfed WT controls and show no significant difference to CCL5 -/-mice on a SD after 8 weeks of diet (Fig. 36A andC). Although HFD-fed CCL5 -/-mice are still significantly different from their WT mice controls at 16 weeks of diet (Fig. 36A), there is no longer a difference in glucose tolerance and fasting glucose levels between CCL5 deficient and WT mice on a HFD at 16 weeks of diet (Fig. 36B, F-H). Furthermore, CCL5 -/-mice displayed no difference in insulin sensitivity at 8 and 16 weeks to WT mice on a SD (Fig. 36I andJ). While WT mice presented with insulin resistance at both 8 and 16 weeks of HFD diet, CCL5 -/-deficiency seemed to prevent this impairment significantly at both time points, despite being equally glucose intolerance at 16 weeks as WT controls (Fig. 36B,I We further wanted to know if insulin signaling was altered at the level of the brain, so we compared the mRNA expression of IRS1 and as shown in Fig. 37A HFD reduced the mRNA levels significantly in WT mice compared to their SD controls but not in CCL5 -/-mice (Fig. 37A-B, E-F). Furthermore, we tested PPARγ expression levels in the hypothalamus, as it was reported to be a determinant of insulin sensitivity [START_REF] Lu | Brain PPAR-γ promotes obesity and is required for the insuling-sensitizing effect of thiazolidinediones[END_REF]. We did not find any difference for hypothalamic PPARγ mRNA expression levels between WT mice on different diets, but CCL5 -/-mice expressed more PPARγ mRNA in the hypothalamus relative to their WT controls and independent of diet (Fig. 37G-H).

The Effect of CCL5 on Central Inflammation and Hypothalamic

Gliosis

To characterize the HFD-induced phenotype of CCL5 -/-and WT mice further, we wanted to know if they are less obese due to less chronic low-grade inflammation that accompanies obesity. First, we determined the hypothalamic mRNA expression levels of inflammatory cytokines and chemokines. We found as expected a significant increase in inflammatory cytokines TNF-a and IL-6 and an increase in CCL5 levels although not significant in HFD-fed WT mice as opposed to their SD controls (Fig. 38A-B).

Different from our expectations, we found a similar significant increase in HFDfed CCL5 -/-mice compared to their controls for TNF-a and a slight but not significant increase in IL-6 expression levels relative to their SD controls (Fig. 38A-B). We did not find any difference in expression levels of inflammatory markers between genotypes independent of diet (Fig. 39A-D).

Obesity is not only associated with central low-grade inflammation but several studies have also identified gliosis in the hypothalamus of obese mice. Hence, we wanted to find out whether CCL5 might act on glia cells and whether the lack of it has a protective effect on HFD-induced hypothalamic gliosis. We therefore examined the mRNA expression of the common astrocyte marker GFAP and microglial marker Iba1 in the hypothalamus after 16 weeks of diet and found that surprisingly WT mice on a HFD had no significant upregulation of both glial markers, although there was a tendency of increase for GFAP, although not significant (Fig. 40A andC). We have not found any HFDinduced upregulation in CCL5 -/-mice, neither (Fig. 40B andD). Interestingly, we found that compared to HFD-fed WT mice, CCL5 -/-had reduced expression of GFAP, which indeed might indicate that CCL5 could act through GFAP + cells such as astrocytes.

The Effect of CCL5 on Peripheral and Systemic Inflammation

At the peripheral level of AT, HFD induced a marked increase in mRNA expression levels of cytokines IL-1β, IL-6 and chemokines CCL2 and CCL5 in perigonadal (PG) AT of WT mice after 16 weeks of diet compared to their SDfed controls (Fig. 41A-E). Similarly, HFD seemed to induce an increased expression of cytokines like IL-1β and a slight increase in IL-6 but not in TNF-a and no significant increase in CCL2 in CCL5 -/-mice compared to SD-fed controls (Fig. 41A-F). Interestingly, when compared between genotypes, we found a significant decrease in TNF-a levels in CCL5 -/-mice on a HFD, although when fed a SD they are significantly increased in compared to WT controls with the respective diet (Fig. 42A).

However, systemically we have not found any significant increase in inflammatory markers apart from CCL5 in sera of HFD-fed WT mice compared to SD-fed mice (Fig. 43A-G). Interestingly, although TNF-a mRNA levels are increased in PG of CCL5 -/-mice on a SD diet, their serum levels of TNF-a are significantly reduced compared to WT controls. Similarly, CCL5 -/-mice show a decline in IL-10 in HFD-and SD-fed mice, although only significant for the former, relative to respective controls (Fig. 43E). Furthermore, serum levels of CCL2 were significantly increased upon HFD feeding in CCL5 deficient mice compared to SD-fed control (Fig. 43F).

CCL5 Deficiency Partly Protects from HFD-Induced Increase in Neuropathic Pain Sensitivity

A very common comorbidity of obesity and diabetes is neuropathic pain. As a model of neuropathic pain, we chose the Hargreaves' test of thermal pain sensitivity, to test whether CCL5 might have an impact on HFD-induced neuropathic pain sensitivity. At 16 weeks of diet, WT mice on a HFD show, as anticipated, a significant reduction in the duration until paw withdrawal upon presentation of a heat source to their hind paw compared to SD-fed control mice. This is indicative of a higher pain sensitivity in HFD-fed mice (Fig. 44).

Compared to that, CCL5 -/-mice show no significant difference in thermal pain sensitivity compared to their SD-fed control group (Fig. 44). We then wanted to know whether the difference in pain sensitivity could be due to a difference inflammation. We thus determined the expression levels of inflammatory markers in samples of DRG and spinal cord samples of mice after 16 weeks of SD or HFD. We did not find any significant increase in inflammatory marker expression in DRG nor any difference in inflammatory marker expression between CCL5 -/-and WT mice independent of diet (Fig. 45 and 46). Interestingly, we found no significant increase in spinal cords of HFDfed WT mice neither, apart from a slight increase in IL-6, although not significant (Fig. 47).

However, against our expectations, when comparing inflammatory cytokine and chemokine expression in spinal cords between CCL5 -/-and WT mice, we found significantly higher levels of TNF-a, IL-1β, and CCL2 in HFD-fed CCL5 -/- mice and significantly increased TNF-a and IL-6 levels in SD-fed CCL5 deficient mice (Fig. 48A-D).

Hypothalamic Expression of CCL5 and CCR5

We have so far confirmed that CCL5 indeed plays a role in the development and maintenance of obesity and diabetes. In addition to that, we showed some evidence, that CCL5 might have a central effect on neuropeptides. As on SD diet also constitutively express the chemokine CCL5 and its receptor also in the ARC that lies directly lateral to the 3V (Fig. 49). We have also noticed expression in the median eminence and the PVN of the hypothalamus as well as in extra hypothalamic sections. In these pictures, we observed that CCR5 mRNA is quite abundantly expressed but localized only to a few cells, whereas CCL5 seems to be expressed more diffusely in many cells but at low amounts. We can further see that although there are cells coexpressing both ligand and receptor, such as in the LH (yellow dots), they are not often co-localized in the same cell, but rather in neighboring cells or even further apart. We further noticed, that while, CCR5 seems to have a perinuclear expression pattern, CCL5 seems to be rather expressed within the cytoplasm. We also investigated whether we see a difference in the expression pattern between WT mice, which were fed a different long-term diet of either SD or HFD. Figure 50 displays images of the ARC nucleus section of the brain laterally to the 3V, with the brain sections in the SD condition being on the left and the HFD-fed mouse brain sections on the right hand side. Interestingly, it seems like that under HFD condition a greater number of cells are expressing both CCL5 (green, upper panel Fig. 50) and its receptor CCR5 (red, lower panel Fig. 50). In addition, it even seems like the expression of CCL5 and CCR5 are upregulated in the cells. Note also the number of cells in the lower

Role of Chemokine Receptor CCR5 in the Development of Obesity

We have seen in the previous section that CCL5 indeed seems to play a role in the development and the maintenance of obesity. We further established that together with its cognate chemokine receptor CCR5, it is expressed in hypothalamic nuclei controlling energy balance and feeding regulation.

Based on these findings, we decided to also elucidate the role of CCR5 in the development of DIO and characterize the phenotype of CCR5 deficient mice as opposed to WT mice.

Chemokine Receptor CCR5 Deficiency Partly Protects Against

HFD-Induced Development of Obesity.

To establish the role of the cognate chemokine receptor CCR5 in the role of DIO we performed the same set of experiments as discussed above for CCL5.

We have fed CCR5 deficient and WT mice with either SD or HFD for 16 weeks and found no difference in weight gain between CCR5 -/-and WT mice when fed a SD diet. However, we have seen a similar trend in significantly reduced BW gain in CCR5 -/-mice as opposed to WT mice on when fed a HFD (Fig. 51A-B). Interestingly, different from the results seen earlier with CCL5 -/-mice, CCR5 -/- mice on did not consume less HFD than WT controls (Fig. 51C-D). In fact, we observed no difference in food intake neither in grams not kcal between genotypes. As expected, both HFD-fed groups consumed significantly less grams compared to their SD controls but the same amount of kcals (Fig. 51C-D). Similarly, we observed the same trend for water intake as for food intake as HFD consuming groups drank less water compared to their SD controls, without any difference between genotypes (Fig. 51E).

Although we did not see any difference in food intake, but observed a hypothalamic expression of CCR5 in the ARC of the hypothalamus of WT mice, we wanted to see whether the lack of CCR5 has a modulatory effect on neuropeptide expression.

Chemokine Receptor CCR5 Has a Neuromodulatory Effect on Hypothalamic Neurons

As for CCL5, we have also found a difference in neuropeptide expression for CCR5 deficient mice. Although we did not observe any difference in neuropeptide expression in mice fed a SD diet, we did find a significant downregulation of NPY expression in CCR5 -/-mice on a HFD compared to both their SD control and their WT control on a HFD (Fig. 52B and Fig 53B). In addition to that, we identified further downregulation in both anorexigenic POMC and orexigenic MCH and AgRP in CCR5 -/-mice fed a HFD compared to their WT controls.

CCR5 Affects Glucose Metabolism in Obese Mice

We tested glucose tolerance of WT and CCR5 -/-mice after 8 and 16 weeks of diet by IP injection of 1.5mg/kg of glucose. While we found no difference in fasting glucose levels or glucose tolerance in both genotypes on a SD diet (Fig. 54A-H), CCR5 -/-mice fed a HFD were significantly more glucose tolerant than WT mice on a HFD and had significantly reduced basal glucose levels after 16h of fasting after 8 weeks of diet (Fig. 54A-D). However, shorter fasting times revealed a significantly higher blood glucose levels in not only WT but also CCR5 -/-mice on a HFD after 8 weeks of diet but not at 16 weeks of diet (Fig 54E and H). Interestingly, while the blood glucose levels of HFD-fed WT mice were not only significantly higher than SD-fed mice, but mostly above the diabetic threshold of 150 mg/dL, CCR5 -/-mice had fasting blood glucose levels that remained below that threshold throughout testing (Fig. 54D-E, G-H).

At 16 weeks of diet, HFD-fed CCR5 -/-mice showed no difference in glucose tolerance compared to the HFD-fed WT control group (Fig. 54B andF).

Regarding insulin resistance, we have not found any difference between genotypes of neither SD-fed nor HFD-fed mice. Although there was no difference between HFD-fed groups at 8 weeks of diet, the WT groups on a HFD was significantly less sensitive to insulin compared to their WT control on a SD, while the HFD-fed CCR5 -/-group was not (Fig. 54I). Furthermore, we determined glucose-stimulated plasma insulin for SD and HFD groups.

Unfortunately, we did not get enough data points for the SD control groups at basal levels due to limits of detection and hemolysis of plasma collected and thus could not determine the potential differences in basal plasma insulin levels (Fig. 54K). However, we could observe that glucose-stimulated insulin secretion was similar in SD-fed groups between genotypes (Fig. 54L).

Moreover, HFD-fed WT mice seemed to have significantly elevated insulin levels throughout the 60 min of testing compared to SD-fed controlled controls, indicative of hyperinsulinemia (Fig. 54L). Compared to their SD controls, CCR5 -/-mice secreted significantly more insulin 15 min after IP glucose injection (1.5mg/kg), which decreased to levels not significant to control mice at time points T30 and T60 (Fig. 54L). We found that plasma levels were significantly lower 60 min after glucose injection in CCR5 -/-compared to WT mice on HFD (Fig. 54L). At the level of the hypothalamus, we found that HFD induced a significant decrease in IRS1 mRNA expression after 16 weeks of diet compared to SD controls in WT mice but not in CCR5 -/-mice on a HFD (Fig. 55A-B). In fact, when comparing genotypes on the same diet, we found that HFD-fed CCR5 -/- mice expressed significantly more IRS1 mRNA than WT mice (Fig. 55F).

Interestingly, CCR5 -/-mice expressed also more PPARγ mRNA in the hypothalamus compared to WT mice when fed a HFD (Fig. 55H) but not on a SD (Fig. 55G). We did not find any difference in mRNA expression between SD and HFD-fed mice when comparing within genotypes (Fig. 55C-D).

The Effect of CCR5 on Central Inflammation and Hypothalamic

Gliosis

At the end of the 16 week diet protocol we examined the hypothalamus of CCR5 -/-and WT mice for the expression of inflammatory cytokine and chemokine expression by qPCR. However, we have not found any difference induced by the HFD in WT mice compared to their SD controls and only a small reduction in mRNA levels of IL-6 and CCL2 (P=0.056 and P=0.087),

although not significant in HFD-fed CCR5 -/-mice compared to their SD control (Fig 56B and D). Interestingly, when compared to their respective diet control, CCR5 -/-mice expressed more TNF-a when on a SD diet and significantly less IL-1β in the HFD group (Fig. 57A andC). A small reduction in mRNA expression levels of IL-6 was also observed in the hypothalamus of HFD-fed CCR5 -/-mice compared to WT mice, although not significant (P=0.065; Fig. 57B).

We also looked at hypothalamic gliosis at 16 weeks of diet, but apart from a slight but not significant increase in GFAP in WT mice compared to their SD controls (P=0.094), we did not found significant differences in GFAP and Iba1 mRNA expression (Fig 58A -H).

The Effect of CCR5 Deficiency on Systemic Inflammation

Among a panel of pro-and anti-inflammatory cytokines and chemokines tested, HFD-fed WT and CCR5 -/-mice showed both a significant increase in serum levels of TNF-a (Fig. 59A). Apart from that, they showed a different inflammatory pattern. Obese WT mice had also significantly increased levels of anti-inflammatory IL-10 compared to their SD-fed controls (Fig. 59E).

Interestingly, CCR5 -/-mice on a SD had significantly higher concentrations of pro-inflammatory cytokines IL-1β and IFN-γ, compared to WT control mice (Fig. 59C-D). Both mice fed a HFD had significantly higher serum levels of TNFa levels compared to SD fed mice, but were no different between genotypes (Fig. 59A andJ). WT HFD vs CCR5 -/-HFD; #, WT SD vs WT HFD; $, CCR5 -/-SD vs HFD; @, WT SD vs CCR5 -/- SD; ns: not significant.

The Effect of CCR5 Deficiency on Diabetes-Associated

Neuropathic Pain

At the end of the DIO protocol, at 16 weeks of diet, we performed a

Hargreaves' test to investigate if CCR5 -/-mice showed altered sensitivity to thermal pain sensitivity, which is common after prolonged HFD in mouse models of obesity and diabetes. Within our expectations, we documented a significant decrease in WT HFD paw withdrawal latency upon stimulation with an infrared heat source, which is indicative of an increased sensitivity to thermal pain. In addition to that, the lack of CCR5 did not significantly increase the thermal pain sensitivity threshold compared to WT mice in SD condition. Similarly, under HFD condition CCR5 -/-mice remained within normal range of thermal pain sensitivity and where not significantly different from their SD control group (Fig. 60).

The Effect of CCL5 and CCR5 on Subcutaneous AT Morphology

Due to common reports implicating CCL5 and CCR5 in AT infiltration and function, in particular in the context of obesity, we decided to take a closer look at SC AT morphology. Hence, we stained fixed sections of SC AT of WT, CCL5 -/-and CCR5 -/-mice, which were fed either a SD or HFD for 16 weeks, with H&E and quantified the morphological distribution of SC adipocyte size.

As depicted in figure 61, all three groups on a HFD have on average larger adipocytes compared to their respective SD-fed controls. Interestingly, although SD-fed mice do not differ in the average size of their SC adipocytes along genotypes, we found a different frequency distribution between those groups (Fig. 61B-C). According to Fig. 61B it seems, that WT mice on a SD have a higher frequency (~25.5%) of very small adipocytes in the range of 1500µm 2 , while adipocyte area of CCL5 -/-mice have the highest frequency (20.3%) at around 3500µm 2 while CCR5 -/-mice have the highest frequency of adipocytes with 18.4% around 2500µm² (Fig. 61B). The frequency of adipocytes of both KO mice is therefore more skewed to the right compared to WT mice on a SD. WT mice under HFD condition have very little adipocytes in the small range and have the highest frequency of adipocytes in the range of 7000µm² (19.4%), while HFD-fed CCL5 -/-mice share the most abundant cell size of 7000µm² but have a higher frequency (26.6%) of those compared to WT HFD-fed mice. CCR5 deficient mice on a HFD seem to have a higher number of even larger adipocytes, as they have around 22.7% of adipocytes in the range of 9000µm². Interestingly though, WT mice on a HFD have a wider range of adipocyte sizes with the largest adipocytes reaching an area of around 21000µm² (0.971%), while the size of adipocytes of CCL5 and CCR5 deficient mice on a HFD seem to stop at around 13000µm² (1.3%) for the former and 14000µm² (4%) for the latter. Furthermore, we tested the amount of free serum glycerol, indicative of the rate of lipolysis in mice of different genotypes under both diet conditions.

Interestingly we found, as depicted in Fig. 61D, that while mice on a SD, were not different in their levels of glycerol, mice deficient in CCL5 and CCR5 on a HFD had significantly higher amounts of free glycerol and therefore seem to have higher rates of lipolysis compared to WT control mice (Fig. 61D). The highest concentration of free glycerol was observed in CCL5 -/-mice under HFD condition (Fig. 61D).

The Effect of Chronic ICV Infusions of Different Concentrations of

CCL5

To further characterize and confirm the results we have seen in previous section, most notably on BW gain, we have performed experiments, where we implanted a guide cannula into the right lateral ventricle of mice and chronically injected different concentrations of CCL5 (10ng, 50ng and 100ng) and aCSF as control. We then determined the effect of the injections on BW, food intake and a panel of inflammatory marker and neuropeptide expression in the hypothalamus. Figure 62A and B display the effects of different concentrations of CCL5 on BW as opposed to aCSF injections. We have found that even though we performed a single aCSF injection to all mice prior to the initiation of different injections, that mice lost BW and recuperated only about a week after initiation of the injections, independent on the agent we injected (Fig. 62A). Furthermore, we observed that injecting recombinant CCL5 at concentrations of 50ng and 100ng but not 10 ng induced a more prominent loss of BW compared to aCSF vehicle injection (Fig. 62A andB). However, only the difference seen between injections of 100ng CCL5 and aCSF was significant. After the first injection all mice eventually recuperated without any significant difference between different concentrations or agents injected and most interestingly, despite the continuous chronic injection of each agent every second day. The result indicates that chronic injections under SD conditions lose their effect after the first initial injection. We examined also the effect of injections of food intake every day, but although there seems to be a tendency of reduction in food intake when injecting 100ng of CCL5, although the effect is not significant (P= 0.067) and thus remains inconclusive (Fig. 62C). Furthermore, we quantified the hypothalamic mRNA expression of inflammatory markers and neuropeptides after chronic injections of different concentrations of CCL5 and found a significant increase in Iba1 expression with chronic injections of 10ng CCL5 compared to aCSF injected mice (Fig. 62C). Although there were some variations for mRNA expression of hypothalamic GFAP, it remained not significant. When we quantified the mRNA expression of neuropeptides, we again so several variations in expression and tendencies that remained at the limit of significance. For example, we found that CCL5 injections of 10, 50 and 100ng of CCL5 increased the expression of AgRP mRNA (P=0.067, P=0.065 and P=0.093 respectively). In addition to AgRP, we found a significant increase in POMC mRNA expression after 50ng injection of CCL5 and a tendency for increase in 100ng injected mice (P=0.058) (Fig. 62C). In a second series of experiments, we wanted to confirm the effect of CCL5 injection on the reduction of BW and food intake and additionally examine whether CCL5 injection also has an impact on glucose homeostasis and thermal pain sensitivity as it was suggested by our data discussed above. In addition to the chronic injection of 0.3ng of CCL5 over the course of 4 weeks, we injected also 0.3ng of the antagonist called Met CCL5 every second day, after one week of aCSF injections for habituation (Fig. 63). The results indicate that chronic injections of CCL5 induce a significant reduction of BW as indicated in figure 63A andB. Interestingly, injection of the antagonist had a similar effect on the reduction of BW compared to aCSF as CCL5 injection.

The effect on food intake was again inconclusive, although it suggests that CCL5 induces a reduction in food intake within 24h, which disappears after 48h. Next, after 3 weeks of chronic aCSF, CCL5 and Met CCL5 injection, we performed a Hargreaves' test to see whether injection of CCL5 induces an increased sensitivity of thermal pain under SD. As expected, our results show that CCL5 injection induces a significant reduction in the threshold for pwl compared to both aCSF and Met CCL5 injection, which did not show the same effect (Fig. 63C). In fact, Met CCL5 injection showed no difference in pwl compared to aCSF injections and was significantly higher compared to CCL5 injected mice. At about 4 weeks after starting repeated injections every second day, we performed a GTT, and ITT and found no difference between basal fasting glucose levels, glucose tolerance or insulin sensitivity compared to aCSF injected mice (Fig. 64A-F). Although we observed a slight increase in insulin sensitivity of Met CCL5 injected mice compared to either aCSF or CCL5 injected mice, this difference was not significant (Fig. 64D). When we analyzed the serum insulin and glucose-stimulated insulin secretion, we did not find any significant difference, although it seemed like CCL5 injected mice had lower basal insulin (non-fasted condition) compared to aCSF and Met CCL5 injected mice, but slightly more insulin upon glucose-stimulation at 30 and 60 min after glucose stimulation (Fig. 64E andG).

Discussion

DIO is one of the most common causes of obesity and associated not only with comorbidities like diabetes and neuropathic pain among others, but also with metabolic disorders and chronic low-grade inflammation. This inflammation affects several metabolic tissues in the periphery but also is very well present in the CNS especially the hypothalamus. Furthermore, many studies have shown the link between inflammation, insulin resistance, obesity and HFD consumption. Several studies have described HFD-induced upregulation of pro-inflammatory cytokines and chemokines as well as reactive gliosis, well before the first signs of obesity appear as well as after long-term HFD consumption (J. P. [START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Kälin | Hypothalamic innate immune reaction in obesity[END_REF].

Although there are many theories to what might cause the intial inflammatory response, some studies have shown evidence that lipids and FFAs themselves induce the activation of inflammatory pathways such as NF-κB or IKKβ through TLR4 [START_REF] Holland | Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice[END_REF]. This inflammatory process in the hypothalamus leads to disruption of the energy balance and might thus lead to hypothalamic resistances to leptin and insulin [START_REF] Clegg | Consumption of a high-fat diet induces central insulin resistance independent of adiposity[END_REF][START_REF] Holland | Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice[END_REF]. The disruption of energy balance regulation and resistance to peripheral hormones alters feeding behavior and EE, which ultimately will precipitate the development of obesity and diabetes among other consequences (De Souza et al., 2005;[START_REF] Kleinridders | MyD88 Signaling in the CNS Is Required for Development of Fatty Acid-Induced Leptin Resistance and Diet-Induced Obesity[END_REF][START_REF] Arruda | Low-grade hypothalamic inflammation leads to defective thermogenesis, insulin resistance, and impaired insulin secretion[END_REF].

The effect of lipid nature on diet-induced obesity

Several animal studies as well as a few human studies point towards a protective role of ω3 in the development of DIO [START_REF] Buckley | Long-chain omega-3 polyunsaturated fatty acids may be beneficial for reducing obesity-a review[END_REF][START_REF] Cintra | Unsaturated fatty acids revert diet-induced hypothalamic inflammation in obesity[END_REF][START_REF] Oliveira | Diets containing α-linolenic (ω3) or oleic (ω9) fatty acids rescues obese mice from insulin resistance[END_REF]. It has been reported that a lower ω6/ω3 ratio or higher intake of ω3 PUFA might be beneficial, because while and serum lipid lowering effects [START_REF] Kelley | Modulation of human immune and inflammatory responses by dietary fatty acids[END_REF][START_REF] Lee | Saturated Fatty Acids, but Not Unsaturated Fatty Acids, Induce the Expression of Cyclooxygenase-2 Mediated through Toll-like Receptor 4[END_REF][START_REF] Cleland | The role of fish oils in the treatment of rheumatoid arthritis[END_REF][START_REF] Gupta | Saturated long-chain fatty acids activate inflammatory signaling in astrocytes[END_REF][START_REF] Yan | Omega-3 Fatty Acids Prevent Inflammation and Metabolic Disorder through Inhibition of NLRP3 Inflammasome Activation[END_REF][START_REF] Leslie | A review of the effect of omega-3 polyunsaturated fatty acids on blood triacylglycerol levels in normolipidemic and borderline hyperlipidemic individuals[END_REF].

In the first part of this study, we sought to establish a mouse model of DIO and to investigate whether a HFD based on different lipid nature and ω6/ω3 ratio has different effects on the development of DIO and associated comorbidities. In line with the literature, our data show that the HFD with a high amount of saturated fat derived from animals (HFD-B) was the most obesogenic diet and rendered mice obese, diabetic and more sensitive to thermal pain as well as upregulated inflammatory markers in the hypothalamus after 12 weeks of diet [START_REF] Maric | The effects of dietary saturated fat on basal hypothalamic neuroinflammation in rats[END_REF].

Although all HFDs based on vegetal lipid sources showed a milder development of DIO, we did not find any difference in BW between diets with same amount of lipids but different ω6/ω3 ratio. Similarly, we have not found any effects between ω6/ω3 ratios on the development of diabetes. Based on our results it seems that the amount of lipids in a HFD is more important in the development of obesity and diabetes, as well as hypothalamic inflammation and ultimately the deregulation of energy balance. To be able to answer the question of whether ω6/ω3 ratio and lipid source is crucial in the development of DIO and associated metabolic syndrome, our model was insufficient and it would require a higher contant of the respective lipids, similar to the HFD-B that contains 40% of lipids.

Interestingly, although there was no difference in BW gain between HFDs with vegetal sources of fat, we found that HFD-SN with the highest and most harmful ratio of ω6/ω3 did upregulate inflammatory markers and glial cell markers in the hypothalamus, which is in line with the literature. Thus it seems like, although ω6/ω3 ratio does not seem to play a major role in body weight gain, it might be responsible for the inflammatory response associated with obesity. It is possible that due to the lower amount of lipids, glucose metabolism impairment and body weight gain might have developed later on if the diet was to be continued longer than 12 weeks for the HFD-SN.

