Claude Bernard -Lyon

M Frédéric Fleury

M Hamda

Ben Hadid

M Didier

Jean-François Mornex

Directeur Général

Services M Pierre

Rolland Composantes Sante

Judith Estrada-Meza
email: judith.estrada-meza@ens-lyon.org

Jasmine Videlo

Clara Bron

Cécile Saint-Béat

Marine Silva

François Duboeuf

Olivier Peyruchaud

Fabienne Rajas

Gilles Mithieux

Amandine Gautier-Stein
email: amandine.gautier-stein@univ-lyon1.fr

Judith Estrada-Meza1

Jasmine Videlo1

Clara Bron1

Adeline Duchampt1

Cécile Saint-Béat1

Marine Silva1

Fabienne Rajas1

Gilles Mithieux1

Amandine Gautier-Stein1

Tamoxifen treatment in the neonatal period affects glucose homeostasis in adult mice in a sex-dependent manner Authors

Keywords: Tamoxifen, Neonatal period, Glucose tolerance, Diabetes, Obesity, Bone weight NTT, neonatal tamoxifen treatment, ER, estrogen receptor, HF/HS, highfat/high-sucrose

Purpose: Tamoxifen is a selective estrogen receptor modulator used in biotechnology to activate the CRE ERT2 recombinase, allowing tissue-specific and temporal control of the somatic mutagenesis to generate transgenic mice. Some studies integrating development and metabolism require a genetic modification induced by tamoxifen administration since the neonatal period. Here, we investigate the effects of a neonatal administration of tamoxifen on energy homeostasis in adult male and female C57BL/6J mice.

115 Methods: C57BL/6J male and female mouse pups received a unique injection of tamoxifen one day after birth and were fed either a standard chow or a high-fat/high-sucrose (HF/HS) diet at 6 weeks of age. We measured weight, body composition, glucose and insulin tolerance, basal metabolism and tibia length and weight in adult mice fed either a standard diet or a HF/HS diet for one and two months.

Results:

The neonatal administration of tamoxifen exerted sex-dependent effects on energy homeostasis. Female mice treated with tamoxifen during the neonatal period showed an acceleration of the onset of overweight and type 2 diabetes in comparison to vehicle-treated littermates, when challenged with a HF/HS diet. These mice exhibited more than a 60% increase in fat mass associated with increased food intake, decreased physical activity and decreased energy expenditure, massive glucose and insulin intolerance, and fasting hyperglycemia and hyperinsulinemia. On the contrary, male mice treated with tamoxifen during the neonatal period exhibited a modest amelioration of glucose and insulin tolerance, and life-long decreased lean mass linked to a decrease in bone weight. These effects also took place in male and female mice fed a standard diet.

Conclusion:

These results suggest that the neonatal administration of tamoxifen exerted a marked and sex-dependent influence on adult energy homeostasis, and must be used with caution for the development of transgenic mouse models regarding studies on energy homeostasis.

as interesting as it was for me, and that it will be pedagogical! I also thank Dr. Julien Courchet and Dr. Sebastien Bouret for having accepted to be members of my "comité de suivi de these", and being part of my thesis jury. Thank you for all your precious suggestions, our discussions and for your very valuable advice regarding the after-PhD! Je tiens à remercier de tout mon coeur tous et toutes les membres du laboratoire U1213, « Nutrition, diabète et cerveau ». C'est un super labo. La bienveillance et l'humour de l'U1213 sont précieux, et vous allez me manquer ! Je remercie Amandine Gautier-Stein pour avoir dirigé ma thèse. Merci parce que tu es une directrice géniale, qui n'a jamais peur de se lancer vers un nouveau projet (j'en suis admirative !) et qui aime apprendre. Un énorme merci de m'avoir confié ce projet et parce que tu m'as permis d'être très indépendante, mais en même temps tu as été toujours disponible pour mes questions, doutes et commentaires. J'apprécie énormément cela. Merci aussi pour tout le temps que tu as consacré à mon manuscrit de thèse, tes critiques constructives et toutes nos discussions scientifiques. Je te remercie également parce que tu m'as aidé quand j'ai eu le plus besoin cette année.

Je remercie aussi Gilles Mithieux pour m'avoir accueillie dans son laboratoire à plusieurs reprises → L2, L3, M2 et ensuite une thèse ! C'est vraiment top l'ouverture que tu as pour accueillir des gens dans ton laboratoire… que ce soit des étudiants pour un petit/long stage, ou du public pour faire connaître la science que nous faisons au laboratoire. Merci aussi pour toutes les discussions, tes remarques, tes suggestions et le management que tu fais au sein de l'U1213.

Merci Fabienne parce je pense que le labo ne serait pas ce qu'il est sans toi. Merci pour tes corrections et suggestions pertinents sur ce manuscrit de thèse, les articles et les réunions de laboratoire. Je ne peux pas finir cette partie sans te remercier aussi pour les photos apéro/spritz sur whatsapp, qui me font toujours sourire ! Merci Justine pour les suggestions que tu m'as fait pour ce manuscrit, et évidemment un grand merci pour tous les gateaux que tu nous fais (mon TAB te remercie également☺). Je remercie également Maud, tu n'es plus au laboratoire, mais merci parce que tu as été ma première encadrante au labo et parce que tu m'as appris ta passion pour la science et la thématique du labo ! Merci Carine pour ton aide à travers ces >4 années. Merci Adéline, parce que tu prends soin de notre sécurité au labo ☺, mais aussi pour ton aide et ta patience avec tous les western blots !! Marie-Ange, j'avoue que je suis fan de ton humour même si parfois ça pique ! Je te remercie donc parce que tu me fais rire, pour nos conversations et bien-sûr toutes les commandes que tu passes pour nous. Merci aux filles, Marine, Alexane, Clara, Cécile et Laure (même si tu n'es plus au labo !). Merci parce qu'au niveau professionnel (IA, animalière ou doctorante) vous êtes top... extra motivées et toujours avec un sourire (ou pas, selon si le CFX marche ou pas ☺). Merci pour tous nos fous rires, pour le bullying aux bureaux, pour nos danses, et pour votre amitié. Un petit merci en plus pour Clara parce que tu m'as beaucoup aidé cette année avec mes manips… j'aurais été dans la miércoles si tu n'étais pas la !! Lorine et Jasmine, je n'ai pas eu le temps de trop vous connaître mais j'ai beaucoup apprécié toutes nos conversations. Je vous souhaite plein de résultats passionnants et bien sûr, beaucoup de patience. Manon et Amber, les nouvelles, j'espère qu'on pourra avoir l'opportunité de se connaître un peu plus les mois à venir☺.

Bien-sûr, mon plus grand remerciement revient à ma famille, mais ils parlent espagnol. Switch ! El "gracias" más grande que tengo para dar es para mis padres, Judith Meza Aulestia y Allan Estrada Morales. Mami, no estaría aquí si no fuera por ti. Gracias porque siempre apoyaste mis sueños (bueno, menos cuando te dije que quería ser Dj o azafata), gracias porque siempre creíste en mí y me enseñaste a siempre dar mi mejor versión. Para mí, siempre fuiste SuperWoman y tu recuerdo calienta mi corazón. Mi querido papi, tampoco estaría aquí si no fuera por ti. Gracias por tu educación. Eres la tranquilidad, la serenidad y el humor que me han ayudado a relativizar cualquier cosa que pase. Gracias por cuidarme aun cuando estamos a distancia. Te amo! Gracias a mis hermanichis muy bonichis Grace y Allan Alberto. Ñaña, gracias por enseñarme que nunca es tarde para seguir tus sueños y por enseñarme a ser valiente. Ñaño, gracias por escucharme, porque sé que siempre puedo contar contigo cuando necesito un consejo o consuelo... fuiste una de mis ayudas más importantes cuando recién me mudé a Francia <3 Un gracias para la cuñi Adri, por ser lo maximiii. Gracias a mis tías y tíos, y a mis primas y primos. Un shout out especial para mi hermosa Gaby, mi chiqui Sophie, y mi primasoff Jaimito (mi padrino de todo evento importante jaja). Los quiero un montón. Finalmente, gracias a mis abuelitas: Abuelita Blanca y Mamita Lucy. A pesar de que ya no están aquí, ustedes son mi raíz más grande; y su fortaleza y fe son ejemplo de vida para mí.

También agradezo enormemente a mis amigas, quienes, sin duda alguna, han sido también mi roca, me han llenado de cariño y me han impulsado a creer en mí. Indeed, mi mamá siempre me decía "tu sí que tienes un buen grupo de amigas, eso me deja tranquila". Gracias a mi lindo JAM. Yo sé que no entienden nada de lo que hago jajaja pero gracias por siempre apoyarme. Michelle, sabes que eres como una segunda hermana para mí. Hemos crecido juntas, pasando por lo bueno y lo malo. Una gran parte de mi te la debo a ti <3, y por cierto, best photographer ever. Mi Pinito, gracias porque el tiempo no pasa entre nosotras, así nos veamos cada 2 años. Gracias por escucharme y por confiar en mí. Mi Menéndez (mi amigui fitness), Inesita (mi Miss Teen forever), Choclita (la mejor mamá) y Ady (nuestra fitness coach), gracias por su amistad y por estar ahí para mí cuando las necesito <3 Las ami. Gracias a mis madrileñas, Roxy/chocochip y Brini y mi Munkyo. Gracias por esta amistad que ha durado tanto, y que espero mantendremos por siempre. Gracias a mi May, por tu sabiduría y lo divertida que eres. Gracias a Jael, porque literal eres una de mis mejores amigas a pesar de que nuestra amistad ha sido sobre todo virtual! Jaelingui, gracias por tus múltiples consejos. Yo sé que lograrás esos proyectos que tienes <3 Gracias a mis équatoriennes en France: Andrea/Boli, ma super témoin, et Anita. Boli, gracias por tu apoyo siempre, y por preocuparte por mí. Je vous aime! Je tiens à remercier également les petits nouveaux de l'ENS : Amaury, Marie, Marion et Mykolas. Je suis sûre que vous faites des super thèses. D'ailleurs, nos soirées « préparation oral master » étaient ouf quand même. Mykolas, love you pumpkin! Je remercie également deux enseignants/chercheurs qui m'ont profondément aidé en France: Dr. Michelle Ottmann et Dr. Hugo Aguilaniu. Je ne sais pas s'ils se rendent compte de ceci, mais il y a des nombreuses années leurs mots d'encouragement et de gentillesse ont été un facteur clé pour mon intégration en France <3.

Un grand merci à ma belle famille. Eric, Anne, Iris, Myriam et Sammy. Merci parce que vous m'avez toujours accueilli avec plein d'amour. En plus, je vous remercie en avance pour tout ce que nous vivrons par la suite ensemble ☺ ! Vous êtes une super famille, et j'ai beaucoup de chance de pouvoir vous appeler ma belle famille <3 Pour finir, merci à mon mari, Thibault. Mon petit monkey (et oui ! il ne faut pas avoir honte ! ☺). Concernant cette thèse, merci parce que tu m'as fait des superbes figures, et parce que tu m'as littéralement nourri pendant la fin de la rédaction de thèse (et tu as pris soin de l 'appartement, etc., etc.). Merci pour ton soutien, ta présence, ton écoute et parce que tu prends soin de moi. No sabemos lo que nos depara el futuro, pero confío porque sé que estaremos juntos. Te amo.

The role of intestinal gluconeogenesis on the neonatal development of hypothalamic feeding circuits

Obesity increases the risk of developing diabetes and cardiovascular disease, and is a major public health challenge. Epidemiological, clinical and experimental data have highlighted the role of the perinatal environment on the development of metabolic diseases. The perinatal period is crucial for development and is therefore a very vulnerable period. Nutritional and hormonal insults arriving during the perinatal period can severely compromise the development of key organs that regulate energy balance, like the hypothalamus, and thus jeopardize life-long metabolic health. Hypothalamic AgRP/NPY (agouti-related peptide and neuropeptide Y) and POMC/CART (pro-opiomelanocortin and cocaine-and amphetamine-regulated transcript) feeding circuits develop during the first three weeks of life in rodents, under the influence of a postnatal surge of the hormone leptin. Leptin promotes axonal outgrowth from the arcuate nucleus by stimulating the STAT3 signaling pathway. A defective development of these axonal projections is associated with an adult onset of metabolic problems.

Intestinal gluconeogenesis (IGN) exerts beneficial effects against obesity and diabetes in adults by targeting the STAT3 signaling pathway in POMC neurons from the arcuate nucleus. Interestingly, IGN doubles during the first two weeks of life in rodents. We therefore investigated whether neonatal IGN controls: 1) the development of AgRP/NPY and POMC/CART axonal projections from the arcuate nucleus; and 2) the susceptibility to develop obesity when exposed to a diet rich in fat and sugar (HFHS).

Using a murine model with an intestine-specific, inducible overexpression of the catalytic subunit of the glucose-6-phosphatase, the mandatory enzyme for gluconeogenesis, we show that the induction of IGN since birth increases the density of arcuate nucleus AgRP axonal projections to the hypothalamic paraventricular nucleus. This effect is specific to the first two weeks of life because the induction of IGN on postnatal day 12 fails to affect AgRP fiber density. The neonatal induction of IGN before postnatal day 12 also protects adult mice from becoming obese when fed a HFHS diet. These data suggest that the neonatal peak of IGN exerts a neurotrophic effect on AgRP axons from the arcuate nucleus, and that inducing IGN during the first two weeks of life protects long-term adiposity.

Key words: Intestinal gluconeogenesis, diabetes, obesity, neonatal development, hypothalamus.

Rôle de la néoglucogenèse intestinale sur le développement néonatal des circuits

hypothalamiques qui régulent la prise alimentaire L'obésité augmente le risque de développer un diabète et des maladies cardiovasculaires, et constitue un défi majeur de santé publique. Des données épidémiologiques, cliniques et expérimentales ont mis en évidence le rôle de l'environnement périnatal sur le développement des maladies métaboliques. La période périnatale est cruciale pour le développement et est donc une période très vulnérable. Des dérégulations nutritionnelles et hormonales survenant pendant la période périnatale peuvent gravement compromettre le développement des organes clés qui régulent la balance énergétique, comme l'hypothalamus, et ainsi compromettre la santé métabolique de l'individu. Les circuits hypothalamiques exprimant agouti-related peptide et neuropeptide Y (AgRP/NPY) et pro-opiomelanocortin et cocaine-and amphetamine-regulated transcript (POMC/CART) et régulant la prise alimentaire se développent au cours des trois premières semaines de vie chez les rongeurs, sous l'influence d'un pic postnatal de l'hormone leptine. Cette hormone stimule la croissance axonale du noyau arqué via la voie de signalisation STAT3. Un développement défectueux de ces projections axonales est associé à des problèmes métaboliques à l'âge adulte.

La néoglucogenèse intestinale (NGI) exerce des effets bénéfiques contre l'obésité et le diabète chez l'adulte en ciblant la voie de signalisation STAT3 dans les neurones POMC du noyau arqué. Fait intéressant, la NGI double pendant les deux premières semaines de vie chez les rongeurs. Nous avons donc étudié si la NGI néonatale contrôle: 1) le développement des projections axonales AgRP/NPY et POMC/CART du noyau arqué; et 2) la susceptibilité de devenir obèse sous un régime alimentaire riche en graisses et en sucres (HFHS).

En utilisant un modèle murin avec une surexpression inductible de la sous-unité catalytique de la glucose-6-phosphatase, enzyme nécessaire à la néoglucogenèse, spécifiquement dans l'intestin, nous montrons que l'induction de la NGI dès la naissance augmente la densité des projections axonales AgRP vers le noyau paraventriculaire hypothalamique. Cet effet est spécifique aux deux premières semaines de vie, car l'induction de la NGI le 12e jour postnatal ne modifie pas la densité des fibres AgRP. L'induction néonatale de la NGI avant le 12e jour postnatal protège les souris adultes de la prise de poids lorsqu'elles sont nourries avec un régime HFHS. Ces données suggèrent que le pic néonatal de la NGI exerce un effet neurotrophique sur les axones AgRP du noyau arqué, et que l'induction de la NGI pendant les deux premières semaines de vie protège l'adiposité de l'individu à long terme.

LIST OF ABBREVIATIONS

ACC : Acetyl-CoA Carboxylase 28, ACTH : adrenocorticotropin hormone AgRP : Agouti-related peptide 46,47,51,55,58,59,91,93,94,95,96,98, AMPK : AMP-activated protein kinase ARH : arcuate nucleus 44, 46, 48, 51, 53, 55, 58, 59, 60, 70, 71, 91, 93, 95, 96, 97, , 47, 48, 51, 52, 55, 59, 60, 70, 91, 93, 94, 95, 96, 98, PVH : paraventricular nucleus of the hypothalamus 46,49,53,59,93,94,95,96,97,98

PREFACE

The prevalence of obesity and type 2 diabetes is increasing at an alarming rate, especially among young adults. A growing body of research data is showing that neonatal exposure to adverse metabolic conditions, such as maternal obesity or diabetes, results in a predisposition of offspring to develop the metabolic syndrome during adulthood [START_REF] Dearden | Early Life Origins of Metabolic Disease: Developmental Programming of Hypothalamic Pathways Controlling Energy Homeostasis[END_REF]. Changes in the neonatal metabolic programming of brain regions involved in energy homeostasis could therefore contribute to the development of obesity and type 2 diabetes [START_REF] Bouret | Nutritional Programming of Hypothalamic Development: Critical Periods and Windows of Opportunity[END_REF]). Among these regions, the hypothalamus is an essential integrative center for the maintenance of energy balance.

An optimal hypothalamic development is imperative for correct regulation of metabolism. Importantly, the neonatal hypothalamic development is assured by different factors that are still being studied, one of the most important being leptin. During the neonatal period, the surge of leptin occurring in the first week of life is particularly needed for neuronal connections between hypothalamic nuclei [START_REF] Bouret | Trophic Action of Leptin on Hypothalamic Neurons That Regulate Feeding[END_REF].

In adults, leptin (secreted by the adipose tissue according to energy stores) decreases food intake and increases energy expenditure [START_REF] Schwartz | Central Nervous System Control of Food Intake[END_REF].

Intestinal gluconeogenesis (IGN) is another function decreasing food intake and increasing energy expenditure in adults. Intestinal gluconeogenesis transmits signals to the hypothalamus in response to the nutritional environment (Soty et al. 2017), through the detection of glucose produced by the portal vein nervous system (Duraffourd et al. 2012).

Induction of IGN can protect against diabetes and obesity induced by a diet rich in fats and sugars [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF][START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF]. In contrast, the absence of IGN is sufficient to induce a pre-diabetic state, in particular by inducing an alteration of the homeostatic function of the hypothalamus [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF]. Interestingly, as leptin does, IGN exhibits a surge of activity during the second week of life [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF].

The purpose of this thesis was to demonstrate that IGN, which seems to mimic the effects of leptin in adults, could therefore exert the same functions than leptin on hypothalamic development and plasticity.

INTRODUCTION

23

Chapter 1. Glucose homeostasis

Homeostasis is defined as a relatively stable state of equilibrium. The roots of the modern conception of homeostasis go back to around the year 1850, with Claude Bernard's beliefs that an organism has the capacity to actively stabilize its body states against external disturbances [START_REF] Bernard | Acute Hyperinsulinemia and Its Reversal by Vagotomy after Lesions of the Ventromedial Hypothalamus in Anesthetized Rats*[END_REF][START_REF] Cooper | From Claude Bernard to Walter Cannon. Emergence of the Concept of Homeostasis[END_REF]. Glucose homeostasis, or blood glucose control, is a physiological need that keeps glycaemia at around 0.9-1 g/L, when it is functioning optimally. On the one hand, more than a few minutes of hypoglycaemia could severely damage organs that are highly metabolically active and that therefore need a constant flux of energy substrate. Consistently, a 6-8% drop in blood glucose is already enough for rats to decide to eat (Louis-Sylvestre and Magnen 1996). On the other hand, prolonged hyperglycemia is a major risk factor for life-threatening conditions like diabetes and diabetes-related complications such as neuropathy, kidney failure, diabetic retinopathy and cardiovascular diseases.

But why is maintaining glucose at a constant level so important from a molecular point of view? Glucose is popularly called "the molecule of life". However, contrary to popular belief, this is not due to its energetic value. Glucose is indeed the only source of energy for erythrocytes [START_REF] Brown | Erythrocyte Metabolism and Enzyme Defects[END_REF]. However, most organs do not rely on the complete oxidation of the six-carbon molecule glucose for energy. Organs that are highly metabolically active like the brain [START_REF] Owen | Brain Metabolism during Fasting*[END_REF], the heart, the kidneys and the gut [START_REF] Windmueller | Identification of Ketone Bodies and Glutamine as the Major Respiratory Fuels in Vivo for Postabsorptive Rat Small Intestine[END_REF] can actually use ketone bodies as fuels when glucose supply from food is scarce, allowing the body to preserve glucose. The need to maintain glucose is not driven only for providing energy. Glucose needs to be maintained at a constant level also because its use in non-oxidative glucose metabolic pathways, like glycolysis and the pentose shunt, provides carbon skeletons for vital functions like nucleotide synthesis and chromosomal duplication during cell renewal (Soty et al. 2017). Glucose homeostasis is thus a physiological function that is constantly and finely regulated.

Nutritional glucose supply varies throughout the life of an animal, ranging from an excessive or a normal food intake, to fasting and starvation. Diverse metabolic processes allowing adaptation to these different circumstances have thus evolved. These metabolic processes are life-long regulated by nutritional, hormonal and environmental cues, and are also programmed during the perinatal period. During the post-prandial period (i.e. right after a meal), the rich influx of energy substrates leads to an increment of glycaemia and the activation of metabolic pathways that stock glucose as glycogen and lipids. On the contrary, when food becomes scarce (such as during fasting), glycaemia is maintained at 0.9-1 g/L via the activation of metabolic pathways involved in endogenous glucose production: beginning with the breakdown of hepatic glycogen stores (i.e. glycogenolysis) and followed by the de novo production of glucose (i.e. gluconeogenesis) in the liver [START_REF] Bernard | Acute Hyperinsulinemia and Its Reversal by Vagotomy after Lesions of the Ventromedial Hypothalamus in Anesthetized Rats*[END_REF], the kidneys [START_REF] Krebs | RENAL GLUCONEOGENESIS[END_REF]) and the intestine [START_REF] Croset | Rat Small Intestine Is an Insulin-Sensitive Gluconeogenic Organ[END_REF][START_REF] Rajas | The Glucose-6 Phosphatase Gene Is Expressed in Human and Rat Small Intestine: Regulation of Expression in Fasted and Diabetic Rats[END_REF]).

The post-prandial period

The pancreas is a mixed gland containing both an exocrine region that secretes digestive enzymes, and an endocrine region (the islets of Langerhans) that produces and releases the hypoglycaemic hormone insulin, the hyperglycaemic hormone glucagon and somatostatin, among others. These three hormones regulate glucose homeostasis.

The post-prandial period is the time frame right after a meal. In the post-prandial period, glycaemia increases and metabolic pathways that allow a decrease in glycaemia are immediately stimulated. The post-prandial period is therefore characterized by an increased plasma insulin/glucagon ratio. As glycaemia increases, glucose is transported into pancreatic β-cells from the Langerhans islets via the glucose transporter 2 (GLUT2), which leads to the exocytosis of vesicles containing the hypoglycaemic hormone insulin. Insulin is an anabolic hormone that stimulates energy storage and inhibits energy release, therefore decreasing glycaemia [START_REF] Gathercole | Hormonal Regulation of Lipogenesis[END_REF]. Circulating insulin stimulates glucose uptake in the liver via GLUT2, and in the muscle and in the adipose tissue via GLUT4. Once in the cell, one glucose molecule is oxidized into two pyruvate molecules in the glycolysis pathway, paving the way for the Krebs cycle and the respiratory chain in the mitochondria.

The excess of glucose is then stored as two forms of energy reserves: glycogen via the glycogenesis pathway mainly in the liver and in the skeletal muscle, and as lipids via the lipogenic pathway mainly in the liver and in the white adipose tissue.

Glycogenesis

Glycogenesis is the synthesis of glycogen. This physiological function takes place in small quantities in several organs like in brain astrocytes [START_REF] Brewer | Brain Glycogen Structure and Its Associated Proteins: Past, Present and Future[END_REF][START_REF] Rich | The Role of Brain Glycogen in Supporting Physiological Function[END_REF] and cardiomyocytes [START_REF] Chandramouli | Myocardial Glycogen Dynamics: New Perspectives on Disease Mechanisms[END_REF]. However, the major sites of an organism's glycogen stores are in the liver and in the skeletal muscle. The glycogen stores present in the skeletal muscle provide glucose only for local use because muscle lacks the mandatory enzyme for endogenous glucose production, glucose-6phosphatase. Glycogen stores in the liver are, in contrast, more quantitatively relevant for glucose homeostasis as it provides glucose, directly released in the bloodstream, through the process of glycogenolysis during the post-absorptive period.

Glycogen is a polymer of glucose molecules present in animals, fungi and bacteria.

Glycogenesis implicates several enzymes, which expression and activity are tightly controlled by hormones via phosphorylation, and by glucose-6-phosphate (G6P) (which will not be detailed here) (Figure 1). As glucose enters the hepatocyte, Hexokinase IV (or Glucokinase) phosphorylates the carbon 6 of glucose, converting it into G6P. Then, a phosphoglucomutase transfers the phosphate group from carbon 6 to carbon 1, forming glucose-1-phosphate. Glucose-1-phosphate is then activated by UDP-glucose pyrophosphorylase by joining it to a nucleotide, which forms uridine diphosphate glucose (UDPG), a nucleotide sugar. Glycogen synthase then adds UDPG to an already existing glycogen branch, by forming an α1→4 bond. Once the glycogen chain has at least ten glucose molecules, the branching enzyme creates an α1→6 bond with another glycogen chain. The sum of these processes results in the polysaccharide structure of glycogen. However, given that glycogen synthase activity requires an existing glycogen chain, this would be problematic whenever glycogen stores are depleted in an animal, such as after a long period of famine, and then the animal eats again. Therefore, an organism requires another very important glycogenesis enzyme called glycogenin. Glycogenin is an initiator protein and, in an autocatalytic process, acts as a receiver of a first-arriving UDPG and can then form a shorter chain of glucose molecules bound by α1→4 bonds [START_REF] Blanco | Medical Biochemistry[END_REF].

Figure 1. Glycogenesis.

Glucose is converted to glucose-6-phosphate by the Glucokinase. Glucose-6-phosphate is then converted to glucose-1-phosphate by the Phosphoglucomutase. A residue of Uridine diphosphate (UDP) is next transferred by the UDP-glucose pyrophosphorylase, leading to the formation of UDP-glucose. UDP-glucose is then attached to the glycogen polymer via the glycogen synthase, releasing UDP and therefore elongating the linear chain of glycogen.

It is important to note that glycogen is a short-term energy store and it can be substantially depleted after a relatively short period of time, such as two days of carbohydrate restriction [START_REF] Acheson | Glycogen Storage Capacity and de Novo Lipogenesis during Massive Carbohydrate Overfeeding in Man[END_REF], two hours of high-intensity training [START_REF] Gonzalez | Liver Glycogen Metabolism during and after Prolonged Endurance-Type Exercise[END_REF] or 24 hours of fasting [START_REF] Nilsson | Liver Glycogen in Man--the Effect of Total Starvation or a Carbohydrate-Poor Diet Followed by Carbohydrate Refeeding[END_REF] in humans. The long-term energy reserve in an organism is its fat mass. In this sense, Acheson and colleagues showed that healthy humans that ate a carbohydrate-rich diet stored more than 15 g/kg of body weight of hepatic glycogen, but after this point, the hepatic metabolism turned mainly towards de novo lipogenesis [START_REF] Acheson | Glycogen Storage Capacity and de Novo Lipogenesis during Massive Carbohydrate Overfeeding in Man[END_REF]).

De novo lipogenesis

Lipogenesis is the synthesis of fatty acids and triglycerides. In healthy animals, lipogenesis has been reported in tissues like the hypothalamus [START_REF] López | Brain Lipogenesis and Regulation of Energy Metabolism[END_REF], the mammary gland [START_REF] Mercer | The Regulation of Lipogenesis in Vivo in the Lactating Mammary Gland of the Rat during the Starved-Refed Transition. Studies Wtih Acarbose, a Glucosidase Inhibitor[END_REF][START_REF] Mohammad | De Novo Synthesis of Milk Triglycerides in Humans[END_REF], the skeletal muscle [START_REF] Gathercole | Hormonal Regulation of Lipogenesis[END_REF], the adipose tissue and the liver. However, both hepatic and adipose tissue lipogenesis have been the most thoroughly described regarding glucose homeostasis. Between both latter organs, it has been shown that, in Humans, hepatic de novo lipogenesis is more stimulated by a carbohydrate-rich diet and contributes substantially more to triglyceride storage when compared to adipose tissue lipogenesis [START_REF] Diraison | Differences in the Regulation of Adipose Tissue and Liver Lipogenesis by Carbohydrates in Humans[END_REF]. For this reason, the hepatic lipogenesis pathway will be the one described below (Figure 2).

As mentioned before, once hepatic glycogen stores reach a certain point, de novo (FAS). Next, Palmitate can be elongated by the successive addition of two carbons at a time by Elongases like the elongation of very long chain fatty acids protein 6 (ELOVL6). These longer-chain fatty acids can then be desaturated in the endoplasmic reticulum by desaturases that add double bonds between the fatty acid carbons such as the Stearoyl-CoA desaturase (SCD1). Three fatty acids are then esterified to glycerol to form triglycerides, before being packaged into Very-Low-Density Lipoproteins (VLDL). VLDL are then secreted by the liver into the bloodstream where they will reach other tissues, such as the white adipose tissue for fat storage [START_REF] Blanco | Medical Biochemistry[END_REF][START_REF] Postic | The Role of the Lipogenic Pathway in the Development of Hepatic Steatosis[END_REF].

lipogenesis
Lipogenesis is mainly regulated by the two hormones insulin and glucagon, but also by glucose. Both hormones regulate lipogenesis by acting at the transcriptional and posttranslational levels. Insulin (and glucose) stimulates lipogenesis, whereas glucagon inhibits it. Right after a meal, lipogenesis is stimulated due to the increase of the insulin/glucagon ratio. Insulin activates the sterol regulatory element binding protein 1 (SREBP1), which binds to the promoters of the main regulatory genes of glycolysis and lipogenesis (coding for ACC, FAS and GLUCOKINASE) [START_REF] Foufelle | New Perspectives in the Regulation of Hepatic Glycolytic and Lipogenic Genes by Insulin and Glucose: A Role for the Transcription Factor Sterol Regulatory Element Binding Protein-1c[END_REF]. Plus, glucose, or its metabolites such as G6P, activates the carbohydrate responsive element binding protein (ChREBP), which also activates the transcription of lipogenic genes (such as ACC, FAS, and SCD1) [START_REF] Abdul-Wahed | Sweet Sixteenth for ChREBP: Established Roles and Future Goals[END_REF]. Importantly, an excessive liver de novo lipogenesis is one of the mechanisms leading to ectopic fat accumulation in the liver [START_REF] Nassir | Pathogenesis and Prevention of Hepatic Steatosis[END_REF][START_REF] Postic | The Role of the Lipogenic Pathway in the Development of Hepatic Steatosis[END_REF]. The resulting hepatic steatosis occurs when intrahepatic lipids reach at least 5% of liver weight [START_REF] Nassir | Pathogenesis and Prevention of Hepatic Steatosis[END_REF], and, in some individuals, it can progress into non-alcoholic steatohepatitis (NASH), which is a condition in which the excess of fat in the hepatocytes is associated with hepatic inflammation and/or fibrosis.

The endocrine system regulates de novo lipogenesis through a concerted action of not only insulin and glucagon, but also an array of other hormones. Glucocorticoids, the growth hormone, estrogens, dehydroepiandrosterone, catecholamines, leptin and glucagon-like peptide-1 (GLP-1) also inhibit de novo lipogenesis and fat accumulation; whereas progesterone, androgens and ghrelin promote this pathway [START_REF] Gathercole | Hormonal Regulation of Lipogenesis[END_REF].

However, the regulation of the secretion of these hormones is complex and will not be described here.

The post-absorptive period

As an organism awaits its next meal, the nutritional glucose supply decreases. This moment is known as the post-absorptive period, or the beginning of the fasting period, which is when the food ingested has already been digested, absorbed and stored. Without nutritional glucose supply, glycaemia naturally tends to decrease because blood glucose continues to be used by tissues. In this scenario, glycaemia is maintained constant by the activation of endogenous glucose production (EGP) (Figure 3A). Endogenous glucose production is the production of glucose by an organism, which is a physiological function tightly regulated by the nutritional state and the endocrine system. In this sense, EGP is inhibited during the post-prandial period by insulin; and stimulated during the postabsorptive period by counterregulatory hormones like glucagon, catecholamines and glucocorticoids. Endogenous glucose production comes from glycogenolysis and gluconeogenesis. A key enzyme, glucose-6-phosphatase (G6Pase), catalyzes the final step of both glycogenolysis and gluconeogenesis (Figure 3B).