In line with other studies the diets with lower ω6/ω3 ratio did no show any upregulation of inflammatory markers in the hypothalamus, unlike the diet based on butter and HFD-SN with an elevated ω6/ω3 ratio [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF][START_REF] Cintra | Unsaturated fatty acids revert diet-induced hypothalamic inflammation in obesity[END_REF][START_REF] Oliveira | Diets containing α-linolenic (ω3) or oleic (ω9) fatty acids rescues obese mice from insulin resistance[END_REF]. Interestingly, other studies report, that supplementing their HFD with ω9 or ω3 FAs ameliorated both hypothalamic inflammation as well as hypothalamic leptin and insulin resistance but also whole body insulin resistance. In addition to that, they found that icv injection of either ω9 or ω3 FAs in HFD-fed mice also reversed neuropeptide expression of POMC and CART. This is similar to our results as non of the diets with beneficial ω6/ω3 ratio had upregulated POMC or CART expression unlike the diet high in saturated FAs (HFD-B) [START_REF] Cintra | Unsaturated fatty acids revert diet-induced hypothalamic inflammation in obesity[END_REF].

However, our model was sufficient to develop a model of DIO with diabetes and thermal pain sensitivity to test the effects of the chemokine CCL5 in the second part of the study in this setting.

The role of chemokine CCL5 in the development of diet-induced obesity

Chemokines like CCL2 and CCL5 and their receptors have been linked to the development of obesity and peripheral insulin resistance as well as the infiltration of immune cells into AT as was described for obesity. Although chemokines and chemokine receptors are known for their role in the peripheral immune system, their constitutive expression in the CNS has been reported and many authors suggest that they might have a physiological role in the CNS [START_REF] Kitabgi | Chemokines: A New Peptide Family of Neuromodulators[END_REF][START_REF] Rostène | Chemokines and chemokine receptors: New actors in neuroendocrine regulations[END_REF][START_REF] Goazigo | Current status of chemokines in the adult CNS[END_REF]. Previous work has shown some first results on CCL5 and CCR5 KO models in SD and HFD conditions, while studying different aspects and showing conflicting results. Kitade and colleagues have shown that young CCR5 -/-mice are seemingly protected from DIO-induced effects on AT infiltration, inflammation and glucose metabolism impairment. In contrast to this, other studies have shown rather contrasting data of impairments in insulin signaling and glucose metabolism, systemically and in peripheral tissues like AT and muscle but also in the hypothalamus [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF][START_REF] Kennedy | Loss of CCR5 results in glucose intolerance in diet-induced obese mice[END_REF][START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF].

In this study, we wanted to shed light on the conflicting results so far in the literature and further add to the existing results in the literature of the role of CCL5 not only in the periphery but also in the brain in the context of energy balance and DIO. Thus, using a genetic invalidation model as well as some pharmacological tools, we attempted to provide the missing link between (neuro) inflammation, energy balance regulation and obesity and its comorbidities by studying the role of CCL5 and its receptor CCR5 in the development of obesity, diabetes and neuropathic pain.

First, we confirmed that serum CCL5 is increased in HFD-induced DIO in adult WT mice after 16 weeks of diet, as was previously reported in both obese humans and mice [START_REF] Wu | T-Cell Accumulation and Regulated on Activation, Normal T Cell Expressed and Secreted Upregulation in Adipose Tissue in Obesity[END_REF][START_REF] Dalmas | Variations in circulating inflammatory factors are related to changes in calorie and carbohydrate intakes early in the course of surgery-induced weight reduction1-3[END_REF][START_REF] Hu | High-fat diet-induced adipokine and cytokine alterations promote the progression of prostate cancer in vivo and in vitro[END_REF].

Furthermore, we have shown that HFD induced a rapid development of DIO, in WT mice on the chosen obesogenic diet based on butter with 35% fat.

We found, using a KO model in a DIO context, that both CCL5 and CCR5 seem to be involved in the pathogenesis of obesity and associated comorbidities like diabetes and neuropathic pain. It seems that the lack of either CCL5 or CCR5 protects from DIO associated weight gain, neuropathic pain and delays the glucose impairment and insulin resistance associated with obesity and the metabolic syndrome. This seems to indicate that CCL5 might exert its function in energy and glucose metabolism via its cognate receptor CCR5. Furthermore, we observed that both CCL5 and CCR5 are not only expressed in the ARC and ME of the hypothalamus, but also seem to have a neuromodulatory role, as seen by the change in neuropeptide expression for example with NPY. Our data suggest that CCL5 might have an effect on BW in a situation of high lipid intake possibly mediated through the modulation of neuronal activity in the hypothalamus and affecting feeding behavior either directly, or indirectly by affecting glucose metabolism and/or insulin signaling. CCL5 seems to have an endocrine function as suggested by many studies in the literature, a function however, that is not evident in KO mice under physiological conditions as they have a rather normal phenotype.

Only subtle changes are visible under physiological conditions, such as differences in neuropeptide expression levels at the mRNA level, which are not enough to cause a distinct phenotype. The effect of CCL5 and its receptor CCR5 is more prominent however in pathophysiological stress conditions like a metabolic challenge of long-term lipid excess.

CCL5 and CCR5 mRNA expression in subpopulation of cells in the nuclei of the hypothalamus

Although several studies have reported the expression of chemokines and their receptors in the CNS including CCL5, we have not found any convincing results among them that show the expression of this chemokine in the hypothalamus. Some studies have reported the expression of CCL5 and CCR5 in human fetal astrocytes and microglia in primary cultures, while other report their expression in neurons [START_REF] He | CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia[END_REF][START_REF] Hu | Inhibition of microglial cell RANTES production by IL-10 and TGF-β[END_REF][START_REF] Bakhiet | RANTES promotes growth and survival of human first-trimester forebrain astrocytes[END_REF][START_REF] Westmoreland | Developmental expression patterns of CCR5 and CXCR4 in the rhesus macaque brain[END_REF][START_REF] Avdoshina | Morphine Induces the Release of CCL5 from Astrocytes: Potential Neuroprotective Mechanism Against the HIV Protein gp120[END_REF][START_REF] Sorce | The chemokine receptor CCR5 in the central nervous system[END_REF]. Hence, we wanted to confirm whether CCL5 is expressed in the region of energy balance and feeding regulation. We have shown that the mRNA of CCL5 and its receptor CCR5 is indeed expressed in a few cells in regions of the hypothalamus, including the ARC, LH, VMH and cells of the ME but also outside the hypothalamus. The perinuclear expression pattern of CCR5 mRNA might indicate its localization in ER that surrounds the nucleus.

CCL5 mRNA expression seems to be more dispersed and difficult to assign to certain DAPI-stained nuclei, which might suggest a cytoplasmic location, possibly even in axons of neurons. However, these are only speculations, which require further experiments.

These results are in line with the literature as one study used RT-PCR to examine RNA expression in different parts of the brain and found CCR5 and CXCR4 mRNA to be expressed in the hypothalamus among other regions of the brain [START_REF] Bajetto | Glial and neuronal cells express functional chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor 1[END_REF]. Furthermore, another study demonstrated the expression of CCR5 in the mouse brain in areas such as cerebellar and cerebral cortex, striatum and hippocampus. Interestingly, they reported that the majority of CCR5 seems to colocalize with neuronal marker NeuN and mainly in the cytoplasm, neuronal perikaryon and processes [START_REF] Avdoshina | Neurotrophins modulate the expression of chemokine receptors in the brain[END_REF]. However, they have not reported anything about the expression or lack thereof in the hypothalamus.

A few recent studies have confirmed some of our data. First, one study reports having tested different antibodies and encountered trouble with them due to unspecific binding. Hence, they opted similar to us, for fluorescence in situ hybridization via RNAScope® technology to assess the expression pattern of CCL5 mRNA in adult rat brain. The study has shown that CCL5 mRNA is expressed throughout the brain with highest expression occurring in major fiber tracts. However, CCL5 probe fluorescent signals were found in other areas of the brain including the hypothalamus and the VTA, confirming previous and our reports [START_REF] Lanfranco | Glial-and Neuronal-Specific Expression of CCL5 mRNA in the Rat Brain[END_REF]. Unfortunately, the study does not specify, nor show the exact location of fluorescent signals in different nuclei of the hypothalamus. In contrast to our results, they show a nuclear localization of CCL5 mRNA, while our results show expression mostly around DAPI-stained nuclei and only a few colocalized with DAPI. However, they found, as did we, that CCL5 is expressed only by subpopulations of cells.

Interestingly, the study reported positive staining for CCL5 mRNA in the region of the periaqueductal grey, an area that has been implicated in pain transduction, which will be discussed further below.

Moreover, the study performed colocalization with markers of astrocytes (GFAP), microglia (Iba1), neurons (NeuN) and oligodendrocytes (Nkx2.2) and observed that while CCL5 mRNA expression was found in all types of cells, it seems to be expressed more in neurons than other cell types, at least in the VTA. Furthermore, they found that only a subset of neurons expressed CCL5, some of which have been identified to be a subpopulation of dopaminergic neurons as indicated by tyrosine hydroxylase (TH) [START_REF] Lanfranco | Glial-and Neuronal-Specific Expression of CCL5 mRNA in the Rat Brain[END_REF].

The differences in our and their result might be due to differences in protocol despite using the same technique as they used free-floating sections in 20µM brain section while we used fixed-frozen sections of 10µM thickness, both of which have different pretreatment protocols for the RNAScope® procedure.

For example, the protocol of the study as reported did not include a protease step while ours did. This might also explain why our combining of RNAScope® technology with immunohistochemistry was not successful, while the study had positive outcomes. Furthermore, species-specific differences between the expression of CCL5 mRNA between mice and rats are also likely.

Another study reported the specific expression of CCR5 exclusively in the ARC of the hypothalamus but not in the VMH or other hypothalamic nuclei, while they demonstrated immunoreactivity of antibodies with CCL5 in both the ARC and VMH of the hypothalamus suggesting an autocrine loop in the ARC [START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF]. This is in part matching our results, as we equally found expression of both CCL5 and CCR5 in the ARC, but different from this study and rather in agreement with the study by Lanfranco and colleagues, we found both ligand and receptor to be expressed throughout the brain and not only specific to the ARC and VMH. Furthermore, we attempted to visualize all three receptors of CCL5 (CCR1, CCR3 and CCR5) using the same and other antibodies as in the study and did not manage to replicate the staining achieved seemingly without any kind of amplification or other additional steps apart from a standard immunofluorescence staining protocol.

Furthermore, the authors report that CCR5 is expressed in POMC and MP2

(neuronal marker used in the study) positive and POMC negative neurons in in vitro primary hypothalamic neuron cultures [START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF].

Our results seem to indicate that long-term consumption of HFD might increase the expression of both the ligand CCL5 and its receptor CCR5 in the hypothalamus of HFD-fed WT mice compared to SD controls. It appears to be the case due to both an increased expression of ligand and receptor mRNA in the same cells, as well as increased number of cells expression both CCL5 and CCR5. However, these first observations have to be confirmed with quantification, and further experiments could be done to further quantify the mRNA expression of different receptors in the hypothalamus of HFD-fed WT mice compared to SD-fed mice via quantitative RT-PCR. Due to the limitation of our experiment, we cannot make any assumption about whether the seemingly increased mRNA levels translate to protein levels. However, this result shows the need for further experiments, investigating protein levels via ELISA/MSD or Western blot in hypothalamic tissue samples or primary cultures of HFD and SD-fed mice. In this way, one could use specific culture conditions of FACS sorted cells to test the protein levels of CCL5 and various receptors in specific cultures of neurons, microglia or astrocytes. Although we have attempted to visualize the expression of CCR1, CCR3 and CCR5 in brain samples of WT mice at the protein level via different receptor-specific antibodies with different protocols, we have not managed to make the antibodies work. Further optimization might be needed or other antibodies have to be tested with appropriate controls from other tissues to visualize protein expression of CCL5 and its receptors. Similarly, we attempted to combine RNAScope® technology with immunoflurescent-based antibodies to colocalize CCR5 and CCL5 mRNA expression with specific cell-markers of neurons (NeuN), microglia (Iba1) and astrocytes (GFAP). However, the only antibody that worked was GFAP. The specific protocol for RNAScope® might be problematic for combinations as the protocol for fixed-frozen sections as we have used includes a proteinase step, which might degrade the epitopes of proteins of interest, and thus are no longer detectable by the antibodies tested. One might, try to optimize by adjusting the protease treatment time, although the result is no longer guaranteed by RNAScope® in case of deviations of the protocol.

The effect of CCL5/CCR5 signaling on HFD-induced body weight gain and feeding behaviour

Our results demonstrated a partial protection of HFD-induced BW gain in CCL5 -/-and CCR5 -/-mice compared to WT mice, which suggests that CCL5 has an impact on BW regulation mediated by CCR5 signaling. This difference in BW gain cannot be attributed to changes in leptin, as leptin levels were not significantly different. These results, although not directly comparable, are in line with results of Kitade and colleagues, who showed no difference in leptin mRNA expression in WAT of CCR5 deficient mice on a HFD compared to WT controls [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF]. This indicates that, according to our data, CCL5

has no visible impact on peripheral leptin secretion of ATs and suggests that CCL5 -/-and WT mice on a HFD have similar amounts of WAT, since leptin levels are usually proportional to fat mass. The difference in BW was highest around 8 weeks of diet and decreased towards 16 weeks of diet. However, serum leptin levels were obtained at the end of the experimental protocol, when the difference in BW between genotypes was although significant, much less pronounced, which could explain the similar levels of leptin. Further experiments are necessary to examine serum leptin levels at ≤8 weeks of diet to see whether CCL5/CCR5 signaling might be implicated in leptin signaling.

Another possibility for the difference in BW could be that CCL5 is involved in hypothalamic leptin signaling and thus CCL5 -/-and CCR5 -/-mice could be protected from hypothalamic leptin resistance and/or impaired leptin uptake, associated with DIO. It is intriguing to speculate, that CCL5 -/-mice might be protected from HFD-induced leptin resistance or impaired leptin uptake into the CNS and thus reduce their food intake, while WT mice are resistant and ingest the same kcal as control mice. Additional experiments are necessary to investigate hypothalamic leptin resistance in CCL5 -/-and CCR5 -/- mice in order to assess these possibilities. However, both our data and the literature are not suggestive of this possibility and far from enough to conclude anything in this regard.

Regarding CCL5 and CCR5 KO mice there is quite some controversy in the literature. All previous studies so far showed no difference in BW gain or food intake between genotypes on either a SD or HFD (for studies that used HFD) nor any difference in leptin mRNA expression in the AT (only [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF] tested for leptin expression) of CCR5 -/-mice or CCL5 -/- [START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF] on a SD diet. This is in part comparable to our result as in SD condition, we have not found any difference between genotypes for BW neither, and no difference in food intake for CCR5 deficient mice on either diet. However, in contrast to aforementioned studies, CCL5 deficient mice on a SD diet had no difference in BW but showed a slight reduction in average food intake over 16 weeks of diet. So far and to the best of our knowledge, no study has yet examined the effects of CCL5 deletion in HFD-induced obesity. We showed that on average CCL5 -/-mice consumed less g and kcal of HFD and gain strikingly less BW compared to WT controls. The reduction in food intake in HFD-fed mice might explain the reduction in BW we see for CCL5 -/-mice.

Interestingly, our data indicate that the difference in BW between CCL5 -/-and WT mice on a HFD is greater than for CCR5 -/-mice. This is an interesting result, especially since CCR5 -/-mice show no effect on feeding behavior. This might suggest that the difference in BW gain between HFD-fed CCL5 -/-and WT mice can be explained only in part by the difference in feeding behavior.

Furthermore, the reduction in food intake that we observed for CCL5 -/-mice might be mediated by one of the other receptors of CCL5. It would be interesting to perform similar DIO protocols for CCR1 and CCR3 deficient mice, to elucidate which receptor is responsible for the effect on feeding.

Hypothalamic neuromodulation by CCL5/CCR5 signaling

Our data on the effect of CCL5 and CCR5 deficiency on hypothalamic neuropeptide expression indicates that CCL5 might exert a neuromodulatory effect on hypothalamic neurons via direct or indirect manner, quite possible through CCR5 signaling. For example, we found a consistent reduction in orexigenic NPY mRNA expression for HFD-fed CCL5 -/-and CCR5 -/-mice. This fits with the reduction in food intake observed for HFD-fed CCL5 -/-mice, but is conflicting with the only study so far, that has observed neuropeptide expression in CCL5 -/-and CCR5 -/-mice and showed an upregulation of hypothalamic NPY, AgRP and POMC mRNA expression in both KO mice compared to WT mice. However, the study by Chou and colleagues, has measured neuropeptide expression only in SD-fed mice. While, we saw an upregulation of hypothalamic orexigenic neuropeptide expression, it was for MCH and ORX in SD-fed CCL5 -/-mice and no changes for CCR5 -/-mice. These differences, including the contrasting results on feeding behavior in CCL5 -/- mice could be due to differences in housing conditions of animal facilities, such as humidity but also in sacrificing protocols (whether mice were fed or fasted), which can affect feeding behavior and neuropeptide expression. In any case, both their and our results have in common that CCL5 and CCR5 deficiency has an effect on hypothalamic mRNA expression of neuropeptides that are implicated in energy, glucose metabolism and feeding regulation. In line with this, we found a marginal increase in AgRP expression and a significant increase in POMC expression after ICV infusion of CCL5, which provides further evidence for the neuromodulatory effect of CCL5. However, our cannula implantation experiment showed surprisingly and against our expectations that ICV CCL5 infusion seems to reduce BW in a dosedependent manner in SD condition. Although only the highest dose (100ng) was significant while the lower dose of 50ng was only marginally increased, which could be explained with a reduction in food intake although our data on food intake is not conclusive (not shown). This result is contradicting the result we found in our HFD study in CCL5 and CCR5 deficient mice. If CCL5 or CCR5 deficiency leads to protection from HFD-induced weight gain, then the chronic infusion of CCL5 into the brain of WT mice should increase food intake and BW over time. However, this seems to not be true. Due to technical difficulties, we had only a small number of mice left for experimentation, as mice started to lose the guide cannula after around 2 weeks, indicating that long-term experimentation with guide cannula implantation into the brain might be difficult. We therefore decided to repeat the experiment and optimize the technique, for example by using a different dental cement for better adhesion and inject apart from CCL5, the antagonist Met CCL5.

Furthermore, we observed that handling of mice during infusion as well as frequent infusion is stressful for mice and might lead to a decrease in BW. Thus, we increased handling and habituated mice for aCSF infusion, before we administered CCL5 and antagonist. However, when we repeated the experiment, we observed a similar effect as in the first experiment. Chronic (every 2 nd day) infusion over several weeks of CCL5 (0.3ng) and Met CCL5 (0.3ng) both reduced the BW of mice on a SD, which is against our expectation. This confirmed the results of CCL5 infusion into brains of mice in reducing the weight gain; however, we expected that the antagonist would have the opposite effect. Although these results are contradicting our HFD studies in KO mice, these results are in line with other studies that have previously performed ICV injections of CCL5 and found that injection of 20ng CCL5 reduced short-term food intake in rats [START_REF] R877 | Chemokines/intercrines and central regulation of feeding[END_REF]. In another study, daily administration of CCL5 (0.92nM) for 5 days had no impact on food intake in rats, but when combined with the neurotoxic HIV glycoprotein 120 IIIB (HIVgp120IIIB), prevented the HIVgp120IIIB-induced reduction in food intake [START_REF] Guzmán | RANTES, MDC and SDF-1α, prevent the HIVgp120-induced food and water intake decrease in rats[END_REF]. This study shows, different from our results and the previously mentioned study that CCL5 might have a role in food intake modulation, but only in a pathological context. To further speculate, that might explain why we did not observe an increase in food intake, as CCL5 might not induce an effect on its own but its effect might rather be evident in a pathological context such as chronic HFD. Another possibility is that CCL5 has a different concentration-depending effect. Since we do not know the exact concentration of CCL5 in the hypothalamus or aCSF of obese mice and it is technically difficult to find out, as very sensitive tests are required since inflammation associated with obesity is a low-grade inflammation, it is hard to know which concentration should be used to achieve an effect. Maybe the concentrations we used in chronic injections (between 10ng and 300ng) might be too high, and could instead mediate an acute inflammation. An acute inflammation could rather reduce food intake as was demonstrated in a previous study by our team, where acute ICV injections of CCL2 reduced BW and food intake in mice, mimicking the effects of LPS-induced inflammation (Le Thuc et al., 2016). The same could be true in this experiment. While the concentration of CCL5 might be too high to mimic the chronic low-grade inflammation, and instead induces an acute inflammatory response, the concentration of antagonist used might impair the effect of endogenous CCL5, which in physiological concentrations is present but not abundant, and induce the actual expected effect of a reduction in BW as seen in our KO mice. However, this experiment is only preliminary and although it seems to suggest that CCL5 has a dual role depending on the context, further experiments and replications are necessary with higher number of animals to explain this phenomenon.

The difference in BW could be explained by the difference in food intake we observed between genotypes on a HFD, at least for CCL5 deficient mice.

While CCL5 deficient mice consumed on average less kcals compared to their HFD-fed WT control, CCR5 deficient mice did not show any difference in food consumption. Thus, the question remains about the reason for the difference in BW. The other obvious possibility would be a difference in EE such as locomotor activity. Maybe CCR5 mice exert more activity and thus burn more calories, which translates into less BW. Another possibility would be that their basal metabolic rate, also a component of EE, is higher.

Unfortunately, we do not have the equipment to measure basal metabolic rate. These questions have thus to be answered in additional future experiments with metabolic cages, that are able to measure the respiratory echange ratio (VO2/CO2) and give indirect indications on metabolism.

In addition to that, the change in NPY neuropeptide expression we saw in both CCL5 and CCR5 deficient mice during HFD challenge, suggests that CCL5 exerts its effect by binding to CCR5 and modulates either directly or indirectly NPY or MCH neuronal activity.

In support of its role as neuromodulator, more evidence has been shown in studies indicating that CCL5 can modulate the release of glutamate from both astrocytes and neurons [START_REF] Musante | RANTES Modulates the Release of Glutamate in Human Neocortex[END_REF][START_REF] Pittaluga | CCL5-Glutamate Cross-Talk in Astrocyte-Neuron Communication in Multiple Sclerosis[END_REF].

We cannot exclude the possibility that microglia or astrocytes, who have been reported to express CCR5 receptor and secrete or produce CCL5 [START_REF] Avdoshina | Morphine Induces the Release of CCL5 from Astrocytes: Potential Neuroprotective Mechanism Against the HIV Protein gp120[END_REF][START_REF] Škuljec | CCL5 induces a pro-inflammatory profile in microglia in vitro[END_REF][START_REF] Sorce | The chemokine receptor CCR5 in the central nervous system[END_REF][START_REF] Lanfranco | Glial-and Neuronal-Specific Expression of CCL5 mRNA in the Rat Brain[END_REF] to be the mediator between CCL5 signaling and modulation of neuronal activity. For example, studies report that direct activation of GFAP-expressing cells through calcium signaling pathways in the ARC leads to increases in food intake, while disruption of calcium signaling in GFAP positive cells results in diminished feeding behavior through the interaction with AgRP/NPY and POMC neurons [START_REF] Chen | Direct modulation of GFAP-expressing glia in the arcuate nucleus bi-directionally regulates feeding[END_REF]. This study indicates that glia might be more than just a support for neurons and have roles to play in the regulation of energy balance. Further experiments are required to shed light on this thematic.

The effect of CCL5/CCR5 signaling on glucose homeostasis

Intriguingly, our results have shown that both CCL5 and CCR5 deficiency seems to delay the HFD-induced glucose metabolism impairment and insulin resistance, as evidenced by a protection from glucose intolerance and insulin resistance in CCL5 -/-and CCR5 -/-mice at 8 weeks of diet. This is accompanied by a reduced overnight fasting blood glucose at 8 weeks of diet in both mice deficient of either CCL5 or CCR5 in HFD condition. Surprisingly, blood glucose levels are significantly increased in CCL5 -/-and CCR5 -/-HFD-fed mice after a short fasting period (6h) at 8 weeks of diet, but different from WT controls, they stay below the diagnostic margin of 150mg/dL for diabetes. In a previous study, authors argued that overnight fasting renders mice catabolic, where they lose a great percentage of their BW, which could improve the outcome of GTT testing at least in CCR5 -/-mice because their lean body mass is correlated with glucose intolerance [START_REF] Kennedy | Loss of CCR5 results in glucose intolerance in diet-induced obese mice[END_REF]. It is unclear so far if the same correlation is true for CCL5 -/-mice on a HFD. In any case, despite elevated fasting blood glucose levels compared to their SD control, CCL5 -/- mice had a more favorable outcome in ITT after a 6h fast both at 8 weeks and 16 weeks of diet compared to HFD-fed WT mice. Similarly, we found that CCR5 -/-mice, although not significantly different from their HFD controls, had no significant difference in their insulin tolerance compared to their SD control group. This suggests that although glucose metabolism might be slightly impaired, they still are able to compensate for this impairment, due to mechanisms that remain unclear and remain in a non-diabetic state different from their WT controls on HFD. It might be possible that a CCL5/CCR5 dependent mechanism might be involved the regulation of hepatic glucose production to compensate for short periods of stress like fasting. CCR5 mRNA expression is expressed and upregulated not only in AT but also in liver cells in DIO mice [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF]. Furthermore, CCL5 and CCR5 have been implicated in the early development of HFD-induced hepatic steatosis and liver fibrosis. Hepatic stellate cells as well as other hepatic cell types can secrete CCL5 and thus contribute to a pro-inflammatory state and steatosis via CCR5 in healthy hepatocytes [START_REF] Li | Steatosis induced CCL5 contributes to early-stage liver fibrosis in nonalcoholic fatty liver disease progress[END_REF][START_REF] Kim | Hepatic stellate cells secrete Ccl5 to induce hepatocyte steatosis[END_REF]. This mechanism that induces hepatic steatosis, which is associated with obesity could also have an effect on hepatic glucose metabolism.

It might be possible that CCL5-CCR5 dependent signaling mechanisms might be involved in the hypothalamic regulation of hepatic glucose metabolism, for example by inhibiting glucose production. Thus, when CCL5/CCR5 is missing, hepatic glucose production "overcompensates" the hypoglycemic state of fasting in an already metabolically stressed environment. This compensation might be lost during prolonged fasting.

Previous studies have reported confounding results regarding the glucose tolerance of CCR5 -/-and CCL5 -/-on SD and HFD. Kitade and colleagues report an improved glucose tolerance and insulin sensitivity despite reduced insulin secretion after 16 weeks of HFD, which is similar to our results. Although our results show a similar protection of CCR5 -/-mice from glucose intolerance in HFD fed mouse, there are some differences. Contrary to their data, we did not see any difference in glucose tolerance or insulin sensitivity in SD-fed CCR5 -/-and WT mice. Furthermore, the improvements they observe are after 16 weeks of diet, whereas our resistance to impairment is only transient and visible at 8 weeks of diet, but no longer present later on, suggesting that the improvement they see is stronger and long lasting. Interestingly, they show similar to us a decrease in glucose-stimulated insulin secretion between HFDfed genotypes, in particular, which is significantly different 60 min after glucose-stimulation. These differences could be due to variations in diet fat content, source and composition. Factors such as lipid nature (saturated vs unsaturated FAs) and ratio of ω-6/ω-3 have proven to be important factors in the pathogenesis of DIO and insulin resistance due to their different capacity to induce inflammation and lipotoxicity [START_REF] Simopoulos | The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases[END_REF][START_REF] Simopoulos | An increase in the Omega-6/Omega-3 fatty acid ratio increases the risk for obesity[END_REF][START_REF] Gupta | Saturated long-chain fatty acids activate inflammatory signaling in astrocytes[END_REF][START_REF] Yan | Omega-3 Fatty Acids Prevent Inflammation and Metabolic Disorder through Inhibition of NLRP3 Inflammasome Activation[END_REF]. Another factor that might lead to variations is the age of diet initiation. While the authors used adolescent mice of 5 weeks, we used 12 weeks old mice. However, in some preliminary age-dependent experiments we found no differences between WT of CCL5 -/-mice when initiating HFD exposure at an age of 4 weeks (not shown).

Furthermore, another study used 10 week old mice to start a HFD and reported the opposite effect, namely that CCR5 -/-have a greater HFDinduced impairment in glucose tolerance compared to WT mice after 16 weeks of diet [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF][START_REF] Kennedy | Loss of CCR5 results in glucose intolerance in diet-induced obese mice[END_REF].

Similarly, Chou and colleagues reported that genetic ablation of CCR5 or CCL5 as well as chronic ICV (3V) infusion of the antagonist Met CCL5 (7 to 14 days) rendered mice intolerant to glucose and insulin resistant compared to either WT mice or aCSF infusion [START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF]. However, they assessed glucose tolerance and insulin sensitivity only under SD condition [START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF]. This is in contrast to our results as we did not observe any impairment or difference in glucose tolerance or insulin resistance in SD-fed mice between CCL5 -/-, CCR5 -/-or Met CCL5 ICV infused mice. These results are conflicting with both our study and the one reported before, even though they used older mice and had a HFD, with a similar kcal value as ours. In contrast, the former study had a different GTT protocol, with a shorter fasting period (5h) and glucose injections according to lean mass, while we fasted our mice overnight, so did the other study, and injected according to BW. Adding to further differences, the study by Chou and colleagues performed an oral GTT (oGTT), as opposed to the IP injected GTT (iGTT) that we, and the aforementioned study, used. While the iGTT is dependent on two factors, pancreatic insulin secretion and insulin sensitivity, it does not account for the incretin-induced potentiation of insulin secretion. Incretins are hormones that are secreted when nutrients pass through the gastrointestinal tract, where glucose is detected and leads to incretin release, which in turn enhances the insulin secretion by the pancreas. Thus, an oGTT provides information of whether an impairment could take place at the level of incretin signaling.

Interestingly, the study by Chou and colleagues tested glucose tolerance in WT mice after infusion (ICV into 3V) of only the antagonist Met CCL5 but not CCL5, which blocked CCL5 signaling and rendered mice glucose intolerant and insulin resistant on a SD. A distinct study has reported that CCL5 can impair insulin secretion in mice by reducing the glucose-stimulated secretion of incretins such as GLP-1 and 2 in the intestines [START_REF] Pais | RANTES (CCL5) reduces glucose-dependent secretion of glucagon-like peptides 1 and 2 and impairs glucose-induced insulin secretion in mice[END_REF]. However, it is unlikely that Met CCL5 infusion would affect peripheral CCL5 action, but rather induce local effects in the hypothalamus. It is unclear yet, if CCL5 can regulate incretin function via efferent nerve fibers from the hypothalamus.

Because this is unlikely, for our purposes an iGTT was sufficient.

In our study, we performed a similar experiment, where we implanted guide cannula into the lateral ventricle of WT mice to infuse every second day either the control solution (aCSF), Met CCL5, or CCL5 and tested 14-21d after, glucose tolerance via iGTT and insulin sensitivity via ITT. Our results differ from the previously reports, as we found no difference in glucose tolerance nor in insulin sensitivity between conditions. There are variations in experimental approaches that could explain the differences seen in our results compared to the other study. First, we used an infusion technique via implanted guide cannula to administer every second day the same antagonist as in the study, however at higher concentrations (300ng as opposed to 10ng/mL in the literature) while the authors of the other study used a more expensive osmotic pump to continuously infuse the molecule. While the osmotic pump is a great technique to continuously infuse a product, not like our choice to inject only every second day, the method has a disadvantage [START_REF] Tanabe | Murine Astrocytes Express a Functional Chemokine Receptor[END_REF][START_REF] Dorf | Astrocytes express functional chemokine receptors[END_REF][START_REF] Van Der Meer | Immunohistochemical Analysis of CCR2, CCR3, CCR5, and CXCR4 in the Human Brain: Potential Mechanisms for HIV Dementia[END_REF]. Recombinant proteins often have a short half-life, which is why the supplier recommends the fresh preparation of the protein before usage. Osmotic pumps however are filled before implantation and the solution containing the protein are subjected to the temperature of the animal, which could affect the stability and half-life of the protein leading to different effects. Second, the difference in concentration of protein injected could lead to different effects. Inflammation was previously shown to affect energy metabolism differently depending on whether it is acute and high-grade or chronic and low grade. For example, while the chemokine CCL2 is well known for its role in the chronic low-grade associated with DIO, weight gain and insulin resistance, a study has shown, that when injected ICV acutely and at high concentrations, CCL2 induces anorectic effects leading to weight loss (Le Thuc et al., 2016). This suggests that chemokines can have different concentration-dependent functions and might explain why we found no difference in glucose tolerance and insulin resistance after long-term ICV infusion of antagonist or CCL5. Third, we injected into the lateral ventricle while the study by Chou et al injected into the 3V, which is more specifically targeting the hypothalamus, even though it cannot be avoided that the product is diffused into other regions. However, we chose deliberately to inject a higher dose to compensate for injecting into the lateral ventricle, knowing that it will be diluted in the CSF of the mouse and the actual dose that might finally reach the hypothalamus is much lower.

The lateral ventricle is also more accessible and inserting a cannula into the lateral ventricle instead of all the way to the ventral part of the brain could avoid excessive damage to the brain. However, an advantage of the Alzet pump is that handling of mice is not necessary for infusion and thus reduces the confounding factor of stress on the animal that can affect glycemia and food intake. Due to the redundancy of the chemokine system as mentioned previously, it might be possible that the effect of the antagonist was compensated by one of the other receptors or chemokines, that have been shown to be expressed in the CNS as well [START_REF] Tanabe | Murine Astrocytes Express a Functional Chemokine Receptor[END_REF][START_REF] Dorf | Astrocytes express functional chemokine receptors[END_REF][START_REF] Van Der Meer | Immunohistochemical Analysis of CCR2, CCR3, CCR5, and CXCR4 in the Human Brain: Potential Mechanisms for HIV Dementia[END_REF]. Furthermore, the concentration of CCL5 and antagonist might not have been the right one to see the expected effect, or the preparation of the protein not adequate for this application. Injecting CCL5 in mice, that already have basal levels of the chemokine but not adapted numbers of receptors to accommodate the higher concentration of ligand, might thus be lost if CCL5 does not have a positive feedback effect to increase the numbers of the receptors. In addition to that, as described in the introduction, certain scavenger receptors exist that might play a role in decreasing the concentration of chemokines in the circulation. Similar mechanisms might be in place in the CNS, to avoid potential adverse effects due to prolonged exposure to high concentrations of chemokines. Glia cells such as microglia could take that role and take up and degrade excess amount of chemokines. Both hypoglycemia and hyperglycemia can be detrimental to health, thus evolution could have favored a highly regulated and redundant mechanism for insulin signaling and secretion to avoid situations such as hypoglycemia, and thus might require more than one chemokine to cause deregulation. Our result does not seem to suggest that centrally administered CCL5 has an impact on peripheral glucose metabolism, at least not in SD condition. However, these results represent only preliminary data, which have to be verified in additional experiments with further optimization and more animals. In fact, it might be possible that the effect of CCL5 or the antagonist will only be evident in a pathological context. Our data is suggestive of this, as CCL5 or CCR5 deficient mice show no difference in glucose tolerance or insulin resistance under SD condition but differences in HFD condition. Hence, it would be interesting to perform cannula-mediated ICV infusion of CCL5 and antagonist in obese mice and CCL5 deficient mice to test whether they could reverse the phenotype observed in this study in WT mice (induce weight loss upon antagonist Met CCL5 administration) and CCL5 -/-mice (induce weight gain upon CCL5 administration). A few selective antagonist have been reported for receptors of CCL5, such as maraviroc for CCR5, CCX354-C for CCR1 and GW766994 for CCR3, which should be tested to identify the specific effects of each receptor via ICV infusion [START_REF] Tak | Chemokine receptor CCR1 antagonist CCX354-C treatment for rheumatoid arthritis: CARAT-2, a randomised, placebo controlled clinical trial[END_REF][START_REF] Marciniak | The Chemokine MIP-1α/CCL3 impairs mouse hippocampal synaptic transmission, plasticity and memory[END_REF][START_REF] Laudati | Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm[END_REF][START_REF] Zhu | Targeting CCR3 to Reduce Amyloid-β Production, Tau Hyperphosphorylation, and Synaptic Loss in a Mouse Model of Alzheimer's Disease[END_REF].

As mentioned above, other factors causing the differences in our data compared to literature in regard to glucose metabolism in HFD and SD-fed KO or WT mice, could be diet composition, type and amount of different lipids. Nevertheless, in spite of the differences between studies, it seems like CCL5-CCR5 signaling plays an important role in central and peripheral insulin signaling, especially in the context of DIO and the associated metabolic syndrome. Other studies are in line with the observed effect of our study, as it was reported that CCL5 reduces the glucose-stimulated secretion of incretins, such as GLP-1 and 2 in L-cells of the intestine via CCR1 signaling and thereby impairs insulin secretion [START_REF] Pais | RANTES (CCL5) reduces glucose-dependent secretion of glucagon-like peptides 1 and 2 and impairs glucose-induced insulin secretion in mice[END_REF]. Another study demonstrated that CCL5 can act as an agonist for another receptor called GPR75, which is expressed in addition to CCL5 in human and mouse pancreatic islet cells and can signal via Gq and PKC to elevate intracellular calcium and lead to insulin secretion. Interestingly, in addition to GPR75 and CCL5, healthy human and mouse islets expressed also other CCL5 receptors such as CCR1 for the former and CCR3 and CCR5 for the latter [START_REF] Liu | The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis[END_REF]. Furthermore, CCR5 expression in the pancreas was reduced by administration of antiinflammatory cytokine IL-4. In addition to this, the temporal expression of chemokine receptors like CCR5 in pancreatic islets was associated with the progression to insulitis and T1DM in mouse models of T1DM [START_REF] Cameron | Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes[END_REF].

In line with this, our results further show a reduction of IRS1 mRNA in response to HFD in obese and diabetic WT mice compared to their SD controls, but no difference between diets in CCL5 -/-and CCR5 -/-mice, and a significant increase for CCR5 -/-mice on a HFD compared to their WT controls on a HFD.

Similarly, hypothalamic PPARγ mRNA expression was increased in CCL5 -/-mice independent of diet, and in CCR5 -/-on HFD, compared to their respective WT control. While insulin is an important regulator of whole body glucose metabolism and inducing the uptake of glucose into different tissues, insulin action in the brain has two effects. First, it reduces food intake and BW when injected ICV in primates or administered intranasally in humans [START_REF] Woods | Chronic intracerebroventricular infusion of insulin reduces food intake and body weight of baboons[END_REF][START_REF] Benedict | Differential sensitivity of men and women to anorexigenic and memory-improving effects of intranasal insulin[END_REF]. Second, it suppresses endogenous hepatic glucose production and improves peripheral glucose metabolism [START_REF] Carey | Evidence for central regulation of glucose metabolism[END_REF][START_REF] Kimura | Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor[END_REF]. Brain-specific deletion of IR, as well as in hypothalamic neurons induces hyperphagia, hypertriglyceridemia, insulin resistance and enhanced DIO in rodents [START_REF] Bruning | Role of brain insulin receptor in control of body weight and reproduction[END_REF][START_REF] Obici | Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats[END_REF].

IRS is an important regulator of insulin signaling downstream of the IR. Insulin binding to IR induces autophosphorylation of the IR, thereby activating it and leading to the phosphorylation of IRS1. Depending on the phosphorylation of IRS1, it can either positively or negatively affect insulin signaling via PI3K/PIP3/Akt pathway. Impairments at the IR or IRS1 in the hypothalamus can lead to insulin resistance or glucose intolerance [START_REF] Kubota | Insulin receptor substrate 2 plays a crucial role in β cells and the hypothalamus[END_REF][START_REF] Lin | Dysregulation of insulin receptor substrate 2 in β cells and brain causes obesity and diabetes[END_REF][START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF]. Modulations at IRS1 thus might indicate that CCL5 can contribute to the insulin signaling regulation in the hypothalamus. Indeed a study has shown that CCR5 seems to associate with the IR in the hypothalamus of mice and thereby modulate pathways implicated in GLUT4 translocation and affect pathways including IRS via S6 kinase and AMPK, a transporter for glucose uptake [START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF]. In line with this, CCL5 has previously been reported to regulate cellular metabolism and glucose uptake via CCR5 activation and AMPK signaling in a PI3K/mTOR-dependent manner in T cells [START_REF] Chan | The chemokine CCL5 regulates glucose uptake and AMP kinase signaling in activated T cells to facilitate chemotaxis[END_REF]. It was shown that CCL5 treatment can both increase metabolites such as glucose-6-phosphate and activate Akt, which in turn lead to an increase in GLUT1 expression in breast cancer cell lines and an increase in glucose uptake, glycolysis and ATP production in a CCR5 and mTOR-dependent way [START_REF] Gao | CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells[END_REF]. Moreover, CCL5

treatment changed cellular metabolic activity to a state of anabolic metabolism with decreases in FA levels while increasing β-oxidation and the metabolite acetyl CoA, and downstream ketone bodies [START_REF] Chan | The chemokine CCL5 regulates glucose uptake and AMP kinase signaling in activated T cells to facilitate chemotaxis[END_REF][START_REF] Chou | CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5[END_REF][START_REF] Gao | CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells[END_REF]. All these studies show that CCL5 has a role in cellular metabolism and affects pathways that coincide with or can affect actors of the insulin signaling pathway. Thus, with the evidence presented including our observation of the modulation of IRS, it is not surprising that CCL5 can affect insulin signaling, most likely via CCR5 in hypothalamic cells. Interestingly, IR is expressed on hypothalamic neurons such as POMC/CART and NPY/AgRP producing neurons. It is intriguing to speculate that CCL5 could modulate insulin signaling due to its ability to modulate the expression of neuropeptides like NPY, as suggested by our data.

PPARγ is another factor that is seemingly affected by CCL5 or CCR5 deletion, under normal conditions for the former and HFD condition for the latter.

PPARγ is a transcription factor, whose expression has been reported in particular in AT but also in kidneys, intestine, skeletal muscle, liver, macrophages and the brain and represents an attractive therapeutic target for metabolic syndrome [START_REF] Dreyer | Positive regulation of the peroxisomal beta-oxidation pathway by fatty acids through activation of peroxisome proliferator-activated receptors (PPAR)[END_REF][START_REF] Lu | Brain PPAR-γ promotes obesity and is required for the insuling-sensitizing effect of thiazolidinediones[END_REF][START_REF] Xu | DBZ is a putative PPARγ agonist that prevents high fat diet-induced obesity, insulin resistance and gut dysbiosis[END_REF]. In fact, it has been implicated as key regulator in adipogenesis, lipogenesis, glucose and lipid homeostasis, insulin sensitivity and inflammation as well as hypothalamic energy balance and food intake regulation [START_REF] Stienstra | PPARs, Obesity, and Inflammation[END_REF][START_REF] Ryan | A role for central nervous system PPAR-γ in the regulation of energy balance[END_REF][START_REF] Janani | PPAR gamma gene -A review[END_REF]. Interestingly, PPARγ expression was found in AgRP/NPY and POMC/CART expressing neuronal cell lines, and treatment with agonist led to excitement of AgRP and inhibition of POMC neuronal activity [START_REF] Diano | Peroxisome proliferation-associated control of reactive oxygen species sets melanocortin tone and feeding in diet-induced obesity[END_REF]. Moreover, while food deprivation in rodents increased hypothalamic AgRP and PPARγ expression, ICV or ip injection of the antagonist of PPARγ had a similar effect in rodents, namely increasing both AgRP and NPY expression and food intake. In line with this, antagonist injection, prevented the food deprivation-induced effect on hypothalamic neurons and food intake, without affecting ghrelin [START_REF] Ryan | A role for central nervous system PPAR-γ in the regulation of energy balance[END_REF][START_REF] Garretson | Peroxisome Proliferator-Activated Receptor Controls Ingestive Behavior, Agouti-Related Protein, and Neuropeptide Y mRNA in the Arcuate Hypothalamus[END_REF].

Activated by dietary lipids and their metabolites, this nuclear receptor induces the transcription of genes important for lipid and glucose metabolism [START_REF] Ryan | A role for central nervous system PPAR-γ in the regulation of energy balance[END_REF]. In WAT it is key in the regulation of adipogenesis, and upon activation promotes lipid storage [START_REF] Xu | DBZ is a putative PPARγ agonist that prevents high fat diet-induced obesity, insulin resistance and gut dysbiosis[END_REF]. It is an attractive potential target for metabolic syndrome, because it was found to be targeted by insulin-sensitizing drugs, used for diabetes treatment. It thus could play an important role in insulin sensitivity/insulin resistance, however it has two interesting side-effects confirming the results discussed herein: increase in subcutaneous WAT and increased food intake [START_REF] Garretson | Peroxisome Proliferator-Activated Receptor Controls Ingestive Behavior, Agouti-Related Protein, and Neuropeptide Y mRNA in the Arcuate Hypothalamus[END_REF][START_REF] Xu | DBZ is a putative PPARγ agonist that prevents high fat diet-induced obesity, insulin resistance and gut dysbiosis[END_REF]. Another interesting study used neuron-specific PPARγ deletion and found that HFD feeding in those mice led to decreased food intake, increased EE and resulted in reduced weight gain [START_REF] Lu | Brain PPAR-γ promotes obesity and is required for the insuling-sensitizing effect of thiazolidinediones[END_REF]. The authors thus concluded that HFD-induced weight gain is in part due to PPARγ signaling in neurons to both increase food intake and limit thermogenesis. This is in opposition however, with our finding that PPARγ expression is increased in the hypothalamus of HFD-fed CCL5 -/-and CCR5 -/-mice, which present with a decreased obese phenotype compared to WT mice that have lower PPARγ expression in the hypothalamus.

It might suggest though that the consumption of HFD can induce PPARγ activation. This in turn might be impaired in WT HFD-fed mice due to chronic exposure to HFD, while the lack of CCL5/CCR5 signaling in KO mice could prevent this disturbance in PPARγ signaling either upstream or downstream and thus avert the increases in food intake and adipogenesis. Another possibility is that PPARγ increase might be a compensatory reaction to a decrease in food intake in CCL5 -/-mice compared to WT mice. Other studies reported that another partial agonist of PPARγ (DBZ), without the side-effects associated with the other compound, could prevent both LPS-stimulated inflammation and lipid accumulation in macrophages [START_REF] Xie | DBZ blocks Lps-induced monocyte activation and foam cell formation via inhibiting nuclear factor-κB[END_REF]. Other studies however, as mentioned previously in the introduction, suggested that PPARγ mediated the lipolysis inducing effect of TNF-a on adipocytes. These studies show that PPARγ might provide a link between inflammation and changes in metabolism.

Another study in agreement with our results, using the same partial agonist for PPARγ, showed that daily gavage of DBZ protected against HFD-induced DIO and associated metabolic impairments such as insulin resistance, while also stimulating BAT browning and maintaining the intestinal barrier integrity [START_REF] Xu | DBZ is a putative PPARγ agonist that prevents high fat diet-induced obesity, insulin resistance and gut dysbiosis[END_REF]. Interestingly, it was reported that constitutive activation of PPARγ in adipocytes leads to improved glucose tolerance and inflammatory profiles in DIO mice, without causing weight gain [START_REF] Sugii | PPARγ activation in adipocytes is sufficient for systemic insulin sensitization[END_REF]. In addition to that, other studies have reported anti-inflammatory properties for PPARγ, which might reduce pro-inflammatory cytokines production via interaction with NF-κB in monocytes [START_REF] Jiang | PPAR-γ agonists inhibit production of monocyte inflammatory cytokines[END_REF].

In summary, these results in addition to our data show that CCL5 and its receptors play an important role in insulin signaling, possibly by modulating Similarly, other chemokines like CCL2 and its receptor CCR2 have been shown

to ameliorate obesity-induced insulin resistance if deleted or inhibited through genetic or pharmacological means or worsen it if overexpressed. These effects on insulin sensitivity seem to be linked to the ability of CCL2 to recruit macrophages into AT in obesity [START_REF] Kamei | Overexpression of monocyte chemoattractant protein-1 in adipose tissues causes macrophage recruitment and insulin resistance[END_REF][START_REF] Kanda | MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity[END_REF][START_REF] Tamura | Inhibition of CCR2 ameliorates insulin resistance and hepatic steatosis in db/db mice[END_REF][START_REF] Tamura | C-C chemokine receptor 2 inhibitor improves diet-induced development of insulin resistance and hepatic steatosis in mice[END_REF]. However, for CCL2 there are also conflicting results in the literature. This indicates that the chemokine family forms a complex network of signaling molecules that interact with each other as well as other signaling molecules to exert their multifactorial functions that are far from limited to immune function. This complexity requires more sophisticated approaches that take into account the multiple interactions of chemokines to delineate the mechanism behind such a multilayered pathology as obesity.

The redundancy of the chemokine system such as the fact that chemokines can bind several receptors, which in turn can bind several ligands suggests that chemokines are far from having a single function as chemoattractants and invites the possibility of different receptors mediating different functions.

Nevertheless, this redundancy also permits the compensation of ligands or receptors if one is lacking, thereby potentially leading to confounding results

or incomplete reversal of phenotypes in such studies. This might explain the confounding results found in different studies.

One of the remaining mysteries behind DIO pathogenesis is the fact that some individuals seem to be more resistant to the development of DIO, while others are more prone to it. In an attempt to solve part of this mystery, one group used a specific model of rats, that are bred into resistant and DIO prone animals. They found that these groups seem indistinguishable from each other in their phenotype on a SD diet but differed in their synaptic arrangement at the level of hypothalamic neuronal input of POMC and NPY neurons. They found that before rats are exposed to HFD that POMC and NPY neurons differ in their number of inhibitory and stimulatory inputs, which might make them more or less susceptible to DIO. Interestingly, they found that HFD induced a loss of synaptic inputs on POMC and NPY neurons, which was either inhibitory or stimulatory, respectively. Furthermore, these lost inputs on neuronal perikarya where replaced by astrocytic processes, which might impair peripheral signals from reaching these neurons and thereby contribute to a resistance to peripheral satiety or hunger signals like leptin, ghrelin or insulin [START_REF] Horvath | Synaptic input organization of the melanocortin system predicts diet-induced hypothalamic reactive gliosis and obesity[END_REF]. This might be one of several reasons for the variations of results demonstrated by different studies, like our results in glucose tolerance from the ones of other studies. Studies from different countries often have inbred colonies of mice, which despite being the same strain of mice, could accumulate subtle but important changes and thus cause differences in their susceptibility to DIO between groups of mice from different laboratories.

The role of CCL5/CCR5 signaling in HFD-induced changes in adipose tissue

Interestingly, DIO induces many changes in AT, like hypertrophic adipocytes and adipogenesis, in an attempt to store the increasing influx of lipids from HFD but also inflammatory changes, like infiltration of immune cells into AT, their activation and polarization to a pro-inflammatory state and production of pro-inflammatory mediators. These changes become evident in an increased amount of AT but also morphological changes of adipocyte number and size. We wanted to know whether the reduction in BW in HFD-fed CCL5 and CCR5 deficient mice is reflected in their WAT. Immunohistological assessment of subcutaneous AT revealed that CCL5 -/-and CCR5 -/-mice have a slightly shifted frequency distribution of adipocytes from many very small adipocytes to less but slightly bigger adipocytes in SD conditions, which is not significant though, when comparing average adipocyte size between genotypes. This finding was different from expectations, as we expected to find smaller adipocytes in CCL5 -/-and CCR5 -/-mice. It suggests that CCL5/CCR5 signaling might play a role in the regulation of some of these changes that translate into a shift in morphology. These changes could include adipogenesis, lipid uptake and storage, or lipolysis. Similarly, small differences are visible between KO mice and WT mice on a HFD. Histological images as well as the frequency distribution seem to indicate that CCL5 -/- mice have more adipocytes that are slightly smaller than WT mice, whereas CCR5 -/-mice have more adipocytes that are slightly bigger than WT mice.

However, both KO mice seem to have a few smaller adipocytes as wells as a lower maximum size of adipocytes than WT mice on a HFD. These changes are interesting, although not significant. However, we found a significant increase in free serum glycerol in both HFD-fed CCL5 -/-and CCR5 -/-mice compared to WT mice on a HFD, which is indicative of increased lipolysis in KO mice. These results could be due to the higher expression of PPARγ mRNA that is known for its role in adipogenesis and could lead to morphological changes through lipolytic activity. This result is surprising as no changes in average adipocyte size were detected between HFD-fed animals, indicating that all of them have increased adipocytes due to the higher load of lipids in their diet. In support of our results, previous studies reported no difference in adipocyte size between HFD-fed CCR5 -/-and WT mice [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF].

The role of CCL5/CCR5 signaling in HFD-induced inflammation

The HFD-induced changes in AT of obese mice are associated with infiltration and accumulation of macrophages and T cells in AT as wells as upregulation of cytokines, most notably, TNF-a, IL-1β and IL-6 as well as chemokines like CCL2, CCL3 and CCL5 and their receptors such as CCR2 and CCR5. The inflammation is not limited to AT, but can be found systemically, as increased immune factors in serum of obese humans and mice, as well as other metabolic tissues and the hypothalamus. Due to the pro-inflammatory nature of CCL5 and its well-known contribution to AT inflammation, we expected a reduced inflammatory profile in AT, serum and hypothalamus of CCL5 -/-and CCR5 -/-mice after HFD, compared to WT mice. In support of previous reports, we found a HFD-induced upregulation of inflammatory markers like IL-6 and IL-1β and CCL5 as well as CCL2 in PG AT, but surprisingly, they were not different between WT and CCL5 -/-mice, apart from a reduction in TNF-a in CCL5 -/-mice on a HFD. We have not found any HFD-induced increase in inflammatory markers apart from CCL2 in CCL5 -/-mice and a trend for increase in WT mice, although not significant. Similar to reports in literature, serum CCL5 was increased in HFD-fed WT mice compared to their SD control.

Surprisingly, CCL5 -/-mice showed no differences in serum levels of cytokines compared to WT mice, but an increase in the chemokine CCL2 compared to their SD control. Although not significant, we saw the same tendency in WT mice on a HFD. Furthermore, IL-10 an anti-inflammatory cytokine is significantly reduced in CCL5 -/-mice on a HFD. This suggests that CCL5 could be involved in the anti-inflammatory feedback response to inflammation, which is reduced in these mice due to CCL5 deficiency.

Surprisingly, we found a different inflammatory profile for CCR5 -/-mice in serum, as we have not determined the AT inflammation for those mice yet. In this group of mice, we found an increase in serum TNF-a levels for HFD-fed mice, without any difference between genotypes.