Glycogenolysis

Glycogenolysis is the breakdown of glycogen. During a short fast, such as 6 hours in rats or 12 hours in humans, hepatic glycogenolysis provides about 70-75% of EGP when the last meal was a standard, starch-based diet [START_REF] Mithieux | Adaptation of Hepatic, Renal and Intestinal Gluconeogenesis During Food Deprivation[END_REF])(Figure 4). Briefly, glycogen phosphorylase liberates glucose-1-phosphate, which is then transformed into G6P by the phosphoglucomutase. Then, G6Pase dephosphorylates G6P into glucose + inorganic phosphate. Glucose is then released into the bloodstream mainly via liver GLUT2 transporters, thereby maintaining glycaemia at a constant level.

As the post-absorptive period continues and a prolonged fasting takes place, glycogen stores risk complete depletion. In this scenario, gluconeogenesis is strongly stimulated in the three gluconeogenic tissues to maintain glycaemia at 0.9-1 g/L.

Figure 3. Endogenous glucose production and the glucose-6-phosphatase complex.

(A) The endogenous glucose production is assured by glycogenolysis, taking place mainly in the liver, and gluconeogenesis, taking place in the liver, the kidneys and the intestine. (B) The glucose-6-phosphatase is composed of two sub-units. The translocase (G6PT) transports glucose-6-phosphate from the cytosol to the endoplasmic reticulum where the catalytic subunit (G6PC) hydrolyses the glucose-6-phosphate into glucose and inorganic phosphate (Pi). Illustration made with Servier Medical Art.

Gluconeogenesis

Gluconeogenesis is the de novo production of glucose from non-carbohydrate substrates (Figure 5). This pathway shares almost all enzymes with the glycolysis pathway but in the opposite sense. However, three enzymatic reactions are irreversible and unique to the gluconeogenesis pathway: ( Importantly, all of the above mentioned enzymes are present in several tissues, except for G6Pase. G6Pase is a functional complex, composed of two sub-units. The translocase (G6PT) is ubiquitous and transports G6P from the cytosol to the endoplasmic reticulum. The catalytic subunit of G6Pase (G6PC, and coded by G6pc) is present only in the liver, the kidneys and the intestine [START_REF] Rajas | The Glucose-6 Phosphatase Gene Is Expressed in Human and Rat Small Intestine: Regulation of Expression in Fasted and Diabetic Rats[END_REF], making them the only gluconeogenic organs (Figure 3A). The expression of G6pc is therefore considered as mandatory for EGP.

All three gluconeogenic organs are necessary for glycaemic control, but each organ exerts a different and complementary role in glucose homeostasis. In this sense, the transition from the post-absorptive period to a prolonged fasting is characterized by a redistribution of glucose production among liver, renal and intestinal gluconeogenesis (Figure 4) (Soty et al. 2017).

Hepatic gluconeogenesis

In quantitative terms, the liver is the organ that produces glucose the most. After hepatic glycogen stores are almost depleted due to a prolonged fasting, the liver synthesizes glucose mainly from lactate (coming from an anaerobic metabolism) and alanine (coming from muscle proteolysis) in an endergonic process, and glycerol (coming from adipose tissue lipolysis) (Soty et al. 2017). Through this pathway, the contribution of the liver to EGP decreases from about 70-75% during glycogenolysis to about 20-25% during a prolonged fasting (Figure 4). When animals are fed a standard starch-based diet, glucose production from the kidney represents about 15% to 20% of the endogenous glucose production (EGP) at the beginning of the post-absorptive period. Kidney glucose production increases to about 50% at 24 hours of fasting [START_REF] Pillot | Protein Feeding Promotes Redistribution of Endogenous Glucose Production to the Kidney and Potentiates Its Suppression by Insulin[END_REF]. Intestinal glucose production accounts for about 5% to 7% of EGP at the beginning of the post-absorptive period and increases to about 20 to 25% at 24 hours of fasting in rats [START_REF] Croset | Rat Small Intestine Is an Insulin-Sensitive Gluconeogenic Organ[END_REF][START_REF] Mithieux | Contribution of Intestine and Kidney to Glucose Fluxes in Different Nutritional States in Rat[END_REF]. Comparable augmentations of the contributions of the intestine and the kidney take place upon eating a protein-enriched diet, a situation in which the intestine accounts for about 20% and the kidney about 45% of EGP at the beginning of the post-absorptive period [START_REF] Pillot | Protein Feeding Promotes Redistribution of Endogenous Glucose Production to the Kidney and Potentiates Its Suppression by Insulin[END_REF]. A comparable contribution of the intestine to about 20% of EGP was measured upon a fiber-enriched diet [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF]. Kidney glucose production was not determined in this situation. (From Soty et al., 2017). Gluconeogenic substrates (glycerol, alanine, lactate, glutamine and propionate, in blue boxes) enter the gluconeogenesis pathway. Enzymes specific to gluconeogenesis are written in blue. Oxaloacetate is transported to the cytosol through the malate/aspartate shuttle. For comparative purposes, enzymes specific to glycolysis are written in yellow.

Although hepatic gluconeogenesis is an important contributor to EGP in physiological conditions, an excess of hepatic gluconeogenesis is actually a hallmark of type 2 diabetes as its excessive stimulation contributes to hyperglycaemia [START_REF] Clore | Glucose-6-Phosphatase Flux in Vitro Is Increased in Type 2 Diabetes[END_REF][START_REF] Magnusson | Increased Rate of Gluconeogenesis in Type II Diabetes Mellitus. A 13C Nuclear Magnetic Resonance Study[END_REF]). To determine the contribution of an increased hepatic glucose production to the onset of diabetes, a study in rats showed that temporally overexpressing the hepatic G6Pase was enough to promote glucose intolerance, insulin resistance and ectopic triglyceride stores [START_REF] Trinh | Perturbation of Fuel Homeostasis Caused by Overexpression of the Glucose-6-Phosphatase Catalytic Subunit in Liver of Normal Rats[END_REF]). Hepatic gluconeogenesis is therefore a target of research teams investigating drugs that reduce glucose production to treat type 2 diabetes patients [START_REF] Petersen | Regulation of Hepatic Glucose Metabolism in Health and Disease[END_REF]. Interestingly, studies have suggested that hepatic gluconeogenesis is not vital for mice. Our laboratory demonstrated that mice lacking G6PC specifically in the liver, and therefore lacking hepatic glucose production only, had normal glycaemia during the fed and the prolonged fasting states because gluconeogenesis was assured by both renal and intestinal gluconeogenesis [START_REF] Mutel | Control of Blood Glucose in the Absence of Hepatic Glucose Production during Prolonged Fasting in Mice: Induction of Renal and Intestinal Gluconeogenesis by Glucagon[END_REF][START_REF] Penhoat | Intestinal Gluconeogenesis Is Crucial to Maintain a Physiological Fasting Glycemia in the Absence of Hepatic Glucose Production in Mice[END_REF]. What is more, mice lacking hepatic glucose production exhibited hypoglycaemia during the post-absorptive period because they were unable to mobilize hepatic glycogen stores, but were actually protected from obesity and diabetes when fed a high-fat/highsucrose diet [START_REF] Abdul-Wahed | A Link between Hepatic Glucose Production and Peripheral Energy Metabolism via Hepatokines[END_REF]. Hepatic glucose production is thus a significant participant of EGP, but its excessive stimulation can be harmful for glucose homeostasis and health.

Renal gluconeogenesis

Whereas hepatic gluconeogenesis decreases during the transition from the postabsorptive period to a prolonged fasting, renal gluconeogenesis is actually increased. Indeed, Mithieux and colleagues showed that renal gluconeogenesis accounted for about 15% of total EGP in rats during the post-absorptive period, but its contribution rose to about 50% when the fast was prolonged to 24 hours (Figure 4) [START_REF] Mithieux | Contribution of Intestine and Kidney to Glucose Fluxes in Different Nutritional States in Rat[END_REF]. Importantly, in the fed and fasted states, renal and hepatic gluconeogenesis differ not only in quantitative terms but in qualitative terms too. In contrast to hepatic gluconeogenesis, the main precursor of renal gluconeogenesis is glutamine (coming from muscle proteolysis) and, to a lesser extent, from lactate [START_REF] Meyer | Renal Substrate Exchange and Gluconeogenesis in Normal Postabsorptive Humans[END_REF][START_REF] Stumvoll | The human kidney as an important producer of glucose[END_REF]. The reaction coming from glutamine is exergonic and produces 4 ATP per mole of glucose synthesized [START_REF] Mithieux | A Novel Role for Glucose 6-Phosphatase in the Small Intestine in the Control of Glucose Homeostasis[END_REF]Soty et al. 2017). Thus, the redistribution from hepatic to renal gluconeogenesis as the fasting state is prolonged allows the body to simultaneously preserve the energetic status of the organism and maintain glycaemia at a constant level (Soty et al. 2017). Interestingly, Penhoat and colleagues demonstrated that in a mouse model invalidated for both liver and intestinal gluconeogenesis, albeit mice had low physiological range glycaemia (0.6 g/L) during a prolonged fasting, it was not a pathophysiological range, suggesting that renal gluconeogenesis can, on its own, assure glycaemic control in the prolonged-fasting state [START_REF] Penhoat | Intestinal Gluconeogenesis Is Crucial to Maintain a Physiological Fasting Glycemia in the Absence of Hepatic Glucose Production in Mice[END_REF]. Furthermore, a deficiency of renal glucose production, such as during chronic kidney disease, promotes a redistribution of EGP among gluconeogenic organs [START_REF] Kaneko | The Role of Kidney in the Inter-Organ Coordination of Endogenous Glucose Production during Fasting[END_REF]. The deficient renal glucose production promotes intestinal gluconeogenesis, probably via the vitamin D system, and leads to glycogen sparing in the liver [START_REF] Kaneko | The Role of Kidney in the Inter-Organ Coordination of Endogenous Glucose Production during Fasting[END_REF].

Besides a prolonged fast, certain diets can also promote a redistribution of glucose production among gluconeogenic organs (Figure 4). This is the case when consuming a protein-enriched diet, which is considered a nutritional strategy for diabetes management as it promotes satiety and glycaemic control [START_REF] Ajala | Systematic Review and Meta-Analysis of Different Dietary Approaches to the Management of Type 2 Diabetes[END_REF][START_REF] Gannon | Effect of a High-Protein, Low-Carbohydrate Diet on Blood Glucose Control in People with Type 2 Diabetes[END_REF]. Indeed, rats fed a protein-enriched diet show a dramatic induction of renal gluconeogenesis, but not hepatic gluconeogenesis [START_REF] Pillot | Protein Feeding Promotes Redistribution of Endogenous Glucose Production to the Kidney and Potentiates Its Suppression by Insulin[END_REF]). These modifications were associated with an increased suppression of hepatic glucose production (glycogenolysis and gluconeogenesis) by insulin, which is in line with the improved hepatic insulin sensitivity observed in animals fed a protein-enriched diet.

Intestinal gluconeogenesis

The liver and the kidney were considered the only gluconeogenic organs for a long time. It was in 1999 that Rajas and colleagues published for the first time that G6pc is also expressed in the intestine of rats and Humans [START_REF] Rajas | The Glucose-6 Phosphatase Gene Is Expressed in Human and Rat Small Intestine: Regulation of Expression in Fasted and Diabetic Rats[END_REF]. As for the kidney, intestinal gluconeogenesis is increased from the post-absorptive period to the prolonged fasting state, at the expense of hepatic gluconeogenesis (Figure 4). Indeed, in the post-absorptive state, intestinal gluconeogenesis accounts for about 5-7% of total EGP, when consuming a standard starch-based diet. As the fasting state is prolonged and the redistribution of glucose production among gluconeogenic organs takes place, intestinal gluconeogenesis increases and contributes in about 20% to EGP [START_REF] Croset | Rat Small Intestine Is an Insulin-Sensitive Gluconeogenic Organ[END_REF].

Importantly, intestinal gluconeogenesis has several specific features that make it particularly interesting for glucose homeostasis and thus, metabolic health. In first place, in contrast to the liver and in similitude with the kidneys, the precursors of intestinal gluconeogenesis are mainly glutamine and, to a much lesser extent, glycerol [START_REF] Croset | Rat Small Intestine Is an Insulin-Sensitive Gluconeogenic Organ[END_REF][START_REF] Mithieux | New Data and Concepts on Glutamine and Glucose Metabolism in the Gut[END_REF]. Most of intestinal gluconeogenesis is thus an exergonic process, allowing the body to simultaneously preserve its energetic status and maintain glycaemia constant [START_REF] Hartmann | Intestinal Glutamine Metabolism[END_REF][START_REF] Mithieux | New Data and Concepts on Glutamine and Glucose Metabolism in the Gut[END_REF]. For information, the intestine can also produce glucose from propionate and succinate [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF][START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF]. In second place, a specific feature of the intestine is that it oxidizes glutamine and glucose incompletely.

An incomplete glutamine and glucose oxidation allows the body to preserve carbon skeletons for essential body functions, as previously explained [START_REF] Mithieux | New Data and Concepts on Glutamine and Glucose Metabolism in the Gut[END_REF]Soty et al. 2017). In third place, intestinal gluconeogenesis allows the body to preserve its gluconeogenic potential because it releases lactate into the bloodstream, which can be used by the liver and, to a lesser extent, the kidney gluconeogenic pathways [START_REF] Hartmann | Intestinal Glutamine Metabolism[END_REF][START_REF] Mithieux | New Data and Concepts on Glutamine and Glucose Metabolism in the Gut[END_REF]Soty et al. 2017).

Importantly, the role of intestinal gluconeogenesis is not restricted to the maintenance of glycaemia during a prolonged fasting. Intestinal gluconeogenesis can also be progressively stimulated during the post-prandial period by specific nutrients, leading to a persistence of intestinal gluconeogenesis during the post-absorptive period [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF][START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF]Duraffourd et al. 2012;[START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF]. Given that the intestine is located upstream of the portal nervous system, it has the capacity to trigger a gut-brain neural signal. This signal launches a neural circuit that plays a major role in the maintenance of energy homeostasis (Soty et al. 2017). This latter feature will be thoroughly described in chapter 3.

To conclude, EGP is an essential part of glucose homeostasis. Indeed, the lack of G6Pase, the key enzyme that catalyzes the final step of both glycogenolysis and gluconeogenesis, leads to a rare disease called glycogen storage disease type 1 (GSD1). GSD1

patients develop severe, life-threatening hypoglycaemia during short fasting periods [START_REF] Rajas | Lessons from New Mouse Models of Glycogen Storage Disease Type 1a in Relation to the Time Course and Organ Specificity of the Disease[END_REF][START_REF] Rajas | Glucose-6 Phosphate, A Central Hub for Liver Carbohydrate Metabolism[END_REF]. Moreover, the regulation of glucose homeostasis requires the concerted action of gluconeogenesis in the liver, kidney and intestine. The capacity that the organism has to redistribute the quantitative contribution of each gluconeogenic organ to total EGP allows for short-term and long-term regulation of glucose homeostasis. Disequilibrium in this distribution participates in the development of type 2 diabetes.

Chapter 2. Central control of food intake

Glucose homeostasis depends on the two pillars of energy balance: food intake and energy expenditure. The regulation of these two pillars requires constant communication between the endocrine system and the nervous system, which is a field of study known as neuroendocrinology. Regarding energy balance, the endocrine system signals the brain about the energetic status of an individual, and the brain then controls hormonal secretion of different tissues in order to regulate energy balance. This chapter will first describe the areas involved in the regulation of food intake and energy expenditure within the hypothalamus and the brainstem, which act as centers that integrate peripheral information about the energetic status of an organism. Then, the major hormonal cues necessary for this regulation will be described.

The hypothalamus

The hypothalamus (Figure 6) is located in the basis of the brain and, as a major integrative center of the body, it links the nervous system with the endocrine system through the pituitary gland. The hypothalamus regulates basic life functions like reproduction, the circadian rhythm, body temperature, immune defense, aggressive behavior, and food intake and energy expenditure. In accordance with its vital roles, the hypothalamus exists in all vertebrate nervous systems, and its neuronal circuits are well conserved across vertebrate species [START_REF] Boswell | Regulation of Agouti-Related Protein and Pro-Opiomelanocortin Gene Expression in the Avian Arcuate Nucleus[END_REF][START_REF] Saper | The Hypothalamus[END_REF].

Influx of nutritional and hormonal cues through the median eminence

The localization of the hypothalamus allows it to constantly receive nutritional and hormonal cues that signal about the energetic status of the organism. The hypothalamus encircles the third ventricle, below which it contains a circumventricular organ called the median eminence (Figure 6). In contrast to most areas of the brain, the median eminence lacks a regular blood-brain barrier, and is highly permeable to blood-born molecules. The structure of the median eminence is mainly made of specialized glial cells called tanycytes which cell bodies line the wall of the third ventricle, and their projections are in contact with fenestrated capillaries from the hypophyseal portal blood system (Figure 7) [START_REF] Balland | Hypothalamic Tanycytes Are an ERK-Gated Conduit for Leptin into the Brain[END_REF][START_REF] Langlet | Tanycyte-like Cells Form a Blood-Cerebrospinal Fluid Barrier in the Circumventricular Organs of the Mouse Brain[END_REF]. The close proximity with fenestrated capillaries is important because these capillaries lack the characteristic tight junction complexes of [START_REF] Dehouck | Plasticity of the bloodhypothalamus barrier: a role in energy homeostasis[END_REF]. Therefore, while the blood-brain barrier protects the brain microenvironment by requiring a polarized and receptor-mediated transport through endothelial cells [START_REF] Neuwelt | Engaging Neuroscience to Advance Translational Research in Brain Barrier Biology[END_REF], the median eminence allows a passive and non-selective diffusion of molecules [START_REF] Prevot | The Versatile Tanycyte: A Hypothalamic Integrator of Reproduction and Energy Metabolism[END_REF]. The structure of this blood-hypothalamus barrier has been shown to be plastic and it changes according to the nutritional status of the organism [START_REF] Langlet | Tanycytic VEGF-A Boosts Blood-Hypothalamus Barrier Plasticity and Access of Metabolic Signals to the Arcuate Nucleus in Response to Fasting[END_REF]. In this sense, the post-absorptive period is associated with an increased permeability of the fenestrated capillaries from the median eminence [START_REF] Langlet | Tanycytic VEGF-A Boosts Blood-Hypothalamus Barrier Plasticity and Access of Metabolic Signals to the Arcuate Nucleus in Response to Fasting[END_REF][START_REF] Prevot | The Versatile Tanycyte: A Hypothalamic Integrator of Reproduction and Energy Metabolism[END_REF], resulting in an improved access of homeostatic cues to the arcuate nucleus of the hypothalamus (ARH). It is important to note that this specific transport feature not only allows the access of blood-born cues into the ARH [START_REF] Schaeffer | Rapid Sensing of Circulating Ghrelin by Hypothalamic Appetite-Modifying Neurons[END_REF], but it also allows the rapid diffusion of neuro-hormones released by hypothalamic neuro-endocrine axons into the hypophyseal portal blood system [START_REF] Ciofi | Brain-Endocrine Interactions: A Microvascular Route in the Mediobasal Hypothalamus[END_REF].

As explained in the first chapter, even a few minutes of severe hypoglycaemia could kill an individual. Given that energy homeostasis requires a highly dynamic, reactive and tightly controlled regulation, it seems reasonable that brain areas controlling energy balance would need to be in constant communication with peripheral cues about the energy homeostasis status of the animal. In this respect, it is reasonable that the hypothalamus would have a facilitated access to circulating homeostatic factors like nutritional and hormonal cues. This is thus possible in part by its direct contact with tanycytes from the median eminence. These data therefore suggest that tanycytes in the median eminence are of crucial importance for the hypothalamic control of food intake (For a review, see [START_REF] Prevot | The Versatile Tanycyte: A Hypothalamic Integrator of Reproduction and Energy Metabolism[END_REF].

Hypothalamic nuclei

The mammalian hypothalamus has more than forty histologically different bilateral nuclei that receive signals about the energy status of the organism and subsequently release factors that influence energy balance. Some of these nuclei are further divided into sub-nuclei [START_REF] Simerly | Anatomical Substrates of Hypothalamic Integration[END_REF]. Experiments using physical lesions of specific hypothalamic nuclei, and studies using conditional, neuron-specific gene targeting strategies have highlighted that the regulation of energy homeostasis engages an interconnected neural network in the hypothalamus involving specific nuclei: the arcuate nucleus (ARH), the ventromedial nucleus (VMH), the dorsomedial nucleus (DMH), the lateral hypothalamic area (LH) and the paraventricular nucleus (PVH) (Figure 6).

The arcuate nucleus and neuropeptides implicated in food intake control

The ARH is located right above the median eminence. Its localization allows the ARH to integrate blood-born signals of hunger and satiety that reflect energy stores of the body, and then send neuro-peptidergic signals to intra-hypothalamic nuclei, as well as the brainstem and the cortico-limbic system, ultimately leading to energy balance regulation [START_REF] Berthoud | Multiple Neural Systems Controlling Food Intake and Body Weight[END_REF]. The neuronal anatomy and neuro-peptide expression of the ARH is generally well conserved across vertebrate taxa [START_REF] Boswell | Regulation of Agouti-Related Protein and Pro-Opiomelanocortin Gene Expression in the Avian Arcuate Nucleus[END_REF].

The ARH contains several neuronal populations that express different neuro-peptides and harbors a part of the central melanocortin system (Figure 6). The central melanocortin system is probably the best characterized neuronal circuit implicated in the regulation of energy balance. It contains: (1) neurons from the ARH containing either proopiomelanocortin and cocaine amphetamine-related transcript peptides (POMC/CART neurons) or Agouti-related peptide and neuropeptide-Y (AgRP/NPY neurons); (2) brainstem POMC neurons from the nucleus of the solitary tract (NTS) in the dorsal vagal complex (DVC); and (3) downstream targets of POMC/CART and AgRP/NPY neurons that express the melanocortin-3 receptor (MC3R) and the melanocortin-4 receptor (MC4R) (For a review, see [START_REF] Cone | Anatomy and Regulation of the Central Melanocortin System[END_REF]. Anorexigenic POMC/CART and orexigenic AgRP/NPY neurons will be the neuronal populations described below.

The first neuronal population is generally defined as co-expressing POMC and CART peptides, although studies suggest that some sub-populations of POMC/CART neurons may also express the inhibitory neurotransmitter ү-aminobutyric acid (GABA) or the excitatory neurotransmitter glutamate [START_REF] Wittmann | Distinct Glutamatergic and GABAergic Subsets of Hypothalamic Pro-Opiomelanocortin Neurons Revealed by in Situ Hybridization in Male Rats and Mice: Glutamate and GABA In Rat and Mouse POMC Neurons[END_REF]. The cleavage of POMC produces the opioid peptide β-endorphin and melanocortin peptides like the adrenocorticotropin hormone (ACTH), and α-, βand ү-melanocyte-stimulating hormones (MSH). The depolarization of POMC/CART neurons leads to the release of melanocortin peptides, like α-MSH, which act as agonists on MC3R and MC4R located in downstream neurons, ultimately leading to a decrease in food intake and an increase in energy expenditure [START_REF] Cone | Anatomy and Regulation of the Central Melanocortin System[END_REF]. Nevertheless, the activation of POMC neurons can also lead to an unexpected increase in food intake [START_REF] Lau | Endocannabinoid Modulation of Homeostatic and Non-Homeostatic Feeding Circuits[END_REF]. Indeed, (endo)cannabinoids or activation of the type 1 cannabinoid receptor (CB1R), which are known to drive feeding [START_REF] Cota | The Endogenous Cannabinoid System Affects Energy Balance via Central Orexigenic Drive and Peripheral Lipogenesis[END_REF], selectively stimulate the release of the opioid peptide β-endorphin and not α-MSH from POMC neurons to induce hyperphagia in sated mice [START_REF] Koch | Hypothalamic POMC Neurons Promote Cannabinoid-Induced Feeding[END_REF].

The second neuronal population co-expresses Agouti-related peptide (AgRP), neuropeptide-Y (NPY) and GABA. Activation of hypothalamic AgRP/NPY neurons promotes feeding, and their inhibition leads to starvation [START_REF] Wu | Loss of GABAergic Signaling by AgRP Neurons to the Parabrachial Nucleus Leads to Starvation[END_REF][START_REF] Wu | GABAergic Signaling by AgRP Neurons Prevents Anorexia via a Melanocortin-Independent Mechanism[END_REF]. AgRP/NPY neurons promote feeding through at least three mechanisms: (1) a selective antagonism of MC3R and MC4R [START_REF] Ollmann | Antagonism of Central Melanocortin Receptors in Vitro and in Vivo by Agouti-Related Protein[END_REF]); ( 2) the NPY-mediated inhibition of post-synaptic neurons located in other hypothalamic nuclei; and (3) the GABAmediated inhibition of the activity of POMC neurons [START_REF] Wu | GABAergic Signaling by AgRP Neurons Prevents Anorexia via a Melanocortin-Independent Mechanism[END_REF].

The regulation of food intake and energy expenditure therefore requires the coordinated and, most of the time, opposing activity of POMC/CART and AgRP/NPY neuronal populations.

The ventral and dorsal medial hypothalamic nuclei

First experiments between 1940 and 1980 showed that producing lesions in the ventromedial nucleus of the hypothalamus (VMH) leads to obesity in rodents [START_REF] Anand | Hypothalamic Control of Food Intake in Rats and Cats[END_REF][START_REF] Brobeck | MECHANISM OF THE DEVELOPMENT OF OBESITY IN ANIMALS WITH HYPOTHALAMIC LESIONS[END_REF]. Feeding activates the VMH, and activation of its neurons regulates parasympathetic and sympathetic autonomic responses [START_REF] Bernard | Acute Hyperinsulinemia and Its Reversal by Vagotomy after Lesions of the Ventromedial Hypothalamus in Anesthetized Rats*[END_REF][START_REF] Cox | Prior Vagotomy Blocks VMH Obesity in Pair-Fed Rats[END_REF][START_REF] Sakaguchi | The Effect of Intrahypothalamic Injections of Glucose on Sympathetic Efferent Firing Rate[END_REF][START_REF] Sakaguchi | Intrahypothalamic Injection of Insulin Decreases Firing Rate of Sympathetic Nerves[END_REF][START_REF] Shimazu | Central Nervous System Regulation of Liver and Adipose Tissue Metabolism[END_REF]. Knocking out the nuclear receptor steroidogenic factor 1 (SF-1), which brain expression is restricted to the VMH, is associated with deregulated insulin and leptin homeostasis, obesity, increased food intake and decreased energy expenditure and physical activity [START_REF] Kinyua | Steroidogenic Factor 1 in the Ventromedial Nucleus of the Hypothalamus Regulates Age-Dependent Obesity[END_REF]. Furthermore, data suggest that, during fasting, MC4R-expressing VMH neurons release the anorexigenic brain-derived neurotrophic factor (BDNF) under the influence of POMC neurons [START_REF] Xu | Brain-Derived Neurotrophic Factor Regulates Energy Balance Downstream of Melanocortin-4 Receptor[END_REF]. However, whether POMC neurons exert a direct or an indirect action on VMH MC4R-expressing neurons remains controversial as POMC neurons only sparsely innervate the VMH [START_REF] King | The Rise, Fall, and Resurrection of the Ventromedial Hypothalamus in the Regulation of Feeding Behavior and Body Weight[END_REF]. Furthermore, the VMH contains glucose-sensing neurons [START_REF] Ono | Glucoresponsive Neurons in Rat Ventromedial Hypothalamic Tissue Slices in Vitro[END_REF][START_REF] Routh | Glucosensing Neurons in the Ventromedial Hypothalamic Nucleus (VMN) and Hypoglycemia-Associated Autonomic Failure (HAAF)[END_REF][START_REF] Song | Convergence of Preand Postsynaptic Influences on Glucosensing Neurons in the Ventromedial Hypothalamic Nucleus[END_REF], which adapt their electrical activity by detecting changes in extracellular glucose levels [START_REF] Fioramonti | Recent Advances in the Cellular and Molecular Mechanisms of Hypothalamic Neuronal Glucose Detection[END_REF]. A part of VMH glucose-sensing neurons responds to hypoglycaemia by stimulating the production of glucagon and catecholamines, thereby increasing glycaemia to maintain it constant [START_REF] Borg | Local Ventromedial Hypothalamus Glucose Perfusion Blocks Counterregulation during Systemic Hypoglycemia in Awake Rats[END_REF]). Although the VMH was considered a major satiety center between 1940 and 1980, its popularity as a regulator of energy balance plummeted due to critics about the experimental methods that had been employed. This slowed down research on the role of the VMH in energy balance control. However, studies are now again uncovering the role of the VMH in energy balance regulation. (For a review about the rise, the fall and the resurrection of the VMH in the regulation of energy balance, see [START_REF] King | The Rise, Fall, and Resurrection of the Ventromedial Hypothalamus in the Regulation of Feeding Behavior and Body Weight[END_REF].

Regarding the dorsomedial nucleus of the hypothalamus (DMH), Bernardis and colleagues demonstrated that rats with lesions in the DMH also developed metabolic problems such as altered food intake and body weight [START_REF] Bellinger | The Dorsomedial Hypothalamic Nucleus and Its Role in Ingestive Behavior and Body Weight Regulation: Lessons Learned from Lesioning Studies[END_REF].

Certain studies have suggested that activation of neurons in the DMH stimulate energy expenditure and decrease food intake. For example, Zhang and colleagues recently demonstrated that the chemogenetic activation (via the DMH expression of the designer receptor hM3D(Gq) and the injection of the designer drug clozapine N-oxide or "CNO") of DMH neurons leads to weight loss [START_REF] Zhang | Activation of Dorsomedial Hypothalamic Neurons Promotes Physical Activity and Decreases Food Intake and Body Weight in Zucker Fatty Rats[END_REF]. This was associated with an increase of physical activity and a decrease of food intake [START_REF] Zhang | Activation of Dorsomedial Hypothalamic Neurons Promotes Physical Activity and Decreases Food Intake and Body Weight in Zucker Fatty Rats[END_REF]. Additionally, Pinol and colleagues showed that the activation of DMH neurons expressing the receptor Bombesinlike receptor 3 (BRS3), which is known for regulating energy homeostasis and heart rate, increases body temperature [START_REF] Piñol | Brs3 Neurons in the Mouse Dorsomedial Hypothalamus Regulate Body Temperature, Energy Expenditure, and Heart Rate, but Not Food Intake[END_REF]. However, other studies have shown that cholinergic neurons from the DMH increase food intake by enhancing GABAergic inhibition of POMC/CART neurons in the ARH [START_REF] Jeong | Cholinergic Neurons in the Dorsomedial Hypothalamus Regulate Food Intake[END_REF][START_REF] Rau | GABAergic Inputs to POMC Neurons Originating from the Dorsomedial Hypothalamus Are Regulated by Energy State[END_REF], and decrease brown adipose tissue activity [START_REF] Jeong | Cholinergic Neurons in the Dorsomedial Hypothalamus Regulate Mouse Brown Adipose Tissue Metabolism[END_REF]. These data seem controversial but they probably illustrate the complexity of the neuronal composition of a hypothalamic nucleus. Nevertheless, these studies do show that the DMH plays a role in food intake and energy expenditure.

The paraventricular nucleus of the hypothalamus

The paraventricular nucleus of the hypothalamus (PVH) is a microcosm within the hypothalamus because it contains a multitude of sub-nuclei containing several neuronal populations that influence the endocrine system, the autonomous nervous system and the behavioral system [START_REF] Berthoud | Multiple Neural Systems Controlling Food Intake and Body Weight[END_REF]) (Figure 8).

The cyto-architecture of the PVH changes in a rostro-caudal manner. Beginning at the rostral limit, the mouse PVH is poorly differentiated in comparison to the rat PVH [START_REF] Biag | Cytoand Chemoarchitecture of the Hypothalamic Paraventricular Nucleus in the C57BL/6J Male Mouse: A Study of Immunostaining and Multiple Fluorescent Tract Tracing[END_REF]. At the midrostral-caudal level, the PVH already adopts its characteristic triangular shape (i.e. the anterior PVH). At its caudal limit, the PVH adopts the shape of a lateral wing that extends through the LH (i.e. the posterior PVH). Two-thirds of the PVH correspond to the triangular, anterior PVH, whereas the last one-third corresponds to the posterior PVH [START_REF] Biag | Cytoand Chemoarchitecture of the Hypothalamic Paraventricular Nucleus in the C57BL/6J Male Mouse: A Study of Immunostaining and Multiple Fluorescent Tract Tracing[END_REF].