Contrary to our expectations, our results indicate, that despite a decrease in TNF-a levels in HFD-fed CCL5 -/-mice, long-term HFD feeding induces inflammation in PG AT but not in serum of both WT and CCL5 -/-mice. In agreement with the literature and previous findings of our team, we found increases in serum levels of CCL5, confirming its role in DIO associated inflammation. Previous studies found that CCL5 mRNA expression in WAT correlated positively with other cytokines like TNF-a and IL-6 in human obese patients [START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF]. Our results are in part contradicting these results, as the lack of CCL5 -/-did not prevent the increase of IL-6, but only of TNF-a.

This indicates that at least in mouse WAT, IL-6 increase as well as other cytokine regulation might be independent of CCL5 signaling. While we have not determined the inflammatory state of CCR5 -/-WAT yet, our results are in part fitting to reports of other studies, who found a decrease in TNF-a levels of HFD-fed CCR5 -/-mice compared to WT mice in WAT but no difference between IL-6 [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF]. This is similar to the results observed in our CCL5 -/-mice. However, another study reported no changes in inflammatory markers in AT of HFD-fed WT and CCR5 -/-mice. While we have not determined the phenotype of immune cells and their infiltration into the WAT of our mice, conflicting reports exist on infiltration of AT macrophages in CCR5 -/-mice.

While one study reports no reduction in the infiltration of macrophages into AT of CCR5 -/-mice on a HFD, another study found that lacking CCR5 on a HFD led to a reduced infiltration of macrophages into AT compared to WT mice [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF][START_REF] Kennedy | Loss of CCR5 results in glucose intolerance in diet-induced obese mice[END_REF]. However, results are more consisting in regard to the reduction in pro-inflammatory macrophages. Moreover, one of the studies has reported apart from a reduction in macrophages on HFD, a reduction in different macrophage markers as well as TNF-a levels in WAT of increased NO secretion while decreasing IL-10 in activated microglia (Škuljec et al., 2011).

So far, to the best of our knowledge, no study has tested hypothalamic inflammation or gliosis in CCR5 -/-or CCL5 -/-mice after long-term HFD feeding.

However, in a previous study, our team (Cansell et al., 2019; see annex), investigated the post-prandial effects of HFD on the hypothalamus and found that lipid-rich diet evokes post-prandial inflammatory and neuromodulatory responses involving both astrocytes and microglia. Interestingly, upregulation of GFAP-expressing cells was found as early as 1h after HFD exposure, which coincides with upregulation of hypothalamic CCL5 expression. This study indicates that HFD can induce inflammatory changes only hours after diet ingestion and implicates CCL5 in this response. It further suggests that the inflammatory response observed, might be part of the physiological regulation of energy balance. Our results suggest that the expression of cytokines and chemokines are dependent on a multifactorial and complex regulation, which cannot be simply explained by one factor deletion.

However, judging from the results presented herein and the reports of the literature in regards to HFD-induced inflammatory processes as well as the in part conflicting data on CCR5 -/-mice, it seems that inflammatory cytokine expression is either upstream of chemokine release or in part independent of chemokine action. Reports from the literature as well as unpublished results of our team (not shown), suggest that HFD induces early inflammation in the hypothalamus, which precedes the development of obesity and associated changes in AT and insulin sensitivity [START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF]. Although it is still under debate, whether hypothalamic inflammation is induced locally or is initiated by infiltrating immune cells, some studies report a HFD-induced 30% increase in the recruitment of peripheral immune cells into the hypothalamus, the majority of which have a phenotype similar to microglia/macrophages [START_REF] Buckman | Obesity induced by a high-fat diet is associated with increased immune cell entry into the central nervous system[END_REF]. Hypothalamic inflammation in DIO is apart from its early onset characterized by similar events as peripheral metabolic tissues, namely accumulation of lipids in the hypothalamus, expression of pro-inflammatory cytokines and chemokines, infiltration of immune cells, change into a pro-inflammatory phenotype of immune cells and in the case of the hypothalamus of astrocytes and microglia as well as an upregulation of their characteristic proteins GFAP and Iba1, resistance to hormones (leptin and insulin) and some studies also suggest apoptosis via ROS production, mitochondrial abnormalities and ER stress.

Our data indicate that CCL5 and CCR5 -/-deficiency does attenuate and delay the development of obesity and insulin resistance, but still shows some cytokine upregulation, in the hypothalamus, AT or circulating levels, which seem to not be consistent. Furthermore, CCL5/CCR5 deficient mice have no significant differences in WAT size and only little effects on the frequency distribution of adipocyte size but significantly increases lipolysis. One possibility for the observed effect is, that the difference in BW is not attributable to the size of adipocytes but could be due to a difference in total amount in AT.

Although not shown here, our data indeed shows a reduction in total AT volume in WAT of CCR5 -/-mice on HFD compared to WT mice, but no difference between CCL5 -/-mice and WT mice after 16 weeks on HFD. The difference in AT volume seems to be mainly due to a decrease In SC AT in CCR5 -/-mice. This finding seems to be in line with the increase in lipolysis found for CCR5 -/-mice. However, the question remains as to what happens with the released FFAs. An increase in the release of FFAs can cause ectopic lipid accumulation and is associated with insulin resistance due to lipotoxicity.

Although both KO mice had improved glucose tolerance and insulin sensitivity, this difference was after around 8 weeks of diet, whereas the body composition X-ray and lipolysis test was performed only at the end of the experimental protocol. After 16 weeks of diet both CCL5 -/-and CCR5 -/-mice showed no difference in glucose tolerance to HFD-fed WT. Furthermore, CCL5 - /-mice although still significant were much closer in BW to WT mice, which might explain why we did not find any significant difference in AT volume for CCL5 -/-mice. Although further experiments are necessary, it seems that lipolysis might be in part responsible for the decrease in SC AT and might explain the development of glucose intolerance for both CCL5 -/-and CCR5 -/- mice. The question remains to why this difference develops only later on. As mentioned before, CCL5 signaling was implicated in cellular metabolism in CCR5-expressing T cells and breast cancer cell lines, where it seemed to regulate glucose uptake and lipid metabolism. Another study reported that CCL5 contributes to GLUT4 translocation in the hypothalamus, a protein that is involved in glucose uptake as well. Since AT and other metabolic organs express and secrete CCR5 and CCL5, it could be possible that CCL5 signaling might be involved in lipid metabolism, especially seeing as macrophages also express CCR5 and have been reported to accumulate lipids, too. In any case, further experiments have to be performed to investigate the body composition at an earlier time in KO mice to see, whether differences in adiposity and adipocyte morphology can be found, while differences in BW are highest. Another possibility might be that the difference in BW could be due to an increase in basal metabolic rate, thermogenesis or locomotor activity and thereby contribute to a reduction in lipid excess and protecting from lipotoxicity-induced glucose metabolism impairment. This could be a possibility concerning the implication of CCL5/CCR5 signaling in lipid metabolism and oxidation. Interestingly, some studies have found that NPYexpressing neurons in the DMN of the hypothalamus are implicated in regulating not only food intake but also EE like locomotor and BAT thermogenic activity. These studies demonstrated that knockdown of NPY in neurons of the DMN resulted in a decrease in BW combined with browning of WAT mediated by the SNS that innervates AT. This was further accompanied by increased thermogenesis in BAT and locomotor activity and associated with improved glucose homeostasis and insulin sensitivity [START_REF] Chao | Knockdown of NPY expression in the dorsomedial hypothalamus promotes development of brown adipocytes and prevents diet-induced obesity[END_REF][START_REF] Bi | Dorsomedial hypothalamic NPY modulation of adiposity and thermogenesis[END_REF]. Our results suggest that CCL5/CCR5 might have a modulatory role on NPY neurons and are expressed in different nuclei of the hypothalamus. Thus, it is intriguing to speculate that CCL5/CCR5 might be implicated in this function of NPY circuitry and could modulate thermogenesis or locomotor activity. This possibility has to be verified in additional experiments.

Furthermore, although surprising and against our hypothesis, the upregulation of some cytokines and chemokines still observed in CCL5 -/-and CCR5 -/-mice is not necessarily indicative of a lack of effect on inflammation in both hypothalamus or peripheral organs. As suggested previously, lipotoxicity leads to inflammation, initially in the hypothalamus and if not resolved, later on also in peripheral metabolic tissues. Lipids have the capacity to activate different inflammatory pathways like TLR or IKKB, and activation of immune cells, which leads to the upregulation of cytokines and chemokines. First, the inflammatory response is a complex response including several different cells and mediators. Chemokines are only one of those mediators and although important, one chemokines might not be able to resolve the inflammatory response completely. Second, studies have shown that cytokines like TNF-a, IL-1β and IFN-γ, can induce the expression of chemokines like CCL5 [START_REF] Sarkar | Expression and regulation of chemokines in murine and human type 1 diabetes[END_REF]. These results suggest that chemokines could be induced secondary to cytokine production and thus rather act as a mediator between cytokines and the downstream inflammatory response leading to alterations in the function of various tissues. This is in line with the results shown in the literature that the deletion of chemokines reduces the infiltration of immune cells and leads to a shift in their phenotype to a less activated or anti-inflammatory phenotype. As chemokines are well known for their chemoattractive role, it makes sense that CCL5 might mediate the inflammatory response by recruiting immune cells like macrophages, monocytes, and T cells and activate them in the periphery but also in the brain. In the brain, glia cells that express chemokine receptors including CCR3 and CCR5 might thus be part of the inflammatory response either upstream or downstream of chemokine action, but as they have been shown to also secrete CCL5, it might also be an autocrine feedback loop, that potentiates the inflammatory response. This is in line with our results that seem to indicate that ICV infusion of CCL5 or deletion of CCL5 modulates the expression of GFAP during a HFD challenge.

Thus, it is still possible that HFD can still induce an initial inflammatory response both in the brain and AT, but that this response is not potentiated and with the lack of one of the downstream mediators like CCL5/CCR5, the attenuated inflammatory response might thus not translate into a disrupted metabolism as seen in obese WT mice. Indeed, as mentioned previously, the lack of one actor of the inflammatory response might, help understand the implication of this specific actor and might explain the partial amelioration of the phenotype but is not sufficient for a full recovery of the healthy phenotype. This is especially the case since the chemokine system is such a redundant system with many overlaps in signaling pathways, ligands and receptors. Thus, experiments have to be more sophisticated and adapted to this complex process of inflammatory regulation in order to answer the remaining questions.

The role of CCL5/CCR5 signaling in thermal pain sensitivity

Lastly, apart from their role in immune function and as potential neuromodulators, chemokines and their receptors such as CCL5, CX3CL1, CCL3, CCR2 among others have also been implicated in hyperalgesia, although the mechanism behind their ability to modulate pain responses is still unknown [START_REF] Zhang | Proinflammatory Chemokines, Such as C-C Chemokine Ligand 3, Desensitize μ-Opioid Receptors on Dorsal Root Ganglia Neurons[END_REF][START_REF] Staniland | Reduced inflammatory and neuropathic pain and decreased spinal microglial response in fractalkine receptor (CX3CR1) knockout mice[END_REF][START_REF] Lee | Decreased pain responses of C-C chemokine receptor 5 knockout mice to chemical or inflammatory stimuli[END_REF].

Chemokine receptor expression has been reported on primary sensory neurons and their activation by chemokines associated with hyperalgesia [START_REF] Lee | Decreased pain responses of C-C chemokine receptor 5 knockout mice to chemical or inflammatory stimuli[END_REF].

Several studies have investigated and associated CCL5 and CCR5 with neuropathic pain in different contexts and phenotypes. Previous reports showed that CCR5 has a possible association with the µ-opioid receptor and thereby can modulate nociceptive pain responses.

In collaboration with J. Noël in the team of E. Lingueglia at IPMC, who specialize in the study of pain, we performed experiments to assess another comorbidity in obese and diabetic patients, namely the occurrence of neuropathic pain. Their team has observed in many experiments that HFD feeding in mice induces neuropathic pain after about 8 weeks of HFD, which becomes evident and measurable in a thermal pain sensitivity test called Hargreaves' Test. We employed this test to confirm the increased pain sensitivity to thermal pain in HFD fed WT mice and tested whether the lack of CCL5 and CCR5 might be implicated in the HFD-induced increase in the sensitivity to pain. Indeed, we found that HFD-fed WT mice become more sensitive to thermal pain after 16 weeks of diet, whereas both CCL5 -/-and CCR5 -/-mice show an amelioration of this hypersensitivity. We further tested whether CCL5 and antagonist Met CCL5 ICV infusion in SD-fed mice could alter the sensitivity to thermal pain. In line with the genetic deletion experiment, we found that CCL5 infusion increased thermal pain sensitivity, while Met CCL5 infused mice showed no difference in their thermal pain sensitivity to aCSF infused mice. Although, this is a preliminary result with low number of mice and has to be repeated together with our genetic deletion study, our results

show that CCL5 and CCR5 seem to have a role in HFD-induced thermal hyperalgesia. We further examined the expression of inflammatory factors in the spinal cord and dorsal root ganglia of CCL5 -/-and CCR5 -/-mice under HFD and SD conditions to answer whether the alteration of thermal pain sensitivity due to CCL5 and CCR5 might be due to HFD-induced inflammation.

However, against our expectations we have not found any HFD-induced upregulation in pro-inflammatory markers in DRG of WT nor CCL5 -/-mice.

Similarly, we did not find inflammatory marker expression in the spinal cords of obese WT mice, but an increase in expression of pro-inflammatory markers like IL-1β, CCL2 and TNF-a in the spinal cords of HFD-fed CCL5 -/-mice and TNF-a in CCL5 -/-mice on a SD. This result is rather surprising and counter-intuitive, and requires further testing to be able to explain this phenomenon, as the number of mice is low and represents the results of only one independent experiment.

The increased inflammatory response could be explained as mentioned above in that CCL5 is a downstream mediator of the inflammatory response that induces the recruitment of immune cells and thereby potentiates the inflammation as well as mediates the dysfunctions induced by inflammation.

Thus, one of the limitation of our study is that we have not tested the infiltration of immune cells and glia cells as well as their change into an activation state in DRG of mice. However, a study has shown similar results to ours, although the experimental model differed. The study used instead of DIO a model of PNL to induce inflammation and neuropathic pain in the injured site in both WT and CCL5 -/-mice. In line with our results the authors report an attenuated response to thermal pain but also mechanical pain in CCL5 -/- compared to WT mice [START_REF] Liou | Absence of C-C motif chemokine ligand 5 in mice leads to decreased local macrophage recruitment and behavioral hypersensitivity in a murine neuropathic pain model[END_REF] Furthermore, they report different from our results that CCL5 deficiency decreased both recruitment of immune cells as well as pro-inflammatory cytokine and increased anti-inflammatory marker production in the sciatic nerves. This was accompanied by a decrease in the mRNA expression of endogenous opioid peptides. In line with our result, another study performed CCL5 administration into the periaqueductal gray (PAG), a critical area for mediating the analgesic effect of opioids, of the brain in rats. They found that this reduced the effect of an anti-nociceptive agent (DAMGO), that acts as agonist of µ-opioid receptor and rendered rats more sensitive to painful stimuli [START_REF] Szabo | Heterologous desensitization of opioid receptors by chemokines inhibits chemotaxis and enhances the perception of pain[END_REF][START_REF] Lee | Decreased pain responses of C-C chemokine receptor 5 knockout mice to chemical or inflammatory stimuli[END_REF]. This study in line with our cannula experiment suggests that the effect of CCL5 could act at the levels of the brain and not only in the periphery and further indicates that the mechanisms behind the effect of CCL5/CCR5 on pain sensitivity might be related to sensitization/desensitization of opioid receptors like the µ-opioid receptor. Further evidence for this hypothesis was provided by a study, demonstrating that opioid receptors are expressed in several regions of the CNS such as cerebral cortex, hippocampus, locus coeruleus, dorsal root ganglia, striatum and the PAG.

They further report that opioid receptors can interact and form heterodimers with chemokine receptors, resulting in desensitization or other effects on both receptors through binding of one of the ligands [START_REF] Chen | Heterodimerization and cross-desensitization between the μ-opioid receptor and the chemokine CCR5 receptor[END_REF].

In partial agreement with our results another study showed that CCR5 deletion in mice lowered nociceptive pain response, and found enhanced expression of µ-opioid receptor in PAG of CCR5 -/-mouse brains compared to WT control [START_REF] Lee | Decreased pain responses of C-C chemokine receptor 5 knockout mice to chemical or inflammatory stimuli[END_REF]. However, different from our experiments they have not found any difference in thermal pain sensitivity between WT and CCR5 -/-mice but rather in chemical-and inflammatory pain [START_REF] Lee | Decreased pain responses of C-C chemokine receptor 5 knockout mice to chemical or inflammatory stimuli[END_REF].

Ip administration of the antagonist Met CCL5, in an inflammatory pain model of nerve damage attenuated the hypersensitivity to pain after PNL, which was concurrent with decreased infiltration of immune cells, inflammatory marker expression and opioid agonists like enkephalins and endorphins [START_REF] Liou | Absence of C-C motif chemokine ligand 5 in mice leads to decreased local macrophage recruitment and behavioral hypersensitivity in a murine neuropathic pain model[END_REF]. These results provided by our study and the literature strongly suggest the involvement of CCL5 in different forms of neuropathic pain sensitivity.

Furthermore, it seems like inflammatory pain is reduced by CCL5 or CCR5 deficiency, as it reduces infiltration of immune cells to the site of damage and the lack of inflammatory response potentiation might diminish hyperalgesia, possibly via interaction with opioid receptors. Furthermore, the evidence provided indicates that the effect of CCL5 might not only be in the periphery but also can act on analgesia pathways at the level of the brain. Previous results have shown impaired T cell and macrophage infiltration into the CNS of hepatitis virus-infected mice that were depleted of CCL5 [START_REF] Lane | A Central Role for CD4+ T Cells and RANTES in Virus-Induced Central Nervous System Inflammation and Demyelination[END_REF]. Similarly, CCL5 could be involved in the sustaining or mediation of hypothalamic inflammation as a downstream mediator, induced by cytokines, and recruiting blood-borne T cells and macrophages into the CNS.

In this way, it could contribute to hypothalamic inflammation, worsening it or

Conclusion & Perspectives

The present study aimed at elucidating the role of chemokines in the CNS. In particular, we were interested in the role of chemokines in the regulation of energy balance and their contribution to its deregulation as is the case in modern disorders like obesity. Several studies have suggested that chemokines are more than just recruiters of leukocytes in the immune system and might be actors in the physiology of brain function. We have taken a global approach in an attempt to characterize the phenotype associated with mice deficient in CCL5 or CCR5 by investigating different aspects of the complex disorder obesity. We have studied both HFD-induced inflammation in the periphery as well as in the hypothalamus and looked at the modulation of neuropeptides in the hypothalamus by diet and by CCL5 deficiency. We assessed central expression of CCL5 and CCR5 as well as glucose metabolism and HFD-induced neuropathic pain, associated with diabetes. Herein we have found that both CCL5 and CCR5 are expressed in hypothalamic nuclei such as the ARC, LH, ME among others in adult mice under SD conditions and their expression seems to be upregulated in HFD condition, although this has to be confirmed. Furthermore, we showed that although deleting either CCL5 or CCR5 did not necessarily change the expression of cytokines it yet greatly improved the HFD-induced obesity phenotype by reducing weight gain and delaying the glucose intolerance and insulin resistance associated with DIO.

In accordance with this, we see also differences at the level of the brain in the expression of genes important for lipid metabolism and insulin signaling and sensitivity. Lastly, we have seen also improvements in another aspect associated with obesity -neuropathic pain. This suggests that although inflammation is crucial for the development of obesity as shown in the literature, chemokines are crucial as a secondary mediator after the initial inflammatory insult, which might contribute by recruiting blood-borne immune cells and/or activate microglia and astrocytes and thereby potentiating the inflammation. Studies have previously shown that CCL5 induces a pro-inflammatory state in microglia and can be secreted by astrocytes and microglia. This is confirmed by our preliminary results showing upregulation of microglia markers after CCL5 injections and in long-term HFD condition by a decrease in astrocyte marker expression in CCL5 deficient mice. To confirm these results, it would be interesting to verify whether CCL5 and CCR5 deficient mice prevent the infiltration of peripheral and local immune cells and their activation including glia cells such as microglia and astrocytes via immunohistochemistry using specific markers for each marker. Furthermore, it would be interesting to inject CCL5 and the antagonist Met CCL5 in CCL5

Introduction

Obesity is a major public health problem that has been highlighted by the World Health Organization. In 2016, almost 40% of the worldwide population was overweight and 13% was obese (http://www.who.int/en/). The main driving force of this body weight disturbance is an energy balance dysregulation, due to kilocalorie consumption overriding energy expenditure [START_REF] Gao | Neuronal control of energy homeostasis[END_REF]. Therefore, chronic exposure to the Western diet rich in saturated fat (commonly known as the high-fat diet (HFD)) is now recognized as a significant contributor to the development of obesity and its associated comorbidities, such as insulin resistance, type 2 diabetes mellitus and other chronic illnesses such as cardiovascular disease or cancer (Hotamisligil, 2006).

Excessive intake of dietary fat induces chronic low-grade inflammation in many metabolic organs including the pancreas, adipose tissue, muscle and liver, as well as in the brain (Lumeng and Saltiel, 2011). HFD consumption increases the expression of inflammatory mediators (De Souza et al., 2005;[START_REF] Thaler | Minireview: Inflammation and obesity pathogenesis: the hypothalamus heats up[END_REF], especially the pro-inflammatory cytokines Interleukin-1 beta (IL-1β) , Tumor Necrosis

Factor alpha (TNF-α) and Interleukin 6 (IL-6), and disrupts insulin and leptin signaling in murine and primate hypothalamus (HT), a brain area involved in energy balance regulation [START_REF] Arruda | Low-grade hypothalamic inflammation leads to defective thermogenesis, insulin resistance, and impaired insulin secretion[END_REF][START_REF] Grayson | Changes in melanocortin expression and inflammatory pathways in fetal offspring of nonhuman primates fed a high-fat diet[END_REF][START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF][START_REF] Zhang | Hypothalamic IKKβ/NF-κB and ER Stress Link Overnutrition to Energy Imbalance and Obesity[END_REF]. HFD-induced inflammatory modifications of brain signals also converge to alter levels of hypothalamic neuropeptides, suggesting a new concept where hypothalamic neuropeptides act as a link between central inflammation, dysregulation of feeding behavior and energy homeostasis, and indicating that these peptides could be potential therapeutic targets for the treatment of obesity (Timper and Bruning, 2017).

Since 2012, several studies have shown that HFD consumption induces astrogliosis and microgliosis in the arcuate nucleus (ARC) of the HT [START_REF] Andre | Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation[END_REF][START_REF] Balland | Short-term high-fat diet increases the presence of astrocytes in the hypothalamus of C57BL6 mice without altering leptin sensitivity[END_REF][START_REF] Baufeld | High-fat diet-induced brain region-specific phenotypic spectrum of CNS resident microglia[END_REF][START_REF] Gao | Hormones and diet, but not body weight, control hypothalamic microglial activity[END_REF][START_REF] Guillemot-Legris | High-fat diet feeding differentially affects the development of inflammation in the central nervous system[END_REF][START_REF] Kim | Microglial UCP2 Mediates Inflammation and Obesity Induced by High-Fat Feeding[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Valdearcos | Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF]. The microglia activation that is induced by lipids and connects dietary fat and brain inflammatory signaling is begging to be seen as the missing link between the hypothalamic response to HFD and susceptibility to obesity [START_REF] Andre | Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation[END_REF][START_REF] Kim | Microglial UCP2 Mediates Inflammation and Obesity Induced by High-Fat Feeding[END_REF][START_REF] Valdearcos | Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF]. Nevertheless, it has recently been shown that HFD-induced inflammation in astrocytes may have a protective effect against the development of obesity [START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF]. Interestingly, hypothalamic inflammation has been described very early in response to HFD, before the onset of obesity [START_REF] Balland | Short-term high-fat diet increases the presence of astrocytes in the hypothalamus of C57BL6 mice without altering leptin sensitivity[END_REF][START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF][START_REF] Kim | Microglial UCP2 Mediates Inflammation and Obesity Induced by High-Fat Feeding[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Waise | One-day high-fat diet induces inflammation in the nodose ganglion and hypothalamus of mice[END_REF].

Exposure to HFD for a 24h period increases gene expression of inflammatory astrocyte and microglia markers as well as astrogliosis in the HT [START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF][START_REF] Dalvi | High fat induces acute and chronic inflammation in the hypothalamus: effect of high-fat diet, palmitate and TNF-alpha on appetite-regulating NPY neurons[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF]. Microgliosis has also been found in the ARC after a 72-hour exposure to HFD [START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF]. These findings tempt us and others to speculate that hypothalamic inflammation may not be limited to chronic exposure to dietary fat, but may occur after consumption of a single meal [START_REF] Cani | Metabolic endotoxemia initiates obesity and insulin resistance[END_REF][START_REF] Emerson | Magnitude and Timing of the Postprandial Inflammatory Response to a High-Fat Meal in Healthy Adults: A Systematic Review[END_REF][START_REF] Ghanim | Increase in plasma endotoxin concentrations and the expression of Toll-like receptors and suppressor of cytokine signaling-3 in mononuclear cells after a high-fat, high-carbohydrate meal: implications for insulin resistance[END_REF][START_REF] Herieka | High-fat meal induced postprandial inflammation[END_REF][START_REF] Kelly | Of microbes and meals: the health consequences of dietary endotoxemia[END_REF][START_REF] Khor | Postprandial oxidative stress is increased after a phytonutrient-poor food but not after a kilojoulematched phytonutrient-rich food[END_REF]. Therefore, we investigated cellular and molecular hypothalamic remodeling in mice that were exposed for the first time to a HFD for short periods of time (1h, 3h or 6h) and we compared it to mice exposed to standard diet (SD).

Materials and methods

Animals.

8-week-old C57Bl/6J male mice (20-25 g, Janvier Labs, France) and 9-10 weekold CX3CR1eGFP/eGFP male mice [START_REF] Jung | Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion[END_REF] ImageJ [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] on maximal intensity projections of these z-stacks of images. After the z-projection, a Region Of Interest (ROI) corresponding to the ARC of the hypothalamus was manually drawn on these images. GFAP and Iba1 staining areas were selected and measured (in µm 2 ) in these ROIs by intensity thresholding and measurement above this threshold. The same process was applied to measure the soma size of microglial cells.

Acute HT slices preparation for microglia recordings.

Male mice aged between 9 and 10 weeks were anesthetized with isoflurane before the brain was harvested and coronal slices through the HT were cut using a Leica VT1200 vibratome in an oxygenated (5% CO given in the legends.

Results

A single high-fat meal increases serum levels of saturated triglycerides and

endotoxins, but not those of cytokines and chemokines.

Obesity and metabolic syndromes are concomitant with a state of chronic systemic inflammation (Lumeng and Saltiel, 2011) and the degree to which triglycerides (TG) increase after HFD consumption correlates with the probability of developing metabolic syndrome [START_REF] Herieka | High-fat meal induced postprandial inflammation[END_REF]Nogaroto et al., 2015). Because the main difference between SD and HFD is lipid content, we measured TG serum levels after short exposure to the diet. By mass spectrometry, we found that HFD consumption did not change the total TG serum levels (Fig. 1A) but led to increase the percentage of TGs containing only saturated fatty acids (Fig. 1B) compared to SD-fed mice. Previous studies have linked HFD consumption to post-prandial systemic inflammation [START_REF] Cani | Metabolic endotoxemia initiates obesity and insulin resistance[END_REF][START_REF] Emerson | Magnitude and Timing of the Postprandial Inflammatory Response to a High-Fat Meal in Healthy Adults: A Systematic Review[END_REF][START_REF] Ghanim | Increase in plasma endotoxin concentrations and the expression of Toll-like receptors and suppressor of cytokine signaling-3 in mononuclear cells after a high-fat, high-carbohydrate meal: implications for insulin resistance[END_REF][START_REF] Herieka | High-fat meal induced postprandial inflammation[END_REF][START_REF] Kelly | Of microbes and meals: the health consequences of dietary endotoxemia[END_REF][START_REF] Khor | Postprandial oxidative stress is increased after a phytonutrient-poor food but not after a kilojoulematched phytonutrient-rich food[END_REF]. Therefore, we measured the serum levels of the pro-inflammatory cytokines IL-1β, IL-6 and TNFα, pro-inflammatory chemokines CCL2 and CCL5 (Chemokine (C-C motif) Ligand 2-5), as well as endotoxin (lipopolysaccharide, 3OH C14:0) after SD and HFD short-term exposure (1h, 3h and 6h). Interestingly, HFD consumption did not increase proinflammatory cytokine or chemokine serum levels compared to SD-fed mice over the 3 time points (Fig. 1C-G). However, 3h and 6h of HFD consumption increased endotoxin serum levels compared to SD-fed mice (Fig. 1H). As anticipated [START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF], an increase in kilocalories consumed was noticeable in mice fed for 6h

with HFD (SD)-fed mice (Supplementary Fig. 1A), while the total quantity in grams of ingested food was similar between groups (Supplementary Fig. 1B). Altogether, these observations prove that short-term HFD exposure does not affect systemic levels of inflammatory markers, but increases endotoxin concentrations and the proportion of saturated TGs in the serum compared to SD-fed mice.

A single high-fat meal induces an exacerbated inflammatory-like gene

response in the hypothalamus.

Given the lack of a peripheral inflammatory response in HFD-fed mice, we subsequently investigated the impact of a HFD on hypothalamic inflammatory markers. Consistent with previous studies [START_REF] Andre | Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation[END_REF][START_REF] Baufeld | High-fat diet-induced brain region-specific phenotypic spectrum of CNS resident microglia[END_REF][START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF][START_REF] Gao | Dietary sugars, not lipids, drive hypothalamic inflammation[END_REF][START_REF] Gao | Hormones and diet, but not body weight, control hypothalamic microglial activity[END_REF][START_REF] Guillemot-Legris | High-fat diet feeding differentially affects the development of inflammation in the central nervous system[END_REF][START_REF] Nadjar | Bioactive lipids as new class of microglial modulators: When nutrition meets neuroimunology[END_REF][START_REF] Thaler | Minireview: Inflammation and obesity pathogenesis: the hypothalamus heats up[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Valdearcos | Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF] we found that gene expression of the proinflammatory cytokine genes IL1β, IL6 and TNFα, as well as chemokine genes CCL2 and CCL5 was significantly increased in HFD-fed mice compared to SD-fed mice (Fig. 2A-E). The novelty of our study relies on the demonstration that an inflammatory-like gene response in the HT is physiologically induced by SD and arises within the first hours of food consumption. Moreover, this inflammatory-like gene response exhibits a diet specific profile defined by cytokine-/chemokine-type, amplitude and time course of the response, and is exacerbated in HFD-fed mice. In fact, the increase in IL-1β and TNFα gene expression appeared after 1h of food exposure (Fig. 2A andC) with a greater increase in HFD-fed mice for IL-1β (Fig. 2A).

Moreover, the increase in CCL2 and CCL5 gene expression also appeared within 1h of food exposure, but was observed exclusively in HFD-fed mice (Fig. 2D andE).

After 3h of food exposure, IL-1β, IL-6, TNFα and CCL2 gene expressions were increased only in HFD-fed mice (Fig. 2A-D). Finally, after 6h of food exposure, proinflammatory cytokine and chemokine gene expression in HFD-fed mice tend to return to baseline. Taken together, these results indicate that short term HFD exposure induces a specific and exacerbated hypothalamic inflammatory-like gene response, different from the one observed in SD-fed mice.

A single high-fat meal induces a specific acute neuropeptide gene expression profile in the hypothalamus

Given the acute and specific response in hypothalamic inflammatory-like gene expression of HFD-fed mice, we subsequently investigated the impact of a single high fat meal on hypothalamic neuropeptide genes. We examined changes in hypothalamic neuropeptide gene expression involved in energy balance regulation in the HT after 1h, 3h and 6h exposure to SD and HFD [START_REF] Gao | Neuronal control of energy homeostasis[END_REF]. Food exposure induces changes in both SD-and HFD-fed mice. However, hypothalamic gene expression levels of orexigenic peptides MCH, AgRP and ORX (Fig. 3C, D and F) and anorexigenic peptide CART (Fig. 3E) were higher in HFD-fed mice compare to SD-fed mice while NPY levels remained equivalent in the 2 groups (Fig. 3A). In contrast, a decrease in anorexigenic peptide POMC gene expression, was observed at 1h only in HFD-fed mice (Fig. 3B). Interestingly, the time course of those changes was very specific for each diet. For example, SD-fed mice presented a transient and modest increase in MCH gene expression after 3h of food exposure compared to basal level (Fig. 3C) while it increased within the first hour of food exposure in HFDfed mice and remained high for 6h (Fig. 3C). AgRP and ORX gene expression levels increased within the first hours of food exposure in both diet groups, with an exacerbated response in HFD-fed mice that lasted for 6h (Fig. 3D andF), unlike the increase in CART gene expression, which did not occur until 3h of food exposure and was only observed in HFD-fed mice (Fig. 3E). Taken together, our results show that short term HFD exposure is sufficient to specifically modulate hypothalamic neuropeptide gene expression involved in energy balance regulation, differently from what is observed in SD-fed mice.

A single high-fat meal induces an acute increase of GFAP gene expression in the hypothalamus.

Hypothalamic inflammation after medium-term (1 to 10 days) and long-term (2 to 32 weeks) HFD exposure is associated with astrogliosis [START_REF] Balland | Short-term high-fat diet increases the presence of astrocytes in the hypothalamus of C57BL6 mice without altering leptin sensitivity[END_REF][START_REF] Baufeld | High-fat diet-induced brain region-specific phenotypic spectrum of CNS resident microglia[END_REF][START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF]. It has yet to be determined whether the same is true at earlier time points. Given that our results

showed an exacerbated hypothalamic inflammatory-like gene response in HFD-fed mice, we tested whether short-term exposure (within 1h, 3h and 6h) to HFD might affect astrocytes in the HT. To this end, we used the astrocyte marker GFAP for genetic and histological analysis. Surprisingly, 1h of food exposure increased GFAP gene expression in the HT of HFD-fed mice but not in SD-fed mice (Fig. 4A).

Moreover, the increase in GFAP gene expression appeared to be transient as expression levels returned to baseline at 3h and 6h (Fig. 4A). We then tested if the increase in GFAP gene expression was associated with changes in astrocyte remodeling in the ARC, as described previously for medium term and long term HFD exposure [START_REF] Balland | Short-term high-fat diet increases the presence of astrocytes in the hypothalamus of C57BL6 mice without altering leptin sensitivity[END_REF][START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF]. SD and HFD-fed mice presented similar GFAP staining in the ARC, as shown in the photomicrographs displayed in Fig. 4C and corroborated by fluorescence quantification in the ARC of mice fed for 1h, 3h and 6h (Fig. 4B). These findings demonstrate that short term HFD exposure, specifically and transiently, upregulates hypothalamic GFAP gene expression.

3.5.

A single high-fat meal induces acute microglial remodeling in the arcuate nucleus of the hypothalamus.

In addition to astrogliosis, hypothalamic inflammation after medium-term (3 to 7 days) and long-term (2 to 20 weeks) HFD exposure is also associated with microgliosis [START_REF] Andre | Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation[END_REF][START_REF] Baufeld | High-fat diet-induced brain region-specific phenotypic spectrum of CNS resident microglia[END_REF][START_REF] Dorfman | Sex differences in microglial CX3CR1 signalling determine obesity susceptibility in mice[END_REF][START_REF] Gao | Dietary sugars, not lipids, drive hypothalamic inflammation[END_REF][START_REF] Gao | Hormones and diet, but not body weight, control hypothalamic microglial activity[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Valdearcos | Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF]. As for astrocytes, we tested whether short-term exposure (1h, 3h and 6h) to HFD might affect microglial cells in the HT. HFD exposure increased microglial marker Iba1 gene expression in the HT at 3h and 6h compared to SD-fed mice (Fig. 5A). We then tested whether Iba1 gene upregulation impacted Iba1 protein levels in the ARC, as previously described for medium term and long term HFD exposure [START_REF] Andre | Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation[END_REF][START_REF] Baufeld | High-fat diet-induced brain region-specific phenotypic spectrum of CNS resident microglia[END_REF][START_REF] Dorfman | Sex differences in microglial CX3CR1 signalling determine obesity susceptibility in mice[END_REF][START_REF] Gao | Dietary sugars, not lipids, drive hypothalamic inflammation[END_REF][START_REF] Gao | Hormones and diet, but not body weight, control hypothalamic microglial activity[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Valdearcos | Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF]. No change in Iba1 immunofluorescence staining was observed (Fig. 5B andD). We also considered whether Iba1 gene expression modification might be associated with a change in microglial activity. Using the patch clamp technique, membrane properties of microglial cells were determined in 3h HFD-fed mice. We did not observe changes in the current-voltage relationship (Fig. 5E), but a significant increase in the cell capacitance in HFD-fed mice (Fig. 5F). We therefore looked more closely into cell morphology. While the histological approach may have had insufficient resolution to detect changes in soma size after 3h of HFD exposure (Fig. 5C and D), we did observe an increase in soma size in 6h HFD-fed mice (Fig. 5C), which is probably linked to the change observed in cell capacitance (Fig. 5F). Collectively, these results

indicate that short term HFD exposure induces fast microglial cells remodeling in the ARC.

Modulation of GFAP-positive cells changes post-prandial hypothalamic

neuropeptide and inflammatory-like gene responses to SD and HFD exposure.

We observed an acute and diet-specific hypothalamic gene response to food exposure for neuropeptides involved in energy balance regulation and proinflammatory markers. This response is associated with an increase in GFAP mRNA levels and microglial remodeling in the HT. We subsequently tested if modulating GFAP-positive cells and microglia could change this hypothalamic gene response. In order to modulate GFAP-positive cell activity we used DREADD (Designer Receptors Exclusively Activated by Designer Drugs) technology [START_REF] Chen | Direct modulation of GFAP-expressing glia in the arcuate nucleus bi-directionally regulates feeding[END_REF][START_REF] Yang | Astrocytes control food intake by inhibiting AGRP neuron activity via adenosine A1 receptors[END_REF]. An Adeno Associated Virus 8 (AAV8) with a GFAP promoter controlling DREADD Gi coupled with mCitrine gene sequence expression in the MBH of mice was used to target GFAP-positive cells. Analysis of mCitrine staining by immunohistochemistry confirmed successful injection (Supplementary Fig. 2A, B andC). Two weeks after virus injection, we treated mice with the specific agonist CNO (Clozapine N-Oxide) at a dose of 1 mg/kg to activate the DREADD Gi and 30 min later we exposed the mice to SD or HFD for 1h. Using this tool, we observed differential inflammatory-like and neuropeptide gene responses to SD and HFD in vehicle (NaCl) group (Fig. 6) compared to our original observation (Fig. 2 and3). This could be explained by different inflammatory states of animals, as discussed later, but doesn't change the originality of our results. CNO injection did not change 1h food consumption in both SD-and HFD-treated mice (Supplementary Fig. 2D andE).

CNO injection did not alter POMC, MCH, AgRP and ORX gene expression response to SD and HFD exposure (Fig. 6B, C, D and F). In contrast, CNO injection, but not vehicle injection, decreased CART hypothalamic gene expression regardless of diet (Fig. 6E). Only NPY gene expression in response to food exposure is changed by the activation of DREADD Gi in GFAP-positive cells in a diet-specific manner (Fig. 6A).

Interestingly, CNO injection, but not vehicle injection, decreased IL-1β, TNFα, and CCL2 gene expression in the HT regardless of diet (Fig. 6G, I and J). Moreover, CNO injection, but not vehicle injection, decreased CCL5 and Iba1 gene expression in HFD-treated mice (Fig. 6K andL). However, variability in CCL5 and Iba1 gene expression levels in SD-fed mice prevented statistically robust conclusions. CNO effect seems similar regardless of the diet on CCL5 gene expression (Fig. 6K), but the decrease in Iba1 gene expression is stronger in HFD-fed mice compared to SDfed mice (Fig. 6L). Finally, IL6 gene expression in response to food exposure is changed by CNO injection in HFD-treated mice but not in SD-treated mice (Fig. 6H).

Collectively, these results show that modulation of GFAP-positive cell activity in the MBH alters hypothalamic neuropeptide and inflammatory-like gene responses to both SD and HFD exposure. Interestingly, our results also demonstrate that specific NPY and IL-6 gene responses to food exposure are altered by the modulation of GFAPpositive cells activity in a diet-dependent manner.

Modulation of microglial activity changes post-prandial hypothalamic

neuropeptide and inflammatory-like gene responses to SD and HFD exposure.

To assess if microglia is involved in the neuropeptide and inflammatory gene responses induced by food exposure, we used PLX5622, which has been previously used to remove microglia from brain [START_REF] Elmore | Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain[END_REF]. PLX5622 is a CSF1R agonist known to regulate microglial density in the brain. Two weeks after PLX5622 treatment, we assessed its efficiency to deplete microglia. PLX5622 almost completely ablated the microglial population as shown by the absence of Iba1 staining in the MBH (Supplementary Fig. 3A andB). We exposed PLX5622-treated and non-treated mice to SD or HFD for 1 h and 3h. Using this tool, we observed differential inflammatory-like and neuropeptide gene responses to SD and HFD in non-treated group (Fig. 7 and Supplementary Fig. 4) compared to our original observation (Fig. 2 and3). This may be due to animal variability and susceptibility to inflammatory states, which does not undermine our results. SD and HFD intake (Supplementary Fig. 3E, F, H and I) and hypothalamic GFAP gene expression (Supplementary Fig. 3G andJ) were not modified by PLX5622-induced microglial depletion. Moreover, PLX5622 treatment did not affect the hypothalamic neuropeptide gene response induced by 1h of food exposure (Supplementary Fig. 4A-F). PLX5622 treatment did not alter CART hypothalamic gene expression response to 3h food exposure neither (Fig. 7E). However, after 3h of food exposure, PLX5622 treatment increased similarly NPY and POMC gene expression regardless the diet (Fig. 7A andB). Concerning the AgRP gene response, result variability in SD-treated mice prevented a statistically robust conclusion, but PLX5622 treatment also seemed to upregulate AgRP gene expression regardless the diet (Fig. 7D).

Interestingly, PLX5622 treatment changed MCH and ORX gene expression response to food exposure in a diet-dependent manner (Fig. 7C andF).

With respect to the impact of microglia depletion on inflammatory gene responses the high variance between the samples unfortunately detracted from any robust conclusion (Supplementary Fig. 4G-J and K; Fig. 7 G-K). Nevertheless, PLX5622 treatment increased TNFα mRNA levels in 1h HFD-treated mice (Supplementary Fig. 4I). Moreover, IL-1β and TNFα mRNA levels were decreased in 3h HFD-fed mice and effect seems similar on SD-fed mice (Fig. 7G andI).

Therefore, the results show that microglia depletion affects hypothalamic neuropeptide and inflammatory-like gene responses to both SD and HFD exposure.

More interestingly, our results also demonstrate that specific MCH and ORX gene responses to food exposure are affected by the absence of microglial cells in a dietdependent manner.

Discussion

Several studies have shown a link between HFD consumption, inflammation and risk factors associated with obesity. It seems certain that long-term exposure to HFD induces chronic low-grade systemic inflammation, which is responsible for the development of insulin resistance, type 2 diabetes mellitus along with other chronic illnesses such as cardiovascular disease or cancer [START_REF] Cani | Metabolic endotoxemia initiates obesity and insulin resistance[END_REF][START_REF] Emerson | Magnitude and Timing of the Postprandial Inflammatory Response to a High-Fat Meal in Healthy Adults: A Systematic Review[END_REF][START_REF] Ghanim | Increase in plasma endotoxin concentrations and the expression of Toll-like receptors and suppressor of cytokine signaling-3 in mononuclear cells after a high-fat, high-carbohydrate meal: implications for insulin resistance[END_REF][START_REF] Herieka | High-fat meal induced postprandial inflammation[END_REF][START_REF] Kelly | Of microbes and meals: the health consequences of dietary endotoxemia[END_REF]Lumeng and Saltiel, 2011;[START_REF] Thaler | Minireview: Inflammation and obesity pathogenesis: the hypothalamus heats up[END_REF]. More recently, hypothalamic inflammation, a process involving neurons, astrocytes and microglia, has been correlated with obesity and chronic HFD consumption [START_REF] Balland | Short-term high-fat diet increases the presence of astrocytes in the hypothalamus of C57BL6 mice without altering leptin sensitivity[END_REF][START_REF] Baufeld | High-fat diet-induced brain region-specific phenotypic spectrum of CNS resident microglia[END_REF][START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF][START_REF] Gao | Hormones and diet, but not body weight, control hypothalamic microglial activity[END_REF][START_REF] Guillemot-Legris | High-fat diet feeding differentially affects the development of inflammation in the central nervous system[END_REF][START_REF] Kim | Microglial UCP2 Mediates Inflammation and Obesity Induced by High-Fat Feeding[END_REF][START_REF] Milanova | Diet-Induced Obesity Disturbs Microglial Immunometabolism in a Time-of-Day Manner[END_REF][START_REF] Thaler | Minireview: Inflammation and obesity pathogenesis: the hypothalamus heats up[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Valdearcos | Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF], underlining the effect of chronic exposure to HFD on the central inflammatory status. Interestingly, brain inflammatory responses in the HT were also observable after only one day of HFD consumption [START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF] or 24h of lipid perfusion to the brain [START_REF] Dalvi | High fat induces acute and chronic inflammation in the hypothalamus: effect of high-fat diet, palmitate and TNF-alpha on appetite-regulating NPY neurons[END_REF], well before the development of obesity. Moreover, inhibition of brain inflammatory responses to HFD leads to energy balance dysregulation [START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF][START_REF] Fernandez-Gayol | Different responses to a high-fat diet in IL-6 conditional knock-out mice driven by constitutive GFAP-Cre and Synapsin 1-Cre expression[END_REF]. In humans, studies have described acute post-prandial inflammation associated to increased levels of circulating inflammatory markers in response to HFD [START_REF] Emerson | Magnitude and Timing of the Postprandial Inflammatory Response to a High-Fat Meal in Healthy Adults: A Systematic Review[END_REF][START_REF] Herieka | High-fat meal induced postprandial inflammation[END_REF].

Altogether, these data suggest that HFD-induced inflammation is a physiological response to cope with excess lipids, which could occur at the peripheral or central levels.

In our study, the calories from fat and sugar in HFD were equivalent (40% each), in contrast to numerous studies where fat content accounts for 60% kilocalories (kcal) while carbohydrate content was only 20% kcal. Therefore, the increase in caloric proportion represented by the lipids is more realistic in our model, and comparable to the typical human diet, where the occurrence of lipids in the presence of elevated carbohydrate content contributes to brain inflammation [START_REF] Andre | Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation[END_REF][START_REF] Gao | Dietary sugars, not lipids, drive hypothalamic inflammation[END_REF][START_REF] Kim | Microglial UCP2 Mediates Inflammation and Obesity Induced by High-Fat Feeding[END_REF]. According to previous studies of systemic post-prandial inflammation, HFD consumption promotes lipopolysacharide trafficking over the intestinal epithelium through trans-cellular and para-cellular transport [START_REF] Valdearcos | Microglia Dictate the Impact of Saturated Fat Consumption on Hypothalamic Inflammation and Neuronal Function[END_REF][START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF]. Analysis of endotoxin levels in the serum associated with inflammatory markers in both serum and HT, affirmed that post-prandial inflammation appears first in the HT. Therefore this study established, for the first time, the existence of a specific post-prandial hypothalamic inflammation response to both SD and HFD exposure linked to a unique modulation of mRNA levels of neuropeptides that are known to be involved in the regulation of energy balance [START_REF] Gao | Neuronal control of energy homeostasis[END_REF]. However, while both SD and HFD consumption increased hypothalamic gene expression of the orexigenic neuropeptides AgRP and ORX, as well as inflammatory markers, IL-1β and TNFα, within the first hours of diet exposure, the amplitude of the response was higher in HFD-fed mice. Moreover, compared to SD exposure in the same time frame, 1h exposure to HFD induced selective expression of MCH orexigenic neuropeptide, as well as pro-inflammatory cytokines, CCL2 and CCL5. A sustained expression of proinflammatory cytokines IL-1β, IL-6, TNFα, chemokine CCL2 and orexigenic neuropeptide MCH was still observable after 3h exposure to food but only in HFD-fed mice. Finally, after 6h of food exposure, the differences observed between SD-and HFD-treated mice tended to disappear, except for the MCH orexigenic neuropeptide mRNA level, which remained higher in the HFD group. This suggests that short-term exposure to a lipid-rich diet triggers hypothalamic gene expression of orexigenic neuropeptides leading to an adaptive behavioral response correlated to increased kilocalorie intake observed 6h post HFD exposure [START_REF] Gao | Neuronal control of energy homeostasis[END_REF]. It is not yet known how these genetic changes occur in orexigenic neurons, although we believe that they may be the consequence of lipid-induced neuroinflammation. We have yet to define the role of the inflammatory brain cells in this response over such a short time frame in this study.

Nevertheless, a recent study demonstrated that one day of lipid perfusion to the brain is sufficient to activate an inflammatory response in NPY/AgRP neurons through TNFα and ER-stress signaling [START_REF] Dalvi | High fat induces acute and chronic inflammation in the hypothalamus: effect of high-fat diet, palmitate and TNF-alpha on appetite-regulating NPY neurons[END_REF]. We could therefore expect such a mechanism to appear even earlier in response to HFD, which could modulate the neuropeptide expression involved in energy balance regulation as we observed in our study.

As previously described, HFD-induced hypothalamic inflammation is associated with glial reactivity [START_REF] Balland | Short-term high-fat diet increases the presence of astrocytes in the hypothalamus of C57BL6 mice without altering leptin sensitivity[END_REF][START_REF] Baufeld | High-fat diet-induced brain region-specific phenotypic spectrum of CNS resident microglia[END_REF][START_REF] Buckman | Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice[END_REF][START_REF] Gao | Hormones and diet, but not body weight, control hypothalamic microglial activity[END_REF][START_REF] Guillemot-Legris | High-fat diet feeding differentially affects the development of inflammation in the central nervous system[END_REF][START_REF] Kim | Microglial UCP2 Mediates Inflammation and Obesity Induced by High-Fat Feeding[END_REF][START_REF] Milanova | Diet-Induced Obesity Disturbs Microglial Immunometabolism in a Time-of-Day Manner[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Valdearcos | Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF][START_REF] Waise | One-day high-fat diet induces inflammation in the nodose ganglion and hypothalamus of mice[END_REF]. First, we focused on GFAP-positive cells. Targeting of cell activity decreases the postprandial pro-inflammatory cytokines IL-1β, IL-6, TNFα and the chemokines CCL2 and CCL5 gene response to both SD and HFD exposure.

Interestingly AAV8-DREADD-Gi-mCitrine injection exclusively in MBH is powerful enough to downregulate pro-inflammatory marker mRNA levels measured in the whole HT, suggesting that MBH GFAP-positive cells play a critical role in hypothalamic post-prandial inflammation. Moreover, we have provided novel evidence that an acute exposure to HFD specifically increases GFAP mRNA levels in HT within the first hours of food consumption. Another observation worth highlighting is that the IL-6 gene response to food exposure is modulated by GFAP-positive cells in a differential way, depending on diet. In fact, modulation of GFAP-positive cellular activity decreases IL-6 mRNA hypothalamic levels only in HFD-treated mice. This is consistent with recent discoveries demonstrating that IL-6 produced by astrocytes is a major actor in central energy balance regulation and plays a protective role against obesity development during HFD exposure [START_REF] Fernandez-Gayol | Different responses to a high-fat diet in IL-6 conditional knock-out mice driven by constitutive GFAP-Cre and Synapsin 1-Cre expression[END_REF][START_REF] Quintana | Astrocyte-specific deficiency of interleukin-6 and its receptor reveal specific roles in survival, body weight and behavior[END_REF]Timper et al., 2017). On the other hand, modulation of GFAP-positive cellular activity does not dramatically affect hypothalamic neuropeptide mRNA levels and does not change HFD intake. This is consistent with previously published results and indicates that modulation of NPY/AgRP neuron activity by GFAP-positive cells in response to HFD occurs over a different time frame from our study [START_REF] Chen | Direct modulation of GFAP-expressing glia in the arcuate nucleus bi-directionally regulates feeding[END_REF][START_REF] Yang | Astrocytes control food intake by inhibiting AGRP neuron activity via adenosine A1 receptors[END_REF].

Because glial reactivity to HFD consumption is also characterized by microglial reactivity, we then focused on microglia. In accordance with previous studies, we did not observe any effect on HFD intake when microglia was depleted [START_REF] Waise | One-day high-fat diet induces inflammation in the nodose ganglion and hypothalamus of mice[END_REF]. We found that 3h of HFD exposure increases hypothalamic mRNA levels of the microglial marker Iba1 compare to SD-fed mice. This rise was associated with soma size enlargement of microglial cells in the ARC of HT only in HFD treated mice.

For microglial cells, the association of a specific activity state with morphological aspects remains controversial [START_REF] Kettenmann | Physiology of microglia[END_REF][START_REF] Tremblay | The role of microglia in the healthy brain[END_REF].

However, what is certain is that soma size enlargement of microglial cells and the modification of Iba1 gene expression initiated by HFD consumption reflects a change in their activity, which might enable protective mechanisms to deal with lipid excess [START_REF] Kim | Microglial UCP2 Mediates Inflammation and Obesity Induced by High-Fat Feeding[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Valdearcos | Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF]. Here we provide novel evidence that microglial reactivity to HFD exposure appears within the first hours of food exposure. Moreover, our approach clearly shows that microglial cells participate in the post-prandial hypothalamic inflammation induced by both SD and HFD, though it is too early to state to what extent and what diet-specificity. In fact, microglial depletion seems to exacerbate hypothalamic inflammatory-like response to 1h HFD exposure, but that amplitude varies between individuals. Because the variability cannot be explained with a correlation with body weight and food intake (data not shown) nor by PLX efficiency (Supplementary Fig.