The PVH contains mainly three categories of neurons (Figure 8). The first category is composed of magnocellular neuroendocrine neurons that produce and release either oxytocin or vasopressin through axonal projections that terminate in capillaries from the posterior lobe of the pituitary (i.e. neurohypophysis) [START_REF] Biag | Cytoand Chemoarchitecture of the Hypothalamic Paraventricular Nucleus in the C57BL/6J Male Mouse: A Study of Immunostaining and Multiple Fluorescent Tract Tracing[END_REF]. The second category is composed of parvicellular neuroendocrine neurons that synthesize an array of hormones and project to the median eminence for further transport to the anterior pituitary lobe (i.e. adenohypophysis). The hormones produced in parvicellular neuroendocrine neurons are the corticotrophin-releasing hormone (CRH), the thyrotropin-releasing hormone (TRH) and somatostatin (SS), which stimulate the subsequent production of hormones in endocrine cells of the adenohypophysis [START_REF] Biag | Cytoand Chemoarchitecture of the Hypothalamic Paraventricular Nucleus in the C57BL/6J Male Mouse: A Study of Immunostaining and Multiple Fluorescent Tract Tracing[END_REF]. The third category is composed of descending pre-autonomic neurons which axonal projections target the lower brainstem and the spinal cord, and presumably influence sympathetic, parasympathetic and somatomotor responses [START_REF] Biag | Cytoand Chemoarchitecture of the Hypothalamic Paraventricular Nucleus in the C57BL/6J Male Mouse: A Study of Immunostaining and Multiple Fluorescent Tract Tracing[END_REF]. Regarding the distribution of these three categories of neurons, the majority of neurons expressing the same neuropeptide tend to be grouped together in such a way that different neuronal populations localize in distinct PVH areas. For details about the neuronal distribution patterns within the PVH, please see Figure 8 [START_REF] Biag | Cytoand Chemoarchitecture of the Hypothalamic Paraventricular Nucleus in the C57BL/6J Male Mouse: A Study of Immunostaining and Multiple Fluorescent Tract Tracing[END_REF])

The lateral hypothalamic nucleus

The lateral nucleus of the hypothalamus (LH) is believed to be the most interconnected area of the hypothalamus. Its axonal projections connect it with the telencephalon, the hindbrain, the spinal cord, and with other intra-hypothalamic nuclei.

Although the LH lacks projections to the endocrine system, it has an outstanding amount of projections to brain regions regulating the autonomic nervous system and the behavioral system [START_REF] Berthoud | Multiple Neural Systems Controlling Food Intake and Body Weight[END_REF]. Studies suggest that the LH may directly integrate information about glycaemia fluctuations because it also contains glucose-sensing neurons [START_REF] Berthoud | Multiple Neural Systems Controlling Food Intake and Body Weight[END_REF]. The LH contains orexin-, NPY-and GABA-expressing neurons that seem to be inhibited by glucose, and melanin-concentrating homone (MCH) neurons that seem to be excited by glucose [START_REF] Burdakov | Physiological Changes in Glucose Differentially Modulate the Excitability of Hypothalamic Melanin-Concentrating Hormone and Orexin Neurons In Situ[END_REF][START_REF] Fioramonti | Recent Advances in the Cellular and Molecular Mechanisms of Hypothalamic Neuronal Glucose Detection[END_REF][START_REF] Gonzalez | Metabolism-Independent Sugar Sensing in Central Orexin Neurons[END_REF][START_REF] Marston | Neuropeptide Y Cells Represent a Distinct Glucose-Sensing Population in the Lateral Hypothalamus[END_REF].

To summarize, the hypothalamus has a strategic localization that allows it to sense peripheral cues, in part, through the median eminence. These peripheral cues influence AgRP/NPY and POMC/CART neurons, among other neuronal populations, in the ARH. The ARH acts as a crossroad between peripheral signals and other hypothalamic nuclei.

Importantly, there is frequent interaction between most hypothalamic nuclei, as well as with other brain areas, including the cortex, the limbic system and the brainstem. All of these interactions aid in the regulation of food intake and energy expenditure, and are still subject of investigation. For example, studies on homeostatic and non-homeostatic pathways have revealed the central role of the endocannabinoid system in food intake regulation [START_REF] Lau | Endocannabinoid Modulation of Homeostatic and Non-Homeostatic Feeding Circuits[END_REF]). On the one hand, (endo)cannabinoids regulate food intake by modulating neuronal activity through CB1Rs that are present in multiple hypothalamic nuclei. On the other hand, (endo)cannabinoids regulate food intake by promoting dopamine release in neurons from the cortico-limbic system in response to palatable food. The endogenous cannabinoid and opioid systems are also key neuronal circuits that respond to the rewarding properties of food (For a review, see [START_REF] Cota | Cannabinoids, Opioids and Eating Behavior: The Molecular Face of Hedonism?[END_REF].

The brainstem

As the hypothalamus, the brainstem also contains a circumventricular organ called the area postrema and has thus facilitated access to peripheral cues about the energy status of an organism. This brain region is also an integrative center, therefore relaying peripheral inputs to forebrain areas in order to regulate energy balance. Concerning the regulation of food intake and energy expenditure, the dorsal vagal complex and the parabrachial nucleus are the most described brainstem structures.

The dorsal vagal complex

The dorsal vagal complex (DVC) integrates both visceral sensory and hormonal signals. Most data concerns DVC neurons. However, recent experiments have shown that astrocytes in the DVC also integrate peripheral satiety signals to control feeding [START_REF] Macdonald | Regulation of Food Intake by Astrocytes in the Brainstem Dorsal Vagal Complex[END_REF]). The DVC is composed of the nucleus of the solitary tract (NTS), the area postrema (AP) and the dorsal motor nucleus of the vagus (dmnX). The DVC receives visceral sensory information mainly through vagal afferents (see Chapter 3) that terminate in the NTS, and to a lesser extent, in the AP and the dmnX [START_REF] Berthoud | Anatomy and Function of Sensory Hepatic Nerves[END_REF]. Connections between the DVC and the cortex exist and are mainly mediated by the NTS, which projects to the parabrachial nucleus, the thalamus, the amygdala, the raphe nucleus and the locus coeruleus [START_REF] Berthoud | Multiple Neural Systems Controlling Food Intake and Body Weight[END_REF].

Interestingly, this complex shares several similarities with the hypothalamus. Besides harboring vagal afferent endings, the DVC also has direct access to peripheral hormonal and nutritional cues through the AP [START_REF] Berthoud | Multiple Neural Systems Controlling Food Intake and Body Weight[END_REF]. Indeed, the DVC is rich in receptors to metabolic hormones like leptin, ghrelin and insulin [START_REF] Buyse | Expression and Regulation of Leptin Receptor Proteins in Afferent and Efferent Neurons of the Vagus Nerve[END_REF][START_REF] Filippi | Insulin Signals Through the Dorsal Vagal Complex to Regulate Energy Balance[END_REF][START_REF] Zhang | Ghrelin Stimulates Neurogenesis in the Dorsal Motor Nucleus of the Vagus: Stimulation of Neurogenesis by Ghrelin[END_REF]). Within the DVC, the NTS contains POMC neurons, although to a lesser extent than the ARH, as well as gluco-sensitive neurons [START_REF] De Bernardis Murat | A Voltage-Dependent Depolarization Induced by Low External Glucose in Neurons of the Nucleus of the Tractus Solitarius: Interaction with KATP Channels[END_REF][START_REF] Mountjoy | Localization of the Melanocortin-4 Receptor (MC4-R) in Neuroendocrine and Autonomic Control Circuits in the Brain[END_REF]. Furthermore, the NTS has a high expression of MC4Rs, which activation by vagal afferent endings has been proposed to partly mediate food intake reduction by the DVC [START_REF] Campos | Central Vagal Afferent Endings Mediate Reduction of Food Intake by Melanocortin-3/4 Receptor Agonist[END_REF][START_REF] Mountjoy | Localization of the Melanocortin-4 Receptor (MC4-R) in Neuroendocrine and Autonomic Control Circuits in the Brain[END_REF].

The parabrachial nucleus

The parabrachial nucleus (PBN) is located in the dorsal pons of the brainstem, and is believed to regulate food intake, taste [START_REF] Spector | Gustatory Function in the Parabrachial Nuclei: Implications from Lesion Studies in Rats[END_REF] and behavioral thermoregulation [START_REF] Yahiro | The Lateral Parabrachial Nucleus, but Not the Thalamus, Mediates Thermosensory Pathways for Behavioural Thermoregulation[END_REF]. In contrast to the DVC, the PBN visceral sensory inputs come mainly from spinal afferents (see Chapter 3) [START_REF] Berthoud | Anatomy and Function of Sensory Hepatic Nerves[END_REF]). Concerning food intake, the medial and the lateral zones of the PBN relay both taste and visceral sensory information to the ARH, the PVH, the VMH and the LH, as well as to the amygdala [START_REF] Berthoud | Multiple Neural Systems Controlling Food Intake and Body Weight[END_REF]. The PBN regulation of food intake involves appetite suppression through calcitonin gene-related peptide (CGRP)-expressing neurons that project to the amygdala [START_REF] Campos | Parabrachial CGRP Neurons Control Meal Termination[END_REF][START_REF] Carter | Genetic Identification of a Neural Circuit That Suppresses Appetite[END_REF][START_REF] Krahn | Effects of Calcitonin Gene-Related Peptide on Food Intake[END_REF].

In conclusion, the central regulation of food intake and energy expenditure involves several brain areas, from areas that integrate peripheral cues about the energetic status of the organism like the hypothalamus and the brainstem to brain areas that integrate signals of aversion and reward such as the cortico-limbic system. Here, some basic points regarding both the hypothalamus and the brainstem were described.

Hormonal regulation of food intake and energy expenditure

The central nervous system regulates food intake and energy expenditure in response to peripheral hormones that reflect the energy status of the organism. The areas described above express receptors to and thus respond to a number of hormones released by the adipose tissue, the gastrointestinal tract and the pancreas (Figure 9).

Hormones released by the adipose tissue

The adipose tissue is a major endocrine organ that secretes numerous peptide and steroid hormones [START_REF] Guerre-Millo | Adipose Tissue Hormones[END_REF]. However, leptin is the most thoroughly described and relevant hormone regarding energy balance regulation until now. Leptin is a These hormones regulate food intake by targeting several areas of the nervous system.

pleiotropic peptide hormone, therefore exerting multiple roles that range from the developmental to the immunological and, of course, the energy balance regulation [START_REF] Stanley | Hormonal Regulation of Food Intake[END_REF]. The major producer of leptin is the white adipocyte, but its production has also been reported in the stomach, the placenta, the brown adipose tissue, the ovaries, the skeletal muscle and the central nervous system [START_REF] Kwon | Leptin Signalling Pathways in Hypothalamic Neurons[END_REF][START_REF] Stanley | Hormonal Regulation of Food Intake[END_REF]. Leptin exerts a potent anorexigenic effect in healthy individuals, and is generally released in conditions of a rich energy status. In this sense, plasma leptin levels increase during the postprandial period and they correlate positively with fat mass. Recent studies suggest that leptin reaches hypothalamic nuclei involved in energy balance control through tanycytes from the median eminence [START_REF] Balland | Hypothalamic Tanycytes Are an ERK-Gated Conduit for Leptin into the Brain[END_REF]. Balland and colleagues showed that circulating leptin goes through the fenestrated capillaries from the hypophyseal portal blood system and reaches tanycytes. Leptin then binds leptin receptors present in tanycyte projections, and is shuttled in a polarized manner through the tanycyte and into the third ventricle, where it can then diffuse into the ARH [START_REF] Balland | Hypothalamic Tanycytes Are an ERK-Gated Conduit for Leptin into the Brain[END_REF].

The alternative mRNA splicing and post-translational processing of the leptin receptor leads to long, short and secreted receptor isoforms [START_REF] Tartaglia | The Leptin Receptor[END_REF][START_REF] Tartaglia | Identification and Expression Cloning of a Leptin Receptor, OB-R[END_REF]. Leptin is believed to mediate its anorexigenic effect on food intake via its long receptor (LepRb) [START_REF] Baumann | The Full-Length Leptin Receptor Has Signaling Capabilities of Interleukin 6-Type Cytokine Receptors[END_REF][START_REF] Rosenblum | Functional STAT 1 and 3 Signaling by the Leptin Receptor (OB-R); Reduced Expression of the Rat Fatty Leptin Receptor in Transfected Cells[END_REF]. The LepRb is mainly expressed in POMC/CART and AgRP/NPY neurons from the ARH [START_REF] Cheung | Proopiomelanocortin Neurons Are Direct Targets for Leptin in the Hypothalamus[END_REF][START_REF] Mercer | Coexpression of Leptin Receptor and Preproneuropeptide Y MRNA in Arcuate Nucleus of Mouse Hypothalamus[END_REF], as well as in the VMH, DMH and LH, and in the preoptic area [START_REF] Fei | Anatomic Localization of Alternatively Spliced Leptin Receptors (Ob-R) in Mouse Brain and Other Tissues[END_REF][START_REF] Stanley | Hormonal Regulation of Food Intake[END_REF]).

In the hypothalamus, leptin signaling through LepRb leads to the phosphorylation of tyrosine kinase Janus Kinase (JAK), leading to the stimulation of different signaling pathways (Figure 10). The Leptin→STAT3 signaling pathway involves the transcription factor signal transducer and activator of transcription 3 (STAT3) [START_REF] Kwon | Leptin Signalling Pathways in Hypothalamic Neurons[END_REF][START_REF] Münzberg | Role of Signal Transducer and Activator of Transcription 3 in Regulation of Hypothalamic Proopiomelanocortin Gene Expression by Leptin[END_REF]. Phosphorylated STAT3 dimerizes and translocates to the nucleus where it downregulates AgRP/NPY expression and upregulates POMC expression in each respective neuronal population (For a review, see [START_REF] Kwon | Leptin Signalling Pathways in Hypothalamic Neurons[END_REF]. The stimulation of POMC/CART neurons and the inhibition of AgRP/NPY neurons allow signaling through MC3R and MC4R

in downstream neurons, ultimately leading to a decrease in food intake. Importantly, Leptin→STAT3 signaling exerts its own negative feedback by inducing the transcription of suppressor of cytokine signaling 3 (SOCS3) [START_REF] Bjørbaek | The Role of SOCS-3 in Leptin Signaling and Leptin Resistance[END_REF][START_REF] Kwon | Leptin Signalling Pathways in Hypothalamic Neurons[END_REF][START_REF] Starr | A Family of Cytokine-Inducible Inhibitors of Signalling[END_REF]. A second leptin-induced signaling pathway is the PI3K-Akt-FoxO1 pathway. This pathway is stimulated by leptin and insulin.

JAK-2 recruits and phosphorylates (activates) the insulin receptor substrate (IRS), which subsequently activates Phosphoinositide 3-kinase (PI3K) [START_REF] Duan | SH2-B Promotes Insulin Receptor Substrate 1 (IRS1)-and IRS2-Mediated Activation of the Phosphatidylinositol 3-Kinase Pathway in Response to Leptin[END_REF][START_REF] Niswender | Insulin and Its Evolving Partnership with Leptin in the Hypothalamic Control of Energy Homeostasis[END_REF]. PI3K is thought to mediate leptin effects on POMC neuroelectrical activity [START_REF] Hill | Acute Effects of Leptin Require PI3K Signaling in Hypothalamic Proopiomelanocortin Neurons in Mice[END_REF]. When dephosphorylated, the transcription factor forkhead box protein O1 (FoxO1) upregulates the transcription of AgRP and downregulates the transcription of POMC. When the PI3K-Akt-FoxO1 pathway is stimulated, PI3K activates Akt which later phosphorylates FoxO1. This leads to an indirect increase of POMC transcription [START_REF] Kitamura | Forkhead Protein FoxO1 Mediates Agrp-Dependent Effects of Leptin on Food Intake[END_REF]. A third leptin-induced signaling pathway is the mTORC1-S6K pathway. The mammalian target of rapamycin complex 1 (mTORC1) is present in more than 90% of AgRP/NPY neurons and in about 40% of POMC neurons [START_REF] Cota | Hypothalamic MTOR Signaling Regulates Food Intake[END_REF][START_REF] Inhoff | Novel Insight in Distribution of Nesfatin-1 and Phospho-MTOR in the Arcuate Nucleus of the Hypothalamus of Rats[END_REF]. Leptin decreases food intake in part via a mechanism that involves the mTORC1-mediated increase of oxidant levels in POMC neurons [START_REF] Haissaguerre | MTORC1-Dependent Increase in Oxidative Metabolism in POMC Neurons Regulates Food Intake and Action of Leptin[END_REF].

Besides these signaling pathways, the regulation of food intake and energy balance by leptin requires the stimulation of other pathways detailed in Figure 10. For example, the inhibition of the AMP-activated protein kinase (AMPK) by leptin is necessary for food intake decrease [START_REF] Minokoshi | AMP-Kinase Regulates Food Intake by Responding to Hormonal and Nutrient Signals in the Hypothalamus[END_REF]).

In addition to leptin hypothalamic signaling, data suggest that leptin also mediates food intake decrease through the brainstem. For example, LepRb signaling in the lateral PBN reduces meal size without affecting locomotor activity or gastric emptying [START_REF] Alhadeff | Leptin Receptor Signaling in the Lateral Parabrachial Nucleus Contributes to the Control of Food Intake[END_REF]. Moreover, LepRb signaling in the NTS from the DVC reduces food intake by decreasing food-seeking behavior [START_REF] Kanoski | Leptin Signaling in the Medial Nucleus Tractus Solitarius Reduces Food Seeking and Willingness to Work for Food[END_REF]. Therefore, leptin decreases food intake by acting at several levels of the central nervous system.

Hormones released by the gastrointestinal tract

The gastrointestinal tract could be considered the largest endocrine organ as it releases about 20 hormones. Concerning gut hormones that regulate energy balance, we will focus on ghrelin, cholecystokinin (CCK), peptide YY (PYY ), oxyntomodulin and glucagonlike peptide-1 (GLP-1). All of these hormones have been suggested to decrease food intake [START_REF] Stanley | Hormonal Regulation of Food Intake[END_REF], except for ghrelin which is the only gastrointestinal hormone signaling hunger described until today (Figure 9). The principal producer of ghrelin is the gastric oxyntic cell. However, the hormone is also produced in the duodenum, the ileum, the cecum and the colon, and is expressed by several hypothalamic neurons [START_REF] Stanley | Hormonal Regulation of Food Intake[END_REF].

Plasma ghrelin levels are regulated by the circadian rhythm and by food intake. Contrary to leptin, ghrelin is released in conditions of a poor energy status. In this sense, plasma ghrelin levels are the highest right before a meal, low during the postprandial period and correlate inversely with adiposity. Regarding the transport of ghrelin into hypothalamic nuclei that regulate feeding, Schaeffer and colleagues showed that ghrelin can also diffuse through the fenestrated capillaries of the median eminence into the ARH [START_REF] Schaeffer | Rapid Sensing of Circulating Ghrelin by Hypothalamic Appetite-Modifying Neurons[END_REF].

Interestingly, a 24 hour fast increases newly formed fenestrated capillaries close to the ARH, suggesting a facilitated transport of ghrelin into the ARH during fasting [START_REF] Prevot | The Versatile Tanycyte: A Hypothalamic Integrator of Reproduction and Energy Metabolism[END_REF].

Ghrelin is the endogenous agonist of the growth hormone secretagogue receptor (GHS-R). The orexigenic effect of ghrelin is thought to be mediated mainly by the GHS-R type 1a. However, the binding of ghrelin to the GHS-R type 1a requires the enzymatic addition of an acyl side chain to its third serine residue by the ghrelin O-acyltransferase [START_REF] Hougland | Ghrelin Octanoylation by Ghrelin O-Acyltransferase: Unique Protein Biochemistry Underlying Metabolic Signaling[END_REF]. The GHS-R type 1a is expressed in numerous regions of the central nervous system, especially in the hypothalamus, and particularly in the ARH. Ghrelin induces feeding in part by binding to the GHS-R type 1a that is present in AgRP/NPY neurons from the ARH [START_REF] Chen | Ghrelin Receptors Mediate Ghrelin-Induced Excitation of Agouti-Related Protein/Neuropeptide Y but Not pro-Opiomelanocortin Neurons[END_REF]. The mechanisms underlying the AgRP/NPY neuronal stimulation of feeding were described in Chapter 2 section 1.2. Indeed, ghrelin administration excites AgRP/NPY neurons, and ghrelin fails to increase food intake in mice lacking AgRP and NPY signaling [START_REF] Chen | Orexigenic Action of Peripheral Ghrelin Is Mediated by Neuropeptide Y and Agouti-Related Protein[END_REF][START_REF] Chen | Ghrelin Receptors Mediate Ghrelin-Induced Excitation of Agouti-Related Protein/Neuropeptide Y but Not pro-Opiomelanocortin Neurons[END_REF]. In addition to the hypothalamus, studies suggest that the orexigenic actions of ghrelin are also mediated through the brainstem. Indeed, the GHS-R type 1a is present in the gastric vagal nerve [START_REF] Date | The Role of the Gastric Afferent Vagal Nerve in Ghrelin-Induced Feeding and Growth Hormone Secretion in Rats[END_REF]; and ghrelin fails to stimulate food intake after ablation of vagal nerves [START_REF] Le Roux | Ghrelin Does Not Stimulate Food Intake in Patients with Surgical Procedures Involving Vagotomy[END_REF][START_REF] Williams | Vagotomy Dissociates Short-and Long-Term Controls of Circulating Ghrelin[END_REF]. Additionally, ghrelin administration increases c-fos in the NTS and the area postrema [START_REF] Lawrence | Acute Central Ghrelin and GH Secretagogues Induce Feeding and Activate Brain Appetite Centers[END_REF].

Besides the ARH and the DVC, ghrelin might also induce feeding by targeting the reward circuits [START_REF] Abizaid | Ghrelin Modulates the Activity and Synaptic Input Organization of Midbrain Dopamine Neurons While Promoting Appetite[END_REF].

All the other hormones produced by the gut increase satiety and reduce food intake by acting at multiple levels of the nervous system [START_REF] Stanley | Hormonal Regulation of Food Intake[END_REF]) (Figure 9). The release of the anorexigenic hormones PYY , GLP-1, oxyntomodulin and CCK is stimulated at differential times during the postprandial period. PYY seems to mediate its anorexigenic effects by stimulating the inhibitory Y2 receptor present in AgRP/NPY neurons from the ARH [START_REF] Teubner | PYY(3-36) into the Arcuate Nucleus Inhibits Food Deprivation-Induced Increases in Food Hoarding and Intake[END_REF], but also by signaling the brainstem via the vagal nerve [START_REF] Abbott | The Inhibitory Effects of Peripheral Administration of Peptide YY(3-36) and Glucagon-like Peptide-1 on Food Intake Are Attenuated by Ablation of the Vagal-Brainstem-Hypothalamic Pathway[END_REF][START_REF] Koda | The Role of the Vagal Nerve in Peripheral PYY3-36-Induced Feeding Reduction in Rats[END_REF]. GLP-1 and oxyntomodulin have been proposed

to decrease food intake via the ARH, probably through a common cellular mechanism involving GLP-1 receptors [START_REF] Riediger | Effects of Glucagon-like Peptide 1 and Oxyntomodulin on Neuronal Activity of Ghrelin-Sensitive Neurons in the Hypothalamic Arcuate Nucleus[END_REF], and by signaling the DVC via the vagal nerve [START_REF] Abbott | The Inhibitory Effects of Peripheral Administration of Peptide YY(3-36) and Glucagon-like Peptide-1 on Food Intake Are Attenuated by Ablation of the Vagal-Brainstem-Hypothalamic Pathway[END_REF][START_REF] Parker | Glucagon and GLP-1 Inhibit Food Intake and Increase c-Fos Expression in Similar Appetite Regulating Centres in the Brainstem and Amygdala[END_REF]. Additionally, GLP-1 exerts a hypoglycaemic effect via GLP-1 receptors located in vagal terminals innervating the portal vein [START_REF] Vahl | Glucagon-like Peptide-1 (GLP-1) Receptors Expressed on Nerve Terminals in the Portal Vein Mediate the Effects of Endogenous GLP-1 on Glucose Tolerance in Rats[END_REF]).

Finally, CCK has been suggested to mediate its anorexigenic effects via the ARH and the PVH [START_REF] Lo | Intraperitoneal CCK and Fourth-Intraventricular Apo AIV Require Both Peripheral and NTS CCK1R to Reduce Food Intake in Male Rats[END_REF], as well as by signaling the NTS via the vagal nerve [START_REF] Lo | Intraperitoneal CCK and Fourth-Intraventricular Apo AIV Require Both Peripheral and NTS CCK1R to Reduce Food Intake in Male Rats[END_REF][START_REF] Smith | Afferent Axons in Abdominal Vagus Mediate Satiety Effect of Cholecystokinin in Rats[END_REF].

Hormones released by the pancreas

The pancreas also secretes anorexigenic hormones that target central areas involved in energy balance control. Among these hormones, the most thoroughly described ones are insulin, amylin and the pancreatic polypeptide. As discussed in Chapter 1, plasma insulin levels increase rapidly during the postprandial period. Insulin decreases food intake and increases sympathetic nerve activity via insulin receptors that are widely distributed in the brain, and particularly in the hypothalamic ARH, DMH, PVH, and the suprachiasmatic and periventricular zones [START_REF] Stanley | Hormonal Regulation of Food Intake[END_REF]. Insulin increases hypothalamic POMC expression and decreases NPY expression [START_REF] Stanley | Hormonal Regulation of Food Intake[END_REF], suggesting that the anorexigenic effect of insulin could be mediated in part by the stimulation of POMC/CART neurons and the inhibition of AgRP/NPY neurons. At the molecular level, studies suggest that insulin feeding suppression is mediated via the mTORC1 signaling pathway [START_REF] Muta | The Role of Hypothalamic MTORC1 Signaling in Insulin Regulation of Food Intake, Body Weight, and Sympathetic Nerve Activity in Male Mice[END_REF], which is coherent given that insulin stimulates the insulin receptor substrate (IRS) (Figure 11). As insulin, amylin is also secreted by pancreatic β-cells during the postprandial period. Amylin seems to promote meal-ending satiation by acting on the area prostema of the DVC, which relays information to the NTS, the lateral PBN and the LH [START_REF] Lutz | The Role of Amylin in the Control of Energy Homeostasis[END_REF]. Amylin also regulates energy balance by targeting calcitonin receptors (CTR), at least in POMC neurons, in the ARH [START_REF] Coester | Amylin/Calcitonin Receptor-Mediated Signaling in POMC Neurons Influences Energy Balance and Locomotor Activity in Chow-Fed Male Mice[END_REF]. Finally, the pancreatic polypeptide is also released following food intake, and mediates its anorexigenic effects by targeting the DVC and Y4 receptors in the ARH [START_REF] Khandekar | The Role of Pancreatic Polypeptide in the Regulation of Energy Homeostasis[END_REF].

The production of all the above mentioned hormones may differ on how rapidly they are released after a meal, and for how long they circulate in the organism [START_REF] Moran | Gut Peptides in the Control of Food Intake[END_REF]. This pattern of release suggests that the gastrointestinal tract hormonal production exerts both an immediate, meal-size reduction role and a longer-lasting, satiating role [START_REF] Moran | Gut Peptides in the Control of Food Intake[END_REF].

Importantly, nutrients also play a role in regulating energy balance by signaling the central nervous system. For example, studies have shown that the amino acid leucine is sensed by POMC neurons in the ARH and by the NTS in the DVC through a mechanism involving the mTOR signaling pathway, which mediates the anorexigenic effect of leucine [START_REF] Blouet | Mediobasal Hypothalamic Leucine Sensing Regulates Food Intake through Activation of a Hypothalamus-Brainstem Circuit[END_REF][START_REF] Cota | Hypothalamic MTOR Signaling Regulates Food Intake[END_REF]. Additionally, lipid-sensing neurons and astrocytes have been described in the hypothalamus, which have been suggested to subsequently modulate hepatic glucose production and lipogenesis [START_REF] Clément | Intracerebroventricular Infusion of a Triglyceride Emulsion Leads to Both Altered Insulin Secretion and Hepatic Glucose Production in Rats[END_REF][START_REF] Magnan | Brain Lipid Sensing and the Neural Control of Energy Balance[END_REF][START_REF] Obici | Central Administration of Oleic Acid Inhibits Glucose Production and Food Intake[END_REF][START_REF] Oomura | Effect of Free Fatty Acid on the Rat Lateral Hypothalamic Neurons[END_REF]. Finally, as previously described, numerous hypothalamic and brainstem areas contain glucose-sensitive neurons, which activation also leads to a subsequent modulation of energy balance.

As mentioned before, intestinal gluconeogenesis triggers a gut-brain neural circuit, starting in the portal vein, which is crucial for metabolic health. This chapter will first describe the suggested routes through which intestinal gluconeogenesis influences central areas that regulate energy balance. Then, it will describe the specific conditions upon which intestinal gluconeogenesis is strongly induced in the post-absorptive state. Finally, there will be a description of the different animal models developed by our laboratory to study this key physiological function.

Mechanisms

Intestinal and portal vein innervation

The gastrointestinal tract is rich in extrinsic and intrinsic innervations. The extrinsic innervation consists of vagal and splanchnic afferents that project into brain areas that regulate energy balance, as well as parasympathetic and sympathetic motor efferents. The intrinsic innervation consists of neurons located entirely in the gastrointestinal wall, known as the enteric nervous system (ENS). For explanatory purposes, mainly vagal and splanchnic afferents, and the ENS will be described.

The vagal innervation

The vagal nerves are composed of afferent and efferent nerve fibers. However, 75%

of the gastrointestinal tract vagal innervation consists of afferents [START_REF] Ratcliffe | Molecular Development of the Extrinsic Sensory Innervation of the Gastrointestinal Tract[END_REF]. The common hepatic branch from vagal afferent fibers mainly innervates the bile ducts, the portal vein, the duodenum, the pancreas, the pylorus and the distal gastric antrum [START_REF] Berthoud | Anatomy and Function of Sensory Hepatic Nerves[END_REF]) (Figure 11), as well as baro-and chemoreceptors from the aortic arch, chemoreceptors in the carotid body and gustatory chemoreceptors in the tongue [START_REF] Grundy | Neuroanatomy of Visceral Nociception: Vagal and Splanchnic Afferent[END_REF]. The cell bodies of vagal afferents are contained in the nodose ganglia (Figure 11), which majority of central projections enter the brainstem [START_REF] Grundy | Neuroanatomy of Visceral Nociception: Vagal and Splanchnic Afferent[END_REF]). In the brainstem, these afferents form synapses with second order neurons mainly in the NTS, which then relay the visceral sensory information to other brain areas including the PBN, the amygdala, and the hypothalamus (Figure 11) [START_REF] Berthoud | Anatomy and Function of Sensory Hepatic Nerves[END_REF]). Vagal efferents are mostly parasympathetic, and emerge mainly from the dorsal and ambiguus nuclei from the Spinal afferent innervation is shown in red broken lines. Spinal afferents are connected to the dorsal root ganglia (DRG) at thoracic levels Th7 through Th13. Thickness of lines represents the relative abundance of innervations, and the diameter of dots represents sensory terminals in the various compartments. Sensory information is then relayed to the parabrachial nucleus (PBN), and to a lesser extent, to the nucleus of the tractus solitarius (NTS) in the dorsal vagal complex (DVC). dmnX: dorsal motor nucleus of the vagus; Amy: amygdala; PVN: paraventricular nucleus of the hypothalamus; LHA: lateral hypothalamic area (From Berthoud, 2004).

medulla oblongata and innervate the heart, the lungs and abdominal viscera (Figure 12) [START_REF] Baker | Neuroanatomy, Vagal Nerve Nuclei (Nucleus Vagus)[END_REF][START_REF] Petko | Neuroanatomy, Nucleus Ambiguus[END_REF].