3

) it is tempting to speculate that the involvement of microglia in HFD-induced hypothalamic inflammation differs between subjects and may be involved in the Up to now, astrocytic or microglial reactive gliosis in the ARC of the HT in response to chronic HFD exposure and their participation in HFD-induced inflammatory processes and functional impact is still controversial and may depend on the nutritional lipid composition [START_REF] Harrison | Profound weight loss induces reactive astrogliosis in the arcuate nucleus of obese mice[END_REF][START_REF] Kim | Microglial UCP2 Mediates Inflammation and Obesity Induced by High-Fat Feeding[END_REF][START_REF] Mclean | A high-fat diet induces rapid changes in the mouse hypothalamic proteome[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF]. Our study offers new insight on the impact of HFD consumption on astrocytic or microglial populations located in areas involved in energy balance regulation. Both cell types may be activated by short-term food exposure and orchestrate inflammatory-like processes in the ARC modulating neuronal function, but in differential time frames and with a diet specificity. Indeed, during short-term HFD exposure, GFAP gene expression is acute and transient, whereas Iba1 gene expression and microglial reactivity occurs after 3h of diet exposure and lasts over 6h. Moreover, the time frame of microglial reactivity to shortterm HFD exposure coincides with the appearance of endotoxin and saturated fatty acid-enriched triglyceride serum content. This supports the hypothesis that saturated fatty acid-induced hypothalamic inflammation may require microglial activation [START_REF] Kim | Microglial UCP2 Mediates Inflammation and Obesity Induced by High-Fat Feeding[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF]. Therefore, our experiments suggest that the increase of lipid content in diet is primarily sensed by GFAP-positive cells, which initiates the hypothalamic inflammatory response followed by hypothalamic microglial cell activation by central and systemic signals that impact energy balance regulation through the modulation of hypothalamic neuropeptide expression.

Despite the originality of our results, we are aware of study limitations. While we focused on the HT because it is a well-known area involved in energy balance regulation [START_REF] Gao | Neuronal control of energy homeostasis[END_REF], we do not exclude the possibility that other brain areas may display post-prandial inflammation associated with neuropeptide modulation. Indeed, several studies have shown that chronic HFD consumption leads to hippocampal inflammatory responses and affects behavioral disorders associated with obesity [START_REF] Waise | One-day high-fat diet induces inflammation in the nodose ganglion and hypothalamus of mice[END_REF][START_REF] Guillemot-Legris | High-fat diet feeding differentially affects the development of inflammation in the central nervous system[END_REF]Gzielo, Kielbinski et al. 2017;Tsai, Wu et al. 2018;Spencer, Basri et al. 2019).

Furthermore, our methodology prevented a detailed analysis of subtle changes that might occur in the several functionally distinct nuclei of the HT. Moreover, to gain insight on the involvement of each cell type, we used two different tools to manipulate GFAP-positive and microglial cell activity which must be called into question.

DREADD technology has already been used to target GFAP-positive cells of the ARC glia in the regulation of neuronal subtype-specific modulation of energy homeostasis [START_REF] Yang | Astrocytes control food intake by inhibiting AGRP neuron activity via adenosine A1 receptors[END_REF][START_REF] Chen | Direct modulation of GFAP-expressing glia in the arcuate nucleus bi-directionally regulates feeding[END_REF]. Given this context, we initiated our study using DREADD Gi as this strategy was proven to achieve cellular inhibition -at least in neuronal cell types (Roth 2016). However, a 2019 study has demonstrated that Gi/o protein-coupled receptor may differentially affect brain cell type, inhibiting neurons and activating astrocytes stimulating gliotransmitter release (Durkee, Covelo et al. 2019). Thus, at this stage while it seems difficult to definitely conclude whether GFAP-positive glial cell activation or inhibition mediates HFD-induced feeding behavior changes, our results clearly support GFAP-positive glial cell involvement in this process. Concerning microglial cells, although new tools using DREADD technology to target microglia have been recently reported (Grace, Wang et al. 2018), we chose PLX5622 as it was the only tool available at the time we initiated the study to modulate microglial cells in the brain. PLX5622 has been shown

to act mainly on microglia in adult whole brains [START_REF] Elmore | Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain[END_REF]Oosterhof, Kuil et al. 2018). Thus, in our study we analyzed total brain microglial depletion effects, and recognize that further analysis needs to be done to specify microglial involvement in hypothalamic post-prandial inflammation. Besides, using those two tools we observed differential inflammatory and neuropeptide gene responses in respective control groups for AAV8-DREADD-Gi-mCitrine or AIN76A diet experiments compared to our original observation without cellular targeting. This could be explained by different inflammatory states of animals: sham and non-treated animals vs injected with virus vs treated with an AIN76A diet containing PLX5622.

While it is obvious that stereotaxic injection of virus has immunological implications that could affect brain inflammatory status, it is worth noting that 76.9% of carbohydrates contained in AIN76A are sucrose, known to play a critical role in dietinduced hypothalamic inflammation [START_REF] Andre | Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation[END_REF][START_REF] Gao | Dietary sugars, not lipids, drive hypothalamic inflammation[END_REF].

Overall, while the effect of medium-term and long-term nutritional lipid exposure has been extensively demonstrated to promote hypothalamic inflammation involving astrocytic and microglial cells [START_REF] Andre | Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation[END_REF][START_REF] Balland | Short-term high-fat diet increases the presence of astrocytes in the hypothalamus of C57BL6 mice without altering leptin sensitivity[END_REF][START_REF] Baufeld | High-fat diet-induced brain region-specific phenotypic spectrum of CNS resident microglia[END_REF][START_REF] Gao | Dietary sugars, not lipids, drive hypothalamic inflammation[END_REF][START_REF] Gao | Hormones and diet, but not body weight, control hypothalamic microglial activity[END_REF][START_REF] Guillemot-Legris | High-fat diet feeding differentially affects the development of inflammation in the central nervous system[END_REF][START_REF] Nadjar | Bioactive lipids as new class of microglial modulators: When nutrition meets neuroimunology[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Valdearcos | Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility[END_REF][START_REF] Valdearcos | Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function[END_REF], little was known about short-term exposure. Here we demonstrate, for the first time, that a moderate increase of nutritional lipid content in diet -representative of current human diets -can exacerbate hypothalamic post-prandial inflammation in a short time and that this response involves hypothalamic GFAP-positive cells and microglial cells. Although this primary response is probably aimed at regulating energy balance, continual nutritional lipid excess may contribute to dysregulating those signals and contribute to the hypothalamic disturbances leading to obesity [START_REF] Thaler | Minireview: Inflammation and obesity pathogenesis: the hypothalamus heats up[END_REF]. Because the amplitude of systemic post-prandial inflammation has been correlated with type 2 diabetes and cardiovascular diseases [START_REF] Emerson | Magnitude and Timing of the Postprandial Inflammatory Response to a High-Fat Meal in Healthy Adults: A Systematic Review[END_REF][START_REF] Milan | Comparisons of the Postprandial Inflammatory and Endotoxaemic Responses to Mixed Meals in Young and Older Individuals: A Randomised Trial[END_REF], our study leads us to believe that exacerbated hypothalamic postprandial inflammation might predispose individuals to obesity and needs to be characterized in order to address this worldwide crisis.

Supplementary data. Supplementary Materials and Methods

Sample Preparation

Reagents:

Acetonitrile (ACN) and isopropropanol (IPA) were purchased from Carlo Erba, France. Chloroform (ChCl3) was purchased from Merck, France. These solvents were LC-MS grade. Methanol (MeOH) was from VWR, France. Formic acid Optima LC-MS quality was purchased from ThermoFisher, France. Ultrapure water was from purelab flex (Veolia Water, France), and formate ammonium (99%) was from Acros Organics, France.

Lipids extraction:

The extraction was performed using 1.5 mL solvent-resistent plastic Eppendorf tubes and 5 mL glass hemolyse tubes to avoid contamination. Methanol, chloroform and water should each be cooled down on wet ice before the lipid extraction.

Lipids were extracted according to a modified Bligh and Dyer protocol.

80 µL of serum was collected in a 1.5 mL Eppendorf tube and 200 µL of methanol was added. After vortexing (30s), the sample was frozen for 20 min at -20°C. The sample was transferred in a glass tube and 100 µL of chloroform was added. The mixture was vortexed for 30s and centrifuged (2500g, 4°C, 10 min). After centrifugation, the supernatant was collected in a new glass tube and 100 µL of chloroform and 100 µL of water were added. The sample was vortexed for 30s and centrifuged (2500g, 4°C, 10 min). 100 µL of the non polar phase was removed, added in a new glass tube and dried under a stream of nitrogen. The dried extract was resuspended in 50 µL of methanol/chloroform 1:1 (v/v) and transferred in an injection vial before liquid chromatography and mass spectrometry analysis.

Instruments and methods

Reverse phase liquid chromatography was selected for separation with an UPLC Supplementary Table 1. Primers used for real time PCR.

Forward and reverse primers were designed using Primer Express software. Genes were analyzed with a SYBR® Green I system.

* ¤ ¤ ¤ A) B) C) D) E) F) G) H) I) J) K)
Supplementary figure 4

FIGURE 1 :

 1 FIGURE 1: ENERGY BALANCE REGULATION BY THE CNS. ................................................................. FIGURE 2: HYPOTHALAMIC CONTROL OF FOOD INTAKE BEHAVIOR. .................................................. FIGURE 3: BODY MASS INDEX. ..................................................................................................... FIGURE 4: HFD-INDUCED ALTERATIONS IN WAT ASSOCIATED WITH OBESITY AND INSULIN RESISTANCE. . FIGURE 5: INFLAMMATORY EFFECTS OF HFD ON DIFFERENT TISSUES IN DIO. ....................................... FIGURE 6: GLUCOSE METABOLISM: GLUCOGENOLYSIS AND GLUCONEOGENESIS. .............................. FIGURE 7: ANATOMICAL LOCATION OF THE HUMAN PANCREAS. ...................................................... FIGURE 8: INSULIN ACTION IN THE WHOLE BODY. ............................................................................ FIGURE 9: INSULIN ACTION IN PERIPHERAL METABOLIC ORGANS. ...................................................... FIGURE 10: INSULIN AND LEPTIN SIGNALING PATHWAY IN THE BRAIN. ................................................ FIGURE 11 : DIAGNOSTIC CRITERIA OF DM. ................................................................................. FIGURE 12: DIFFERENT STAGES OF T1DM. ..................................................................................... FIGURE 13: PATHOGENESIS OF T1DM. ......................................................................................... FIGURE 14: DIFFERENT STAGES OF T2DM DEVELOPMENT. ................................................................ FIGURE 15: MECHANISM BEHIND INSULIN RESISTANCE IN T2DM. ..................................................... FIGURE 16: MECHANISM OF PERIPHERAL INSULIN RESISTANCE IN METABOLIC ORGANS. ...................... FIGURE 17 : HYPERGLYCEMIA-INDUCED GLUCOTOXICITY PATHWAYS. .............................................. FIGURE 18: DIFFERENT TYPES AND PATTERNS OF DN. ....................................................................... FIGURE 19: MECHANISM BEHIND THE PATHOPHYSIOLOGY OF DN. ................................................... FIGURE 20: CHEMOKINE STRUCTURE AND CHARACTERISTIC FEATURES EXEMPLIFIED BY MONOMERIC HUMAN CXCL12. ............................................................................................................. FIGURE 21 : THE SIGNALING CASCADE OF CCL5 COUPLING TO GPR75. ....................................... FIGURE 22 : STRUCTURE OF CCR5 7TM CHEMOKINE RECEPTOR. ................................................... FIGURE 23: SIGNAL TRANSDUCTION PATHWAYS OF CCR5. .......................................................... FIGURE 24: SCHEMATIC REPRESENTATION OF OUR EXPERIMENTAL APPROACH FOR THE FIRST EXPERIMENT. ...................................................................................................................................... FIGURE 25: SCHEMATIC REPRESENTATION OF THE EXPERIMENTAL APPROACH EMPLOYED IN THIS STUDY. ...................................................................................................................................... FIGURE 26: SCHEMATIC REPRESENTATION OF UNILATERAL CANNULA PLACEMENT IN THE LATERAL VENTRICLE. ...................................................................................................................... FIGURE 27: PROTOCOL OF CHRONIC INJECTIONS. ....................................................................... FIGURE 28: THE EFFECTS OF LIPID NATURE ON BODY WEIGHT GAIN AND LEPTINEMIA. ......................... FIGURE 29: THE EFFECTS OF LIPID NATURE ON HYPOTHALAMIC NEUROPEPTIDE EXPRESSION. ............... FIGURE 30: THE EFFECTS OF LIPID NATURE ON GLUCOSE HOMEOSTASIS. .......................................... FIGURE 31: THE EFFECT OF LIPID NATURE AND COMPOSITION ON HYPOTHALAMIC INFLAMMATION AND GLIOSIS. .......................................................................................................................... FIGURE 32: THE EFFECT OF LIPID NATURE AND COMPOSITION ON THERMAL PAIN SENSITIVITY AT 12W OF DIET. ............................................................................................................................... FIGURE 33: EFFECT OF CCL5 DEFICIENCY ON DIO DEVELOPMENT. ................................................ FIGURE 34: EFFECT OF CCL5 DEFICIENCY ON HYPOTHALAMIC NEUROPEPTIDE EXPRESSION COMPARED BETWEEN DIETS. ................................................................................................................ FIGURE 35: EFFECT OF CCL5 DEFICIENCY ON HYPOTHALAMIC NEUROPEPTIDE EXPRESSION COMPARED BETWEEN GENOTYPES. .......................................................................................................

Figure 1 :

 1 Figure 1: Energy balance regulation by the CNS.(Adapted from[START_REF] Morton | Neurobiology of food intake in health and disease[END_REF]).

Figure 2 :

 2 Figure 2: Hypothalamic control of food intake behavior. (Adapted from (Le Thuc et al., 2017)).

  developed countries. It is affecting around 13% off the population worldwide, corresponding to over 650 million obese individuals. In addition to that, of the whole world population 1.9 billion adults (39%) were overweight in 2016. With 41 million children under 5 years old being overweight or obese, childhood obesity constitutes one of the major health challenges of the 21st century (WHO, 2018). With the recent change in lifestyle to a more sedentary life and unhealthy food choices, as well as the growing population of older people, the numbers are bound to keep increasing dramatically. With around 2.8 million deaths it is at the fifth position for being the major and leading risk for deaths globally ('WHO | Global health risks', 2015).

Figure 3 :

 3 Figure 3: Body mass index. Calculation of BMI to classify the state of obesity. (Adapted from Mexico Bariatric Center, BMI chart).

Figure 4 :

 4 Figure 4: HFD-induced alterations in WAT associated with obesity and insulin resistance. (Adapted from (González-Muniesa et al., 2017)).

  phase proteins like plasminogen activator inhibitor 1 (PAI-1), lipocalin and serum amyloid A (SAA) among others. Adipokines include also cytokines like interleukin-1 (IL-1β) and its receptor antagonist IL-1Ra, IL-6 IL-10, IL-7 and tumor necrosis factor-a (TNF-a); chemokines like interleukin-8 (IL-8), CCL2, 3, 4, and

  are glycoproteins, which are produced and secreted on demand by many different cell types in response to different triggers like infection, injury but also other signals like cellular stress and metabolic stress. They play important roles in different functions but are most famous for their role in the immune response and as part of the innate immune response can activate other immune cells via specific cytokine receptors on the membrane of target cells. Similarly, they are implicated in the differentiation of lymphocytes and hematopoiesis among other functions. Cytokines include, TNF-a, interleukins (IL), chemokines, and interferons (IFN). Cytokines can be further classified into functional groups depending on their effect, such as proinflammatory (TNF-a, IL-1β, IL-6) and anti-inflammatory cytokines (IL-4, IL-10 and IL-13).

Figure 5 :

 5 Figure 5: Inflammatory effects of HFD on different tissues in DIO.HFD-induced development of obesity and metabolic syndrome involves many different tissues and players. Chronic HFD feeding in DIO leads to excess lipid accumulation in AT and eventually ectopic lipid accumulation in other tissues, once the capacity of AT is reached and increases serum levels of FFAs. Excess of lipid accumulation impairs the proper neutralization and storage of lipid metabolites and results in lipotoxicity. This in turn leads to infiltration of immune cells into AT as well as other tissues, a change in phenotype from a pro-inflammatory to anti-inflammatory state and induces the increased production of inflammatory mediators. HFD induces an increase in the permeability of intestines and changes in microbiota leading to gut dysbiosis, and the increased release of lipopolysaccharide (LPS) by gut bacteria resulting in endotoxemia and further potentiation of the inflammatory response. Furthermore, the BBB increases its permeability, increasing the flux of nutrients into the brain as well as the infiltration of immune cells. The increased accumulation of lipids in the hypothalamus induces neuroinflammation, the increase in immune mediators as well as the activation of glia cells. Hypothalamic inflammation can ultimately result in changes in neuronal activity, as well as neuronal damage or apoptosis. (Adapted from (Guillemot-Legris and Muccioli, 2017)).

Figure 6 :

 6 Figure 6: Glucose metabolism: Glucogenolysis and gluconeogenesis.(Adapted from[START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF]).

Figure 7 :

 7 Figure 7: Anatomical location of the human pancreas.(Adapted from[START_REF] Shi | Anatomy, Histology, and Function of the Pancreas[END_REF]).

Figure 8 :

 8 Figure 8: Insulin action in the whole body. Ach, Aceytlcholine; SST, somatostatin; (Adapted from (Tokarz, MacDonald and Klip, 2018)).

Figure 9 :

 9 Figure 9: Insulin action in peripheral metabolic organs.(Adapted from[START_REF] Samuel | The pathogenesis of insulin resistance: Integrating signaling pathways and substrate flux[END_REF]).

Figure 10 :

 10 Figure 10: Insulin and leptin signaling pathway in the brain.(Adapted from[START_REF] Morton | Central nervous system control of food intake and body weight[END_REF]).

  characteristic: hyperglycemia. Interestingly, in 2000 it was reported that about 150 million people have diabetes and it was estimated that by 2025, 300 million people would be affected. However, only 17 years later the numbers exceeded any estimate and have been more than doubled, as in 2017 the International Diabetes Federation (IDF) reported already about 425 million adults to be diabetic worldwide, which underestimates the actual number due to a large number of asymptomatic and yet undiagnosed patients (World Health Organization. (2019);[START_REF] Zimmet | Global and societal implications of the diabetes epidemic[END_REF] International Diabetes Federation, 2017). While the population increased by ~16% in the 17 years, the cases of diabetes augmented by staggering ~183% in this time frame and thus are most likely attributed less to a rise in population size but more to changes in lifestyle and environmental factors and maybe advances in diagnostic methods. In addition to that, 1.1 million children between the age of 14 and 19 are considered to have T1DM (International Diabetes Federation, 2017). Apart from the two major forms of DM, type 1 diabetes mellitus (T1DM) and T2DM there are various other forms such as gestational diabetes mellitus (GDM) and others. While all forms of diabetes share similar characteristics like dysfunction and destruction of β-cells of pancreatic islets, which results in either a dysfunction of insulin secretion (T1DM) or insulin sensitivity (T2DM and other forms) or both and ultimately leading to hyperglycemia, the mechanism leading up to it and the age of onset differ between types of diabetes. Due to the different pathogenesis mechanisms of different forms of obesity, (they respond each to different treatments)a different treatment has to be adapted for each form individually (International Diabetes Federation, 2017).

  World Health Organization (WHO) has established different diagnostic criteria to diagnose patients with diabetes or a pre-condition (impaired fasting glucose (IFG) or glucose tolerance (IGT)) that can lead to diabetes if not properly treated through medication or altering unhealthy lifestyle factors (Fig. 11) (World Health Organization, 2019; International Diabetes Federation, 2017).

Figure 11 :

 11 Figure 11 : Diagnostic Criteria of DM. The WHO has established diagnostic criteria for diabetes mellitus. Patients have to be presents with at least one of the criteria described in the first column to be diagnosed with DM. IGT, impaired glucose tolerance; IFG, impaired fasting glucose. (Adapted from IDF Diabetes Atlas, Eight Edition, 2017.)

Figure 12 :

 12 Figure 12: Different stages of T1DM.(Adapted from[START_REF] Katsarou | Type 1 diabetes mellitus[END_REF]).

Figure 13 :

 13 Figure 13: Pathogenesis of T1DM.T1DM is an autoimmune-mediated disease that is initiated upon antigenpresentation of DC and activated B cells that interact with and activate CD4+ and CD8+ T cells. Furthermore, contact with β-cell autoantigens drives the production of autoantibodies against islet peptides. Activated T cells can infiltrate the islets and lead to loss of β-cells due to damage and destruction. (Adapted from[START_REF] Katsarou | Type 1 diabetes mellitus[END_REF]).

Figure 14 :

 14 Figure 14: Different stages of T2DM development.(Adapted from[START_REF] Xie | Precision medicine in diabetes prevention, classification and management[END_REF]).

Figure 15 :

 15 Figure 15: Mechanism behind insulin resistance in T2DM. (Adpated from (DeFronzo et al., 2015)).

Figure 16

 16 Figure 16: Mechanism of peripheral insulin resistance in metabolic organs. Inflammation and insulin resistance in WAT leads to infiltration by ATM through chemotactic attraction by chemokines leads to inflammation through cytokine production. Cytokines and impaired insulin action then increase lipolysis and release of FAs. Ectopic lipid accumulation and delivery of FAs in skeletal muscle leads to impaired glucose transport into muscle cells through increases in DAG. This in turn activates PKCθ and leads to insulin signaling impairment and ultimately decreased glucose uptake. Glucose is diverted to the liver and together with the increased flux of FAs from WAT increases both hepatic lipid synthesis through TG production.In addition, it raises levels of acetyl-CoA, which drive gluconeogenesis through activation of PC activity and glycerol and ultimately increase hepatic glucose production. This induces hyperglycemia. Hepatic increase in DAG activates PKCε, which inhibits insulin signaling via IRS serine phosphorylation and decrease in glycogen synthesis. (Adapted from[START_REF] Samuel | The pathogenesis of insulin resistance: Integrating signaling pathways and substrate flux[END_REF]).

Figure 17 :

 17 Figure 17 : Hyperglycemia-induced glucotoxicity pathways.(Adapted from[START_REF] Luo | Hyperglycemic Stress and Carbon Stress in Diabetic Glucotoxicity[END_REF]).

Figure 18 :

 18 Figure 18: Different types and patterns of DN. Diabetic patients can develop different patterns of neuropathy subdivided into (a) distal symmetric polyneuropathy, small-fibre-predominant neuropathy or treatmentinduced neuropathy; (b) radiculoplexopathy or radiculopathy; (c) mononeuropathy; (d) autonomic neuropathy or treatment-induced neuropathy. The most common type is DSP, which has a similar distribution pattern as small-fibre-predominant neuropathy. (Adapted from[START_REF] Feldman | Diabetic neuropathy[END_REF]).

Figure 19 :

 19 Figure 19: Mechanism behind the Pathophysiology of DN. The upper panel indicates the cell types affected by the mechanisms leading to cell damage and ultimately to DN. These are vascular endothelial cells, neurons as well as glial cells such as Schwann cells. The lower panel shows the different mechanisms suggested to contribute to the development of DN. In yellow are the mechanisms involved in DN in T1DM, in blue are presented the mechanisms involved in T2DM associated DN and in green mechanisms that can apply to both conditions.Abbreviations: AGE, advanced glycation end products; LDL, low-density lipoprotein; HDL, high-density lipoprotein; FFA, free fatty acids; ROS, reactive oxygen species (red star); ER, endoplasmic reticulum; PI3K, phosphatidylinositol 3-kinase. (Adapted from[START_REF] Callaghan | Diabetic neuropathy: clinical manifestations and current treatments[END_REF]).

Figure 20 :

 20 Figure 20: Chemokine structure and characteristic features exemplified by monomeric human CXCL12. X-ray structure. (Adapted from (Legler and Thelen, 2016)).

Figure 21 :

 21 Figure 21 : The signaling cascade of CCL5 coupling to GPR75.(Adapted from[START_REF] Pease | Tails of the unexpected -An atypical receptor for the chemokine RANTES/CCL5 expressed in brain[END_REF]).

Figure 22 :

 22 Figure 22 : Structure of CCR5 7TM chemokine receptor. The structure of CCR5 includes different sites of regulation and HIV binding sites. ECL: Extracellular loop. (Adapted from Lapalco, C. 2011).

  the G βγ subunit leads to the release of the dimeric subunit and the activation of PLCβ, which in turn activates PKCε. Chemokine receptors can act also via more distant signaling pathways such as the MAPK pathway and other pathways including janus kinases (JAK), STATs and NF-κB pathways resulting in a plethora of effects (Réaux-Le[START_REF] Goazigo | Current status of chemokines in the adult CNS[END_REF].

Figure 23 :

 23 Figure 23: Signal transduction Pathways of CCR5.(Adapted from[START_REF] Wu | Chemokine Coreceptor Signaling in HIV-1 Infection and Pathogenesis[END_REF]).

  of receptors can regulate their activity and expression level. Firstly, after ligand-mediated signal transduction, CCR5 can be internalized via interaction with β-arrestins, which act as adaptor proteins to initiate endocytosis by linking the phosphorylated receptor to clathrins of clathrin-coated pits and the AP-2 adaptor complex. Binding to the phosphorylated receptor β-arrestins uncouples the receptor from G proteins to prevent their binding and activation. Other clathrin-or β-arrestinindependent endocytosis pathways have been reported involving cholesterol-enriched raft microdomains which engage with palmitoylated Cterminal cysteine domains of the receptor. After internalization, CCR5

5. 2 . 6 .

 26 Role of CCL5 Signaling in NASH/NAFLSD CCL5 has been shown to participate in diseases that display chronic inflammation, some of which are discussed here. One other example of the important role of CCL5 in another chronic inflammatory disease associated with obesity is NASH/NAFLSD and liver fibrosis.

5. 2 . 7 .

 27 Role of CCL5/CCR5 Signaling in HIV CCR5 acts as primary HIV-1 co-receptor to facilitate entry of HIV into cells expressing CCR5. For example, M-tropic HIV (macrophage trophic HIV preferentially uses CCR5 as co-receptor for cell entry, whereas T cell (T-) tropic

2 .

 2 Investigate the role of CCL5/CCR5 in the central regulation of energy balance: a. Does CCL5 signaling have an effect on neuropeptides involved in the regulation of energy balance? b. Do ICV CCL5 and Met CCL5 (CCL5 antagonist) injections have an effect on food intake, blood glucose and insulin regulation? c. Are CCL5 and its receptor CCR5 expressed in the hypothalamus, in particular in the ARC? 3. Identify the role of CCL5/CCR5 in the development and maintenance of DIO and associated comorbidities including T2DM and neuropathic pain using the mouse model of DIO identified in part one of the study. a. Characterize the inflammatory and metabolic phenotype of CCL5 -/-mice on a standard diet (SD) and high-fat diet (HFD) b. Characterize the inflammatory and metabolic phenotype of CCR5 -/-mice on a standard diet (SD) and high-fat diet (HFD)

Figure 24 :

 24 Figure 24: Schematic representation of our experimental approach for the first experiment.

Figure 25 :

 25 Figure 25: Schematic representation of the experimental approach employed in this study.In this study, we used a adult transgenic mice harboring a deletion of the chemokine CCL5 or its receptor CCR5 in the context of DIO and surveilled several parameters during the course of 16 weeks of diet.