The splanchnic innervation

The splanchnic nerves are also composed of afferent and efferent nerve fibers. The afferent splanchnic terminals from the portal region, the bile ducts and the portal vein connect with the celiac superior mesenteric ganglion [START_REF] Berthoud | Anatomy and Function of Sensory Hepatic Nerves[END_REF]. From there, the afferents transport visceral sensory information to cell bodies present in the dorsal root ganglia (Figure 11), which central projections enter the spinal cord to synapse with second order neurons that signal the brain through the spino-mesencephalic, spino-reticular or spino-thalamic tracts [START_REF] Berthoud | Anatomy and Function of Sensory Hepatic Nerves[END_REF]) (Figure 11). Contrary to vagal afferents, the majority of splanchnic afferents ultimately activate the PBN [START_REF] Berthoud | Anatomy and Function of Sensory Hepatic Nerves[END_REF]. Splanchnic efferent fibers are mostly sympathetic, except for the pelvic splanchnic nerves which are parasympathetic (Figure 12) [START_REF] Mccausland | Anatomy, Back, Splanchnic Nerve[END_REF]. The sympathetic splanchnic efferents contain five primary nerves on both sides of the spinal cord: cardiopulmonary, thoracic, lumbar, sacral and pelvic. The efferents that innervate the abdomen come from the thoracic and the lumbar splanchnic nerves that originate from pre-ganglionic neurons (from T5 to L2). The thoracic splanchnic efferents are divided into three categories: the greater thoracic nerves (T5→T9) which form synapses in the celiac ganglion; and the lesser thoracic nerve (T10→T11) and the least thoracic nerve (T12) which form synapses in the superior mesenteric ganglion. The lumbar splanchnic nerves (L1→L2) form synapses in the inferior mesenteric ganglion [START_REF] Mccausland | Anatomy, Back, Splanchnic Nerve[END_REF]. Ultimately, these splanchnic efferents innervate the stomach, the intestine, the liver, the spleen and the kidneys to influence various parameters like motility and secretion.

The enteric nervous system

The gastrointestinal tract also harbors sensory neurons and glia that are entirely located in its wall. In the adult, the ENS contains cholinergic neurons that express the enzyme choline acetyltransferase (ChAT), and nitrergic neurons that express the enzyme The sympathetic nervous sytem is illustrated in green lines, and the parasympathetic nervous system in red lines. Adapted from the doctorate thesis manuscript of Dr. Filipe de Vadder.

neuronal nitric oxide synthase (nNOS) [START_REF] Foong | Postnatal Development of the Mouse Enteric Nervous System[END_REF]). On the one hand, cholinergic neurons are excitatory and function as motor neurons, sensory neurons and interneurons [START_REF] Foong | Postnatal Development of the Mouse Enteric Nervous System[END_REF]. On the other hand, nitrergic neurons are inhibitory and function as motor neurons and interneurons [START_REF] Foong | Postnatal Development of the Mouse Enteric Nervous System[END_REF]. These neurons regroup into aggregates of fibers called nerve plexuses. The gastrointestinal tract contains two nerve plexus: the inner submucosal plexus (or Meissner plexus) and the outer myenteric plexus (or Auerbach plexus) (Figure 13) [START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF]. The submucosal plexus contains both sensory and motor neurons that regulate the activity of glands and the smooth muscle from the mucosa. The myenteric plexus is located between the circular and the longitudinal muscle, and regulates the motility of the gastrointestinal tract [START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF]. These plexuses are connected to the central nervous system by visceral afferents and by both sympathetic and parasympathetic efferents (Furness 2016).

Detection of glucose produced by the intestine and the portal glucose signal

The hepatic portal vein (from now on called portal vein) collects blood from the gastrointestinal tract, the pancreas and the spleen before irrigating the liver; and it is connected to the brain via vagal and splanchnic afferents [START_REF] Berthoud | Anatomy and Function of Sensory Hepatic Nerves[END_REF]). This strategic localization allows the portal vein to detect nutrients like oligopeptides from protein digestion (Duraffourd et al. 2012), fatty acids [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF], gastrointestinal hormones like GLP-1 [START_REF] Vahl | Glucagon-like Peptide-1 (GLP-1) Receptors Expressed on Nerve Terminals in the Portal Vein Mediate the Effects of Endogenous GLP-1 on Glucose Tolerance in Rats[END_REF], and glucose homeostasis cues, before transmitting information to the brain. Moreover, the localization of the portal vein allows it to sense discrete changes in glycaemia before other vessels in the body [START_REF] Croset | Rat Small Intestine Is an Insulin-Sensitive Gluconeogenic Organ[END_REF][START_REF] Donovan | Primacy of Liver Glucosensors in the Sympathetic Response to Progressive Hypoglycemia[END_REF][START_REF] Donovan | Importance of Hepatic Glucoreceptors in Sympathoadrenal Response to Hypoglycemia[END_REF][START_REF] Hevener | Novel Glucosensor for Hypoglycemic Detection Localized to the Portal Vein[END_REF]Soty et al. 2017). For example, at the end of the post-absorptive period, glycaemia is slightly lower in the portal vein than in the systemic circulation because of the considerable glucose utilization by the gut [START_REF] Croset | Rat Small Intestine Is an Insulin-Sensitive Gluconeogenic Organ[END_REF]. Likewise, right after a meal (or during the fasted to fed transition), glycaemia is slightly higher in the portal vein because it receives nutritional glucose supply from the gut before it is stocked as glycogen in the liver (Soty et al. 2017;[START_REF] Strubbe | Blood Glucose Levels in Portal and Peripheral Circulation and Their Relation to Food Intake in the Rat[END_REF]. Like the brain, the portal vein contains glucose-sensing units. Portal glucose sensing is a key component of the gut-brain neural circuit controlled by intestinal gluconeogenesis.

During the post-prandial period, a standard, starch-based diet leads to the arrival of a big glucose flux into the portal vein. In contrast, the induction of intestinal gluconeogenesis during the post-absorptive period and prolonged fasting results in the release of a discrete amount of glucose into the portal vein [START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF]. The portal glucose infusion at a rate that mimics intestinal gluconeogenesis is detected by the sodium-coupled glucose cotransporter 3 (SGLT3) but not by GLUT2 (Figure 14) [START_REF] Delaere | The Role of Sodium-Coupled Glucose Co-Transporter 3 in the Satiety Effect of Portal Glucose Sensing[END_REF]. The latter would be involved in the detection of higher glucose fluxes [START_REF] Thorens | Molecular Physiology of Glucose Transporters[END_REF].

Although the SGLT3 receptor is a member of the sodium-coupled glucose transporters family, SGLT3 acts as a glucose sensor instead of as a glucose/sodium co-transporter [START_REF] Diez-Sampedro | A Glucose Sensor Hiding in a Family of Transporters[END_REF].

The SGLT3 detection of glucose produced by the intestine would lead to an afferent nervous signal, from now on called "portal glucose signaling" (Figure 14). Indeed, portal glucose sensing is blunted with capsaicin, a neurotoxic molecule that destroys afferent nerves (vagal and splanchnic). However, the chirurgical ablation of vagal nerves does not alter actions known to be induced by portal glucose signaling, such as the counter-regulation of hypoglycemia or limiting food intake [START_REF] Delaere | The Role of Sodium-Coupled Glucose Co-Transporter 3 in the Satiety Effect of Portal Glucose Sensing[END_REF][START_REF] Fujita | Celiac-Superior Mesenteric Ganglionectomy, but Not Vagotomy, Suppresses the Sympathoadrenal Response to Insulin-Induced Hypoglycemia[END_REF].

These data suggest that vagal innervation is not necessary for portal glucose signaling. On the contrary, portal glucose fails to counter-regulate hypoglycaemia or limit food intake in rodents with a suppression of the vanilloid receptor type 1, which is expressed mainly in the splanchnic nerves [START_REF] Ward | Distribution of the Vanilloid Receptor (VR1) in the Gastrointestinal Tract[END_REF]. Furthermore, portal glucose also fails to decrease feeding after dissection of the celiac superior mesenteric ganglion, which is part of the splanchnic afferent route [START_REF] Delaere | The Role of Sodium-Coupled Glucose Co-Transporter 3 in the Satiety Effect of Portal Glucose Sensing[END_REF][START_REF] Fujita | Celiac-Superior Mesenteric Ganglionectomy, but Not Vagotomy, Suppresses the Sympathoadrenal Response to Insulin-Induced Hypoglycemia[END_REF]). Together, these data suggest that portal glucose signaling targets central areas controlling energy balance via splanchnic afferents.

Central targets of intestinal gluconeogenesis

Data from our laboratory suggest that the satiating effects elicited by portal glucose signaling are mediated by the central nervous system. A portal glucose infusion mimicking Intestinal gluconeogenesis leads to the production of glucose, which is released in the portal vein ( 1). This glucose is detected by SGLT3 sensors in the portal vein ( 2), and induces a nerve signal (the portal glucose signal) that subsequently activates brain regions responsible for regulating energy balance (the brainstem and the hypothalamus) and reward circuits (3). A central reflex arc then increases satiety, and leads to weight loss and improvements in blood sugar control and insulin sensitivity (4). For clarity, not all nerve connections have been shown in this illustration. PBN: parabrachial nucleus; DVC: dorsal vagal complex; SGLT3: glucose/sodium cotransporter 3. Adapted from the doctorate thesis manuscript of Dr. Flore Sinet.

intestinal gluconeogenesis induces the expression of the activation neuronal marker c-fos in several brain areas controlling energy balance, such as numerous cortico-limbic areas implicated in reward, the mitral and granular layers of the olfactory bulb, the arcuate nucleus from the hypothalamus (ARH), and the lateral parabrachial nucleus (PBN) (Figure 14) [START_REF] Delaere | Portal Glucose Influences the Sensory, Cortical and Reward Systems in Rats[END_REF][START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF].

As discussed in chapter 2, section 3, the phosphorylation of STAT3 is a critical component of the hypothalamic leptin signaling pathway. Hypothalamic STAT3 phosphorylation following leptin treatment is decreased by 50% in mice lacking intestinal gluconeogenesis; and this is rescued by a portal glucose infusion [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF]. These data suggest that intestinal gluconeogenesis enhances hypothalamic leptin sensitivity.

Interestingly, intestinal gluconeogenesis induces STAT3 phosphorylation in the ARH even in Ob/Ob mice, which lack leptin [START_REF] Zhang | Positional Cloning of the Mouse Obese Gene and Its Human Homologue[END_REF], suggesting that intestinal gluconeogenesis induces the STAT3 signaling pathway independently of leptin [START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]. Regarding the neuronal populations implicated, our results suggest that intestinal gluconeogenesis leads to an increase of STAT3 phosphorylation at least in POMC neurons of the ARH independently of leptin [START_REF] Delaere | Portal Glucose Influences the Sensory, Cortical and Reward Systems in Rats[END_REF][START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]). Altogether, these results imply that the effects mediated by intestinal gluconeogenesis implicate the activation of POMC neurons in the ARH.

Neurons expressing the calcitonin gene-related peptide (CGRP) are mainly found in the splanchnic nerves innervating the portal vein and in the lateral PBN [START_REF] Berthoud | Anatomy and Function of Sensory Hepatic Nerves[END_REF][START_REF] Campos | Parabrachial CGRP Neurons Control Meal Termination[END_REF][START_REF] Kashiba | Cell Size and Cell Type Analysis of Calcitonin Gene-Related Peptide-Containing Cutaneous and Splanchnic Sensory Neurons in the Rat[END_REF][START_REF] Krahn | Effects of Calcitonin Gene-Related Peptide on Food Intake[END_REF]. CGRP has been suggested to suppress hunger [START_REF] Carter | Genetic Identification of a Neural Circuit That Suppresses Appetite[END_REF][START_REF] Krahn | Effects of Calcitonin Gene-Related Peptide on Food Intake[END_REF]. Interestingly, the hunger-curbing effect of a portal glucose infusion is blunted in Cgrp knockout mice, and is rescued by an intracerebroventricular injection of CGRP [START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]. In parallel, the increase of STAT3 phosphorylation in ARH POMC neurons induced by a portal glucose infusion is also blunted in Cgrp knockout mice, and is also rescued by an intracerebroventricular injection of CGRP [START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]. These data suggest that intestinal gluconeogenesis could exert its satiating and POMC molecular effects via CGRP.

To summarize, these data suggest that glucose produced by intestinal gluconeogenesis is detected by portal SGLT3 sensors, which (mainly) via splanchnic afferents control the STAT3 signaling pathway in the ARH, via a CGRP-dependent mechanism. CGRP-expressing neurons are highly dense in the portal vein, in sensory fibers arising from the dorsal root ganglia, and also in the PBN [START_REF] Campos | Parabrachial CGRP Neurons Control Meal Termination[END_REF][START_REF] Iyengar | The Role of Calcitonin Gene-Related Peptide in Peripheral and Central Pain Mechanisms Including Migraine[END_REF][START_REF] Ody | Postnatal Development of the Rat Portal Vein: Correlation with Occurrence of Peptidergic Innervation[END_REF]. Therefore, studies are needed to determine which CGRP neurons (peripheral and/or central) are specifically required in the gut-brain neural circuit controlled by intestinal gluconeogenesis (Figure 14).

The induction of intestinal gluconeogenesis

Intestinal gluconeogenesis can trigger the portal glucose signal during the postabsorptive period under certain conditions such as when induced by specific nutrients, like a diet rich in proteins or soluble fibers (Figure 15), and during the neonatal period.

Protein-enriched diets

Protein-enriched diets are known to decrease hunger and subsequent food intake.

Our laboratory showed that intestinal gluconeogenesis is the mandatory mechanistic link in the protein-induced decrease of food intake [START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF]) (Figure 15). Indeed, feeding rats a protein-enriched diet increases the expression and the activity of three gluconeogenic enzymes: glutaminase, phosphoenol pyruvate kinase (PEPCK-c) and G6Pase in the small intestine, as well as portal glucose release [START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF]. At the molecular level, the incomplete digestion of dietary protein leads to the release of oligopeptides into the portal vein (Duraffourd et al. 2012). Here, oligopeptides antagonize µopioid receptors located in terminals from peri-portal nerves to initiate a nervous signal via vagal and splanchnic afferents (Duraffourd et al. 2012). Brain c-fos immunolabeling further allowed us to show that the antagonism of portal µ-opioid receptors activates the NTS, PBN, and the hypothalamus (Duraffourd et al. 2012). This central activation was associated with the subsequent stimulation of the expression of gluconeogenic genes in the intestine (Duraffourd et al. 2012), therefore activating intestinal gluconeogenesis and launching the portal glucose signaling-induced satiety. Finally, consuming a protein-enriched diet fails to decrease food intake or body weight in mice with an intestine-specific knock-out of G6pc (I.G6pc -/-mice), confirming a causal link of intestinal gluconeogenesis in the effect of dietary protein [START_REF] Penhoat | Protein-Induced Satiety Is Abolished in the Absence of Intestinal Gluconeogenesis[END_REF]).

Diets enriched with soluble fibers

As for protein-enriched diets, the consumption of fibers also improves metabolic health. Consuming a fiber-enriched diet reduces the risk of developing type 2 diabetes and cardiovascular disease both in lean and obese subjects [START_REF] Mendeloff | Dietary Fiber and Human Health[END_REF][START_REF] Ray | Long-Term Effects of Dietary Fiber on Glucose Tolerance and Gastric Emptying in Noninsulin-Dependent Diabetic Patients[END_REF][START_REF] Robertson | Prior Short-Term Consumption of Resistant Starch Enhances Postprandial Insulin Sensitivity in Healthy Subjects[END_REF]. The fermentation of soluble fibers by gut commensal bacteria yields the short-chain fatty acids propionate and butyrate, as well as succinate [START_REF] Vadder | Microbiota-Produced Succinate Improves Glucose Homeostasis via Intestinal Gluconeogenesis[END_REF], among diverse other molecules. Importantly, the role of short-chain fatty acids on the metabolic benefits elicited by dietary fiber have been largely recorded [START_REF] Layden | Short Chain Fatty Acids and Their Receptors: New Metabolic Targets[END_REF]), but the role of succinate on host metabolism was unexplored until recently [START_REF] Vadder | Microbiota-Produced Succinate Improves Glucose Homeostasis via Intestinal Gluconeogenesis[END_REF]). Regarding the short-chain fatty acids, these molecules directly or indirectly induce intestinal gluconeogenesis via complementary mechanisms, and this induction is necessary for their beneficial metabolic effects (Figure 15). Indeed, dietary soluble fibers or shortchain fatty acid consumption increases the expression and/or the activity of gluconeogenic enzymes in the intestine [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF]). On the one hand, butyrate directly activates gene expression in the enterocytes through a cAMP-dependent mechanism [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF]. Propionate, on the other hand, is detected by the free fatty acid receptor 3 (FFAR3)

present in peri-portal afferents. This interaction initiates a nervous signal that ultimately leads to the increase of vasoactive intestinal peptide (VIP) signaling in the enteric submucosal plexus and the subsequent cAMP-mediated up-regulation of G6pc expression in enterocytes [START_REF] De | Vasoactive Intestinal Peptide Is a Local Mediator in a Gut-Brain Neural Axis Activating Intestinal Gluconeogenesis[END_REF]. Regarding the effects of succinate on the metabolism of the host, De Vadder and colleagues showed that dietary succinate improves glucose homeostasis in wild-type mice [START_REF] Vadder | Microbiota-Produced Succinate Improves Glucose Homeostasis via Intestinal Gluconeogenesis[END_REF]). Dietary succinate functions as an intestinal gluconeogenic substrate, inducing the glucose flux produced by the intestine, without changes in the expression of gluconeogenic enzymes [START_REF] Vadder | Microbiota-Produced Succinate Improves Glucose Homeostasis via Intestinal Gluconeogenesis[END_REF]. It is important to note that neither a soluble fiber-enriched diet nor a diet enriched with shortchain fatty acids or with succinate succeeded at improving glucose homeostasis and body weight in I.G6pc -/-mice [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF][START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF], proving a causal role of intestinal gluconeogenesis on the metabolic actions of these nutritional molecules.

As explained and illustrated in Figure 15, protein and soluble fiber digests initiate a neural circuit in the brain, which then, progressively, induces intestinal gluconeogenic enzymes during the late post-prandial period/early post-absorptive period. Therefore, the intestinal gluconeogenic activity is fully increased and persists during the post-absorptive period (Duraffourd et al. 2012;[START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF]). These results are in line with previous data showing that portal glucose signaling decreases the size of the next meal [START_REF] Baird | Intake Suppression after Hepatic Portal Glucose Infusion: All-or-None Effect and Its Temporal Threshold[END_REF], which is a hallmark of an effect taking place during the post-absorptive period. Indeed, if the feeding effect of portal glucose signaling took place during the postprandial period, portal glucose signaling would lead to meal termination instead. The fact that intestinal gluconeogenesis is induced during the post-absorptive period is important because at this time many hunger-curbing signals (like gastric distension and an increase in circulating anorexigenic hormones like CCK and GLP-1 [START_REF] Janssen | Review Article: The Role of Gastric Motility in the Control of Food Intake[END_REF][START_REF] Kairupan | Role of Gastrointestinal Hormones in Feeding Behavior and Obesity Treatment[END_REF])) have ended. At that point, the portal glucose signal induced by intestinal gluconeogenesis becomes the only satiety signal to be sensed by the brain and can thus be fully efficient for decreasing hunger (For a review, see Soty et al., 2017).

The postnatal period

Intestinal gluconeogenesis is also strongly induced during the postnatal period (Figure 16) [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]. The transition from the intrauterine to the extrauterine life is characterized by a transient but drastic neonatal hypoglycaemia due to the sudden absence of placental glucose supply [START_REF] Girard | Adaptations of Glucose and Fatty Acid Metabolism during Perinatal Period and Suckling-Weaning Transition[END_REF]. This hypoglycaemia has also been reported in the portal vein of the neonate [START_REF] Girard | Adaptations of Glucose and Fatty Acid Metabolism during Perinatal Period and Suckling-Weaning Transition[END_REF]). The maternal glucose supply does not get a lot better after birth because maternal milk is rich in lipids (about 60%) and low in carbohydrates (less than 10%) [START_REF] Girard | Adaptations of Glucose and Fatty Acid Metabolism during Perinatal Period and Suckling-Weaning Transition[END_REF]. Therefore, to maintain glucose levels, the newborn rapidly activates the pathways of EGP, including gluconeogenesis. For a long time, neonatal gluconeogenesis was believed to be assured by the liver and the kidneys only [START_REF] Girard | Adaptations of Glucose and Fatty Acid Metabolism during Perinatal Period and Suckling-Weaning Transition[END_REF]. However, early studies from the eighties suggested that gluconeogenesis might occur in the intestine during the suckling period too.

The mRNA levels and/or activity of PEPCK-c [START_REF] Hahn | Phosphoenolpyruvate Carboxykinase in the Small Intestine of Developing Rodents[END_REF][START_REF] Watford | Phosphoenolpyruvate Carboxykinase of Rat Small Intestine: Distribution and Regulation of Activity and MRNA Levels[END_REF], pyruvate carboxylase [START_REF] Hahn | Pyruvate Carboxylase, Phosphate-Dependent Glutaminase and Glutamate Dehydrogenase in the Developing Rat Small Intestinal Mucosa[END_REF] and fructose di-phosphatase [START_REF] Hahn | Phosphoenolpyruvate Carboxykinase in the Small Intestine of Developing Rodents[END_REF] are high during the suckling period and then decrease at weaning in rats. It was about a decade later that Chatelain and colleagues reported that although mRNA levels and the activity of G6Pase were low in the fetal rat jejunum, they increased right after birth, reaching a peak at post-natal day 5 (P5) and then decreased again [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]. Interestingly, the level of G6pc mRNA at P5 was as high as the levels found in the liver of 48 hour fasted adult rats [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]. The peak of intestinal gluconeogenesis occurring during the postnatal period in rats [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]) has also been reported in pigs [START_REF] Darcy-Vrillon | Glucose, Galactose, and Glutamine Metabolism in Pig Isolated Enterocytes during Development[END_REF], mice [START_REF] Ménard | Ultrastructural Localization of Intestinal Glucose-6-Phosphatase Activity during the Postnatal Development of the Mouse[END_REF][START_REF] Menard | Glucose-6-Phosphatase Activity in Mouse Small Intestine during Postnatal Development[END_REF] and guinea pigs [START_REF] Anderson | Gluconeogenesis in Jejunal Mucosa of Guinea Pig[END_REF]) (Figure 16).

Mouse models to study intestinal gluconeogenesis

To study the role of intestinal gluconeogenesis independently of nutrition, our laboratory has developed original murine models that lack or overexpress G6PC specifically in the intestine.

Invalidation of intestinal gluconeogenesis

The animal model

Mice that lack intestinal gluconeogenesis (I.G6pc -/-) were obtained via the inducible CRE/lox biotechnology strategy [START_REF] Penhoat | Protein-Induced Satiety Is Abolished in the Absence of Intestinal Gluconeogenesis[END_REF], which allows us to control the genetic modification in a time-dependent and tissue-specific manner [START_REF] Feil | Ligand-Activated Site-Specific Recombination in Mice[END_REF][START_REF] Metzger | Siteand Time-Specific Gene Targeting in the Mouse[END_REF][START_REF] Sauer | Targeted Insertion of Exogenous DNA into the Eukaryotic Genome by the Cre Recombinase[END_REF]. Two loxP sequences were introduced in the introns surrounding the exon 3 of G6pc, which encodes an essential amino acid of the catalytic site of G6Pase (Figure 17). The Cre ERT2 recombinase was placed under the control of the villin promoter, which is expressed specifically in the intestine (El [START_REF] Marjou | Tissue-Specific and Inducible Cre-Mediated Recombination in the Gut Epithelium[END_REF] in order to induce an intestine-specific genetic deletion [START_REF] Penhoat | Protein-Induced Satiety Is Abolished in the Absence of Intestinal Gluconeogenesis[END_REF]. The inducible CRE/lox system was created by fusing the CRE recombinase with a mutated ligand-binding domain of the human estrogen receptor (ER), which allows for activation of the Cre recombinase only upon binding of the synthetic ER ligand 4hydroxytamoxifen (a metabolite of tamoxifen), but not by physiological estrogen [START_REF] Feil | Ligand-Activated Site-Specific Recombination in Mice[END_REF][START_REF] Metzger | Siteand Time-Specific Gene Targeting in the Mouse[END_REF][START_REF] Sauer | Targeted Insertion of Exogenous DNA into the Eukaryotic Genome by the Cre Recombinase[END_REF]. The tamoxifen administration in 5 week-old mice induces a conformational change in the Cre ERT2

recombinase, which then translocates to the nucleus. Once in the nucleus, the Cre ERT2 recombinase recognizes the loxP sites and excises the flanked DNA sequence, leading to a truncated, non-functional G6Pase. EGP in I.G6pc -/-mice is assured by the liver and the kidneys [START_REF] Penhoat | Protein-Induced Satiety Is Abolished in the Absence of Intestinal Gluconeogenesis[END_REF]) (Figure 17).

The metabolic phenotype of I.G6pc -/-mice: a pre-diabetic state I.G6pc -/-mice develop severe metabolic alterations under a standard diet; and develop more rapidly a type 2 diabetes phenotype under a high-fat/high-sucrose diet. In this respect, mice lacking intestinal gluconeogenesis exhibit fasting hyperglycaemia and hyperinsulinemia as well as glucose intolerance and hepatic insulin resistance. I.G6pc -/-mice also exhibit a defective glucose-stimulated insulin secretion, suggesting a deteriorated pancreatic function [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF]. These data suggest that intestinal gluconeogenesis is necessary to maintain a healthy glucose control. Furthermore, I.G6pc -/-mice have about 70% more hepatic triglycerides content, associated with an increased hepatic expression of genes implicated in the lipogenesis pathway and lipid uptake, in comparison to wild-type mice [START_REF] Vily-Petit | Intestinal Gluconeogenesis Prevents Obesity-Linked Liver Steatosis and Non-Alcoholic Fatty Liver Disease[END_REF]. These data suggest that the absence of intestinal gluconeogenesis is thus enough to induce hepatic steatosis, which is a hallmark of type 2 diabetes (Smith and Adams 2011). Importantly, leptin administration failed to decrease food intake or to induce hypothalamic STAT3 phosphorylation in I.G6pc -/-mice, suggesting that the absence of intestinal gluconeogenesis is associated with hypothalamic leptin resistance and thus an altered hypothalamic function [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF]. Importantly, all the alterations mentioned were corrected by rescuing intestinal gluconeogenesis through the portal infusion of glucose at a rate that mimics intestinal gluconeogenesis, highlighting the causal relationship between portal glucose sensing and the studied parameters [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF][START_REF] Vily-Petit | Intestinal Gluconeogenesis Prevents Obesity-Linked Liver Steatosis and Non-Alcoholic Fatty Liver Disease[END_REF]).

Intestinal gluconeogenesis is therefore a crucial physiological function for energy balance control in both a normal and an overload nutritional state.

Induction of intestinal gluconeogenesis

The animal model

Mice that overexpress intestinal gluconeogenesis (I.G6pc overexp ) were obtained via the constitutive CRE/lox biotechnology strategy (Vily-Petit et al. 2020), which allows us to control the genetic modification in a tissue-specific manner [START_REF] Sauer | Targeted Insertion of Exogenous DNA into the Eukaryotic Genome by the Cre Recombinase[END_REF][START_REF] Soriano | Generalized LacZ Expression with the ROSA26 Cre Reporter Strain[END_REF][START_REF] Zambrowicz | Disruption of Overlapping Transcripts in the ROSA Geo 26 Gene Trap Strain Leads to Widespread Expression of -Galactosidase in Mouse Embryos and Hematopoietic Cells[END_REF]. The basis of this overexpression strategy is the insertion in the Rosa26 locus of both a STOP cassette flanked between two loxP sequences, and the complementary DNA of G6pc [START_REF] Soriano | Generalized LacZ Expression with the ROSA26 Cre Reporter Strain[END_REF][START_REF] Zambrowicz | Disruption of Overlapping Transcripts in the ROSA Geo 26 Gene Trap Strain Leads to Widespread Expression of -Galactosidase in Mouse Embryos and Hematopoietic Cells[END_REF]. This locus exhibits a strong transcriptional activity but is blocked here by the stop cassette (Figure 18) [START_REF] Soriano | Generalized LacZ Expression with the ROSA26 Cre Reporter Strain[END_REF][START_REF] Zambrowicz | Disruption of Overlapping Transcripts in the ROSA Geo 26 Gene Trap Strain Leads to Widespread Expression of -Galactosidase in Mouse Embryos and Hematopoietic Cells[END_REF]. As for I.G6pc -/-mice, the CRE recombinase is also expressed under the control of the villin promoter to induce an intestine-specific genetic modification (El [START_REF] Marjou | Tissue-Specific and Inducible Cre-Mediated Recombination in the Gut Epithelium[END_REF]). However, in contrast to the inducible CRE/lox system, this Cre recombinase has not been fused to ERT2, and is thus always active. The stop cassette is therefore constantly excised by the CRE recombinase, allowing a strong transcription of G6pc specifically in the intestine since embryogenesis [START_REF] Maunoury | Villin Expression in the Visceral Endoderm and in the Gut Anlage during Early Mouse Embryogenesis[END_REF][START_REF] Maunoury | Developmental Regulation of Villin Gene Expression in the Epithelial Cell Lineages of Mouse Digestive and Urogenital Tracts[END_REF].

I.G6pc overexp mice exhibit a four-fold overexpression of G6PC, and a three-fold increased I.G6pc overexp mice showed an inferior hepatic expression of genes implicated in proinflammatory and pro-fibrotic pathways in comparison to wild-type mice (Vily-Petit et al.

2020

). Together, these data suggest that overexpressing intestinal gluconeogenesis, independently of nutrition, is enough to protect the metabolism of an organism from the alterations induced by an obesogenic diet.

It is important to emphasize that results obtained with our transgenic murine models have validated the results obtained with specific diets regarding the beneficial effects of intestinal gluconeogenesis. For information, all results were obtained using male rodents.

Chapter 4. Developmental aspects of the gut-brain axis in healthy individuals

This chapter will cover a brief description of the development of some of the main components of the gut-brain axis: the enteric nervous system, the portal vein and its afferent innervations, and hypothalamic feeding circuits.

Preamble

During embryonic development, gastrulation is the forming of three germ layers: the ectoderm, the mesoderm and the endoderm [START_REF] Swartley | Histology Atlas of the Developing Mouse Hepatobiliary Hemolymphatic Vascular System with Emphasis on Embryonic Days 11.5-18.5 and Early Postnatal Development[END_REF]. The ectoderm gives rise to the neural plate border, which then gives rise to the neural crest and the cranial placodes

The development of the enteric nervous system

The fetus and the newborn need a functional gut to digest and absorb blood-derived and milk-derived nutrients, respectively. In the adult, the enteric nervous system (ENS) plays a key role in the digestive function because it regulates gastrointestinal motility, fluid secretion, nutrient absorption and blood flow. The enteric nervous system develops mainly during fetal life and continues to mature during the post-natal period in rodents. Indeed, research in mice shows that most enteric neurons are formed during gestation and the early post-natal life [START_REF] Pham | Time of Origin of Neurons in the Murine Enteric Nervous System: Sequence in Relation to Phenotype[END_REF]. Interestingly, despite being present since E14.5, enteric neurons do not mediate intestinal motility until E18.5. Indeed, neuronal-mediated motility starts at E18.5 in the duodenum, and approximately one week after birth in the colon [START_REF] Roberts | Development of Colonic Motility in the Neonatal Mouse-Studies Using Spatiotemporal Maps[END_REF][START_REF] Roberts | The First Intestinal Motility Patterns in Fetal Mice Are Not Mediated by Neurons or Interstitial Cells of Cajal: Gastrointestinal Motility in Fetal Mice[END_REF]. This delay has been attributed to the maturing of enteric neurons and their neuronal network [START_REF] Roberts | The First Intestinal Motility Patterns in Fetal Mice Are Not Mediated by Neurons or Interstitial Cells of Cajal: Gastrointestinal Motility in Fetal Mice[END_REF]. In fetal mice, the motility of the duodenum and the colon is assured by nonneuronal, myogenic propagating contractions that are sometimes called ripples [START_REF] Roberts | The First Intestinal Motility Patterns in Fetal Mice Are Not Mediated by Neurons or Interstitial Cells of Cajal: Gastrointestinal Motility in Fetal Mice[END_REF].

Regarding the development of the structured organization of the mature ENS, the vagal neural crest cells first settle in what will become the myenteric ganglia. Pham and colleagues showed that almost all myenteric neurons are already born in the small intestine by postnatal day 10 (P10) [START_REF] Foong | Postnatal Development of the Mouse Enteric Nervous System[END_REF][START_REF] Pham | Time of Origin of Neurons in the Murine Enteric Nervous System: Sequence in Relation to Phenotype[END_REF][START_REF] Uesaka | Development of the Intrinsic and Extrinsic Innervation of the Gut[END_REF]. In parallel, a subpopulation of the developing myenteric ganglia migrates inward to form the submucosal ganglia [START_REF] Payette | Neurofilament Expression in Vagal Neural Crest-Derived Precursors of Enteric Neurons[END_REF][START_REF] Uesaka | Development of the Intrinsic and Extrinsic Innervation of the Gut[END_REF]. Indeed, submucosal neurons tend to be born later than myenteric neurons [START_REF] Pham | Time of Origin of Neurons in the Murine Enteric Nervous System: Sequence in Relation to Phenotype[END_REF]. The principal functions of the two plexuses are peristalsis control (for the myenteric ganglia) and regulating secretion, fluid exchange and blood flow (for submucosal ganglia) [START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF].