  Mice were starved overnight (16h) or 6h respectively and distributed into single cage housing for the duration of the test. Mice were left 30 min for habituation at red light conditions, before basal glycaemia values were established by measuring the blood of tail vein using a glucose meter (Bayer Contour® XT and test strips (Contour® Next test strips). Glucose (1.5 g/kg, Sigma-Aldrich) or human insulin (0.5 U/kg, Humalog, Lilly) diluted in NaCl 0.9% was injected intraperitoneal (ip) as per recommended procedures and blood glucose was measured again 15, 30, 45, 60, 90 and 120 min post-injection.

6 (

 6 5 mM desoxyribonucleotide triphosphate mix, 5 mM dithiothreitol, 40U RNAsin (Promega, France). The reaction was incubated 5 min at 25 °C, then 50 min at 50 °C then inactivated 15 min at 70 °C. Real-time quantitative PCR was performed using the SYBR® Green method (Roche France) with the LightCycler 480 sequence detector system (Roche Diagnostics France) for amplification of mouse IL-1β (Interleukin 1 beta), IL-Interleukin 6), TNF-α (Tumor necrosis factor alpha), CCL2, CCL5 (Chemokine (C-C motif) ligand 2-5), Iba1 (Ionized calcium Binding Adaptor molecule 1), GFAP (Glial Fibrillary Acidic Protein), MCH (Melanin-Concentrating Hormone), ORX (OReXine), POMC (Pro-OpioMelanoCortin), CART (Cocaine-and Amphetamine-Regulated Transcript), NPY (Neuropeptide Y), AgRP (Agouti-Related Peptide), IRS1 (Insulin Receptor Substrate 1), PPARγ (Peroxisome Proliferator-Activated Receptor gamma) and GAPDH (GlycerAldehyde Phosphate DesHydrogenase) mRNA. GAPDH was used as housekeeping gene

  cannula mice were anesthetized by ip injection of a ketamine-xylazine mix (80mg/kg -12 mg/kg) in addition to topical lidocaine application. Mice were implanted unilaterally with a 26 stainless steel gauge guide cannula (Plastics One Inc, Roanoke, Virginia, USA) using a Kopf stereotaxic instrument (David Kopf Instruments, Tujunga, USA) to allow icv injection. Unilateral implantation was made into the right lateral ventricle (stereotaxic coordinates relative to Bregma: X: +1 mm; Y: -0.34 mm; Z: -2.5 mm below the surface of the skull (Fig. 26) according to "The Mouse Brain in Stereotaxic Coordinates", Second Edition, by Paxinos & Franklin. The injection site was drilled open and the cannula guide was inserted according to the stereotactic coordinates into the brain. Dental cement mix (Dentalon® plus powder and liquid, PHYMEP, France) was used to maintain cannula guide anchored to a stainless steel screw (1.6mm length screw for mice, PHYMEP, France), which was fixed contralaterally to the skull, left to dry, before being sutured using coated Vicryl™ surgical suture (6-0, Polyglactin 910, ref: V302H, Ethicon) and applying betadine on the closed scalp. The cannula guide was closed with a small cap (4mm PED for 26G guide, no projection, PHYMEP, France). At the end of surgical procedures, mice received 1 mg/kg ip injection of atipamezole, 5 mg/kg subcutaneously injected ketoprofen and were left to recover for 2 weeks housed in pairs, while being handled, accustomed to the placement of the 26-gauge internal cannula and their BW monitored daily.

For

  the first experiment of repeated injections of CCL5 with different concentrations, a single aCSF infusion was performed to habituate mice before starting the actual experiments. For the second experiment of repeated injections of aCSF, CCL5 and antagonist Met CCL5, mice were infused every second day using a Hamilton syringe (1701 SN 10µL, 22G/30mm/PS3; PHYMEP, France), which was placed into an adaptor.

Figure 26 :

 26 Figure 26: Schematic representation of unilateral cannula placement in the lateral ventricle. (Adapted from "The Mouse Brain in Stereotaxic Coordinates", Second Edition, by Paxinos & Franklin.)

Figure 27 :

 27 Figure 27: Protocol of chronic injections. Abbreviations: w: weeks after the start of different injections (red arrow). d: days; aCSF: artificial cerebrospinal fluid; GTT: glucose tolerance test; ITT: insulin tolerance test.

1 . 1 .

 11 DIO to test the effect of different long-term HFDs based on different lipid nature on the development of obesity and the associated comorbidities such as diabetes and neuropathic pain. The following part will depict the results obtained of feeding young 4 weeks old C56BL6/J mice (WT) with different HFD based on either soja/corn (HFD-SC), sunflower (HFD-SN), rapeseed oil (HFD-C) or butter (HFD-B) and compared to SD-fed mice and characterizing their phenotype during the development of obesity for 12 weeks. Additionally, the different lipid composition of the HFDs resulted in a different ω6/ω3 ratio. We wanted to know whether the ratio of ω6/ω3 in the HFDs had a different impact on obesity development and inflammatory state of mice. The Effect of Lipid Nature on the Development of Obesity and Food Intake Feeding age-and weight-matched mice with different HFDs with different ω6/ω3 ratio for 12 weeks of time lead to a different development of obesity between the different HFDs. The diet with the highest percentage of lipids (35%) based on butter (HFD-B) as a lipid source, is as expected, the most obesogenic diet among the HFDs and leads to a rapid weight gain and development of obesity, starting to be significantly different from SD fed mice at 3 weeks of diet (Fig. 28A). Compared to that, all other HFDs with 20% lipids independent of lipid source showed the same rate of weight gain, which was markedly reduced compared to HFD-B fed mice. They developed obesity only slow and were significantly different form SD-fed mice only starting from 8 weeks of diet for HFD-SC (ω6/ω3=7.3), 9 weeks for HFD-C (ω6/ω3 =2.3) and 10 weeks for HFD-SN (ω6/ω3=17.3)(Fig 28A).

Figure 28 :

 28 Figure 28: The effects of lipid nature on body weight gain and leptinemia. A: Evolution of average BW during 12 weeks of experimental protocol of DIO containing different lipids. B: Quantification of (A) as area under the curve -AUC. C: Serum leptin concentration measured by ELISA after 12 weeks of different HFD. Data are reported as the means ±SEM (n=10/group for A and B; n=5-6/group for C). Twoway ANOVA or one-way ANOVA followed by Dunnett's post hoc test or Mann Whitney test. * P<0.05 *, P<0.01 **, P<0.001 ***, P<0.0001 ****, all vs SD; ns: not significant.

Figure 29 :

 29 Figure 29: The effects of lipid nature on hypothalamic neuropeptide expression. Real-time PCR quantification of relative mRNA expression of hypothalamic neuropeptides such as anorexigenic neuropeptides POMC (A) and CART (D), and orexigenic neuropeptides NPY (B), AgRP (E), MCH (C) and ORX (F) in WT mice after 12 weeks of SD or one of four HFDs containing different lipids. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to SD-fed control group. Values are the mean ± SEM (n=7 mice/ group). Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc test; P<0.05 *, P<0.01 **, P<0.001 ***, all vs SD; ns: not significant.

Figure 30 :

 30 Figure 30: The effects of lipid nature on glucose homeostasis. Effect of different lipid nature on A: ipGTT after 12 weeks of diet and B: quantification of (A) represented as area under the curve (AUC). C: Effect of different lipid nature on insulin sensitivity after 12 weeks of diet. D: Basal blood glucose levels after 16h of fasting after 12 weeks of diet. Dashed line indicates the threshold for diabetic levels of blood glucose at 150 mg/dL. Values are means ± SEM (n=10/group). Statistical significance was determined with two-way ANOVA or one-way ANOVA with Dunnett's post hoc test or Kruskal-Wallis test with Dunn's post hoc test. * P<0.05 *, P<0.01 **, P<0.001 ***, P<0.0001 ****, all vs SD; ns: not significant.

Figure 31 :

 31 Figure 31: The effect of lipid nature and composition on hypothalamic inflammation and gliosis. Real-time PCR quantification of hypothalamic mRNA expression of inflammatory cytokines (A) TNF-a, IL-6 (B), and IL-1β (C), chemokines, CCL5 (D) and CCL2 (E); and glia cell markers (F) for Iba1 positive microglia and (G) for GFAP positive astrocytes after 12 weeks of diet in WT on either SD or one of four HFDs based on different lipids. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to SD-fed control group. Values are represented as means ± SEM (n=6-7 mice/group). Statistical significance was determined with Kruskal-Wallis test with Dunn's post hoc test. P<0.05 *, P<0.01 **, P<0.001, ***, P<0.0001 ****. All vs SD; ns: not significant.

Figure 32 :

 32 Figure 32: The effect of lipid nature and composition on thermal pain sensitivity at 12w of diet. Estimation of of thermal pain sensitivity by paw withdrawal latency in seconds as determined with Hargreaves' test in WT mice fed a SD or one of four HFDs based on different lipid nature. Values are represented as means ± SEM (n=9-15mice/group). Statistical significance was determined with Kruskal-Wallis test with Dunn's post hoc test. (n=9-15 mice); P<0.05 *, P<0.01 **, P<0.001, ***, P<0.0001 ****, all vs SD; ns: not significant.

Figure 33 :

 33 Figure 33: Effect of CCL5 deficiency on DIO development. A: Evolution of average BW at 16 weeks of experimental protocol of DIO. B: Quantification of (A) as area under the curve -AUC. C: Average food consumption during 16 weeks represented as grams per mouse per day. D: Average food consumption during 16 weeks represented as kcal per mouse per day. E: Average H2O consumption during 16 weeks in grams per mouse per day. F: Serum leptin concentration measured by ELISA after 16 weeks of HFD. Data are reported as the means ±SEM (n=14-25/group for A and B)(n=2-3 experiments/group for C-E)(n=8-9/group for F). Two-way ANOVA followed by Tukey post hoc test, one-way ANOVA or Mann Whitney Test for comparisons between two groups. * P<0.05 *, P<0.01 **, P<0.001 ***, P<0.0001 ****, WT HFD vs CCL5 -/-HFD; #, WT SD vs WT HFD; $, CCL5 -/-SD vs HFD; @, WT SD vs CCL5 -/-SD; ns: not significant.

Figure 34 :

 34 Figure 34: Effect of CCL5 deficiency on hypothalamic neuropeptide expression compared between diets. Real-time PCR quantification of relative mRNA expression of hypothalamic neuropeptides such as anorexigenic neuropeptides POMC (A) and CART (B), and orexigenic neuropeptides NPY (C), AgRP (D), MCH (E) and ORX (F) after 16 weeks of diet in WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to SD-fed control groups. Values are the mean ± SEM (n=6-9 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 35 :

 35 Figure 35: Effect of CCL5 deficiency on hypothalamic neuropeptide expression compared between genotypes. Real-time PCR quantification of mRNA expression of hypothalamic neuropeptides such as anorexigenic neuropeptides POMC (A) and CART (B), and orexigenic neuropeptides NPY (C), AgRP (D), MCH (E) and ORX (F) after 16 weeks of diet in WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to WT control groups. Values are represented as means ± SEM (n=6-9 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

  and J). Interestingly, CCL5 -/-mice on a HFD were more sensitive to insulin than WT controls, despite showing less glucose-stimulated insulin secretion compared to WT controls on a HFD, which display hyperinsulinemia upon glucose stimulation but no significant difference at baseline (Fig 36J-L).

Figure 36 :

 36 Figure 36: Glucose homeostasis perturbations in WT and CCL5 -/-mice after 8 and 16 weeks of SD or HFD. Effect of genetic CCL5 deletion on A: IP GTT after 8 weeks of diet and B: 16 weeks of diet. C and F: Quantification of (A) and (B) represented as area under the curve (AUC). D: and G: Basal blood glucose levels after 16h of fasting after 8 and 16 weeks of diet, respectively. E: and H: Basal blood glucose levels after 6h of fasting after 8 and 16 weeks, respectively. I and J: Effect of CCL5 deficiency on insulin tolerance in mice fed a SD or HFD for 8 weeks (I) and 16 weeks (J). K: Basal fasted insulin levels at 16 weeks of diet measured via ELISA and L: acute glucose-stimulated insulin secretion

Figure 37 :

 37 Figure 37: Effect of CCL5 deficiency on hypothalamic insulin signaling. A and B: Effect of 16 weeks of diet on hypothalamic mRNA expression of IRS1 in WT (A) and CCL5 -/-(B) mice. C and D: Effect of 16 weeks of SD or HFD on hypothalamic mRNA expression of PPARγ in WT (C) and CCL5 -/-(D) mice. E and F: Effect of CCL5 deficiency on hypothalamic IRS1 expression between genotypes at 16 weeks of SD (E) and HFD (F) diet. G and H: Effect of CCL5 deficiency on hypothalamic PPARγ expression between genotypes at 16 weeks of SD (G) and HFD (H) diet. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to respective controls. Values are represented as means ± SEM (n=6-9 mice/ group). Statistical significance was determined using Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 38 :

 38 Figure 38: Effect of CCL5 deficiency on hypothalamic neuroinflammation compared between diets. Real-time PCR quantification of hypothalamic mRNA expression of inflammatory markers TNF-a (A), IL-6 (B), and IL-1β (C), and the chemokines, CCL2 (D) and CCL5 (E) after 16 weeks of diet in WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to SD-fed control groups. Values are represented as means ± SEM (n=6-9 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 39 :

 39 Figure 39: Effect of CCL5 deficiency on hypothalamic neuroinflammation compared between genotypes. Real-time PCR quantification of hypothalamic mRNA expression of inflammatory markers such as the cytokines: TNF-a (A), IL-6 (B), and IL-1β (C), and the chemokine CCL2 (D) after 16 weeks of diet in WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to WT control groups. Values are represented as means ± SEM (n=6-9 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 40 :

 40 Figure 40: Effect of CCL5 deficiency on DIO associated hypothalamic gliosis. Effect of 16 weeks of SD or HFD diet on hypothalamic mRNA expression of glial markers quantified by qPCR. A and B: Relative hypothalamic expression of astrocyte marker GFAP in WT (A) and CCL5 -/-(B) mice and comparing between mice fed a SD (E) and HFD (F) diet. C and D: Relative hypothalamic expression of microglial marker Iba1 in WT (C) and CCL5 -/-(D) mice and comparing between mice fed a SD (G) and HFD (H) diet for 16 weeks. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to respective controls. Values are represented as means ± SEM (n=7-8 mice/ group). Statistical significance was determined using Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant; GFAP: glial fibrillary acidic protein; Iba1: ionized calcium binding adaptor molecule 1.

Figure 41 :

 41 Figure 41: Effect of CCL5 deficiency on peripheral inflammation compared between diets. Real-time PCR quantification of mRNA expression of inflammatory markers TNF-a (A), IL-6 (B), and IL-1β (C), and the chemokines, CCL2 (D) and CCL5 (E) after 16 weeks of diet in perigonadal AT of WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to WT control groups. Values are represented as means ± SEM (n=3-10 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 42 :

 42 Figure 42: Effect of CCL5 deficiency on peripheral inflammation compared between genotypes. Real-time PCR quantification of mRNA expression of inflammatory markers such as the cytokines: TNF-a (A), IL-6 (B), and IL-1β (C), and the chemokine CCL2 (D) after 16 weeks of diet in perigonadal AT of WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to SD-fed control groups. Values are represented as means ± SEM (n=3-10 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 43 :

 43 Figure 43: Effect of CCL5 deficiency on systemic peripheral inflammation. Serum levels of pro-inflammatory cytokines TNF-a (A), IL-6 (B), and IL-1β (C), IFN-γ (D), anti-inflammatory cytokine IL-10 (E) and the chemokines, CCL2 (F) and CCL5 (G) after 16 weeks of diet in WT and CCL5 -/-mice on either SD or HFD. Serum CCL5 (G) concentration quantified via ELISA in DIO WT vs control mice. Serum CCL5 was measured for CCL5 -/-mice on both diets as well, but were beyond or at the limit of detection and therefore not shown. Values are represented as means ± SEM (n=12-14 mice/ group; for IL-1: n=1-8, for CCL5: n=8-9). Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc test or Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 44 :

 44 Figure 44: Effect of CCL5 deficiency on thermal pain sensitivity in DIO mice. Estimation of mechanical perception of thermal pain sensitivity by paw withdrawal latency in seconds as determined with Hargreaves' test in WT and CCL5 -/-mice on either a SD or HFD for 16 weeks. Values are represented as means ± SEM (n=12-21/ group). Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc test; P<0.05 #, P<0.01 ##, P<0.001, ###, P<0.0001 ####; ns: not significant.

Figure 45 :

 45 Figure 45: Effect of CCL5 deficiency on inflammatory marker expression in DRG compared between diets. Real-time PCR quantification of mRNA expression of inflammatory markers TNF-a (A), IL-6 (B), and IL-1β (C), and the chemokines, CCL2 (D) and CCL5 (E) after 16 weeks of diet in DRG of WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to WT control groups. Values are represented as means ± SEM (n=4-5 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 46 :

 46 Figure 46: Effect of CCL5 deficiency on inflammatory marker expression in DRG compared between genotypes. Real-time PCR quantification of mRNA expression of inflammatory markers such as the cytokines: TNF-a (A), IL-6 (B), and IL-1β (C), and the chemokine CCL2 (D) after 16 weeks of diet in DRG of WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to SD-fed control groups. Values are represented as means ± SEM (n=4-5 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 47 :

 47 Figure 47: Effect of CCL5 deficiency on inflammatory marker expression in spinal cords compared between diets. Real-time PCR quantification of mRNA expression of inflammatory markers TNF-a (A), IL-6 (B), and IL-1β (C), and the chemokines, CCL2 (D) and CCL5 (E) after 16 weeks of diet in spinal cords of WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to WT control groups. Values are represented as means ± SEM (n=4-5 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 48 :

 48 Figure 48: Inflammatory marker expression in spinal cords of DIO WT and CCL5 -/- mice compared between genotypes. Real-time PCR quantification of mRNA expression of inflammatory markers such as the cytokines: TNF-a (A), IL-6 (B), and IL-1β (C), and the chemokine CCL2 (D) after 16 weeks of diet in spinal cords of WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to SD-fed control groups. Values are represented as means ± SEM (n=4-5 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

  figure. The middle panel of the figure shows the expression of both molecules also in the ventromedial hypothalamus. The lower panel reveals that WT mice

Figure 49 :

 49 Figure 49: Hypothalamic expression of CCL5 and receptor CCR5 in SD-fed WT mice. Representation of Immunohistochemical brain sections of WT mice on a SD diet stained with RNAScope® technology using a sonde labeling CCL5 (green) and CCR5 (red) in the LH, VMH and ARC. The outermost panel on the right represents the enlarged section labeled with a white square in the merged picture on the left. Scale bar indicating either 50µm or 20µm as depicted in the images. LH: lateral hypothalamus; VMH: ventromedial hypothalamus; ARC: arcuate nucleus; 3V: 3 rd ventricle.

Figure 51 :

 51 Figure 51: Effect of CCR5 deficiency on DIO development. A: Evolution of average BW at 16 weeks of experimental protocol of DIO. B: Quantification of (A) as area under the curve -AUC. C: Average food consumption during 16 weeks represented as grams per mouse per day. D: Average food consumption during 16 weeks represented as kcal per mouse per day. E: Average H2O consumption during 16 weeks in grams per mouse per day. F: Serum leptin concentration measured by ELISA after 16 weeks of HFD. Data are reported as the means ± SEM (n=13-16/group for A and B; n=2 experiments/group for C-E). Two-way ANOVA followed by Tukey post hoc test, one-way ANOVA or Mann Whitney Test for comparisons between two groups. * P<0.05 *, P<0.01 **, P<0.001 ***, P<0.0001 ****, WT HFD vs CCL5 -/-HFD; #, WT SD vs WT HFD; $, CCL5 -/-SD vs HFD; ns: not significant.

Figure 52 :

 52 Figure 52: CCR5 deficiency has a neuromodulatory effect on hypothalamic neurons. Real-time PCR quantification of relative mRNA expression of hypothalamic neuropeptides such as anorexigenic neuropeptides POMC (A) and CART (B), and orexigenic neuropeptides NPY (C), AgRP (D), MCH (E) and ORX (F) after 16 weeks of diet in WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to SD-fed control groups. Values are the mean ± SEM (n=5-8 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 53 :

 53 Figure 53: CCR5 deficiency has a neuromodulatory effect on hypothalamic neurons. Real-time PCR quantification of mRNA expression of hypothalamic neuropeptides such as anorexigenic neuropeptides POMC (A) and CART (B), and orexigenic neuropeptides NPY (C), AgRP (D), MCH (E) and ORX (F) after 16 weeks of diet in WT and CCL5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to WT control groups. Values are represented as means ± SEM (n=5-9 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 54 :

 54 Figure 54: Glucose homeostasis perturbations in WT and CCR5 -/-mice after 8 and 16 weeks of SD or HFD.

Figure 55 :

 55 Figure 55: Effect of CCR5 deficiency on hypothalamic insulin signaling. A and B: Effect of 16 weeks of diet on hypothalamic mRNA expression of IRS1 in WT (A) and CCR5 -/-(B) mice. C and D: Effect of 16 weeks of SD or HFD on hypothalamic mRNA expression of PPARγ in WT (C) and CCR5 -/-(D) mice. E and F: Effect of CCR5 deficiency on hypothalamic IRS1 expression between genotypes at 16 weeks of SD (E) and HFD (F) diet. G and H: Effect of CCR5 deficiency on hypothalamic PPARγ expression between genotypes at 16 weeks of SD (G) and HFD (H) diet. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to respective controls. Values are represented as means ± SEM (n=5-8 mice/ group). Statistical significance was determined using Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 56 :

 56 Figure 56: Effect of CCR5 deficiency on hypothalamic neuroinflammation compared between diets. Real-time PCR quantification of hypothalamic mRNA expression of inflammatory markers TNF-a (A), IL-6 (B), and IL-1β (C), and the chemokines, CCL2 (D) and CCR5 (E) after 16 weeks of diet in WT and CCR5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to SD-fed control groups. Values are represented as means ± SEM (n=6-9 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 57 :

 57 Figure 57: Effect of CCR5 deficiency on hypothalamic neuroinflammation compared between genotypes. Real-time PCR quantification of hypothalamic mRNA expression of inflammatory markers such as the cytokines: TNF-a (A), IL-6 (B), and IL-1β (C), and the chemokines CCL2 (D) CCR5 (E) after 16 weeks of diet in WT and CCR5 -/-mice on either SD or HFD. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to WT control groups. Values are represented as means ± SEM (n=6-9 mice/ group). Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant.

Figure 58 :

 58 Figure 58: Effect of CCR5 deficiency on DIO associated hypothalamic gliosis. Effect of 16 weeks of SD or HFD diet on hypothalamic mRNA expression of glial markers quantified by qPCR. A and B: Relative hypothalamic expression of astrocyte marker GFAP in WT (A) and CCR5 -/-(B) mice and comparing between mice fed a SD (E) and HFD (F) diet. C and D: Relative hypothalamic expression of microglial marker Iba1 in WT (C) and CCR5 -/-(D) mice and comparing between mice fed a SD (G) and HFD (H) diet for 16 weeks. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to respective controls. Values are represented as means ± SEM (n=5-8 mice/ group). Statistical significance was determined using Mann-Whitney Test; P<0.05 *, P<0.01 **, P<0.001 ***; ns: not significant; GFAP: glial fibrillary acidic protein; Iba1: ionized calcium binding adaptor molecule 1.

Figure 59 :

 59 Figure 59: Effect of CCR5 deficiency on systemic peripheral inflammation. Serum levels of pro-inflammatory cytokines TNF-a (A), IL-6 (B), IL-1β (C), IFN-γ (D) and anti-inflammatory cytokine IL-10 (E) after 16 weeks of diet in WT and CCR5 -/-mice on either SD or HFD. Values are represented as means ± SEM (n=12-14 mice/ group; for IL-1β: n=1-8, for CCR5: n=8-9). Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc test; * P<0.05 *, P<0.01 **, P<0.001 ***, P<0.0001 ****,WT HFD vs CCR5 -/-HFD; #, WT SD vs WT HFD; $, CCR5 -/-SD vs HFD; @, WT SD vs CCR5 -/- SD; ns: not significant.

Figure 60 :

 60 Figure 60: Effect of CCR5 deficiency on thermal pain sensitivity in DIO mice. Estimation of mechanical perception of thermal pain sensitivity by paw withdrawal latency in seconds as determined with Hargreaves' test in WT and CCR5 -/-mice on either a SD or HFD for 16 weeks. Values are represented as means ± SEM (n=10-14/group). Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc test; P<0.05 #, P<0.01 ##, P<0.001, ###, P<0.0001 ####. WT SD vs WT HFD; ns: not significant.

Figure 61 :

 61 Figure 61: Effect of CCL5 and CCR5 deficiency on AT morphology and lipolysis. H&E staining of representative sections of subcutaneous AT (A) and their quantification in percent frequency of adipocyte area. Upper panel showing the subcutaneous AT of WT, CCL5 -/-, CCR5 -/-under SD condition and lower panel

Figure 62 :

 62 Figure 62: Effect of ICV administration of different concentrations of CCL5 on changes in BW. A: Graph depicting the % difference in BW relative to baseline before aCSF injection. Arrows indicate injections via implanted cannula of either aCSF, or recombinant CCL5 (10ng, 50ng, 100ng). First arrow indicated the first aCSF injection to all mice for habituation. B: Quantification of (A) as area under the curve (AUC). Values are means ± SEM (n=4-6/group). C: Quantification of hypothalamic mRNA expression relative to aCSF injected mice. All mRNA species were quantified relative to Gapdh housekeeping gene expression by ΔCT method and presented as fold change relative to respective controls. Statistical significance was determined using two-way ANOVA with Dunn's post hoc test and two-way ANOVA with Dunnet's post hoc test and Unpaired t-test with Welch's correction; P<0.05 *, P<0.01 **, P<0.001, ***, P<0.0001 ****. All vs aCSF; ns: not significant.

Figure 63 :

 63 Figure 63: Effect of ICV CCL5 and antagonist Met CCL5 administration on changes in BW, food intake and thermal pain sensitivity. Effect of ICV administration of aCSF, CCL5 (0.3µg) and Met CCR5 (0.3µg) on A: BW gain, B: Table representing statistical significance for (A). C: Hargreaves' thermal pain sensitivity test. Statistical significance was determined with Kruskal-Wallis test with Dunn's post hoc test. P<0.05 *, P<0.01 **, P<0.001, ***, P<0.0001 ****. All vs aCSF; ns: not significant.

Figure 64 :

 64 Figure 64: Effect of ICV CCL5 and antagonist administration on glucose homeostasis. Effect of ICV administration of aCSF, CCL5 (0.15µg/µL) and Met CCL5 (0.15µg/µL) on A: GTT, B: Quantification of (A) as AUC, C: Basal blood glucose levels after 16h of fasting, D: ITT, E: Plasma insulin concentration measured by ELISA, F: Basal blood glucose levels after 6h of fasting, and G: Acute glucose-stimulated insulin secretion test. Data are represented as means ± SEM (n=5-7/group). Statistical significance was determined with Friedman's test or Kruskal-Wallis test with Dunn's post hoc test. Dashed line indicates the threshold for diabetic levels of blood glucose at 150 mg/dL. ns: not significant.