Some studies have suggested that intestinal motility patterns seem to be mature at mid postnatal life in both the duodenum and the colon [START_REF] Foong | Postnatal Development of the Mouse Enteric Nervous System[END_REF].

The development of the ENS requires a plethora of mechanisms including signaling molecules, the glial cell-derived neurotrophic factor (GDNF), serotonin (5-HT), axon guidance and rejection cues, and the gut microbiota [START_REF] Foong | Postnatal Development of the Mouse Enteric Nervous System[END_REF][START_REF] Uesaka | Development of the Intrinsic and Extrinsic Innervation of the Gut[END_REF].

Regarding the latter, the gut microbiota starts being formed after birth, when bacteria colonize the gastrointestinal tract. Studies show that mice lacking microbiota (i.e. germ-free mice) exhibit an abnormal neuronal composition and contractibility of the ENS [START_REF] Collins | Intestinal Microbiota Influence the Early Postnatal Development of the Enteric Nervous System[END_REF], suggesting that gut microbiota is necessary for optimal ENS development. In a recent study, De Vadder and colleagues proposed that gut microbiota stimulates the ENS postnatal development by activating the release of the enteric neurotrophic factor serotonin (5-HT) [START_REF] Vadder | Gut Microbiota Regulates Maturation of the Adult Enteric Nervous System via Enteric Serotonin Networks[END_REF][START_REF] Li | Essential Roles of Enteric Neuronal Serotonin in Gastrointestinal Motility and the Development/Survival of Enteric Dopaminergic Neurons[END_REF].

As discussed in chapter 3, in adult mice, glucose produced by the intestine is sensed in the portal vein. Given that intestinal gluconeogenesis is strongly induced during the postnatal period, studies investigating the development of the portal vein are of key interest in this manuscript.

The development of the portal vein

The portal vein develops from the vitelline vein during gestation, around E12.5 in mice [START_REF] Crawford | Histology Atlas of the Developing Mouse Hepatobiliary System with Emphasis on Embryonic Days 9.5-18.5[END_REF][START_REF] Swartley | Histology Atlas of the Developing Mouse Hepatobiliary Hemolymphatic Vascular System with Emphasis on Embryonic Days 11.5-18.5 and Early Postnatal Development[END_REF]. During embryogenesis, the earliest signs of vascular development are believed to appear during gastrulation (E6.0), when angioblasts in the mesoderm differentiate into endothelial cells [START_REF] Swartley | Histology Atlas of the Developing Mouse Hepatobiliary Hemolymphatic Vascular System with Emphasis on Embryonic Days 11.5-18.5 and Early Postnatal Development[END_REF].

During the early development, the embryonic liver venous drainage consists of three paired veins: 1) the umbilical veins from the chorion, carrying oxygenated blood from the placenta to the embryo; 2) the vitelline veins from the yolk sac, draining the gastrointestinal tract, and

3) the cardinal veins from the embryo, draining the embryonic head, neck, and body [START_REF] Crawford | Histology Atlas of the Developing Mouse Hepatobiliary System with Emphasis on Embryonic Days 9.5-18.5[END_REF]) (Figure 21A). At E9.0-E9.5, the liver starts to develop and forms the hepatic bud at the foregut-midgut junction (Figure 21B). At E11.5, significant changes occur in the vascular system as the liver continues to grow. The vitelline veins infiltrate and ramify in the septum transversum (the primitive diaphragm), forming the hepatic sinusoids and drains into the sinus venosus (the primitive heart), whereas the umbilical veins develop into

an intra-and an extra-hepatic branch (Figure 21C). Then, the extra-hepatic branches of the umbilical vein regress (Figure 21D). In respect to the vitelline veins, three parts regress around this period: the caudal part of the right vitelline vein, the cranial part of the left vitelline vein and the caudal-ventral anastomosis (Figure 21D) [START_REF] Carneiro | All about Portal Vein: A Pictorial Display to Anatomy, Variants and Physiopathology[END_REF]). At (Figure 21E) [START_REF] Crawford | Histology Atlas of the Developing Mouse Hepatobiliary System with Emphasis on Embryonic Days 9.5-18.5[END_REF]. The degree of maturation of the portal vein at birth differs among species [START_REF] Ody | Postnatal Development of the Rat Portal Vein: Correlation with Occurrence of Peptidergic Innervation[END_REF]). In the rat, although already existent at birth, the portal vein pursues its maturation process during the postnatal period and achieves its final vessel structure at around P28 [START_REF] Ody | Postnatal Development of the Rat Portal Vein: Correlation with Occurrence of Peptidergic Innervation[END_REF].

Another key component of the gut-brain axis controlled by intestinal gluconeogenesis in adult mice is portal glucose signaling through afferents innervating the portal vein. For this reason, the timing of descent of both vagal and splanchnic afferents to viscera is of key interest in this manuscript, with a particular focus in the branching of the portal vein.

The development of hepatic vagal and splanchnic innervation

In the adult, the cell bodies of vagal and splanchnic afferents reside in different ganglia: the nodose ganglia and the dorsal root ganglia, respectively. Both ganglia have different developmental origins. Cranial placodes gives rise to neurons in the nodose ganglia, whereas neural crest cells from the thoracic level give rise to neurons in the dorsal root ganglia (Park and Saint-Jeannet 2010; Ratcliffe 2011) (Figure 20). The axons from neurons in both ganglia outgrow toward liver and gastrointestinal tract zones during development.

Vagal innervation

The timing of descent of vagal afferents has been studied in much greater detail in comparison to splanchnic afferents. In rats, labeling of vagal sensory neurons is observed in the nodose ganglia at least since E12, which were the youngest rat fetuses examined [START_REF] Rinaman | Establishment of Vagal Sensorimotor Circuits during Fetal Development in Rats[END_REF][START_REF] Uesaka | Development of the Intrinsic and Extrinsic Innervation of the Gut[END_REF]. In mice, vagal axons are already present in the stomach at E11, the esophagus at E12, the duodenum at E14, and the distal part of the small intestine at E16 [START_REF] Ratcliffe | Molecular Development of the Extrinsic Sensory Innervation of the Gastrointestinal Tract[END_REF]). In the stomach, vagal axonal bundles showed an adult-like distribution at E16.6 (Ratcliffe 2011).

To my knowledge, no studies have reported the timing of the outgrowth of vagal afferents specifically to the portal vein. However, Rinaman and colleagues also demonstrated in rats that vagal sensory neurons are present by E12 in the abdomen. Interestingly, for this experiment, they inserted crystals of the lipophilic tracer 1,1′-dioctadecyl-3,3,3′3,3′tetramethyl-indocarbocyanine perchlorate (DiI) in an abdominal region containing the esophagus, the stomach, the pancreas, the liver, the intestines, and the cecum [START_REF] Rinaman | Establishment of Vagal Sensorimotor Circuits during Fetal Development in Rats[END_REF][START_REF] Uesaka | Development of the Intrinsic and Extrinsic Innervation of the Gut[END_REF]). Therefore, one cannot exclude that some of these terminations could come from the hepatic portal vein. Although studies are needed to determine the timing of vagal innervation of the portal vein specifically, these data show that vagal visceral innervation takes place already during gestation in rodents. Furthermore, nerve bundles in the portal vein are present at least since P5 in rats [START_REF] Ody | Postnatal Development of the Rat Portal Vein: Correlation with Occurrence of Peptidergic Innervation[END_REF]. Plus, in a recent study, Tanimizu and colleagues labeled with an anti-neural class III β-tubulin antibody the liver tissue sections of P7 mouse pups and observed nerve fibers around the portal vein [START_REF] Tanimizu | Intrahepatic Bile Ducts Guide Establishment of the Intrahepatic Nerve Network in Developing and Regenerating Mouse Liver[END_REF]. Additionally, labeling with the pan neuronal markers PGP9.5 and neurofilament and the glial marker S-100 allowed to reveal that hepatic portal tract innervation is observed since gestational week 12 in humans, and its density increases toward term [START_REF] Delalande | Hepatic Nervous System Development[END_REF]. Altogether, these data suggest that vagal innervation of the portal vein could already be present during the postnatal period.

Splanchnic innervation

As previously discussed, the cell bodies of visceral splanchnic afferents reside in the dorsal root ganglia. Literature data show that dorsal root axon projections develop during gestation. Indeed, between E12.5 and E13.5, afferent axons develop node-like structures and form collateral branches from these "nodes" [START_REF] Ozaki | Initial Trajectories of Sensory Axons toward Laminar Targets in the Developing Mouse Spinal Cord[END_REF]. At E13.5, a few afferent axons are present in the dorsal gray matter from the spinal cord [START_REF] Ozaki | Initial Trajectories of Sensory Axons toward Laminar Targets in the Developing Mouse Spinal Cord[END_REF]. Afferent projections to the superficial and the deep areas of the dorsal horn are visible at E15.5 [START_REF] Ozaki | Initial Trajectories of Sensory Axons toward Laminar Targets in the Developing Mouse Spinal Cord[END_REF]. These data confirm the presence of dorsal root ganglia and some of their spinal-cord incoming nerves already during gestation.

To my knowledge, no retrograde studies have been conducted to determine the timing of descent of splanchnic visceral afferents specifically. However, the CGRP peptide is considered an excellent marker for portal splanchnic afferents [START_REF] Berthoud | Anatomy and Function of Sensory Hepatic Nerves[END_REF][START_REF] Kashiba | Cell Size and Cell Type Analysis of Calcitonin Gene-Related Peptide-Containing Cutaneous and Splanchnic Sensory Neurons in the Rat[END_REF]. Interestingly, CGRP innervation in the portal vein is already highly dense at birth and it remains stable during the first two weeks after birth in rats [START_REF] Ody | Postnatal Development of the Rat Portal Vein: Correlation with Occurrence of Peptidergic Innervation[END_REF][START_REF] Thiévent | Potential Role of the Neuropeptide CGRP in the Induction of Differentiation of Rat Hepatic Portal Vein Wall[END_REF]. Portal vein CGRP innervation density then decreases until P28 [START_REF] Ody | Postnatal Development of the Rat Portal Vein: Correlation with Occurrence of Peptidergic Innervation[END_REF].

Then, after weaning, it increases gradually until reaching the same density observed at birth in the adult rat [START_REF] Ody | Postnatal Development of the Rat Portal Vein: Correlation with Occurrence of Peptidergic Innervation[END_REF]. These data suggest that splanchnic afferents are already present in the portal vein during the postnatal period.

The trajectory of axonal outgrowth is subject to axon guidance and rejection cues, neurotrophins, the brain-derived neurotrophic factor (BDNF), and many more molecules.

This complex scenario results in a very precise afferent outgrowth toward specific places in viscera [START_REF] Ratcliffe | Molecular Development of the Extrinsic Sensory Innervation of the Gastrointestinal Tract[END_REF]). However, this subject exceeds the interest of this manuscript and will not be discussed here. More information can be found in the following reviews: [START_REF] Delalande | Hepatic Nervous System Development[END_REF][START_REF] Ratcliffe | Molecular Development of the Extrinsic Sensory Innervation of the Gastrointestinal Tract[END_REF].

In summary, studies have shown that components of the gut-brain axis triggered by intestinal gluconeogenesis develop during fetal life: the portal vein, and neurons from the nodose and the dorsal root ganglia. Importantly, although information is incomplete, studies do suggest that the portal vein might already contain vagal and splanchnic terminals during the early postnatal period and is already innervated at least since P5 in rats.

The hypothalamic development

As discussed in chapter 2, the hypothalamus is a key integrant of the gut-brain neural circuit and is a central target of intestinal gluconeogenesis. This section will first describe the three major steps of the development of the hypothalamic neuronal circuit: 1) neuronal birth and cell specification, 2) axonal outgrowth and 3) synaptogenesis. Then, the endocrine regulation of this development will be described (Figure 22).

Major stages of hypothalamic development

Neuronal birth and cell specification

The hypothalamic primordium derives from cells from the ventral neural tube around E9 in mice. Cells from hypothalamic nuclei are derived from the proliferative zone of the third ventricle (For a review, read [START_REF] Bouret | Development of Hypothalamic Circuits That Control Food Intake and Energy Balance[END_REF]. Studies using the incorporation of the radioactive nucleoside [ 3 H]thymidine suggest that most hypothalamic cells are born between E13 and E16 in rats, and between E10 and E14 in mice (Figure 22A) [START_REF] Ifft | An Autoradiographic Study of the Time of Final Division of Neurons in Rat Hypothalamic Nuclei[END_REF][START_REF] Shimada | Time of Neuron Origin in Mouse Hypothalamic Nuclei[END_REF]. More recent studies have used bromeodeoxyuridine (BrdU), which can be co-administered with phenotypic markers. This technique has therefore allowed researchers to determine the birth of specific hypothalamic neuronal subpopulations. In the arcuate nucleus, the majority of POMC-expressing neurons seems to be born by E10.5 but they acquire their terminal peptidergic phenotype during mid gestation [START_REF] Padilla | Pomc-Expressing Progenitors Give Rise to Antagonistic Neuronal Populations in Hypothalamic Feeding Circuits[END_REF]. About half of embryonic POMC-expressing precursors differentiate into non-POMC expressing neurons [START_REF] Padilla | Pomc-Expressing Progenitors Give Rise to Antagonistic Neuronal Populations in Hypothalamic Feeding Circuits[END_REF]. In this respect, a subpopulation of POMC-expressing neurons differentiates into NPY-expressing neurons [START_REF] Croizier | Central Dicer-MiR-103/107 Controls Developmental Switch of POMC Progenitors into NPY Neurons and Impacts Glucose Homeostasis[END_REF][START_REF] Padilla | Pomc-Expressing Progenitors Give Rise to Antagonistic Neuronal Populations in Hypothalamic Feeding Circuits[END_REF]. Then, NPY neurons acquire their terminal AgRP peptidergic phenotype during the first three weeks of the postnatal period [START_REF] Nilsson | Maturation of the Hypothalamic Arcuate Agouti-Related Protein System during Postnatal Development in the Mouse[END_REF]. A low signal of AgRP mRNA is visible at P0, which increases at P5, and continues increasing gradually at P10, P15 and P21, reaching the characteristic adult levels by P21 [START_REF] Nilsson | Maturation of the Hypothalamic Arcuate Agouti-Related Protein System during Postnatal Development in the Mouse[END_REF]. Expression of the neuropeptides POMC, CART, AgRP and NPY is low in the ARH at P4 but increases considerably since around P10 and is the highest around P14 [START_REF] Cottrell | Developmental Changes in Hypothalamic Leptin Receptor: Relationship with the Postnatal Leptin Surge and Energy Balance Neuropeptides in the Postnatal Rat[END_REF]).

Axon development

Axon development could be divided into three major steps. The first step is axon specification during neuronal polarization. Neurons recently born become bipolar and exhibit a trailing process that will become the axon. The second step is axonal outgrowth which starts during neuronal migration. During this step, the trailing process extends rapidly while outgrowing toward its respective targets. The third step is axonal branching, which allows an axon to connect to several post-synaptic targets, and pre-synaptic differentiation. In the mouse cortex, the neuronal trailing process develops approximately from E11 to E18, axonal outgrowth occurs during the first post-natal week, and axonal branching takes place during the second and third post-natal weeks (For a review, read [START_REF] Lewis | Cellular and Molecular Mechanisms Underlying Axon Formation, Growth, and Branching[END_REF].

In the rodent hypothalamus, axonal outgrowth has been reported to take place during the first three post-natal weeks. By using the lipophilic tracer DiI, Bouret and colleagues showed that axons from the DMH connect to the PVH and the LH by P6 (Bouret 2004a).

Axons from the VMH connect to the LH by P10 (Bouret 2004a). In contrast to axonal

Synaptogenesis

Synaptogenesis is the creation of synapses between neurons. In rats, the first synapses in the ARH are observed in 18-day old fetuses [START_REF] Koritsánszky | Cytoand Synaptogenesis in the Arcuate Nucleus of the Rat Hypothalamus during Fetal and Early Postnatal Life[END_REF]. By using electron miscroscopy, Arai and Matsumoto showed that axodendritic and axosomatic synapses are present but very few by P5 in the ARH of rats [START_REF] Arai | Synapse Formation of the Hypothalamic Arcuate Nucleus during Post-Natal Development in the Female Rat and Its Modification by Neonatal Estrogen Treatment[END_REF]. The number of these synapses increases gradually from then to adulthood, but seems to reach adult-like levels around P45 [START_REF] Bouret | Development of Hypothalamic Circuits That Control Food Intake and Energy Balance[END_REF]. Compared to the number of synapses at P5, ARH synapses are multiplied by four at P20, and by 16 in 20-week-old rats [START_REF] Arai | Synapse Formation of the Hypothalamic Arcuate Nucleus during Post-Natal Development in the Female Rat and Its Modification by Neonatal Estrogen Treatment[END_REF]. Regarding specific neuronal populations in the ARH, a recent study showed that, by P13, the number of glutamatergic inputs to AgRP/NPY neurons is comparable to that observed in adult mice [START_REF] Baquero | Developmental Changes in Synaptic Distribution in Arcuate Nucleus Neurons[END_REF]. Presynaptic inputs to AgRP/NPY neurons in the ARH are predominantly excitatory during the first three weeks of life in mice. The switch to inhibitory inputs within this circuitry does not occur until P21 as a result of an increasing expression of ATP-dependent potassium (KATP) channels (Juan [START_REF] Solis | Postnatal Undernutrition Delays a Key Step in the Maturation of Hypothalamic Feeding Circuits[END_REF]. In line with these data, the quantity of GABAergic inputs to AgRP/NPY neurons is low during the early post-natal period [START_REF] Baquero | Developmental Changes in Synaptic Distribution in Arcuate Nucleus Neurons[END_REF]. In adult mice, AgRP/NPY neurons have comparable levels of both GABAergic and glutamatergic inputs [START_REF] Baquero | Developmental Changes in Synaptic Distribution in Arcuate Nucleus Neurons[END_REF].

Electrophysiology experiments show that synapses between POMC axons and the PVH are not structurally and functionally mature until puberty [START_REF] Melnick | Developmental Switch in Neuropeptide Y and Melanocortin Effects in the Paraventricular Nucleus of the Hypothalamus[END_REF]).

Endocrine regulation of the development of hypothalamic feeding circuits

The development of hypothalamic neuronal circuits controlling life-long energy balance is a process that requires signaling by several hormones. Interestingly, some hormones that regulate feeding and energy expenditure during adulthood actually exert a developmental role during the perinatal period. Leptin, ghrelin, insulin and amylin are four of the "metabolic hormones" that play a major role in the perinatal development of hypothalamic feeding circuits.

Leptin

In adults, leptin is mainly secreted by adipocytes while a variety of tissues produce leptin during embryonic development. In mice, high levels of leptin mRNA are found in the fetal liver and cartilage-bone structures on E13, then in cardiac tissue on E16 [START_REF] Hoggard | Leptin and Leptin Receptor MRNA and Protein Expression in the Murine Fetus and Placenta[END_REF]. The leptin levels secreted by dams are high during pregnancy and depend on their metabolic status, but whether leptin crosses the placenta remains controversial [START_REF] Bouret | Development of Hypothalamic Circuits That Control Food Intake and Energy Balance[END_REF]. Mice plasma leptin levels surge during the postnatal period, reaching a peak between P8 and P12 (Figure 23B) [START_REF] Ahima | Postnatal Leptin Surge and Regulation of Circadian Rhythm of Leptin by Feeding. Implications for Energy Homeostasis and Neuroendocrine Function[END_REF][START_REF] Devaskar | Developmental Changes in Ob Gene Expression and Circulating Leptin Peptide Concentrations[END_REF]. This leptin surge is correlated with higher expression of Leptin mRNA in white and brown adipose tissues [START_REF] Devaskar | Developmental Changes in Ob Gene Expression and Circulating Leptin Peptide Concentrations[END_REF].

During the postnatal period, the "satiety hormone" leptin does not affect food intake but exerts a developmental role. Indeed, it is only after P28 that leptin decreases food intake in mice [START_REF] Baquero | Developmental Switch of Leptin Signaling in Arcuate Nucleus Neurons[END_REF][START_REF] Mistry | Leptin Alters Metabolic Rates before Acquisition of Its Anorectic Effect in Developing Neonatal Mice[END_REF]. In 1999, Ahima and colleagues showed that Ob/Ob mice (leptin deficient mice) had lighter brains and an altered expression of synaptic proteins [START_REF] Ahima | Regulation of Neuronal and Glial Proteins by Leptin: Implications for Brain Development 1[END_REF], which suggested a role for leptin in brain development. Analysis of hypothalamic POMC and AgRP neuronal projections during the neonatal period shows that the "neonatal leptin peak" exerts a neurotrophic effect on neuronal circuits from the ARH (Bouret 2004b). Indeed, Ob/Ob mice exhibit a permanent disruption of axonal projections from the ARH to the PVH, the DMH and the LH (Bouret 2004b). These axonal projections are restored in Ob/Ob mice treated with leptin from P4 to P12 (Bouret 2004b), showing a causal link between neonatal leptin and axonal outgrowth from the ARH. Interestingly, the leptin treatment failed to restore ARH projections when it was administered during adulthood (Bouret 2004b), showing that the neurotrophic effect exerted by leptin is restricted to the postnatal period. Additionally, acute leptin administration in neonates induces Pomc and Socs3 and decreases Npy expression in the ARH [START_REF] Proulx | Leptin Regulates Appetite-Related Neuropeptides in the Hypothalamus of Developing Rats without Affecting Food Intake[END_REF].

From a mechanistic point of view, studies show that leptin receptors are already present in the brain during gestation [START_REF] Hoggard | Leptin and Leptin Receptor MRNA and Protein Expression in the Murine Fetus and Placenta[END_REF], and that LepRb mRNA is highly expressed in the ARH, the VMH, the DMH and the LH in neonatal mice (Figure 22B) [START_REF] Caron | Distribution of Leptin-Sensitive Cells in the Postnatal and Adult Mouse Brain[END_REF]. Furthermore, leptin administration leads to STAT3, ERK and AKT phosphorylation in P10 mice [START_REF] Bouret | Distinct Roles for Specific Leptin Receptor Signals in the Development of Hypothalamic Feeding Circuits[END_REF] expressing LepRb respond to the direct trophic action of leptin [START_REF] Caron | Distribution of Leptin-Sensitive Cells in the Postnatal and Adult Mouse Brain[END_REF]).

Ghrelin

The "hunger hormone" ghrelin also plays a major role in the development of hypothalamic feeding circuits during the perinatal period. In rats, Ghrl mRNA is detected at E12, and detectable acylated (the active form) and deacylated (the inactive form) levels are present in blood by E17 [START_REF] Torsello | Ontogeny and Tissue-Specific Regulation of Ghrelin MRNA Expression Suggest That Ghrelin Is Primarily Involved in the Control of Extraendocrine Functions in the Rat[END_REF]. In contrast to adults, fetal ghrelin may be produced by pancreatic islets [START_REF] Chanoine | Ghrelin Gene Expression Is Markedly Higher in Fetal Pancreas Compared with Fetal Stomach: Effect of Maternal Fasting[END_REF]. However, pancreatic Ghrl expression progressively decreases from birth to weaning whereas stomach Ghrl expression increases to reach adult-like levels by 3 to 5 weeks of life [START_REF] Hayashida | Ghrelin in Neonatal Rats: Distribution in Stomach and Its Possible Role[END_REF][START_REF] Torsello | Ontogeny and Tissue-Specific Regulation of Ghrelin MRNA Expression Suggest That Ghrelin Is Primarily Involved in the Control of Extraendocrine Functions in the Rat[END_REF]. In vitro studies have suggested that ghrelin could influence hypothalamic cell proliferation and differentiation during fetal life [START_REF] Inoue | Transitional Change in Rat Fetal Cell Proliferation in Response to Ghrelin and Des-Acyl Ghrelin during the Last Stage of Pregnancy[END_REF][START_REF] Watanabe | Ghrelin Suppresses Proliferation of Fetal Neural Progenitor Cells, and Induces Their Differentiation into Neurons[END_REF].

Furthermore, ghrelin also plays a role in postnatal ARH axonal elongation. As for leptin, mouse plasma ghrelin levels are high during the postnatal time window. However, ghrelin plasma levels increase around P10 and reach adult levels by P14 (Figure 23B) [START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF], a time during which plasma leptin levels are declining [START_REF] Ahima | Postnatal Leptin Surge and Regulation of Circadian Rhythm of Leptin by Feeding. Implications for Energy Homeostasis and Neuroendocrine Function[END_REF].

Administering ghrelin during the postnatal period decreases AgRP/NPY and POMC/CART projections, whereas blocking ghrelin from P4 to P22 leads to an abnormal increase of AgRP and α-MSH immunoreactivity in the PVH [START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF]. As for leptin, the blunting of neurite outgrowth by ghrelin is observed by a direct exposure of ARH explants to ghrelin [START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF]. Moreover, mice lacking ghrelin develop an exaggerated amount of ARH projections to the PVH [START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF]. Therefore, ghrelin seems to stabilize the outgrowth of ARH axons from mid postnatal life to weaning.

From a mechanistic angle, the ghrelin receptor (GHSR) is already present in the ARH during the postnatal period (Figure 23B). Indeed, P6 and P10 ARH Ghsr mRNA levels are comparable to those found in adults [START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF]. Furthermore, ghrelin treatment at P6 already increases ERK phosphorylation in the ARH, which is one of the signaling pathways induced by ghrelin, in AgRP/NPY and POMC/CART neurons [START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF]. This suggests that the ghrelin receptor and one of its downstream signaling pathways are already functional during the neonatal period. Importantly, ghrelin treatment during the postnatal period leads to less ARH leptin-induced STAT3 phosphorylation, suggesting an altered leptin signaling [START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF]. Postnatal ghrelin therefore seems to stabilize ARH axonal outgrowth by interfering with leptin signaling.

Insulin

The "hypoglycaemic hormone" insulin is known to exert a developmental role during development. In mice, insulin is detected during embryogenesis at E9 but the low number of insulin-positive cells in the pancreas at E9 suggests that insulin levels may have an extrapancreatic origin [START_REF] Deltour | Differential Expression of the Two Nonallelic Proinsulin Genes in the Developing Mouse Embryo[END_REF][START_REF] Spaventi | Insulin and Insulin-like Growth Factor I (IGF I) in Early Mouse Embryogenesis[END_REF]. Insulin expression is indeed also observed in the central nervous system [START_REF] Deltour | Differential Expression of the Two Nonallelic Proinsulin Genes in the Developing Mouse Embryo[END_REF]. Maternal insulin cannot cross the placenta, but maternal glucose can cross it and contribute to the fetal insulin levels by stimulating fetal pancreatic secretion [START_REF] Baumann | Placental Glucose Transfer and Fetal Growth[END_REF]. In addition, the insulin receptor is already present in the brain during the perinatal period (Figure 23B) [START_REF] Kar | Quantitative Autoradiographic Localization of [125I] Insulin-like Growth Factor I,[125I] Insulin-like Growth Factor II, and [125I] Insulin Receptor Binding Sites in Developing and Adult Rat Brain[END_REF] and insulin binds most of hypothalamic nuclei already during fetal life [START_REF] Leloup | Intrauterine Hyperglycemia Increases Insulin Binding Sites but Not Glucose Transporter Expression in Discrete Brain Areas in Term Rat Fetuses[END_REF]. Changes in maternal glycaemia may thus alter perinatal insulinemia of the offspring. Both hyperinsulinemia and hypoinsulinemia during the perinatal period are associated with a disrupted development of hypothalamic feeding circuits. Indeed, pups with neonatal hyperinsulinemia exhibit an aberrant hypothalamic neuronal number and morphological alterations of hypothalamic nuclei [START_REF] Plagemann | Hypothalamic Insulin and Neuropeptide Y in the Offspring of Gestational Diabetic Mother Rats[END_REF][START_REF] Plagemann | Morphological Alterations of Hypothalamic Nucleidue to Intrahypothalamic Hyperinsulinism in Newborn Rats[END_REF]. Furthermore, offspring of insulin-deficient dams exhibit a reduction of ARH axonal projections to the PVH [START_REF] Steculorum | Maternal Diabetes Compromises the Organization of Hypothalamic Feeding Circuits and Impairs Leptin Sensitivity in Offspring[END_REF]. The latter study also found that offspring from insulin-deficient dams had a reduced amount of STAT3 phosphorylation following leptin treatment during the postnatal period [START_REF] Steculorum | Maternal Diabetes Compromises the Organization of Hypothalamic Feeding Circuits and Impairs Leptin Sensitivity in Offspring[END_REF], which opens up the possibility that insulin may influence ARH axonal outgrowth by affecting signaling of other developmental hormones. It is important to note that deciphering the developmental role of insulin on hypothalamic circuits is challenging because it is difficult to evaluate the effects of insulin independently of glycaemia fluctuations in vivo. In this sense, the observations mentioned above could be due to differences in glycaemia instead of differences in insulin levels. However, in an in vitro experiment, Toran-Allerand and colleagues showed that addition of both estradiol and insulin to rodent hypothalamic cultures increases neurite outgrowth [START_REF] Toran-Allerand | Estrogen and Insulin Synergism in Neurite Growth Enhancement in Vitro: Mediation of Steroid Effects by Interactions with Growth Factors?[END_REF]. This neurotrophic role of insulin was more recently studied in vivo. Vogt and co-authors showed that offspring of mothers fed a high-fat diet during lactation exhibit reduced α-MSH and AgRP projections to the PVH, the DMH and the LH. Interestingly, knocking down the insulin receptor specifically in hypothalamic POMC neurons rescues α-MSH projections to the PVH posterior [START_REF] Vogt | Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding[END_REF]). Altogether, these data stronly suggest that neuronal insulin signaling exerts a neurotrophic role per se.

Amylin

Recent studies have also suggested that the pancreatic hormone amylin is necessary for optimal hypothalamic development [START_REF] Johnson | Early Postnatal Amylin Treatment Enhances Hypothalamic Leptin Signaling and Neural Development in the Selectively Bred Diet-Induced Obese Rat[END_REF][START_REF] Lutz | Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus[END_REF]). Lutz and coauthors recently proposed that amylin could also influence ARH axonal outgrowth both directly and indirectly. On the one hand, the direct effect would consist on enhancing leptin ERK signaling specifically in POMC neurons in the ARH, leading to α-MSH axonal outgrowth from the ARH to the PVH. On the other hand, the indirect effect would consist on enhancing interleukin 6 (IL-6) production by microglia, which would then stimulate leptin STAT3 signaling specifically in AgRP neurons [START_REF] Johnson | Early Postnatal Amylin Treatment Enhances Hypothalamic Leptin Signaling and Neural Development in the Selectively Bred Diet-Induced Obese Rat[END_REF][START_REF] Lutz | Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus[END_REF]).

To conclude, hypothalamic feeding circuits develop under the concerted action of several hormones that interact within the hypothalamus, and influence both neurons and microglia.

Metabolic hormones like leptin, ghrelin, insulin and amylin therefore modulate neurite outgrowth during the postnatal period. The hypothalamus remains plastic throughout life, although to a lesser degree than during the perinatal period [START_REF] Sominsky | Hormonal and Nutritional Regulation of Postnatal Hypothalamic Development[END_REF]).

Failure to optimally regulate glucose homeostasis and energy balance leads to the onset of metabolic disorders. These last decades have seen an alarming increase of cases of metabolic syndrome at a global scale, which means that both adults and children of both developed and developing countries are affected. This chapter will briefly cover what the metabolic syndrome is and will rather focus on the developmental programming of the metabolic syndrome. Then, it will describe the nutritional insults arriving during the perinatal period; and lastly, how these nutritional insults can compromise optimal organ development, therefore rendering individuals more susceptible to develop the metabolic syndrome later in life.

The metabolic syndrome and its developmental programming

The metabolic syndrome is not a disease but a cluster of cardiovascular disease risk factors [START_REF] Saklayen | The Global Epidemic of the Metabolic Syndrome[END_REF], and its definition varies slightly among health organizations. The International Diabetes Federation defines the metabolic syndrome as having a waist larger than 80 cm for women and 94 cm for men, plus minimum two of the following conditions:

diabetes, low high-density lipoprotein (HDL) cholesterol, high blood triglycerides, and high blood pressure [START_REF] Saklayen | The Global Epidemic of the Metabolic Syndrome[END_REF]. Thus, the definition of "metabolic syndrome" can differ in the literature but always reflects a defect in the metabolism of energy resources.

In 2019, approximately 463 million adults (20-79 years) were living with diabetes worldwide (Figure 23). If current trends persist, this number will rise to 700 million in the year 2045 (International Diabetes Federation Atlas, 9 th edition, 2019). Importantly, a 2015 global survey of obesity showed that 108 million children were already obese, with a rate of increase even higher than that for adults [START_REF] Ebbeling | Childhood Obesity: Public-Health Crisis, Common Sense Cure[END_REF][START_REF] Saklayen | The Global Epidemic of the Metabolic Syndrome[END_REF]. Another important point is that diabetes and obesity are diseases now present in low, middle and high-income countries (Saklayen 2018) (Figure 23). The worldwide increasing prevalence of metabolic syndrome is thus alarming and represents a serious challenge for public health.

The thrifty phenotype hypothesis

Epidemiological data, clinical studies and experimental research have highlighted the role of the perinatal exposure to nutritional stresses on the development of metabolic disorders [START_REF] Cottrell | Developmental Programming of Energy Balance and the Metabolic Syndrome[END_REF]. To explain why offspring exposed to perinatal stresses are more vulnerable to develop the metabolic syndrome in later life, Hales and Barker proposed the "thrifty phenotype hypothesis", nearly three decades ago [START_REF] Hales | The Thrifty Phenotype Hypothesis[END_REF]. The hypothesis is that exposure to a deficit of nutrient supply during the perinatal period leads to adaptations in the developing fetus and/or infant that will maximize their metabolic efficiency when storing and using energy resources [START_REF] Cottrell | Developmental Programming of Energy Balance and the Metabolic Syndrome[END_REF].

This metabolic programming would be beneficial if the undernutrition lived during the perinatal period persisted throughout life; however, if long-term nutritional conditions were richer, this metabolic programming would actually predispose the organism to develop obesity and diabetes [START_REF] Cottrell | Developmental Programming of Energy Balance and the Metabolic Syndrome[END_REF].

Feeding alterations during the perinatal period

The most reported and studied perinatal stresses have been nutritional insults, which can affect the perinatal hormonal milieu of the offspring. Studies have repeatedly shown that exposure to undernutrition or overnutrition during gestation and/or lactation predisposes the offspring to glucose control disorders and obesity during adulthood. Of course, the perinatal nutrition of a growing organism depends on the nutrition of the mother. During gestation, the placenta supplies nutrients and gases to the fetus, while receiving fetal waste products [START_REF] Belkacemi | Maternal Undernutrition Influences Placental-Fetal Development1[END_REF]. During this period, a sub-optimal maternal nutrition can affect fetal nutrition in two ways. The first one is by altering the development and function of the placenta, therefore compromising maternal-fetal exchanges [START_REF] Belkacemi | Maternal Undernutrition Influences Placental-Fetal Development1[END_REF][START_REF] Daniels | Associations of Maternal Diet and Placenta Leptin Methylation[END_REF]). The second one is by affecting the amount of nutrient supply to the fetus, which indirectly leads to an alteration of the fetal endocrine system and epigenetic changes in fetal gene activity [START_REF] Belkacemi | Maternal Undernutrition Influences Placental-Fetal Development1[END_REF]. During lactation, maternal nutrition can affect the nutrient composition of milk (macronutrients, micronutrients and hormones) [START_REF] Bautista | Changes in Milk Composition in Obese Rats Consuming a High-Fat Diet[END_REF][START_REF] Bravi | Impact of Maternal Nutrition on Breast-Milk Composition: A Systematic Review[END_REF][START_REF] Erliana | The Function and Alteration of Immunological Properties in Human Milk of Obese Mothers[END_REF][START_REF] Lemas | Alterations in Human Milk Leptin and Insulin Are Associated with Early Changes in the Infant Intestinal Microbiome[END_REF][START_REF] Agnoux | Perinatal Protein Restriction Affects Milk Free Amino Acid and Fatty Acid Profile in Lactating Rats: Potential Role on Pup Growth and Metabolic Status[END_REF], thereby also affecting the postnatal diet of the offspring.

Perinatal undernutrition

Metabolic programming studies first focused on the consequences of maternal undernutrition. First data associating perinatal undernutrition with a higher susceptibility to develop metabolic disorders during adulthood come from individuals who were in utero during periods of famine like the Dutch Hunger Winter and the Chinese Famine [START_REF] Li | Exposure to the Chinese Famine in Early Life and the Risk of Hyperglycemia and Type 2 Diabetes in Adulthood[END_REF][START_REF] Ravelli | Glucose Tolerance in Adults after Prenatal Exposure to Famine[END_REF]. Individuals born during or right after these famine periods had developed glucose control disturbances such as glucose intolerance, hyperglycaemia and type-2 diabetes [START_REF] Li | Exposure to the Chinese Famine in Early Life and the Risk of Hyperglycemia and Type 2 Diabetes in Adulthood[END_REF][START_REF] Ravelli | Glucose Tolerance in Adults after Prenatal Exposure to Famine[END_REF]). Studies using animal models have provided further support for these data. Calorie restriction during the perinatal period has been associated with low weight at birth, and glucose intolerance, insulin resistance, obesity, hyperinsulinemia and hyperleptinemia in adult rats [START_REF] Garofano | Effect of Ageing on Beta-Cell Mass and Function in Rats Malnourished during the Perinatal Period[END_REF][START_REF] Vickers | Fetal Origins of Hyperphagia, Obesity, and Hypertension and Postnatal Amplification by Hypercaloric Nutrition[END_REF]. Importantly, not only the amount of calories has been found to be important in the developmental programming of metabolic disorders. Iso-caloric diets with restricted amounts of protein have also been linked to low birth weight, retarded growth, and life-long greater abdominal adiposity and disturbances of glucose control parameters [START_REF] Barnett | Impaired Insulin Secretion in Perfused Pancreases Isolated from Offspring of Female Rats Fed a Low Protein Whey-Based Diet[END_REF][START_REF] Blumfield | Dietary Balance during Pregnancy Is Associated with Fetal Adiposity and Fat Distribution[END_REF][START_REF] Herring | Impacts of Maternal Dietary Protein Intake on Fetal Survival, Growth, and Development[END_REF][START_REF] Moullé | Effects of Nutrient Intake during Pregnancy and Lactation on the Endocrine Pancreas of the Offspring[END_REF][START_REF] Snoeck | Effect of a Low Protein Diet during Pregnancy on the Fetal Rat Endocrine Pancreas[END_REF]). Indeed, proteins are essential during development because they supply precursors for de novo protein synthesis in the growing fetus/neonate.

Perinatal overnutrition

Although the first metabolic programming studies focused on the consequences of maternal undernutrition, studies are now focusing also on the consequences of maternal overnutrition. Indeed, as the rate of global obesity and diabetes increases alarmingly, the number of children born to obese mothers (and fathers) is escalating too. Observational studies in humans have shown that children born to obese or overweight mothers are more likely to become overweight or obese and develop diabetes [START_REF] Crozier | Weight Gain in Pregnancy and Childhood Body Composition: Findings from the Southampton Women's Survey[END_REF][START_REF] Gale | Maternal Size in Pregnancy and Body Composition in Children[END_REF][START_REF] Hochner | Associations of Maternal Prepregnancy Body Mass Index and Gestational Weight Gain With Adult Offspring Cardiometabolic Risk Factors: The Jerusalem Perinatal Family Follow-Up Study[END_REF][START_REF] Lawlor | Epidemiologic Evidence for the Fetal Overnutrition Hypothesis: Findings from the Mater-University Study of Pregnancy and Its Outcomes[END_REF]). These studies have been supported by experimental studies using animal models. In rats, offspring born to dams fed a highfat/high-sucrose diet during gestation and lactation exhibit greater adiposity, glucose intolerance, steatohepatitis, mitochondrial dysfunction and increased hepatic lipogenesis during adulthood [START_REF] Bruce | Maternal High-Fat Feeding Primes Steatohepatitis in Adult Mice Offspring, Involving Mitochondrial Dysfunction and Altered Lipogenesis Gene Expression[END_REF][START_REF] Chen | Maternal and Postnatal Overnutrition Differentially Impact Appetite Regulators and Fuel Metabolism[END_REF]. Furthermore, rats born to dams fed a high-fat diet during the perinatal period also exhibit increased body weight, glucose intolerance and an altered fatty acid metabolism in the liver [START_REF] Seet | Maternal High-Fat-Diet Programs Rat Offspring Liver Fatty Acid Metabolism[END_REF][START_REF] White | Maternal Obesity Is Necessary for Programming Effect of High-Fat Diet on Offspring[END_REF]. Interestingly, pups born to dams fed a high-fat diet start independent feeding sooner than pups born to dams fed a standard chow [START_REF] Kojima | Maternal High-Fat Diet Increases Independent Feeding in Pre-Weanling Rat Pups[END_REF], suggesting that perinatal overnutrition could also affect feeding mechanisms since a young age. Besides maternal high-calorie feeding, an excess of specific nutrients like carbohydrates, saturated fats or proteins also seems to influence the developmental programming of the metabolic syndrome [START_REF] Moullé | Effects of Nutrient Intake during Pregnancy and Lactation on the Endocrine Pancreas of the Offspring[END_REF]. In this respect, rats that ingest a high-protein milk formula during lactation have increased weight and exhibit increased insulinemia, leptinemia and triglycerides during adulthood [START_REF] Coupé | Hypothalamus Integrity and Appetite Regulation in Low Birth Weight Rats Reared Artificially on a High-Protein Milk Formula[END_REF].

Undoubtedly, extensive literature exists on the association between perinatal undernutrition or overnutrition and the development of the metabolic syndrome during adulthood. In this line, research investigating the mechanisms underlying this association has greatly increased during the last couple of decades. As discussed in chapter 4, the perinatal period is the most crucial time for development. The development of an organism starts in utero and the maturation of several organs is pursued during the postnatal period.

The perinatal period is therefore also a very vulnerable time, in which nutritional insults can affect the development of several organs and subsequently impair their functioning. The perinatal exposure to undernutrition or overnutrition impairs the development of key organs that regulate energy homeostasis like the hypothalamus, the pancreas, the gastrointestinal tract and the adipose tissue (Figure 24).

Perinatal nutritional insults affect organ development

Impaired hypothalamic development

As discussed in chapter 4, hormones like leptin, ghrelin and insulin orchestrate the postnatal development of hypothalamic feeding circuits. Studies suggest that perinatal exposure to undernutrition or overnutrition affects signaling of these hormones in the pup, either by modifying hormonal circulating levels or central sensitivity to them. This represents a possible mechanism leading to higher susceptibility to develop metabolic disorders during adulthood [START_REF] Bouret | Organizational Actions of Metabolic Hormones[END_REF].

In a rat model of intrauterine growth restriction, followed by postnatal rapid catchup growth, rats showed a decrease in leptin sensitivity and became fat during adulthood [START_REF] Coupé | Postnatal Growth after Intrauterine Growth Restriction Alters Central Leptin Signal and Energy Homeostasis[END_REF]). Yura and co-authors also showed that mice pups exposed to an intrauterine growth restriction developed obesity and central leptin resistance during adulthood, associated with a premature neonatal leptin surge during the postnatal period [START_REF] Yura | Role of Premature Leptin Surge in Obesity Resulting from Intrauterine Undernutrition[END_REF]. Interestingly, inducing a premature leptin surge in control mice was enough to induce central leptin resistance and obesity during adulthood [START_REF] Yura | Role of Premature Leptin Surge in Obesity Resulting from Intrauterine Undernutrition[END_REF],

suggesting that a modification of the neonatal leptin peak could be enough to alter life-long metabolism. The premature leptin surge was also enough to increase NPY and POMC labeling in the PVH of mice [START_REF] Yura | Role of Premature Leptin Surge in Obesity Resulting from Intrauterine Undernutrition[END_REF]. Moreover, Delahaye et al. also showed that maternal undernutrition alters the neonatal peak of leptin, producing a disruption of the development of hypothalamic feeding circuits [START_REF] Delahaye | Maternal Perinatal Undernutrition Drastically Reduces Postnatal Leptin Surge and Affects the Development of Arcuate Nucleus Proopiomelanocortin Neurons in Neonatal Male Rat Pups[END_REF]. These data suggest that prenatal undernutrition can modify neonatal leptin levels, subsequently altering NPY and POMC hypothalamic circuits and causing metabolic disorders during adulthood. Furthermore, protein restriction during pregnancy has been shown to alter mother milk composition.

Indeed, dams nourished a protein-restricted diet during gestation and lactation produce milk with a lower content of essential fatty acids that play a neurodevelopmental role [START_REF] Hornstra | Essential Fatty Acids in Mothers and Their Neonates[END_REF][START_REF] Agnoux | Perinatal Protein Restriction Affects Milk Free Amino Acid and Fatty Acid Profile in Lactating Rats: Potential Role on Pup Growth and Metabolic Status[END_REF].

In the case of perinatal overnutrition, research has shown that it causes significant changes in hypothalamic feeding circuits. Indeed, rodent pups born to obese dams exhibit reduced hypothalamic Npy and increased Pomc mRNA expression [START_REF] Chen | Maternal and Postnatal Overnutrition Differentially Impact Appetite Regulators and Fuel Metabolism[END_REF]) and an impaired development of AgRP and POMC ARH projections [START_REF] Bouret | Hypothalamic Neural Projections Are Permanently Disrupted in Diet-Induced Obese Rats[END_REF][START_REF] Vogt | Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding[END_REF]). These changes seem to be due to an impaired signaling of leptin, ghrelin, and insulin.

Indeed, pups born to obese dams exhibit an amplified and prolonged neonatal leptin peak, associated with decreased hypothalamic leptin signaling during the postnatal period [START_REF] Bouret | Hypothalamic Neural Projections Are Permanently Disrupted in Diet-Induced Obese Rats[END_REF][START_REF] Kirk | Maternal Obesity Induced by Diet in Rats Permanently Influences Central Processes Regulating Food Intake in Offspring[END_REF]. Furthermore, at least the impaired ARH POMC projections could be attributed to an excessive insulin signaling during the postnatal period because pups born to overnourished dams exhibit hyperinsulinemia, and blocking insulin signaling specifically in POMC neurons restores ARH POMC projections to the PHVpost [START_REF] Vogt | Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding[END_REF].

Interestingly, Kim et al. recently showed that offspring born to obese dams exhibit a more permeable blood-brain barrier of the ARH at birth. Pups had greater diffusion into the ARH of the albumin-binding molecule, Evans Blue, a higher amount of endothelial cell fenestrations and a decrease of ARH Vimentin staining (a tanycyte marker) (Kim et al. 2016a). The authors proposed that maternal obesity can render the developing hypothalamus more vulnerable to a modification of levels of circulating hormones via an altered blood-brain barrier. However, another study performed in mice raised in small liters, a well-described mouse model of postnatal overnutrition, showed that pups exhibit decreased levels of ghrelin production, decreased hypothalamic ghrelin receptor expression and decreased ghrelin uptake from the periphery to the brain, which were due to a decrease in transcellular transport of ghrelin by tanycytes [START_REF] Collden | Neonatal Overnutrition Causes Early Alterations in the Central Response to Peripheral Ghrelin[END_REF]). An altered specific transport by tanycytes rather than an increased brain barrier permeability seems thus responsible for changes in hormonal sensitivity of ARH neurons induced by perinatal overnutrition.

Besides affecting the hypothalamic neuronal circuit, perinatal overnutrition also affects hypothalamic astrocytes as offspring born to obese dams exhibit higher astrocyte proliferation in the hypothalamus (Kim et al. 2016b). Nevertheless, a causal link between astrocyte proliferation and the development of the metabolic syndrome has not been established yet.

Impaired development of other organs

Pancreas

The pancreas is another pivotal organ for energy homeostasis regulation because it secretes the glycaemia-regulating hormones insulin, glucagon, and somatostatin. In rodents, the pancreas is immature at birth and considerably grows during the last two weeks of the postnatal period [START_REF] Moullé | Effects of Nutrient Intake during Pregnancy and Lactation on the Endocrine Pancreas of the Offspring[END_REF]. On the one hand, perinatal undernutrition is associated with an impaired capacity of β-cells to regenerate through neo-genesis and therefore with a subsequent reduction of β-cell mass in rats [START_REF] Garofano | Effect of Ageing on Beta-Cell Mass and Function in Rats Malnourished during the Perinatal Period[END_REF]). These rats exhibited insulinopenia and glucose intolerance during adulthood [START_REF] Garofano | Effect of Ageing on Beta-Cell Mass and Function in Rats Malnourished during the Perinatal Period[END_REF]. Furthermore, prenatal protein restriction is also associated with reduced β-cell proliferation and Langerhans islet size, as well as reduced vascularization of the endocrine pancreas [START_REF] Snoeck | Effect of a Low Protein Diet during Pregnancy on the Fetal Rat Endocrine Pancreas[END_REF]). On the other hand, perinatal overnutrition is associated with hyperinsulinemia and glucose intolerance during adulthood, as well as with Langerhans islet hypertrophy and an increased β-cell mass to pancreatic mass ratio during the postnatal period and into adulthood [START_REF] Bringhenti | Maternal Obesity during the Preconception and Early Life Periods Alters Pancreatic Development in Early and Adult Life in Male Mouse Offspring[END_REF][START_REF] Gregorio | Maternal High-Fat Diet Is Associated with Altered Pancreatic Remodelling in Mice Offspring[END_REF]. Moreover, a perinatal excess of glucose and sucrose, saturated fatty acids or proteins also affect the structure and function of the pancreas [START_REF] Moullé | Effects of Nutrient Intake during Pregnancy and Lactation on the Endocrine Pancreas of the Offspring[END_REF]. Perinatal nutrition may control pancreatic function through the epigenetic modification of genes involved in the differentiation of pancreatic cells [START_REF] Moullé | Effects of Nutrient Intake during Pregnancy and Lactation on the Endocrine Pancreas of the Offspring[END_REF][START_REF] Park | Development of Type 2 Diabetes Following Intrauterine Growth Retardation in Rats Is Associated with Progressive Epigenetic Silencing of Pdx1[END_REF][START_REF] Plagemann | Hypothalamic Proopiomelanocortin Promoter Methylation Becomes Altered by Early Overfeeding: An Epigenetic Model of Obesity and the Metabolic Syndrome: Nutritionally Induced Alterations of POMC Promoter Methylation[END_REF]), but also through the regulation of parasympathetic pancreatic innervation [START_REF] Croizier | Leptin Controls Parasympathetic Wiring of the Pancreas during Embryonic Life[END_REF]. These data strongly suggest that nutritional insults arriving during the perinatal period can seriously compromise the development of the endocrine pancreas, which is a key regulator of glucose homeostasis.

The gastrointestinal tract

Perinatal nutritional insults also seem to affect the structure and the function of the gastrointestinal tract. Rats born to undernourished dams exhibit higher Ghrl mRNA expression in the stomach, associated with higher plasma ghrelin levels both at birth and during adulthood [START_REF] Nagata | Altered Gene Expressions of Ghrelin, PYY, and CCK in the Gastrointestinal Tract of the Hyperphagic Intrauterine Growth Restriction Rat Offspring[END_REF]. During adulthood, these rats also showed decreased expression of Pyy and Cck mRNAs [START_REF] Nagata | Altered Gene Expressions of Ghrelin, PYY, and CCK in the Gastrointestinal Tract of the Hyperphagic Intrauterine Growth Restriction Rat Offspring[END_REF], suggesting that the secretion of peptides regulating feeding in the adult animal were altered by perinatal nutritional conditions. Perinatal malnutrition also affects the structure of the enteric nervous system.

Indeed, a recent study showed that mice born to obese dams exhibit an increase of glial proliferation and a decrease of myenteric nitrergic neurons, prior to becoming obese [START_REF] Mcmenamin | High-Fat Diet During the Perinatal Period Induces Loss of Myenteric Nitrergic Neurons and Increases Enteric Glial Density, Prior to the Development of Obesity[END_REF]. Nevertheless, the causal link between these alterations in the enteric nervous system and the development of obesity is missing.

Furthermore, an increasing amount of research has reported that perinatal overnutrition leads to a defective intestinal barrier, associated with an increased susceptibility to intestinal inflammation [START_REF] Babu | Maternal High-Fat Diet Results in Microbiota-Dependent Expansion of ILC3s in Mice Offspring[END_REF][START_REF] Gohir | High-fat Diet Intake Modulates Maternal Intestinal Adaptations to Pregnancy and Results in Placental Hypoxia, as Well as Altered Fetal Gut Barrier Proteins and Immune Markers[END_REF][START_REF] Xue | Maternal Obesity Induces Gut Inflammation and Impairs Gut Epithelial Barrier Function in Nonobese Diabetic Mice[END_REF][START_REF] Yan | Maternal Obesity Induces Sustained Inflammation in Both Fetal and Offspring Large Intestine of Sheep[END_REF]. Some studies suggest that these barrier defects could be due to changes in gut microbiota observed in pups exposed to perinatal overnutrition [START_REF] Babu | Maternal High-Fat Diet Results in Microbiota-Dependent Expansion of ILC3s in Mice Offspring[END_REF][START_REF] Ma | High-Fat Maternal Diet during Pregnancy Persistently Alters the Offspring Microbiome in a Primate Model[END_REF][START_REF] Soderborg | Author Correction: The Gut Microbiota in Infants of Obese Mothers Increases Inflammation and Susceptibility to NAFLD[END_REF].

Adipose tissue

Experiments using rodents have suggested that maternal nutrition could affect adipocyte diameter in the offspring. Rat pups born to undernourished dams exhibit larger adipocytes, which is associated with a decrease in insulin-stimulated glucose uptake and in the expression of proteins implicated in the insulin signaling pathway [START_REF] Thompson | Metabolic Programming of Adipose Tissue Structure and Function in Male Rat Offspring by Prenatal Undernutrition[END_REF]. Additionally, perinatal protein restriction is also associated with a decrease in the expression of insulin signaling proteins in adipocytes from rats [START_REF] Martin-Gronert | Cell-Autonomous Programming of Rat Adipose Tissue Insulin Signalling Proteins by Maternal Nutrition[END_REF].

Interestingly, rat pups born to streptozotocin-induced diabetic dams and mice pups born to obese dams also exhibit larger adipocytes [START_REF] Gregorio | Maternal High-Fat Diet Is Associated with Altered Pancreatic Remodelling in Mice Offspring[END_REF][START_REF] Oliveira | Neonatal Streptozotocin-Induced Diabetes in Mothers Promotes Metabolic Programming of Adipose Tissue in Male Rat Offspring[END_REF].

Adipocytes from pups born to diabetic dams have an increase in insulin-stimulated glucose uptake and in glucose incorporation into lipids, as well as higher protein content of GLUT4, insulin receptors and acetyl-CoA carboxylase [START_REF] Oliveira | Neonatal Streptozotocin-Induced Diabetes in Mothers Promotes Metabolic Programming of Adipose Tissue in Male Rat Offspring[END_REF], suggesting an increased lipogenic capacity. These data show an alteration of lipogenesis and/or lipid stocking in pups following perinatal malnutrition. Nevertheless, further investigation is needed to determine whether there exists a causal link between these alterations and the onset of the metabolic syndrome in the adult.

In conclusion, a perinatal nutrition with inadequate quantities of calories or of specific macronutrients can severely impair the development of key organs that regulate glucose homeostasis and energy balance, in part, via the modification of circulating levels of hormones like leptin, ghrelin and insulin and their central signaling. Besides what has been covered in this chapter, the developmental programming of the metabolic syndrome also implicates other mechanisms like epigenetic modifications (For a review, read [START_REF] Bouret | Gene-Environment Interactions Controlling Energy and Glucose Homeostasis and the Developmental Origins of Obesity[END_REF]. Organs with a defective structure cannot function optimally, which compromises lifelong glucose homeostasis and energy balance control. This complex scenario would naturally render the individual more susceptible to develop metabolic disorders. The perinatal environment is therefore a crucial period in life upon which the metabolic health of an individual depends.

Intestinal gluconeogenesis (IGN) controls a gut-brain neuronal circuit that is crucial in metabolic health. A key element of this gut-brain neuronal circuit is the hypothalamus, which is an essential regulator of energy balance. Importantly, hypothalamic feeding circuits are still maturing during the postnatal time frame in rodents. This developmental process depends on a surge of postnatal leptin signaling, which stimulates axonal outgrowth from the arcuate nucleus (ARH) in part via ARH STAT3 signaling. Interestingly, IGN is strongly induced during the early postnatal period in rodents. And, in adults, it recapitulates some beneficial effects of leptin on food intake and energy homeostasis but also stimulates ARH STAT3 signaling. These two observations led us to hypothesize that IGN could also regulate ARH axonal elongation during the early postnatal period.

In this study, our major goal was to determine the role of IGN on the postnatal development of hypothalamic feeding circuits in mice. To achieve this, we sought to overexpress IGN during the early postnatal period. We used a new murine model with a tamoxifen-dependent inducible overexpression of the catalytic sub-unit of the glucose-6phosphatase (G6pc gene) specifically in the intestine. This new murine model required that we administered tamoxifen at birth, which had not been done before in our laboratory.

Therefore, we sought to decipher the effects associated with a neonatal administration of tamoxifen per se prior to determining the role of intestinal gluconeogenesis on the neonatal development of hypothalamic feeding circuits.

The first part of my thesis consisted in characterizing the long-term metabolic effects of a neonatal administration of tamoxifen in both male and female C57BL/6J mice. On postnatal day 1 (P1), we injected 20 µg of tamoxifen in the stomach of male and female pups.

At 6 weeks of age, mice chow was changed to a high-fat/high-sucrose diet. Then after 1 month on this diet, we started analyzing several metabolic parameters such as body composition, glucose and insulin tolerance, insulinemia and basal metabolism. In parallel, we performed the same analyses in male and female mice fed a standard diet throughout life.

These results led to the first article of this thesis.

The second part of my thesis consisted in deciphering the role of IGN on the postnatal development of hypothalamic feeding circuits in mice. We sought to answer three main questions: (1) Does IGN control the postnatal development of hypothalamic AgRP and POMC circuits? ( 2) Is the effect on hypothalamic neuronal circuits associated with a long-term effect on mice metabolism? And ( 3) are these effects restricted to the neonatal time window?

To answer these questions we induced the overexpression of G6pc specifically in the intestine since P1. We analyzed the consequences of this neonatal IGN induction on pups' hypothalamic feeding circuits and life-long metabolism. In parallel, to determine whether these effects are restricted to the postnatal peak of IGN, we performed the same analyses in mice with an intestinal G6pc overexpression right after the postnatal peak of IGN. These results led to the second article of this thesis.

Introduction

Obesity, insulin resistance and type 2 diabetes have substantially increased during the last two decades, representing a serious challenge for worldwide public health (O'Neill and O'Driscoll 2015; [START_REF] Smith | Obesity Statistics[END_REF]. Importantly, human and animal-based studies strongly suggest that the increasing prevalence of obesity and type 2 diabetes might be partially due to environmental stresses occurring during the perinatal period (i.e. gestation and lactation), which is a concept known as the developmental programming of adult health and disease [START_REF] Bouret | Gene-Environment Interactions Controlling Energy and Glucose Homeostasis and the Developmental Origins of Obesity[END_REF][START_REF] Vickers | Developmental Programming of the Metabolic Syndrome -Critical Windows for Intervention[END_REF]. Studies in rodents have identified the neonatal period (i.e. the first weeks after birth) as a critical time window during which several organs, including organs controlling life-long metabolic health, continue developing [START_REF] Bouret | Gene-Environment Interactions Controlling Energy and Glucose Homeostasis and the Developmental Origins of Obesity[END_REF], making their development vulnerable to environmental stresses, and thus potentially compromising the optimal regulation of lifelong energy homeostasis. Therefore, a global need to understand the neonatal factors contributing to the developmental programming of adult metabolic diseases has led to a growing amount of research on genes that exert a neonatal/developmental role.

Transgenic mouse models have been a valuable tool for elucidating the role of several genes that regulate life-long energy homeostasis [START_REF] Plum | Transgenic and Knockout Mice in Diabetes Research: Novel Insights into Pathophysiology, Limitations, and Perspectives[END_REF]. One of the most efficient and popular strategies to generate transgenic mice is the CRE/lox system. Briefly, the CRE/lox strategy consists of flanking a key region of a gene of interest by loxP sites that will be recognized and excised by a CRE recombinase, thereby generating a mutated allele [START_REF] Metzger | Siteand Time-Specific Gene Targeting in the Mouse[END_REF]. To temporally control the site-specific mutagenesis, the inducible, tamoxifen-dependent, CRE/lox system was created. For this, the CRE recombinase was fused with a mutated ligand-binding domain of the human estrogen receptor (ER), which allows for activation of the CRE only upon binding of the synthetic ER ligand 4-hydroxytamoxifen (a metabolite of tamoxifen), and not by physiological estrogen [START_REF] Metzger | Siteand Time-Specific Gene Targeting in the Mouse[END_REF].

The resulting CRE ERT2 recombinase is the most upgraded version of the CRE/lox recombination system for tissue-specific and temporal control of the somatic mutagenesis when generating transgenic mouse models [START_REF] Feil | Inducible Cre Mice[END_REF].

With the growing popularity of the CRE ERT2 recombinase tool among investigators, several research teams have been reporting secondary effects associated with the administration of tamoxifen, especially on the reproductive system of male and female mice [START_REF] Iguchi | Effects of Sex Hormones on Oncogene Expression in the Vagina and on Development of Sexual Dimorphism of the Pelvis and Anococcygeus Muscle in the Mouse[END_REF]Nohara, Liu, et al. 2013;[START_REF] Patel | Low-Dose Tamoxifen Treatment in Juvenile Males Has Long-Term Adverse Effects on the Reproductive System: Implications for Inducible Transgenics[END_REF][START_REF] Williams | Neonatal Exposure to Potent and Environmental Oestrogens and Abnormalities of the Male Reproductive System in the Rat: Evidence for Importance of the Androgen-Oestrogen Balance and Assessment of the Relevance to Man[END_REF]). Concerning research on obesity and diabetes, studies have shown that tamoxifen administration during adulthood used to develop transgenic mouse models affects glucose and lipid metabolism and insulin secretion [START_REF] Ceasrine | Tamoxifen Improves Glucose Tolerance in a Delivery-, Sex-, and Strain-Dependent Manner in Mice[END_REF][START_REF] Cole | Tamoxifen Induces Triacylglycerol Accumulation in the Mouse Liver by Activation of Fatty Acid Synthesis[END_REF][START_REF] Hesselbarth | Tamoxifen Affects Glucose and Lipid Metabolism Parameters, Causes Browning of Subcutaneous Adipose Tissue and Transient Body Composition Changes in C57BL/6NTac Mice[END_REF][START_REF] Klöting | Tamoxifen Treatment Causes Early Hepatic Insulin Resistance[END_REF]. In parallel, protocols now exist for inducing the CRE/lox recombination system just after birth [START_REF] Lizen | Perinatal Induction of Cre Recombination with Tamoxifen[END_REF], which requires the administration of tamoxifen to mouse pups. However, to our knowledge, no studies on the life-long metabolic effects associated with a neonatal administration of tamoxifen have been reported. Given the pertinence concerning research on the developmental factors affecting life-long metabolic health, we examined several metabolic parameters in male and female C57BL/6J mice after neonatal treatment with the drug. We chose this genetic background because the C57BL/6J mouse is the most used strain in obesity and metabolism research, since it is prone to diet-induced obesity and develops severe insulin resistance with high-fat diets [START_REF] Gout | Leptin Infusion and Obesity in Mouse Cause Alterations in the Hypothalamic Melanocortin System[END_REF]Surwit et al. 1988Surwit et al. , 1995)). We analyzed weight, body composition, food intake, glucose homeostasis, and energy expenditure, in adult male and female C57BL/6J mice that received an injection of tamoxifen one day after birth.

Methods

Animals

Wild-type C57BL/6J mice, from Charles River Laboratories (France), were used to maintain a breeding at the animal facility of Lyon 1 University (Animalerie Lyon Est Conventionnelle: ALECS). All mice were group housed under controlled temperature conditions (22°C), with a 12-hours light/12-hours dark cycle. Litters were balanced to 7-9 pups (male and female) per dam. Half of pups received an intra-gastric injection of tamoxifen (20 µg, Sigma-Aldrich diluted in sunflower oil) at postnatal day 1, and the other half was injected intra-gastrically with 20 µL of sunflower oil (vehicle) [START_REF] Lizen | Perinatal Induction of Cre Recombination with Tamoxifen[END_REF]. At weaning, mice were divided into males and females, and each sex was further divided into a tamoxifen treated group (further called NTT: neonatal tamoxifen treatment) and a control littermate group (n= 4-7 per treatment and per sex) and had ad libitum access to water and to a standard chow diet (A04; SAFE; Augy, France). At 6 weeks of age, mice were either maintained on A04 or switched to a high-fat/high-sucrose diet (HF/HS diet) (consisting of 36.1% fat, 35% carbohydrates (50% maltodextrine + 50% sucrose), 19.8% proteins) [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF]. Unless otherwise described, experiments were performed in 5-months-old C57BL/6J female mice fed AO4, and on C57BL/6J male and female mice fed a HF/HS diet for one (2-months-old) and two months (3-months-old). I.G6pc1 -/-mice were obtained as previously described [START_REF] Penhoat | Protein-Induced Satiety Is Abolished in the Absence of Intestinal Gluconeogenesis[END_REF]. I.G6pc1 -/-mouse pups were injected intra-gastrically with tamoxifen or sunflower oil at postnatal day 1 to induce postnatal gene deletion and were housed under the same conditions as previously described.