  IRS1 and PPARγ expression in hypothalamic neurons like NPY and indirectly or directly affect food intake and other components, leading to changes in BW and cellular metabolism of macronutrients. In the context of HFD-induced obesity, lipids could chronically activate PPARγ and thereby contribute to hypothalamic dysfunction like leptin resistance and insulin resistance as well as deregulation of food intake and energy balance. At the same time, it could increase adipogenesis in the periphery in an attempt to store the incoming excess of lipids, thereby saturating the limited capacities of AT, leading to inflammation and the development of metabolic syndrome. It seems like the link between PPARγ and CCL5 signaling could be found in macrophages, as they both express CCL5 receptors and PPARγ, and both seem to be important in the regulation of cellular macromolecule metabolism. Although there are some parallels between CCL5 and PPARγ signaling, and both seem to play important roles in DIO and associated insulin resistance, their potential interaction and the mechanisms behind it remain unclear and require further experiments to shed some light into the potential link between these two molecules.

  Our study has added to our knowledge the first evidence of the importance of CCL5/CCR5 signaling in the modulation of HFD-induced hyperalgesia associated with obesity and diabetes. Based on our results and previous studies we hypothesize that CCL5 can act via CCR5 but also other receptors in nuclei of the hypothalamus to affect the activity of neurons such as NPY-expressing neurons either by directly modulating their activity via chemokine receptors or indirectly through modulating glia cell activation. Moreover, CCL5 seems to act on IR expressing neurons in the hypothalamus to regulate glucose homeostasis and insulin sensitivity. CCL5/CCR5 signaling might thus affect food intake either through the modulation of neuropeptide expression in neural networks important for energy balance and feeding regulation or indirectly by affecting insulin and glucose homeostasis. Furthermore, is seems to act in the periphery in immune cells, AT, pancreas and intestine to regulate cellular metabolism, immune function and insulin secretion via incretins. The multitude of different results seems to suggest that chemokines such as CCL5 have different functions based on their concentration, for example in acute and chronic inflammation chemokines seem to have potentially opposing effects. Furthermore, their function might be different depending on tissue, cell and receptor interaction.

  rendering it chronic as infiltrated cells can in turn induce cytokine and chemokine production. Depending on the intensity of the stimulus, CCL5 signaling could mediate acute or chronic inflammation. Similar to the controversial and dual effect observed for CCL2 in both acute inflammation, where it induces fever and weight loss, but also is important for the development of obesity, CCL5 signaling could induce different effects with different outcomes, such as weight loss in acute hypothalamic inflammation and weight gain in chronic inflammation. In particular, one could imagine different outcomes for different CCL5-receptor interactions. It would be interesting to investigate the effects of CCL5 signaling in different KO models for each receptor or even a double KO of two of the receptors of CCL5 to pinpoint the specific effect of each receptor of CCL5. To circumvent the problem of receptor or ligand promiscuity, one could even try to combine receptor or ligand double knockouts with antibody-mediated inhibition of other ligands for the same receptor and vice versa as CCR5 binds to other chemokines apart from CCL5. In any case, this would require more complex and time-consuming but very intriguing experiments.In summary, our study has taken a broad approach to investigate different aspects of the role of chemokine CCL5 and its receptor in the context of HFDinduced obesity and associated pathologies like diabetes and neuropathic pain. We have contributed to advancing the knowledge on the multiple functions of chemokines by showing that CCL5 and CCR5 are both important actors in HFD-induced obesity and associated metabolic syndrome and can act at different locations to contribute to weight gain, insulin resistance, thermal pain sensitivity and modulation of neuronal activity to affect energy balance regulation. We thus have provided evidence confirming that chemokines can indeed have a multitude of functions apart from their role in immune system and that the redundancy of the chemokine system and the complexity of the immune system requires more sophisticated experiments than just one factor elimination to unravel the exact mechanism behind chemokine action.

  in a pathological context by potentiating the inflammatory response in the hypothalamus or peripheral tissues by recruiting other immune cells (Fig.65).

Figure 65 :

 65 Figure 65: Proposed mechanism of action of CCL5 in the regulation of energy balance in the hypothalamus.

  predisposition of an individual to the HFD-induced hypothalamic disturbances involved in obesity. Moreover, our experiments showed that microglial depletion modulated gene response to food exposure of neuropeptides involved in energy balance regulation. Not only did it increase NPY and POMC hypothalamic mRNA levels in both SD-and HFD-treated mice, but it also differentially affected MCH and ORX gene responses to diet type, suggesting that microglial responses to food exposure could specifically act on neurons involved in energy balance regulation in a diet-dependent manner.

  Figure1

  Figure7

  system (Ultimate 3000, ThermoFisher). Lipid extracts of serum from mice were separated on an Accucore C18 150x2.1, 2.5µm column (ThermoFisher) operated at 400 µl/min flow rate. The injection volume was 3µl of diluted lipid extract. Eluent solutions were ACN/H2O 50/50 (V/V) containing 10mM ammonium formate and 0.1% formic acid (solvent A) and IPA/ACN/H2O 88/10/2 (V/V) containing 2mM ammonium formate and 0.02% formic acid (solvent B). A step gradient program was used: 0 min 35% B, 0.0-4.0 min 35 to 60% B, 4.0-8.0 min 60 to 75% B, 8.0-16.0 min 97%, 16.0-16.1 100% B This final elution is retained for 6 minutes and finally the column was reconditioned at 35% B for 4.0 min.The UPLC system was coupled with a Q-exactive orbitrap Mass Spectrometer (Thermofisher, CA); equipped with a heated electrospray ionization (HESI) probe. This spectrometer was controlled by the Xcalibur software and was operated in electrospray positive mode. MS spectra were acquired at a resolution of 70 000 (200 m/z) in a mass range of 250 1200 m/z with an AGC target 1e6 value and a maximum injection time of 250 ms. 15 most intense precursor ions were selected and isolated with a window of 1 m/z and fragmented by HCD (Higher energy C-Trap Dissociation) with normalized collision energy (NCE) of 25 and 30 eV. MS/MS spectra were acquired in the ion trap with an AGC target 1e5 value, in a mass range of 200-2000 m/z, the resolution was set at 35 000 at 200 m/z combined with an injection time of 80 ms.Data were reprocessed using LipidSearch 4.1.16 (Thermo Fisher). In this study, the product search mode was used and the identification was based on the accurate mass of precursor ions and MS2 spectral pattern. Mass tolerance for precursor and fragments was set to 5 ppm and 8 ppm respectively. m-score threshold was selected at 5 and ID quality filter was fixed at grades A, B and C. [M+H] + , [M+Na] + and [M+NH4] + adducts were searched.

Food

Table 1 : Composition of diets used for the first part of this study.

 1

			BUTTER	RAPESEED	SOJA/CORN	SUNFLOWER
		)	(HFD-B)	(HFD-C)	(HFD-SC)	(HFD-SN)
	Energie (kcal/g)	3.4	5.3	4.4	4.5	4.5
	Proteins (%)	25.2	20.0	23.0	18.2	20.1
	Carbohydrates (%)	61.3	36.7	43.0	46.8	46.8
	Lipids (%)	5.1	36.0	20.0	20.0	20.0
			Anhydrous	Rapeseed	Anhydrous	Anhydrous
			butter	oil 20%	butter 6.1%	butter 5.1%
			33.3 %		Soja oil 1.3%	Crisco 5.0%
	Details of lipid		Soja oil		Crisco 6.0%	Lard 5.0%
	composition of		2.5%		Lard 6.0%	Sunflower
	HFD				Corn oil	oil 4.77%
					0.5%	Cholesterol
					Cholesterol	0.15%
					0.15%
	Energy
	provided by	13.5	58.8	40.5	40.3	40.3
	lipids (%)
	ω6/ω3 ratio	6.5	8.0	2.3	7.3	17.3

Table 2 : Composition of the diets used in the second part of the experiment.

 2

	Standard Diet	High Fat Diet
	SAFE A03	HF260 (HFD)

at the onset of the dark cycle for 12 weeks. For the second experiment of our study, at the beginning of the dark cycle (T=0h) either standard diet (SD; 3 395 kcal/kg of which 25.2% are from proteins, 13.5% fat and 61.3% from carbohydrates; Safe #A03) or high-fat diet (HFD; 5 283 kcal/kg; 20.0% from proteins, 58.8% from fat and 36.7% from carbohydrates, enriched in anhydrous butter 33.3% + soybean oil 2.5%; Safe #U8978P V0019) (Table

2

) were delivered for 16 weeks. Food intake was measured and changed every second day. At the end of the experimental procedure, mice were sacrifized and brain, AT as well as blood samples were taken.

Table 3 : Primers used for real time PCR.

 3

  measured via ELISA at 16 weeks of diet. Dashed line indicates the threshold for diabetic levels of blood glucose at 150 mg/dL. Values are means ± SEM (n=8-10/group for A-J and n=6-9 for G+H). Statistical significance was determined with two-way ANOVA with Tukey's post hoc test or Kruskal-Wallis test with Dunn's post hoc test. * P<0.05 *, P<0.01 **, P<0.001 ***, P<0.0001 ****, WT HFD vs CCL5 -/-HFD; #, WT SD

	vs WT HFD; $, CCL5-/-SD vs HFD. ns, not significant; 8w: 8 weeks of diet; 16w: 16
	weeks of diet.

  Effect of genetic CCR5 deletion on A: IP GTT after 8 weeks of diet and B: 16 weeks of diet. C and F: Quantification of (A) and (B) represented as area under the curve (AUC). D: and G: Basal blood glucose levels after 16h of fasting after 8 and 16 weeks of diet, respectively. E: and H: Basal blood glucose levels after 6h of fasting after 8 and 16 weeks, respectively. I and J: Effect of CCR5 deficiency on insulin tolerance in mice fed a SD or HFD for 8 weeks (I) and 16 weeks (J). K: Basal fasted insulin levels at 16 weeks of diet measured via ELISA at 16 weeks of diet and L: acute glucosestimulated insulin secretion measured via ELISA at 16 weeks of diet. Dashed line indicates the threshold for diabetic levels of blood glucose at 150 mg/dL. Values are means ± SEM (n=5-8/group for A+C-E+I and n=8-9 for B+F-H+J; n= 1-8 for K+L). Statistical significance was determined with two-way ANOVA with Tukey's post hoc test or Kruskal-Wallis test with Dunn's post hoc test. * P<0.05 *, P<0.01 **, P<0.001 ***, P<0.0001 ****, WT HFD vs CCR5 -/-HFD; #, WT SD vs WT HFD; $, CCR5 -/-SD vs HFD; @, WT SD vs CCR5 -/-SD. ns, not significant. Dashed line indicates the threshold for diabetic levels of blood glucose at 150 mg/dL; ns: not significant; 8w: 8 weeks of diet; 16w: 16 weeks of diet.

  -/-, CCR5 -/-and WT mice on a HFD to see whether it would reverse the phenotype seen in our study. Likewise, because Met CCL5 antagonizes the effect of CCL5 on all the receptors, our results should be completed with experiments using specific antagonists for each receptor. Further experiments to complete our results by studying earlier time points in the development of obesity are necessary, as hypothalamic inflammation occurs early in the development of obesity. Our data should be supplemented with tests of serum leptin levels in It would be indeed important to confirm our results with Alzet pumps, which provide a more consistent infusion of product while reducing confounding factors such as stress.

	In summary, we conclude that chemokines like CCL5 are important actors in
	the deregulation of energy balance, whether it is in an acute way as we have
	shown with CCL2, which mediates the LPS-induced anorexic effect in the
	hypothalamus (see annex, (Le Thuc et al., 2016)), or in the chronic low-grade
	inflammation as seen with obesity. Furthermore, other authors suggest that
	chemokines might have a role as neuromodulators in a physiological context.
	Our results are consistent with this hypothesis as well as another article that we
	have submitted recently, which I have contributed to, too (Cansell et al.,
	2019; see Annex). It seems fitting to speculate that chemokines like CCL5 can
	act as neuromodulator in physiological conditions either by acting directly on
	neurons or indirectly through chemokine receptors expressed on astrocytes
	and microglia, or
	CCR5 mice as well as testing whether CCL5 and CCR5 deficient mice are
	leptin resistant.
	Our results further indicate that CCL5 might act on orexigenic neurons to
	affect food intake, which seems to be CCR5 independent and rather
	involving one of the other receptors that CCL5 might bind to. Thus it would be
	interesting, to perform similar experiments with mice deficient in the other
	receptors for CCL5, namely CCR1 and CCR3 in a HFD context to identify,
	which receptor mediates the effects of CCL5 on food intake, BW and glucose
	metabolism regulation. While our results indicate that CCL5 deficiency seems
	to improve glucose tolerance and insulin resistance in a HFD context, other
	studies suggest that CCL5 receptors may be associated with the IR in the

hypothalamus and thereby affect insulin signaling directly. However, our results are not conclusive if glucose metabolism is improved due to a potential attenuation of downstream inflammation or due to effects on insulin-expressing cells such as NPY/AgRP and/or POMC/CART neurons as is indicative with our results.

  If samples fulfilled normal distribution and variance equality criteria, comparisons between groups were carried out using an unpaired t test for single comparison and a two-way ANOVA for multiple comparisons and interaction. If samples did not follow a normal distribution or had different variances, comparisons

	equipped with a ZORBAX SB-C18 C18 50 x 2.1 mm 1.8 µm (Agilent Technologies,
	France) maintained at 45°C with a binary set of eluents (eluent A: 1 M ammonium
	formate/formic acid/water 5/1/996 v/v/v and eluent B: 1 M ammonium formate/formic
	acid/water/acetonitrile 5/1/44/950 v/v/v) at a flow rate of 0.4 mL/min. An 8 min eluent
	gradient was established as follows: from 0 to 0.5 min, the mobile phase composition between groups were carried out using a non-parametric Mann-Whitney U test for
	was maintained at 45% B; then the proportion of B was increased linearly up to 100% single comparison and non-parametric Kruskal-Wallis test with Dunn's correction for
	over 2.5 min and maintained for 5 additional minutes. The column was equilibrated multiple comparisons. When appropriate, comparison with the theoretical mean
	with 45% B for 5 min after each sample injection. MS/MS analysis was performed on control (1 or 100%) was done using a non-parametric Wilcoxon signed rank test. A p-
	a 6490 triple quadruple mass spectrometer (Agilent Technologies, France) equipped value of 0.05 was considered statistically significant. All tests were performed using
	with a JetStream-Ion funnel ESI source in the negative mode (gas temperature GraphPad Prism 7.02 and Microsoft Office Excel. Numbers of animals and cells are
	290°C, gas flow 19 L/min, nebulizer 20 psi, sheath gas temperature 175°C, sheath
	gas flow 12 L/min, capillary 2000 V, V charging 2000 V). Nitrogen was used as the
	collision gas. The mass spectrometer was set up in the selected reaction monitoring
	(SRM) mode for the quantification of selected ions as follows: for 3-
	hydroxytetradecanoic acid, precursor ion 243.2 Da, product ion 59 Da, fragmentor
	380 V, collision cell 12 V, cell acceleration 2 V; for 3-hydroxytridecanoic acid,
	precursor ion 229.2Da, product ion 59 Da, fragmentor 380 V, collision cell 12 V, cell
	acceleration 2 V.
	2.10. Triglyceride analysis.
	The detailed protocol is provided in the Supplementary data, Supplementary
	2 and 95% O 2 ) ice-cold protective Materials and Methods. Briefly, lipids from mouse serum were extracted according to
	extracellular solution containing (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH 2 PO 4 , 30 a modified Bligh and Dyer protocol with a mixture of methanol/chloroform (2:1). The
	NaHCO3, 20 HEPES, 2 thiourea, 25 D-glucose, 5 sodium ascorbate, 3 sodium lipid extract was then separated on a C18 column in an appropriate gradient. Mass
	pyruvate, 10 MgCl 2 , and 0.5 CaCl 2 (pH 7.3, 320 mOsm). Slices were then incubated spectrometry data were acquired with a Q-exactive mass spectrometer
	in the same protective extracellular solution for 7 min at 34°C and then incubated for (ThermoFisher, France) operating in data dependent MS/MS mode (dd-MS2). Finally,
	30 min in artificial cerebro-spinal fluid (aCSF; pH 7.3, 310 mOsm) at 34°C containing lipids were identified using LipidSearch software v4.1.16 (ThermoFisher, France) in
	(in mM): 135 NaCl, 2.5 KCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 D-glucose, 1 sodium product search mode.
	pyruvate, 1 MgCl 2 , and 2 CaCl 2 . The slices were then maintained at room
	temperature (RT, 22°C) for 0.5-5 h in the regular oxygenated aCSF. After incubation, 2.11. Statistical analysis.
	individual slices selected for best visibility of the ARC were transferred to a recording Displayed values are means ± SD. To test if the data set was well-modeled, a

chamber on the stage of an Olympus microscope (BX50WI) with a 40x water immersion objective, equipped with a CCD camera (Hamamatsu ORCA2-AG, France). Slices were constantly perfused at RT with oxygenated aCSF (5 mL/min).

Kolmogorov-Smirnov normality test was conducted (with Dallal-Wilkinson-Lillie for P value). The ROUT method (robust regression and outlier removal) was used to identify outliers with a Q coefficient equal to 1%. Variance equality was tested using a F-test.

Acknowledgments

Acknowledgments We thank Plexxikon and Dr. Brain West for PLX5622. This work has been supported by the CNRS, the Fondation pour la Recherche Médicale (DEQ20150331738), the Medisite Foundation and by the French government, managed by the National Research Agency (ANR), through the UCA JEDI Investments in the Future project with the reference number ANR-15-IDEX-01, the LABEX SIGNALIFE program with the reference number ANR-11-LABX-0028-01 and La Fondation Nestlé France. We thank Dr. JP. Pais de Barros from the Lipidomic Platform uBourgogne (INSERM UMR1231 / LabEX LipSTIC) for 3OH C14:0 blood analysis. We thank Abby Cuttriss (Office of International Scientific Visibility, Université Côte d'Azur), Dr. Merel Rijnsburger (VU University Medical Center Amsterdam) and Pr. Christophe Magnan (Paris Diderot University) for editing the manuscript.

corner of the HFD condition, that CCL5 has a perinuclear expression and is co-expressed with its receptor CCR5 in the outer corner of the ME. HFD-fed WT mice that received a bone-marrow transplant from CCR5 -/-mice, which was associated with improved glucose tolerance [START_REF] Kitade | CCR5 plays a critical role in obesity-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status[END_REF]. This indicates that infiltration of CCR5-expressing macrophages is an important contributor to AT inflammation and glucose intolerance in DIO.

Similarly, in line with previous reports, we found upregulation of inflammatory cytokines in the hypothalamus of HFD-fed mice compared to SD controls, but no difference between CCL5 -/-and WT mice. However, results were a little bit different for hypothalamic cytokine and chemokine expression for CCR5 -/- mice. We found a decrease in IL-1β expression as well as a marginal but not significant decrease in IL-6 mRNA levels in the hypothalamus of CCR5 -/-after 16 weeks of HFD compared to WT mice. Due to the implication of glia cells in hypothalamic inflammation and the association with hypothalamic gliosis, we tested the expression of the commonly used markers of astrocytes (GFAP) and microglia (Iba1) after 16 weeks of HFD in CCL5 -/-and CCR5 -/-mice.

Interestingly, although we have not seen a significant increase in glial marker expression in hypothalami of HFD-fed mice but only a marginal and not significant increase in GFAP in HFD-fed WT mice, we found a significant reduction in GFAP expression in HFD-fed CCL5 -/-mice compared with WT controls, but not in CCR5 -/-mice. This result suggests that CCL5 might affect GFAP expressing glia cells, but this effect might be a CCR5-independent, although astrocytes have been reported to express CCR5 [START_REF] Sorce | The chemokine receptor CCR5 in the central nervous system[END_REF]. Adding to these results, chronic ICV injections of CCL5 induced an upregulation of Iba1 in our experiment. Although it is a preliminary result with low number of animals, due to technical difficulties in the cannula implantation technique, is suggests that CCL5 might have a modulatory role on glia cells such as astrocytes and microglia. In support of this, astrocytes, neurons and microglia have been reported to express and release CCL5 but also receptors of CCL5, suggesting an autocrine or paracrine signaling mechanism [START_REF] Guillemin | Quinolinic acid upregulates chemokine production and chemokine receptor expression in astrocytes[END_REF][START_REF] Škuljec | CCL5 induces a pro-inflammatory profile in microglia in vitro[END_REF][START_REF] Sorce | The chemokine receptor CCR5 in the central nervous system[END_REF][START_REF] Louboutin | Relationship Between the Chemokine Receptor CCR5 and Microglia in Neurological Disorders: Consequences of Targeting CCR5 on Neuroinflammation, Neuronal Death and Regeneration in a Model of Epilepsy[END_REF][START_REF] Lanfranco | Glial-and Neuronal-Specific Expression of CCL5 mRNA in the Rat Brain[END_REF].

Furthermore, a study found that CCL5 stimulated both chemotaxis and

Annexes

During the course of my thesis, I had the opportunity to assist and participate in several national and international conferences and present the results of my project in poster and oral communications as depicted in the following pages. At some of these events, I had the opportunity to be awarded the best poster or oral communication prize. Furthermore, I had the possibility to participate in the organization of the International Neuroscience Conference and as an invited chair.

Abstract

Obesity and metabolic syndromes are concomitant with a state of chronic systemic inflammation. In humans, obesity was associated with brain inflammation and glial cell proliferation. Moreover, recent studies in rodents have shown that proliferation of microglia and astrocytes occurs within the first 24 hours of high-fat diet (HFD) consumption, well before the onset of obesity. This proliferation was mainly observed in the hypothalamus, a crucial structure for controlling the adaptive metabolic and behavioral responses that regulate body weight. Therefore, in this study, we sought to understand the cell-based mechanisms involved in the post-prandial hypothalamic inflammatory response to a standard diet (SD) and HFD. We investigated the effects of short-term SD and HFD feeding within 1, 3 and 6 hours on cellular and molecular hypothalamic remodeling in mice. A short 1-hour HFD exposure increased gene expression of an astrocyte marker (GFAP) in the hypothalamus, whereas expression of the microglial marker (Iba1) only increased after 3 hours of HFD exposure.

Morphological modifications of microglial cells were observed in the arcuate nucleus of the hypothalamus after 6 hours of HFD consumption. This remodeling was associated with higher expression of pro-inflammatory genes and differential activation of hypothalamic neuropeptides involved in energy balance regulation in HFD-fed mice compared to SD fed-mice. The use of DREADD technology and PLX5622 to modulate GFAP-positive or microglial cells activity, respectively, suggested the involvement of both glial cell types in hypothalamic post-prandial inflammation, but in a different time frame and with a diet specificity. These results in adult mice demonstrate, for the first time, that the increase of nutritional lipid content in diet promotes hypothalamic post-prandial inflammation, which is known to be involved in the central regulation of energy balance, and that this response involves hypothalamic GFAP-positive glial cells and microglia.

Keywords: Energy Balance, Lipids, Obesity, Inflammation, Hypothalamus, Microglia, Astrocyte, Cytokines, Chemokines, Neuropeptides.

Stereotactic virus injections.

Mice were anesthetized by intraperitoneal (i.p.) injection of a ketamine-xylazine mix (80 mg/kg -12 mg/kg). They were then placed on a stereotaxic frame. AAV8/GFAP-HA-hM4G(Gi)-mCitrine (Translational Vector Core, France) virus was injected bilaterally into the medio basal HT (MBH). Stereotactic coordinates relative to bregma were: x: ±0.3 mm; y: -1.5 mm; z: +6 mm. Injections were applied at a rate of 0.5 μL/min for 1 min per side. At the end of the surgical procedures, mice received 1 mg/kg i.p. atipamezole and 5 mg/kg subcutaneous (s.c.) ketoprofen.

RNA isolation and quantitative PCR.

Total RNA from HT was isolated using Fast Prep apparatus (Q-Biogene, France) as previously described (Le Thuc et al., 2016). First-strand cDNAs were synthesized from 2 μg of total RNA with 200 U of SuperScript III reverse transcriptase (SuperScriptIII, Invitrogen, France) in the appropriate buffer in the presence of 25 ng/µL oligo-dT primers, 0. Supplementary Table 1) were purchased from Eurogentec (France).

Cytokine and chemokine quantification.

A V-Plex multiplex assay (Meso Scale Discovery, USA) and a mouse CCL5 ELISA Ready-SET-Go (eBiosciences, France) were used to measure the levels of inflammatory mediators in mice serum according to the manufacturer's protocol.

Immunohistochemical analysis.

Brains were harvested from mice perfused with 4% paraformaldehyde in phosphate buffer saline (PBS) and post-fixed in the same fixative overnight at 4°C.

Electrophysiological recordings.

Visually-identified eGFP-expressing microglial cells, located at least 50 µm below the slice surface, were patched in whole-cell configuration in the ARC. Micropipettes Series resistance (Rs) as well as the cell membrane resistance (Rm), capacitance (Cm) were determined from the current response to a 10 ms depolarizing pulse of 10 mV that was performed at the beginning of each recording. These currents were filtered at 10 kHz and collected using PClamp 10 (Molecular Devices, USA) at a frequency of 100 kHz. To measure the I/V relationship, we used hyperpolarizing and depolarizing steps (from -150 to +40 mV for 50 ms), and currents were low-pass filtered at 5 kHz and collected at a frequency of 20 kHz. Currents were analyzed off line using Clampfit 10.7 (Molecular Devices, USA). To measure capacitance, currents were first fitted with a double exponential function in order to determine the time constant tau (τweighted) defined as τweighted = (A1*τ1+A2*τ2)/(A1+A2) and then capacitance was calculated as Cm=(τweighted(Rm+Rs))/((Rm*Rs)).

LPS mass quantitation by LC-MSMS.

Lipopolysaccharide (LPS) mass concentration was determined by direct quantitation of 3-hydroxytetradecanoic acid (or 3 hM) by LC/MS/MS as previously described by Pais de Barros et al. [START_REF] Pais De Barros | Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay[END_REF]. Quantitative LPS analysis using HPLC/MS/MS was performed as well as the lysate assay of its All authors discussed results and/or reviewed the manuscript.

Conflict of interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

Supplementary data associated with this article can be found in this online version. Quantification of mRNA encoding the microglial marker Iba1 (A) in the hypothalamus of 8-week-old C57Bl/6J male mice fed for 1h, 3h and 6h with either standard diet (SD) or high fat diet (HFD) N=9-12. Quantification of immunohistochemical detection of Iba1 protein (stained area, m2) (B) in coronal sections of the arcuate nucleus of the hypothalamus (ARC) (30 m, -1.70mm relative to Bregma) from 8-week-old C57Bl/6J male mice fed for 1h, 3h and 6h with either SD or HFD N=4-7.

Quantification of average microglial soma size (C) in ARC of 8-week-old C57Bl/6J male mice fed for 1h, 3h and 6h with either SD or HFD N=4-7 n=8-21.

Immunohistochemical detection of Iba1 protein in ARC of 8-week-old C57Bl/6J male mice fed for 1h, 3h and 6h with either SD or HFD (D). Electrophysiological recording of current-voltage relationships (E) and membrane capacitance (F) of microglia cells whole-cell recorded in ARC of 9-10 week-old CX3CR1eGFP/eGFP male mice fed for 3h with either SD (N=3, n=30) or HFD (N=4 n=26). All mRNA levels were quantified

Supplementary Figures Legends:

Supplementary