All procedures were performed in accordance with the principles and guidelines of the European Convention for the Protection of Laboratory Animals. The regional animal care committee (CEEA-55, Lyon 1 University, France) approved all the experiments presented.

Food intake and body weight measurements

Mouse body weight and food intake were measured once and twice per week, respectively, since weaning. As mice were housed in groups, the total food intake was divided by the quantity of mice per cage.

Glucose and insulin tolerance tests

Fasting glycaemia was measured, and glucose and insulin tolerance tests were performed in 6-hour fasted C57BL/6J male and female mice after one (2-months-old mice) and two months (3-months-old mice) on a HF/HS diet, and in 6-hour fasted 5-months-old C57BL/6J female mice fed a standard chow diet. After an intra-peritoneal injection of glucose (1 g/kg of body weight) or insulin (0.75 U/kg of body weight), blood was withdrawn from the tail vein and glycaemia was monitored for 120 minutes using an « Accu-Chek Go » glucometer (Roche Diagnotics).

Body composition and basal metabolism studies

Plasma insulinemia

After a 6-hour fast, blood was withdrawn from the submandibular vein and collected in ethylene diamine tetraacetic acid (EDTA) and plasma was conserved at -80°C. Insulin was assayed using an insulin mouse ELISA kit (Mercodia Cat# 10-1247-01, RRID: AB_2783837).

Gene expression analyses

Mice were killed by cervical dislocation after a 6-hour fast. Tissues were immediately removed and frozen in liquid nitrogen using tongs previously cooled and kept at -80°C. Fifty mg of frozen tissue were homogenized with the Fast Prep® system and total RNAs were isolated with the TRIzol reagent protocol (Invitrogen). Reverse transcription was performed on 1 µg of mRNA using the Qiagen QuantiTect Reverse Transcription kit. Real-time qPCRs were performed using sequence-specific primers with Takyon TM No Rox SYBR® MasterMix dTTP Blue (Eurogentec). The expression of mRNAs was normalized to the mouse ribosomal protein L32 transcript expression using the 2-ΔΔCt method. Primer sequences are indicated in Table 1.

Tibia analyses

After killing, the left leg was removed and fixed in 70% ethanol until use. Tibia were gently cleaned of all soft tissue. Tibia were weighted, and their length was determined using a digital caliper.

Statistical analyses

Results are reported as means ± standard error of the mean (SEM). Normal distribution was verified for all analyses. Two-group comparisons were analyzed using unpaired t-tests. The effect of two independent variables on a dependent variable was analyzed using two-way ANOVA, followed by multiple comparisons, and corrected for multiple comparisons with a SIDAK test. The differences were considered statistically significant at *p<0.05, **p<0.

Results

Neonatal tamoxifen administration affects weight and body composition in a sexdependent manner

To assess the effects of a neonatal administration of tamoxifen on life-long metabolism, we used a validated procedure for achieving efficient recombination of floxed alleles at perinatal stages [START_REF] Lizen | Perinatal Induction of Cre Recombination with Tamoxifen[END_REF]. Briefly, male and female C57BL/6J mouse pups received a single intra-gastric injection of 20 µg of tamoxifen one day after birth (NTT mice). This efficiently activated the CRE ERT2 recombinase in an original mouse model that we developed, in which we knockout the intestinal glucose-6-phosphatase subunit 1, coded by the G6pc1 gene (Figure 1). At the adult age, we challenged male and female C57BL/6J mice, treated or not treated by tamoxifen, with a HF/HS diet, known to induce obesity and diabetes in C57BL/6J mice [START_REF] Gout | Leptin Infusion and Obesity in Mouse Cause Alterations in the Hypothalamic Melanocortin System[END_REF]Surwit et al. 1988Surwit et al. , 1995)). NTT male mice showed decreased body weight and weighted about 10 % less than vehicle-treated littermates after 4 weeks on a HF/HS diet (Figure 2A) despite having equivalent food intake (Figure 2B). We then used nuclear magnetic resonance to determine mouse body composition to better understand which body mass part was altered. NTT male mice exhibited a 9% decrease in body lean mass compared to vehicle-treated littermates, with neither an alteration of body fat mass (Figure 2C), nor a different body composition when body fat and lean mass were normalized to body weight (Figure 2D), suggesting that the reduced lean mass directly corresponds to the diminished body weight. On the contrary, NTT female mice were about 20% heavier than vehicle-treated littermates (Figure 2E) and exhibited a continuous increase in food intake (Figure 2F) after 4 weeks on a HF/HS diet. NTT female mice exhibited a massive alteration of body composition; with 68% increased fat mass and 11.5% decreased lean mass when compared to vehicle-treated littermates (Figure 2G). These alterations remained when fat mass and lean mass were normalized to body weight (Figure 2H). Given that excessive adiposity can be linked with hepatic triglyceride accumulation and altered hepatic lipid metabolism [START_REF] Smith | Non-Alcoholic Fatty Liver Disease[END_REF], we determined whether the dramatic increase in body fat observed in NTT female mice was associated with alterations in their hepatic lipid metabolism. However, no difference in liver weight between NTT and vehicle-treated female mouse groups was observed (Figure 2I-J). Furthermore, the hepatic expression levels of genes involved in de novo lipogenesis (Figure 2K) and β-oxidation (Figure 2L) were similar in both groups. These data suggest that, despite inducing an increased fat mass, the neonatal administration of tamoxifen to female mice does not alter their hepatic lipid metabolism.

Altogether, these results strongly suggest that a neonatal tamoxifen administration affects life-long body weight and body composition in male and female mice, in a sex-dependent manner.

Neonatal tamoxifen administration affects glucose homeostasis in a sex-dependent manner

An alteration of weight and body composition is an important risk factor for impaired blood glucose control and type 2 diabetes [START_REF] Fletcher | Risk Factors for Type 2 Diabetes Mellitus[END_REF][START_REF] Leitner | Obesity and Type 2 Diabetes: Two Diseases with a Need for Combined Treatment Strategies -EASO Can Lead the Way[END_REF]. We assessed glucose tolerance and insulin sensitivity after one and two months on a HF/HS diet in adult male and female mice that were treated with tamoxifen or its vehicle at birth.

After one month on a HF/HS diet, NTT male mice showed improved insulin tolerance compared to vehicle-treated littermates (Figure 3A), but there were not any effects on either glucose tolerance (Figure 3B) or on 6-hour fasting glycaemia (Figure 3C). After two months on a HF/HS diet, NTT male mice did not exhibit any effect on insulin tolerance (Figure 3D) but showed improved glucose tolerance compared to vehicle-treated littermates (Figure 3E). Furthermore, NTT male mice had fasting glycaemia that was similar to that of vehicle-treated littermates (Figure 3F) but showed a tendency to decreased insulinemia after a 6-hour fast (Figure 3G). Altogether, these results suggest that NTT male mice exhibit a modest protection against the impairments of glucose homeostasis induced by a HF/HS diet when compared to their vehicle-treated littermates.

On the contrary, after one month on a HF/HS diet, NTT female mice showed extensive insulin (Figure 4A) and glucose intolerance (Figure 4B) in comparison to vehicle-treated littermates. NTT female mice also exhibited more than a 25% increase of 6-hour fasting glycaemia (Figure 4C). Increasing the duration of the HF/HS diet worsened glucose tolerance in vehicle-treated littermates but not in NTT female mice (Figure 4D). Thus, after two months on a HF/HS diet, insulin sensitivity was no longer significantly different between both groups (Figure 4E). However, NTT female mice still showed decreased glucose tolerance in comparison to vehicle-treated littermates (Figure 4F). Importantly, despite the rise in glucose intolerance in vehicle-treated littermates (Figure 4D), NTT female mice still exhibited 6-hour fasting hyperglycaemia (Figure 4G) and more than a 60% increase in 6hour fasting insulinemia in comparison to vehicle-treated littermates (Figure 4H). It is interesting to note that NTT female mice fed a standard diet throughout life were also heavier than their vehicle-treated littermates (Figure 5A). NTT female mice fed a standard diet also exhibited around 50% increased body fat mass and 50% decreased body lean mass (Figure 5B) and had increased 6-hour fasting glycaemia (Figure 5C) and lower glucose tolerance (Figure 5D) in comparison to vehicle-treated littermates. Plasma insulinemia was not altered by the neonatal tamoxifen treatment in female mice fed a standard diet (data not shown). Together, these results strongly suggest that a neonatal administration of tamoxifen is sufficient to alter life-long energy homeostasis and blood glucose control in female mice in a standard nutritional state, and to accelerate the onset of overweight and diabetes in female mice challenged with a HF/HS diet during adulthood.

Decreased energy expenditure and physical activity in female mice treated with tamoxifen in the neonatal period

We then examined mechanisms that could explain the different body weight and body composition of NTT male and female mice. An altered body weight could be the consequence of an altered energy expenditure and/or physical activity. Thus, we used indirect calorimetry to assess energy expenditure and physical activity in NTT male and female mice.

NTT and vehicle-treated male mice exhibited equivalent energy expenditure (Figure 6A)

and physical activity (Figure 6B), suggesting that the diminished body weight of NTT male mice could not be attributed to an enhanced energy expenditure or physical activity. On the contrary, NTT female mice had a 20% decrease in energy expenditure (Figure 6C), and a 60% decrease in physical activity (Figure 6D) in comparison to vehicle-treated littermates.

Thus, in addition to an increased food intake (Figure 2F), the massively reduced physical activity and energy expenditure observed in NTT female mice might have also contributed 123 to their increased fat mass. Importantly, NTT female mice fed a standard chow diet throughout life also exhibited lower energy expenditure than vehicle-treated littermates (Figure 6E), suggesting that the effect of a neonatal administration of tamoxifen on energy expenditure is independent of food quality.

Decreased bone weight in male mice treated with tamoxifen in the neonatal period

NTT male mice had 9% less lean mass than their vehicle-treated siblings (Figure 2C), but they showed body composition normalized to total body weight (Figure 2D) and basal metabolism (Figure 6A-B) that were similar to those of their vehicle-treated littermates. We thus questioned whether a growth problem could underlie the inferior lean mass observed in NTT male mice. For this purpose, we measured the length of tibia in post-mortem tamoxifen and vehicle-treated male mice, which revealed no difference between both groups (Figure 7A). However, male mice with a neonatal administration of tamoxifen had tibia 9% lighter than those of vehicle-treated littermates (Figure 7B). This effect was restricted to male mice because NTT female mice had tibia length (17.91 mm. ±0.08 vs. 16.65 mm. ±0.19) and weight (45 mg.±0.41 vs. 44.5 mg.±1.76) equivalent to those of their vehicle-treated littermates. Importantly, NTT male mice fed a standard diet throughout life also showed decreased weight gain (about 15% less) (Figure 7C), associated with tibias of the same length (Figure 7D) but 22% lighter than those of their vehicle-treated littermates (Figure 7E). Altogether, these results suggest that a decrease in bone weight could be the most probable mechanism underlying the decreased lean mass observed in NTT male mice under standard and HF/HS diet conditions.

Discussion

Here, we show that a single intra-gastric injection of 20 µg of tamoxifen, a selective estrogen receptor modulator, administered one day after birth is enough to alter life-long energy homeostasis in a drastic and sex-dependent manner in male and female C57BL/6J mice.

Tamoxifen administration has been associated with sexual dysfunction (Saghatchian and Lesur 2019), reproductive tract abnormalities and carcinomas [START_REF] Newbold | Uterine Carcinoma in Mice Treated Neonatally with Tamoxifen[END_REF][START_REF] Patel | Low-Dose Tamoxifen Treatment in Juvenile Males Has Long-Term Adverse Effects on the Reproductive System: Implications for Inducible Transgenics[END_REF][START_REF] Taguchi | Reproductive Tract Abnormalities in Female Mice Treated Neonatally with Tamoxifen[END_REF]. Plus, several studies using female rodents have associated a neonatal administration of tamoxifen with detrimental effects on central regions regulating the reproductive function [START_REF] Agça | Modulation of Estrogen Receptors during Development Inhibits Neurogenesis of Precursors to GnRH-1 Neurones: In Vitro Studies with Explants of Ovine Olfactory Placode[END_REF][START_REF] Gonzales | Anatomically-Specific Actions of Oestrogen Receptor in the Developing Female Rat Brain: Effects of Oestradiol and Selective Oestrogen Receptor Modulators on Progestin Receptor Expression[END_REF][START_REF] Ichimura | Neonatal Exposure to SERMs Disrupts Neuroendocrine Development and Postnatal Reproductive Function through Alteration of Hypothalamic Kisspeptin Neurons in Female Rats[END_REF]. However, to our knowledge, this is the first study that has investigated long term effects on energy homeostasis associated with a neonatal administration of tamoxifen.

Mouse weight follow-up revealed opposite effects on NTT male and female mice. These weight alterations arrived during the juvenile period (at the onset of puberty), at 7 weeks of age. Plus, the neonatal administration of tamoxifen did not affect neonatal food intake (results not shown), suggesting that life-long weight effects would be independent of a modification of neonatal weight gain or food intake. The sexual dimorphism observed in body weight was associated with different, sex-dependent effects on body composition, suggesting that sex-dependent mechanisms could underlie the metabolic differences observed in male and female mice exposed to tamoxifen during the neonatal period.

Only NTT female mice had a dramatic increase in fat accumulation, which was coherent with their sedentariness and decreased energy expenditure. Indeed, NTT female mice exhibited more than a 60% decrease of physical activity and a 20% decrease of energy expenditure.

Naturally, this lack of physical activity could have contributed to the increment of their fat mass; yet it could have also been an after-effect of the increasing fat mass, as obesity diminishes mobility by altering skeletal muscle function [START_REF] Tallis | The Effects of Obesity on Skeletal Muscle Contractile Function[END_REF].

Increased adiposity to the detriment of lean mass has already been shown 5 weeks after a tamoxifen administration to 11-week-old mice. However, these effects were assessed in male mice and linked with a change in adipose tissue biology [START_REF] Hesselbarth | Tamoxifen Affects Glucose and Lipid Metabolism Parameters, Causes Browning of Subcutaneous Adipose Tissue and Transient Body Composition Changes in C57BL/6NTac Mice[END_REF].

NTT female mice also developed impaired blood glucose control, and 6-hour fasting hyperglycaemia and hyperinsulinemia, which feature an insulin-resistance state. Tamoxifen has already been associated with insulin resistance 24 days after administration in adult female mice [START_REF] Klöting | Tamoxifen Treatment Causes Early Hepatic Insulin Resistance[END_REF]. Regarding a neonatal administration, to our knowledge, no study has directly linked a neonatal tamoxifen treatment with glucose intolerance at the adult age, but few studies have shown that NTT female rodents exhibit during adulthood hepatic and serum enzymology alterations, involving hepatic CYP3A [START_REF] Kawai | Modulation of Hepatic CYP2A1, CYP2C11, and CYP3A9 Expression in Adult Rats by Neonatal Administration of Tamoxifen[END_REF][START_REF] Murakami | Effect of Neonatal Exposure of 17beta-Estradiol and Tamoxifen on Hepatic CYP3A Activity at Developmental Periods in Rats[END_REF]) and serum cholinesterase [START_REF] Lamartiniere | Sex-Differentiated Enzyme Levels and Oestrogen Uptake in Adult Rats Treated Neonatally with Tamoxifen or CI-628[END_REF]. It is of note that 125 these enzymatic alterations have been independently associated with metabolic disorders like diabetes [START_REF] Cucuianu | Serum Cholinesterase Activity Correlates with Serum Insulin, C-Peptide and Free Fatty Acids Levels in Patients with Type 2 Diabetes[END_REF][START_REF] Lunkes | Serum Cholinesterase Activity in Diabetes and Associated Pathologies[END_REF][START_REF] Jamwal | Nonalcoholic Fatty Liver Disease and Diabetes Are Associated with Decreased CYP3A4 Protein Expression and Activity in Human Liver[END_REF][START_REF] Patoine | Modulation of CYP3a Expression and Activity in Mice Models of Type 1 and Type 2 Diabetes[END_REF]).

Nevertheless, our results report for the first time direct evidence of overweight and glucose intolerance in adult female mice treated with tamoxifen during the neonatal period.

Tamoxifen induces hepatic steatosis and deregulation of hepatic lipid metabolism pathways in adult women and female mouse models [START_REF] Cole | Tamoxifen Induces Triacylglycerol Accumulation in the Mouse Liver by Activation of Fatty Acid Synthesis[END_REF][START_REF] Larosche | Tamoxifen Inhibits Topoisomerases, Depletes Mitochondrial DNA, and Triggers Steatosis in Mouse Liver[END_REF][START_REF] Lelliott | Transcript and Metabolite Analysis of the Effects of Tamoxifen in Rat Liver Reveals Inhibition of Fatty Acid Synthesis in the Presence of Hepatic Steatosis[END_REF][START_REF] Nishino | Effects of Tamoxifen on Hepatic Fat Content and the Development of Hepatic Steatosis in Patients with Breast Cancer: High Frequency of Involvement and Rapid Reversal after Completion of Tamoxifen Therapy[END_REF]). To our surprise, the overweight and insulin resistance phenotype of NTT female mice was not associated with defects in lipid metabolic pathways in the liver.

Interestingly, it has also been shown that neonatal exposition to testosterone also induces drastic sex-dependent metabolic differences in male and female mice (Nohara et al. 2013;Nohara et al. 2013). Whereas female mice develop massive fat accumulation and insulin resistance, male mice actually develop a mild metabolic phenotype without cardiometabolic consequences (Nohara, Liu, et al. 2013). Neonatal testosterone administration to female mice programs features of insulin resistance and prediabetes and predisposes adult female mice to increased adiposity (Nohara et al. 2013). These deregulations were interpreted as a masculinization of their metabolism (Nohara et al. 2013). Importantly, testosterone can be transformed into estrogen by brain aromatases, and some masculinizing effects following androgen exposure are suggested to be due to aromatases and, therefore, to a local brain production of estrogens (Wu et al. 2009). These data raise the possibility that a selective ER modulator, such as tamoxifen, could interact with central ERs since the neonatal period. In striking contrast to the dramatic metabolic effects observed in NTT female mice, NTT male mice only had a modest effect on their general metabolic state. Although research teams have reported an important amelioration of glucose tolerance on male mice administered with tamoxifen during adulthood [START_REF] Ahn | Tamoxifen Suppresses Pancreatic β-Cell Proliferation in Mice[END_REF][START_REF] Ceasrine | Tamoxifen Improves Glucose Tolerance in a Delivery-, Sex-, and Strain-Dependent Manner in Mice[END_REF], we show here that NTT male mice only had a slight amelioration of glucose and insulin tolerance. NTT male mice showed food intake, fat mass, energy expenditure and physical activity comparable to those of vehicle-treated littermates. Studies connecting body weight to glycaemic control and diabetes focus on fat accumulation and obesity, rather than on weight per se [START_REF] Bastard | Recent Advances in the Relationship between Obesity, Inflammation, and Insulin Resistance[END_REF][START_REF] Wilding | The Importance of Weight Management in Type 2 Diabetes Mellitus[END_REF]. It is thus unlikely that the decreased weight per se could explain the improved glycaemic control observed in NTT male mice. The sole explanation we found for the decreased lean mass observed in NTT male mice was that they had tibia about 9% lighter, consistent with a total body weight about 9% lighter than control mice at the date we determined their body composition.

Our study suggests that administering tamoxifen one day after birth to male mice is enough to affect life-long body weight via an altered bone weight, independently of food quality.

Importantly, the effects on bone weight associated with a neonatal administration of tamoxifen also took place in male mice fed a standard diet. In line with our data, Uesugi and coll. demonstrated in 1993 that tamoxifen inhibits ossification of the neonatal mouse pubis [START_REF] Uesugi | Morphometric Analysis of the Pelvis in Mice Treated Neonatally with Tamoxifen[END_REF]; and Fukazawa and coll. showed that NTT adult male mice had lighter bone ash than control mice, which was associated with decreased calcium, phosphorous and osteoblasts [START_REF] Fukazawa | Effect of Neonatal Exposure to Diethylstilbestrol and Tamoxifen on Pelvis and Femur in Male Mice[END_REF]. Furthermore, the neonatal administration of testosterone or estrogen to C57BL/6J male mice is associated with lower lean mass and bone density and a lower resistance to bone fracture after puberty [START_REF] Connelly | Neonatal Estrogen Exposure Results in Biphasic Age-Dependent Effects on the Skeletal Development of Male Mice[END_REF]Nohara, Liu, et al. 2013). Interestingly, males with aromatase deficiency, leading to estrogen deficiency, have impaired skeletal maturation and bone metabolism too [START_REF] Merlotti | Aromatase Activity and Bone Loss in Men[END_REF]). Altogether, these data suggest that an altered estrogen action could be at the core of the negative effects exerted by a neonatal tamoxifen treatment on male bone weight.

To summarize, our work shows that the neonatal administration of tamoxifen is associated with life-long sex-dependent physiological effects in male and female mice. NTT female mice developed dramatic metabolic disturbances resembling overweight and diabetes, whereas NTT male mice had a modest improvement of glycaemic control, and decreased bone weight associated with decreased body weight. Interestingly, these features mimic the consequences of a neonatal injection of testosterone or estrogen, which differently affect male and female mice. In consequence, we highly encourage researchers using the tamoxifen dependent CRE/lox recombination system during the neonatal period to use appropriate controls when investigating metabolism or bone biology. DNA extracted from the intestine of I.G6pc1 -/-mice receiving an intra-gastric injection of tamoxifen at day 1 was amplified using specific G6pc1 primers [START_REF] Mutel | Control of Blood Glucose in the Absence of Hepatic Glucose Production during Prolonged Fasting in Mice: Induction of Renal and Intestinal Gluconeogenesis by Glucagon[END_REF]. Fragments of 1189 and 595 bp correspond to G6pc1 lox and G6pc1 del alleles, respectively. E: exon; bp: base pairs. hypothesized that the neonatal pic of IGN also regulates the development of hypothalamic feeding circuits.

Methods:

We genetically induced neonatal IGN by overexpressing the catalytic sub-unit of the glucose-6-phosphatase one day after birth (I overexp G6pc P1 ). The neonatal development of

Introduction

The incidence of obesity and diabetes continues to increase at an alarming rate. In 2019, approximately 463 million adults were living with diabetes worldwide (International diabetes federations atlas), and 108 million children were obese in 2015 [START_REF] Ebbeling | Childhood Obesity: Public-Health Crisis, Common Sense Cure[END_REF][START_REF] Saklayen | The Global Epidemic of the Metabolic Syndrome[END_REF]. Diabetes and obesity are metabolic disorders that reflect the inability of an organism to appropriately metabolize its energy resources. This public health challenge drives a need to understand the factors implicated in the development of metabolic diseases. It has been suggested that an optimal hypothalamic development is necessary for accurate life-long regulation of energy balance (Levin 2000). In mice, ARH neuronal projections mainly develop during the first three postnatal weeks (Bouret 2004a) under the influence of hormones that regulate feeding in the adult, such as leptin (Bouret 2004b[START_REF] Bouret | Organizational Actions of Metabolic Hormones[END_REF]. In rodents, plasma leptin levels surge from postnatal day 8 (P8) to P12 [START_REF] Ahima | Postnatal Leptin Surge and Regulation of Circadian Rhythm of Leptin by Feeding. Implications for Energy Homeostasis and Neuroendocrine Function[END_REF][START_REF] Devaskar | Developmental Changes in Ob Gene Expression and Circulating Leptin Peptide Concentrations[END_REF], and promotes ARH axonal outgrowth in part by signaling through the long form of the leptin receptor (LepRb) and via the phosphorylation of signal transducer and activator of transcription 3 (STAT3) (Bouret 2004b;[START_REF] Bouret | Distinct Roles for Specific Leptin Receptor Signals in the Development of Hypothalamic Feeding Circuits[END_REF].

Interestingly, the de novo production of glucose by the intestine (i.e. intestinal gluconeogenesis, or "IGN") also increases during the first two postnatal weeks in rats [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]. In adults, this function controls a gut-brain neural circuit that is crucial for maintaining metabolic health [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF][START_REF] Vily-Petit | Intestinal Gluconeogenesis Prevents Obesity-Linked Liver Steatosis and Non-Alcoholic Fatty Liver Disease[END_REF]. Indeed, mice that lack IGN develop a pre-diabetic phenotype [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF], whereas mice with a constitutive induction of IGN are protected from developing diabetes, obesity and hepatic steatosis when fed a high-fat/high-sucrose diet (HF/HS diet) [START_REF] Vily-Petit | Intestinal Gluconeogenesis Prevents Obesity-Linked Liver Steatosis and Non-Alcoholic Fatty Liver Disease[END_REF]. IGN leads to the release of glucose into the portal vein, producing a "portal glucose signal" that activates the hypothalamus [START_REF] Delaere | Portal Glucose Influences the Sensory, Cortical and Reward Systems in Rats[END_REF][START_REF] Delaere | Hypothalamic Integration of Portal Glucose Signals and Control of Food Intake and Insulin Sensitivity[END_REF][START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF][START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]. Interestingly, IGN enhances hypothalamic leptin signaling and it also stimulates STAT3 phosphorylation independently of leptin [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF][START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]. Consequently, we hypothesized that IGN, which plays a role on food intake in adults, could also regulate the development of ARH AgRP and POMC feeding circuits through STAT3 signaling during the neonatal period.

In this study, we investigated the development of hypothalamic AgRP and POMC feeding circuits and adult metabolism in mice with a genetic induction of IGN since birth. In parallel, we studied the same parameters in mice with a genetic induction of IGN right after the neonatal peak, in order to determine whether the effects were restricted to the early neonatal period. Our results suggest that IGN exerts a neurotrophic effect on AgRP projections into the anterior part of the PVH specifically during the first two weeks of life;

and that inducing IGN specifically during the early neonatal period protects mice metabolism when fed a HF/HS diet during adulthood.

Methods

Animals

We used only male mice. I overexp G6pc P1 and I overexp G6pc P12 male mice were obtained by crossing Rosa26 Stopcassette-lox-G6pc with Villin-cre ERT2 mice, in a C57Bl/6J genetic background.

Litters were balanced to 7-9 pups per dam. Half of pups received an intra-gastric injection of tamoxifen (20 µg, Sigma-Aldrich) at postnatal day 1 (P1) or P12 to induce the genetic modification. The other half was injected intra-gastrically with 20 µL of tamoxifen's vehicle, sunflower oil at P1 or P12, respectively [START_REF] Lizen | Perinatal Induction of Cre Recombination with Tamoxifen[END_REF]. Male wild-type mice (C57Bl/6J, Charles River Laboratories, France) were also used, and followed the same tamoxifen or vehicle treatment previously described. All mice were group housed in the animal facility of All procedures were performed in accordance with the principles and guidelines of the European Convention for the Protection of Laboratory Animals. The regional animal care committee (CEEA-55, Lyon 1 University, France) approved all the experiments presented.

Gene expression analysis

Mouse pups aged of 1, 3, 5, 7, 8, 9 and 10 days were taken from dams and decapitated. The proximal jejunum was immediately removed, frozen in liquid nitrogen and kept at -80°C until use. Tissues were homogenized with the Fast Prep® system and total RNAs were isolated from tissues with the TRIzol reagent protocol (Invitrogen). Reverse transcription was performed on 1 µg of mRNA using the Qiagen QuantiTect Reverse Transcription kit.

Real-time qPCRs were performed using sequence-specific primers with Takyon TM No Rox SYBR® MasterMix dTTP Blue (Eurogentec). The expression of mRNAs was normalized to the mouse ribosomal protein L32 transcript expression using the 2-ΔΔCt method.

G6PC1: Fw: TTACCAAGACTCCCAGGACTG ; Rev: GAGCTGTTGCTGTAGTAGTCG Ribosomal protein L32: Fw: CAAGGAGCTGGAGGTGCTGC; Rev: CTGCTCTTTCTACAATGGC

Glucose-6-phosphatase enzymatic activity

Mouse pups aged of 1, 3, 5, 7, 9 and 15 days were taken from dams and decapitated. The proximal jejunum was removed and treated similarly to tissues treated for gene expression analyses. The glucose-6-phosphatase (G6Pase) activity was measured as previously described [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]. Briefly, all the tissue (around 20 mg) was homogenized in 9

volumes of Hepes Sucrose before glucose-6-phosphatase activity was measured. The specific glucose-6-phosphatase activity was substracted from total phosphatase activity. The results are expressed as micromoles of hydrolyzed substrate per minute.

Immunofluorescence assays

Brain fixation of mice aged of 20 or 80 days was conducted as previously described (Soty et al. 2020). Fixed brains were frozen for 1 min. in C5H12 at -40°C, and 40 µm sections were cut at -25°C. Sections were kept in PBS/0.25% NaN3 until processed for immunofluorescence.

We chose one free-floating section per hypothalamic nucleus of interest with a loop. Briefly, we carried out 3 PBS 1X-Tx0.4% washes, incubated the sections in 10mM citric acid pH 6.0

for 20 min at 80°C, and saturated (PBS1X-Tx0.4%, BSA 5%, goat serum 5%) for 2-4h at room temperature (RT). Incubation with primary antibodies (Rabbit AgRP 1/4000 and POMC 1/10000, Phoenix Pharmaceuticals) was done during 2 nights at 4°C, followed by incubation with secondary antibodies (Goat α-rabbit IgG Alexa fluor 488, 1/500, Life Technologies) for 2h at RT, and Hoescht 33342 (1/5000) for 5 min. Sections were washed 3X and mounted with Life ProLong Gold antifade reagent (Life Technologies). All photographs were taken with an objective 20X (0,8 M27) DIC using a confocal miscrocope Zeiss LSM 880 Airyscan NLO.

Fiber density quantitative analyses

A single image corresponding to the central position in the Z-plane of each nucleus of interest was acquired with an objective 20X (0,8 M27) DIC using a confocal miscrocope Zeiss LSM 880 Airyscan NLO. Image file names were coded prior to quantification to ensure non-biased treatment. Quantification was performed with Image J. For quantitative analyses of AgRP and POMC fiber densities in each hypothalamic nucleus of interest, the area of quantification was selected manually as a region of interest (ROI). Quantification was performed on a manually thresholded image (allowing separation of immunoreactive fibers from the background). We divided the integrated density value (which reflects the sum of pixels in the thresholded image) by the area of the ROI. For quantitative analyses of POMC cell bodies in the ARH, POMC-immunoreactive cell bodies were manually counted. Only one image was quantified per hypothalamic nucleus and per mice.

Body weight and body composition measurements

Mice body weight was measured once per week since weaning. During the perinatal period, mouse pups were weighted at P7, P11, P15 and P20. Fat and lean mass were determined with Nuclear Magnetic Resonance spectroscopy (Minispec LF90 II; BRUKER Society, Wissembourg, France). All procedures were performed by the ANIPHY platform from Lyon 1 University.

Western blot analysis

Mouse pups aged of 9 and 15 days were taken from dams and decapitated. The hypothalamus was removed and kept at -80°C until use. Western blot analyses were performed as previously described. Primary antibodies: AgRP (1/1000, Interchim); POMC (1/1000, Cell Signaling); PSTAT3 and STAT3 (1/2000, Cell Signaling). Secondary antibodies: goat α-rabbit

IgG linked to peroxidase (1/1000, Biorad). Stain-free gels were used (Biorad, ref. #1610181).

Plasma insulinemia and leptinemia

Blood was withdrawn from the trunk when mouse pups were decapitated, or from the submandibular vein from 6h fasted adult mice, then collected in ethylene diamine tetraacetic acid (EDTA) and conserved at -80°C. Insulin and leptin were assayed using insulin (Mercodia, ref. #10-1247-01) and leptin (Enzo, ref #ADI-900-019A) mouse ELISA kits.

Lactation

Mouse pups aged of 7, 11 and 15 days were separated from dams during 2 hours and then weighted. Pups were then reunited with dams for 1 hour, after which pups were weighted again. The difference between both weights was considered as milk lactated by pups.

Statistical analyses

Results are reported as means ± standard error of the mean (SEM). Normal distribution was verified for all analyses. Two-group comparisons were analysed using unpaired t-tests. The effect of two independent variables on a dependent variable was analysed using two-way ANOVA, followed by multiple comparisons and corrected for multiple comparisons with a SIDAK test. The differences were considered statistically significant at *p<0.05, **p<0.01 and ***p<0.001. Statistical details of experiments can be found in the figure legends. Statistical analyses were performed with GraphPad Prism 6 software.

Results

Validation of the neonatal induction of IGN

The neonatal peak of IGN was previously described in rats [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]. Therefore, we first characterized the neonatal peak of IGN in C57Bl/6J mouse pups (the genetic background of our animal model), which takes place between P3 and P9 (Supplementary figure 1). To assess the function of IGN during the neonatal period, we used a CRE/lox biotechnology strategy to develop a murine model with an inducible and intestine-specific overexpression of the catalytic sub-unit of the glucose-6-phosphatase (encoded by G6pc).

The glucose-6-phosphatase (G6Pase) is the mandatory enzyme of endogenous glucose production. For this, we inserted both a STOP cassette flanked between two loxP sequences, and the complementary DNA sequence of G6pc in the Rosa26 locus [START_REF] Vily-Petit | Intestinal Gluconeogenesis Prevents Obesity-Linked Liver Steatosis and Non-Alcoholic Fatty Liver Disease[END_REF] (Figure 1A). This locus exhibits a strong transcriptional activity, which is blocked here by the STOP cassette. In parallel, the Cre ERT2 recombinase was placed under the control of the villin promoter, which expression is restricted to the intestine (El [START_REF] Marjou | Tissue-Specific and Inducible Cre-Mediated Recombination in the Gut Epithelium[END_REF]). The metabolite of tamoxifen, 4-hydroxytamoxifen, activates the Cre ERT2 recombinase, which then excises the flanked STOP cassette, allowing for intestinal overexpression of G6pc [START_REF] Metzger | Siteand Time-Specific Gene Targeting in the Mouse[END_REF]. In this study, we first induced the intestinal G6pc overexpression at P1 in male pups (I overexp G6pc P1 mice). The amplification of the excised STOP cassette is visible specifically in the intestine of I overexp G6pc P1 pups and persists during adulthood (Figure 1B).

I overexp G6pc P1 pups exhibited a 10-fold increase of G6pc expression in comparison to control mice already at P3 (Figure 1C). Furthermore, intestinal G6Pase activity of I overexp G6pc P1 pups was increased more than 3-fold at P7, P9 and P15 in comparison to control mice (Figure 1D). reduction of POMC immunoreactivity in the PVHant compared to control littermates, whereas no differences were observed in the other nuclei studied (Figure 2A,C). The drug tamoxifen per se has been reported to affect brain neuronal populations implicated in reproduction [START_REF] Agça | Modulation of Estrogen Receptors during Development Inhibits Neurogenesis of Precursors to GnRH-1 Neurones: In Vitro Studies with Explants of Ovine Olfactory Placode[END_REF][START_REF] Ichimura | Neonatal Exposure to SERMs Disrupts Neuroendocrine Development and Postnatal Reproductive Function through Alteration of Hypothalamic Kisspeptin Neurons in Female Rats[END_REF]. Therefore, we verified whether the neonatal tamoxifen administration to wild-type pups affects AgRP and POMC fiber immunoreactivity. Tamoxifen administration to P1 wild-type pups did not affect AgRP nor POMC fiber density in any of the studied hypothalamic nuclei (Supplementary figure 2).

We then determined whether the effects on fiber density observed in the PVHant of I overexp G6pc P1 pups persisted throughout life. Both AgRP and POMC fiber immunoreactivity were equivalent in adult I overexp G6pc P1 mice and control littermates (Supplementary figure 3), suggesting that there was a compensation of both AgRP and POMC fiber density between P20 and adulthood. Altogether, these data suggest that, during the neonatal period, IGN exerts a neurotrophic role on AgRP fibers and decreases POMC fibers projecting to the PVHant, which can be compensated later in life. 148

Mice with a neonatal induction of intestinal gluconeogenesis exhibit a better body composition under a HF/HS diet

To determine the role of neonatal IGN on metabolism, we first investigated whether the neonatal induction of IGN affects neonatal weight, lactation and glycaemia during the neonatal period. I overexp G6pc P1 pups showed control-like weight (Supplementary figure 4A), lactation (Supplementary figure 4B) and even glycaemia (Supplementary figure 4C), despite having an increased intestinal glucose production. Next, to uncover the role of the neonatal induction of IGN on life-long metabolism, we challenged I overexp G6pc P1 mice and control littermates with a HF/HS diet since they were 6 weeks old. A HF/HS diet is known for inducing obesity and diabetes in C57Bl/6J mice. After two months on a HF/HS diet, I overexp G6pc P1 mice were 23% lighter than control mice (Figure 3A). Moreover, I overexp G6pc P1 mice exhibited about a 30% decrease of body fat mass (Figure 3B) and a 10% increase of body lean mass in comparison to control littermates (Figure 3C). Importantly, the administration of tamoxifen per se does not affect male mice body composition (Estrada-Meza et al., under preparation). High circulating leptin levels and leptin resistance are hallmarks of obesity [START_REF] Bouhajja | Affinity Kinetics of Leptin-Reactive Immunoglobulins Are Associated with Plasma Leptin and Markers of Obesity and Diabetes[END_REF][START_REF] Izquierdo | Leptin, Obesity, and Leptin Resistance: Where Are We 25 Years Later?[END_REF][START_REF] Jung | Molecular Mechanisms of Central Leptin Resistance in Obesity[END_REF][START_REF] Kettaneh | High Plasma Leptin Predicts an Increase in Subcutaneous Adiposity in Children and Adults[END_REF]). To address whether the neonatal induction of IGN protects adult mice from these alterations, we measured plasma leptin levels and hypothalamic STAT3 phosphorylation levels in mice. I overexp G6pc P1 mice showed a tendency to have reduced plasma leptin levels (Figure 3D) and exhibited increased hypothalamic STAT3 phosphorylation (Figure 3E) in comparison to control littermates. Indeed, STAT3 is part of the signaling pathway of leptin and it becomes phosphorylated upon activation of the long isoform of the leptin receptor (LepRb) [START_REF] Kwon | Leptin Signalling Pathways in Hypothalamic Neurons[END_REF]. These results suggest that the neonatal induction of IGN maintains adult hypothalamic leptin sensitivity. Overall, these data suggest that the neonatal induction of IGN preserves body composition and hypothalamic function in mice challenged with a HF/HS diet during adulthood.

The effect of IGN on hypothalamic AgRP and POMC fibers is restricted to the neonatal period

To investigate whether the effects associated with the neonatal induction of IGN would be restricted to the neonatal period, we then induced intestinal G6pc at P12 in male pups (I overexp G6pc P12 mice), meaning right after the naturally occurring neonatal peak of IGN. We conducted the same AgRP and POMC brain immunolabeling protocol that we had previously performed in I overexp G6pc P1 mice and their respective controls. Strikingly, in contrast to I overexp G6pc P1 pups, P20 I overexp G6pc P12 pups had control-like AgRP and POMC fiber density in all studied nuclei (Figure 4A,B,C). Importantly, the administration of tamoxifen to P12 wildtype pups did not alter AgRP or POMC fiber density (Supplementary figure 5). These data suggest that the regulation of AgRP and POMC fibers projections to the PVHant by IGN would be restricted to the first two weeks of life.

Inducing IGN after P12 fails to protect adult mice body composition under a HF/HS diet

To assess whether inducing IGN after its neonatal surge still affects life-long metabolism, we challenged both I overexp G6pc P12 and control mice with a HF/HS diet since they were 6 weeks old. Again, in contrast to I overexp G6pc P1 mice, adult I overexp G6pc P12 mice had control-like weight gain (Figure 5A), body fat (Figure 5B) and lean mass (Figure 5C). Furthermore, plasma leptin levels and the hypothalamic STAT3 phosphorylation state were equivalent in both I overexp G6pc P12 and control mice (Figure 5D,E). Wild-type mice that received a tamoxifen administration at P12 did not exhibit any tamoxifen-induced effects on weight and body composition (Supplementary figure 6). These results suggest that the protection induced by IGN on body composition and the hypothalamic function of mice nourished with a HF/HS diet is restricted to the first two weeks of life.

Neonatal IGN improves the ratio of STAT3 phosphorylation to plasma leptin in the hypothalamus.

To uncover the mechanisms underlying how neonatal IGN would control AgRP and POMC fiber projections into the PVHant, we first determined whether the increase of AgRP and the decrease of POMC immunoreactivity were linked to changes in the amount of AgRP and POMC peptides. Western blot analyses in the hypothalamus of P9 and P15 mouse pups revealed that I overexp G6pc P1 had the same amount of both AgRP (Figure 6A) and POMC (Figure 6B) peptides in comparison to control littermates. These results suggest that the increase of AgRP and the decrease of POMC immunoreactivity in the PVHant observed in I overexp G6pc P1 (Figure 2B) did not reflect a modification of hypothalamic AgRP and POMC protein content. We then sought to determine the molecular mechanism underlying the neuronal effects controlled by neonatal IGN. During the first two weeks of life in mice, increasing levels of leptin stimulate AgRP and POMC axonal elongation from the ARH to all studied nuclei, in part, via STAT3 phosphorylation [START_REF] Ahima | Postnatal Leptin Surge and Regulation of Circadian Rhythm of Leptin by Feeding. Implications for Energy Homeostasis and Neuroendocrine Function[END_REF]Bouret 2004b;[START_REF] Bouret | Distinct Roles for Specific Leptin Receptor Signals in the Development of Hypothalamic Feeding Circuits[END_REF][START_REF] Devaskar | Developmental Changes in Ob Gene Expression and Circulating Leptin Peptide Concentrations[END_REF]. In adult mice, IGN potentiates hypothalamic leptin signaling and increases ARH STAT3 phosphorylation independently of leptin [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF][START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]. We thus measured the STAT3 phosphorylation state in the hypothalamus of mice aged of 9 and 15 days, a time corresponding to the neonatal peak of leptin and to the increased ARH axonal outgrowth. No differences were observed between both groups (Figure 6C). What is more, I overexp G6pc P1 mice exhibited two times less plasma leptin concentration at P9 (Figure 6D). Interestingly, the ratio of hypothalamic STAT3 phosphorylation to plasma leptin was 2-fold higher in I overexp G6pc P1 pups in comparison to control littermates (Figure 6E). These results suggest that the neonatal induction of IGN could either improve hypothalamic leptin sensitivity in mouse pups or increase STAT3 phosphorylation independently of leptin.

Discussion

IGN controls a gut-brain neural circuit that is crucial for maintaining metabolic health [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF][START_REF] Vily-Petit | Intestinal Gluconeogenesis Prevents Obesity-Linked Liver Steatosis and Non-Alcoholic Fatty Liver Disease[END_REF]. Our laboratory has previously shown that IGN increases strongly under specific conditions such as a diet rich in proteins (Duraffourd et al. 2012;[START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF][START_REF] Penhoat | Protein-Induced Satiety Is Abolished in the Absence of Intestinal Gluconeogenesis[END_REF] or soluble fibers [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF][START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF], after a gastric bypass [START_REF] Troy | Intestinal Gluconeogenesis Is a Key Factor for Early Metabolic Changes after Gastric Bypass but Not after Gastric Lap-Band in Mice[END_REF], and during the first two weeks of life in rodents [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]. However, the role of the neonatal increase of IGN has remained uncharacterized. Here we suggest that, during the neonatal period, IGN exerts a neurodevelopmental role.

Using a nutrient-independent induction of IGN since birth, we showed that the neonatal induction of IGN leads to an increase of AgRP fibers and a decrease of POMC fibers connecting the PVHant. Our data are similar to the results observed in amylin knockout mice, which exhibit an increase of AgRP fiber density and a decrease of the POMC-derived peptide, α-MSH, fiber density in the PVH [START_REF] Lutz | Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus[END_REF]. However, the results we obtained from I overexp G6pc P1 mice could also be linked to the neonatal tamoxifen treatment per se. Indeed, tamoxifen-treated wild-type pups show a decrease of POMC-immunoreactive cells in the ARH in comparison to vehicle-treated wild-type pups, which was also observed in I overexp G6pc P1 pups (Supplementary figure 7). Moreover, neonatal testosterone administration reduces hypothalamic POMC expression in mice, without affecting AgRP expression [START_REF] Nohara | Early-Life Exposure to Testosterone Programs the Hypothalamic Melanocortin System[END_REF]. Interestingly, testosterone that reaches the brain is transformed into estrogen by the aromatase enzyme [START_REF] Roselli | Distribution and Regulation of Aromatase Activity in the Rat Hypothalamus and Limbic System*[END_REF][START_REF] Selmanoff | Aromatization and 5α-Reduction of Androgens in Discrete Hypothalamic and Limbic Regions of the Male and Female Rat 1[END_REF][START_REF] Tabatadze | Quantitative Analysis of Long-Form Aromatase MRNA in the Male and Female Rat Brain[END_REF]. As tamoxifen is a selective estrogen receptor modulator, it could affect hypothalamic POMC neurons through estrogen receptors. Therefore, the results on POMC fiber density need to be interpreted with caution.

The nutrient-independent induction of IGN since birth led to an increase of AgRPimmunoreactive projections into the PVHant. The PVHant has received great attention concerning energy balance because it has outputs regulating the endocrine system, the autonomic nervous system and the behavioral system [START_REF] Biag | Cytoand Chemoarchitecture of the Hypothalamic Paraventricular Nucleus in the C57BL/6J Male Mouse: A Study of Immunostaining and Multiple Fluorescent Tract Tracing[END_REF]. Hence, neonatal IGN seems to modulate AgRP in one of the most influential hypothalamic nuclei. Intestinal gluconeogenesis could increase AgRP axonal outgrowth per se and/or the quantity of AgRP peptides. Indeed, a protein-enriched diet, which strongly activates IGN, leads to a compensatory increase of hypothalamic AgRP mRNA levels in adults [START_REF] Pillot | Role of Hypothalamic Melanocortin System in Adaptation of Food Intake to Food Protein Increase in Mice[END_REF]).

However, we did not measure an increase in the amount of AgRP peptides in the hypothalamus of I overexp G6pc P1 . Consistently, although chemogenetic activation of AgRP neurons drives feeding in mice since P15 [START_REF] Baquero | Developmental Switch of Leptin Signaling in Arcuate Nucleus Neurons[END_REF][START_REF] Zimmer | Functional Ontogeny of Hypothalamic Agrp Neurons in Neonatal Mouse Behaviors[END_REF]), I overexp G6pc P1 mice showed control-like neonatal and post-weaning weight. Thus, the increase of AgRP immunoreactivity in the PVHant of I overexp G6pc P1 pups is probably not linked to an increase of AgRP peptides that would drive food intake and weight gain. The increase of AgRP immunoreactivity would therefore reflect an increase of axons containing AgRP. Importantly, AgRP axons in the PVH originate from the ARH only [START_REF] Grove | Postnatal Development of the Hypothalamic Neuropeptide Y System[END_REF], whereas NPY axons originate also from the brainstem [START_REF] Sahu | Evidence That NPY-Containing Neurons in the Brainstem Project into Selected Hypothalamic Nuclei: Implication in Feeding Behavior[END_REF]). Consequently, it is reasonable to use AgRP immunolabeling in order to identify ARH AgRP axonal projections.

Our results showed that the neurotrophic effect exerted by IGN on AgRP projections into the PVHant takes place only during the first two weeks of life. Thus, we propose that IGN exerts a developmental role specifically during the early neonatal period. The neonatal time frame is a hypoglycaemic-tending environment [START_REF] Girard | Adaptations of Glucose and Fatty Acid Metabolism during Perinatal Period and Suckling-Weaning Transition[END_REF]. The neonate therefore has to rapidly activate its endogenous glucose production (EGP), which is assured by glycogenolysis in the liver [START_REF] Margolis | Glycogen Metabolism in Neonatal Liver of the Rat[END_REF], and gluconeogenesis in the liver, the kidneys and the intestine [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF][START_REF] Girard | Adaptations of Glucose and Fatty Acid Metabolism during Perinatal Period and Suckling-Weaning Transition[END_REF][START_REF] Hahn | Pyruvate Carboxylase, Phosphate-Dependent Glutaminase and Glutamate Dehydrogenase in the Developing Rat Small Intestinal Mucosa[END_REF][START_REF] Watford | Phosphoenolpyruvate Carboxykinase of Rat Small Intestine: Distribution and Regulation of Activity and MRNA Levels[END_REF]. Consequently, one could argue that the most evident neonatal role of IGN is to provide glucose for the growing neonate. However, it is possible that the neonatal EGP is mostly assured by the liver. Indeed, rat fetuses stock large quantities of hepatic glycogen prior to birth, and the liver mobilizes its glycogen stores rapidly during the early neonatal period [START_REF] Margolis | Glycogen Metabolism in Neonatal Liver of the Rat[END_REF]. Furthermore, the activity of the glucose-6-phosphatase, the mandatory enzyme of EGP, is more than 7-times higher in the liver than in the intestine of the newborn [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]. Moreover, a neonatal diminution of hepatic gluconeogenesis is enough to produce hypoglycaemia in neonates [START_REF] Cotter | Impairments of Hepatic Gluconeogenesis and Ketogenesis in PPARα-Deficient Neonatal Mice[END_REF], further highlighting the role of the liver on the regulation of glycaemia in the neonate. Finally, not only glycaemia was unaltered in I overexp G6pc P1 pups, but it is also unaffected in mice with a constitutive deletion of IGN (data not shown). Thus, maintaining glycaemia is probably not a major role of neonatal IGN.

Importantly, the fact that I overexp G6pc P1 pups exhibited control-like glycaemia implies that the increase of AgRP projections into the PVHant was not a consequence of an excess of glucose.

Additionally, plasma insulin levels were equivalent in both P9 I overexp G6pc P1 and control littermates (0.54±0.06 vs. 0.44±0.13). Interestingly, P9 I overexp G6pc P1 pups exhibited controllike hypothalamic STAT3 phosphorylation despite showing reduced leptin levels. An alteration of neonatal leptin levels in plasma of rodents exposed to perinatal malnutrition has repeatedly been associated with an insufficient development of ARH projections [START_REF] Delahaye | Maternal Perinatal Undernutrition Drastically Reduces Postnatal Leptin Surge and Affects the Development of Arcuate Nucleus Proopiomelanocortin Neurons in Neonatal Male Rat Pups[END_REF][START_REF] Kirk | Maternal Obesity Induced by Diet in Rats Permanently Influences Central Processes Regulating Food Intake in Offspring[END_REF][START_REF] Yura | Role of Premature Leptin Surge in Obesity Resulting from Intrauterine Undernutrition[END_REF]. However, a reduction of circulating leptin levels in P9 I overexp G6pc P1 pups failed to decrease hypothalamic STAT3 phosphorylation, and to disrupt AgRP fiber development. Hence, IGN could either enhance hypothalamic leptin sensitivity or stimulate STAT3 phosphorylation during the neonatal period. In adult mice, the calcitonin gene-related peptide (CGRP) is a link in the metabolic benefits exerted by IGN [START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]. Furthermore, an intracerebroventricular injection of CGRP increases ARH STAT3phosphorylation [START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF], suggesting that CGRP could directly influence the ARH. Interestingly, mice with a deletion of the receptor components RAMP1/3 exhibit decreased AgRP and POMC-derived α-MSH fiber density in the PVHant [START_REF] Lutz | Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus[END_REF], suggesting that RAMP1/3 also plays a neurodevelopmental role. It is noteworthy that RAMP1 is a receptor component of CGRP, while RAMP3 has been shown to interact with the CGRP receptor to augment its affinity [START_REF] Dickerson | Role of CGRP-Receptor Component Protein (RCP) in CLR/RAMP Function[END_REF]. Thus, CGRP could be the missing link between IGN and the development of AgRP projections to the PVHant.

Studies have shown that both an excessive and an insufficient development of ARH axonal projections or neurogenesis are associated with metabolic problems during adulthood [START_REF] Chang | Maternal High-Fat Diet and Fetal Programming: Increased Proliferation of Hypothalamic Peptide-Producing Neurons That Increase Risk for Overeating and Obesity[END_REF][START_REF] Mcnay | Remodeling of the Arcuate Nucleus Energy-Balance Circuit Is Inhibited in Obese Mice[END_REF][START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF][START_REF] Vogt | Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding[END_REF][START_REF] Yura | Role of Premature Leptin Surge in Obesity Resulting from Intrauterine Undernutrition[END_REF]. However, during adulthood, I overexp G6pc P1 mice had 30% lower body fat and 10% more body lean mass after being fed a HF/HS diet for two months. Therefore, the protective metabolic effects observed in adult I overexp G6pc P1 mice were probably not due to the increase of AgRP projections into the PVHant. To our surprise, inducing IGN after the neonatal peak (i.e. P12) failed to protect the body composition of mice on a HF/HS diet, suggesting some sort of neonatal imprinting exerted by the neonatal induction of IGN. Central leptin resistance is often a hallmark of obesity and diabetes [START_REF] Gout | Leptin Infusion and Obesity in Mouse Cause Alterations in the Hypothalamic Melanocortin System[END_REF][START_REF] Jung | Molecular Mechanisms of Central Leptin Resistance in Obesity[END_REF] and depends, at least in part, by exposure to perinatal obesogenic conditions [START_REF] Bouret | Development of Hypothalamic Circuits That Control Food Intake and Energy Balance[END_REF].

Hence, the enhanced leptin signaling observed in I overexp G6pc P1 mice (both during the neonatal period and during adulthood) could be the neonatal mechanism resulting in the improved body composition when fed a HF/HS diet during adulthood.

In conclusion, with our investigation, we propose: first, that IGN exerts a neurotrophic role on AgRP projections into the PVHant, specifically during the first two weeks of life; second, that the induction of IGN during the early neonatal period could induce an increase of hypothalamic leptin sensitivity, which would protect the metabolism of an organism when exposed to a HF/HS diet. This study opens a door to exploring the therapeutic potential of inducing IGN in cases of perinatal malnutrition. Our second study suggests that IGN exerts a neurotrophic role on AgRP projections from the ARH to the PVHant during the early postnatal period. Additionally, our second study suggests that inducing IGN during the early postnatal period could be beneficial for long-term metabolism.

Neonatal tamoxifen administration: highlighting the crucial role of sex hormones

during the neonatal period on adult energy homeostasis?

Main results

The neonatal tamoxifen administration to female and male C57BL/6J pups aged of 1 day led to strikingly different long-term effects on metabolism and bone weight between both sexes. Female mice that were treated with tamoxifen on P1 were predisposed to obesity and diabetes, under normal and obesogenic nutritional conditions. Plus, they did not show an altered bone weight. Male mice treated with tamoxifen on P1 showed, in contrast, a mild amelioration of glucose control, and diminished life-long bone weight. Our results highlight the existence of sex differences concerning bone development and the programming of metabolism.

The testosterone surge and sexual differentiation

One of the major hormonal differences between females and males during development is a surge of testosterone that appears during the first hours after birth only in males. This neonatal testosterone surge has been described in male mice, rats, horses and human neonates [START_REF] Corbier | The Neonatal Testosterone Surge: A Comparative Study[END_REF]. The testosterone surge arises from the differentiation of the male gonads into testes, driven by Sry (a gene coded only on the Y chromosome), and is thought to drive sexual differentiation in part by activating the androgen receptor [START_REF] Corbier | The Neonatal Testosterone Surge: A Comparative Study[END_REF]. However, estrogens are also required for sexual differentiation in male mice [START_REF] Bodo | Both Estrogen Receptor-Alpha and -Beta Are Required for Sexual Differentiation of the Anteroventral Periventricular Area in Mice[END_REF][START_REF] Scordalakes | Aggression and Arginine Vasopressin Immunoreactivity Regulation by Androgen Receptor and Estrogen Receptor Alpha[END_REF], which seems counterintuitive because estrogens produced by the gonads are much lower in males [START_REF] Merlotti | Aromatase Activity and Bone Loss in Men[END_REF]Wu et al. 2009). Actually, testosterone is converted into estrogen in extraglandular sites (like the brain) via the enzyme aromatase. This locallyproduced estrogen also controls dimorphism in males [START_REF] Naftolin | The Metabolism of Androgens in Central Neuroendocrine Tissues[END_REF]Wu et al. 2009). It is noteworthy that brain aromatase activity is generally higher in males than in females [START_REF] Naftolin | The Metabolism of Androgens in Central Neuroendocrine Tissues[END_REF]. In summary, several data have shown that testosterone, estrogen and aromatase are implicated in the development of sexual dimorphisms.

Experiments using a neonatal administration of testosterone have suggested that testosterone and estrogen play an important role on "masculinizing" both the brain and the metabolism of mice (which might not be independent one from the other) [START_REF] Nohara | Early-Life Exposure to Testosterone Programs the Hypothalamic Melanocortin System[END_REF]Nohara, Liu, et al. 2013;[START_REF] Nohara | Developmental Androgen Excess Programs Sympathetic Tone and Adipose Tissue Dysfunction and Predisposes to a Cardiometabolic Syndrome in Female Mice[END_REF]). In the ARH, the neonatal testosterone administration to female mice decreases expression and protein quantity of POMC to levels of control male mice [START_REF] Nohara | Early-Life Exposure to Testosterone Programs the Hypothalamic Melanocortin System[END_REF]. POMC is a key down-regulator of food intake [START_REF] Cone | Anatomy and Regulation of the Central Melanocortin System[END_REF]. Interestingly, during my thesis, I showed that the administration of tamoxifen on P1 decreases POMC neuronal count in the ARH of male mice aged of 20 days.

The neonatal testosterone administration to female mice also predisposed them to increased food intake and greater adiposity, reaching levels observed in control male mice [START_REF] Nohara | Early-Life Exposure to Testosterone Programs the Hypothalamic Melanocortin System[END_REF][START_REF] Nohara | Developmental Androgen Excess Programs Sympathetic Tone and Adipose Tissue Dysfunction and Predisposes to a Cardiometabolic Syndrome in Female Mice[END_REF]). These results are also similar to our results, in which female mice treated with tamoxifen on P1 ate more and became overweight. The effects exerted by neonatal tamoxifen reproduce some of the central and the peripheral effects exerted by neonatal testosterone on female mice. As tamoxifen is a selective estrogen receptor modulator, these data suggest that administering tamoxifen neonatally could "masculinize" the female metabolism, potentially by "masculinizing" the brain. Given that testosterone can be converted into estrogen by aromatases, there exists the possibility that the neonatal "masculinization" of the brain and metabolism is mediated, at least in part, by estrogens. Interestingly, neonatal exposition to estrogen in female mice specifically induces central leptin resistance and a compensatory increase in Mc4r expression, which may account for the increase of adiposity induced by neonatal testosterone administration [START_REF] Nohara | Early-Life Exposure to Testosterone Programs the Hypothalamic Melanocortin System[END_REF]. To conclude on the masculinization induced by neonatal tamoxifen, we should study hypothalamic feeding circuits in female mice exposed to tamoxifen on P1, and compare control female mice, tamoxifen-treated female mice and control male mice.

Estrogen also seems to be at the core of bone development. Estrogen receptors (ER) are present in bone tissue [START_REF] Migliaccio | Developmental Exposure to Estrogens Induces Persistent Changes in Skeletal Tissue[END_REF]. Furthermore, in male bone, the aromatase activity is very high and locally produces estrogen [START_REF] Merlotti | Aromatase Activity and Bone Loss in Men[END_REF]. Human aromatase deficiency is a rare disease caused by a loss-of-function mutation in the gene coding the aromatase (CYP19A1) [START_REF] Bulun | Aromatase Deficiency and Estrogen Resistance: From Molecular Genetics to Clinic[END_REF]. Patients have estrogen deficiency and exhibit severe impairment of skeletal maturation and bone metabolism [START_REF] Bulun | Aromatase Deficiency and Estrogen Resistance: From Molecular Genetics to Clinic[END_REF], consistent with a case of a loss-of-function mutation of the gene coding for ERα (ESR1) [START_REF] Smith | Estrogen Resistance Caused by a Mutation in the Estrogen-Receptor Gene in a Man[END_REF].

During my thesis, we observed that the neonatal administration of tamoxifen affected longterm bone weight in male mice, but not in female mice. Our data is consistent with studies

showing that the neonatal exposure to estrogen results in lower weight associated with lower bone mineral density in adult male mice but not in adult female mice [START_REF] Connelly | Neonatal Estrogen Exposure Results in Biphasic Age-Dependent Effects on the Skeletal Development of Male Mice[END_REF][START_REF] Kaludjerovic | Diethylstilbesterol Has Gender-Specific Effects on Weight Gain and Bone Development in Mice[END_REF]. Therefore, neonatal tamoxifen might affect bone weight in mice via an estrogen-dependent mechanism. However, we did not measure ER signaling or aromatase activity in bones of male and female mice treated with tamoxifen on P1.

Neonatal tamoxifen exposure exerted different effects in male and female mice. In female mice, neonatal tamoxifen exposure reproduced in part the effects of neonatal estrogen, suggesting that tamoxifen might induce brain ER activity. In male mice, neonatal tamoxifen exposure reproduced in part the effects of estrogen deficiency, suggesting that tamoxifen might inhibit bone ER activity. These data are consistent with a differential effect of tamoxifen on ER activity, which can be either agonist or antagonist of the ER depending on the tissue [START_REF] Abot | The AF-1 Activation Function of Estrogen Receptor α Is Necessary and Sufficient for Uterine Epithelial Cell Proliferation In Vivo[END_REF][START_REF] Guillaume | Selective Activation of Estrogen Receptor α Activation Function-1 Is Sufficient to Prevent Obesity, Steatosis, and Insulin Resistance in Mouse[END_REF][START_REF] Riggs | Selective Estrogen-Receptor Modulators --Mechanisms of Action and Application to Clinical Practice[END_REF].

Our study could raise questions regarding existing studies having used the tamoxifendependent CRE/lox recombination system during the early neonatal period to study metabolism or bone biology. However, to my knowledge, there is only one such study. The authors focused on adipose tissue metabolism and did not analyze the same metabolic parameters that we did [START_REF] Li | The Nuclear Orphan Receptor COUP-TFII Plays an Essential Role in Adipogenesis, Glucose Homeostasis, and Energy Metabolism[END_REF]). Thus, although appropriate controls were absent in this study, I cannot conclude on whether their results are tamoxifen-dependent. Our study will indeed warn scientists to use appropriate controls if they seek to induce the CRE/lox recombination system during the neonatal period to study metabolism or bone biology.

Besides this importance, I believe that our study also has clinical relevance. This thesis highlights the importance of sex hormones on neonatal development; and suggests that altering sex-hormone levels during the neonatal development could be enough to alter longterm metabolism on female mice, and long-term bone weight in male mice. The use of tamoxifen to treat breast cancer is contraindicated in pregnant women [START_REF] Barthelmes | Tamoxifen and Pregnancy[END_REF]) because tamoxifen has previously been associated with teratogenic effects (birth defects) such as congenital malformations in infants [START_REF] Cullins | Goldenhar's Syndrome Associated with Tamoxifen given to the Mother during Gestation[END_REF][START_REF] Tewari | Ambiguous Genitalia in Infant Exposed to Tamoxifen in Utero[END_REF]. However, tamoxifen is not the only potential source of estrogen receptor modulators. A 2012 study found androgen activity in 35% of tested water sources from 14 states in the United States [START_REF] Stavreva | Prevalent Glucocorticoid and Androgen Activity in US Water Sources[END_REF], suggesting that perinatal exposure to androgens is a possibility.

Neonatal intestinal gluconeogenesis

Does intestinal gluconeogenesis (IGN) control the postnatal development of hypothalamic AgRP and POMC circuits? Main results

The second part of my thesis consisted in determining the role of IGN on the neonatal development of hypothalamic feeding circuits. The nutritionally-independent, genetic induction of IGN affected the development of AgRP and POMC projections into the PVHant, the only two neuronal populations that we studied. Neonatal IGN exerted a neurotrophic effect on AgRP projections, and an unexpected decrease of POMC projections. These results

were restricted to the first two weeks of life.

An increase of AgRP and a decrease of POMC projections into the PVHant

AgRP projections connecting the PVH come only from the ARH [START_REF] Grove | Postnatal Development of the Hypothalamic Neuropeptide Y System[END_REF], making it a reasonable means to measure ARH AgRP fibers. To address whether our observations represented a change in peptide quantity instead of a change in axon density, we performed western blot analyses. Our western blot analyses showed that AgRP and POMC protein quantity was unaffected in mice with a neonatal induction of IGN. However, a more precise way to determine whether neonatal IGN exerts a neurotrophic role on ARH axonal outgrowth would be to implant crystals of the retrograde tracer DiI in the ARH of mice, and then quantify fiber density that has projected into each hypothalamic nucleus of interest.

We were surprised to see an opposing effect on AgRP and POMC projections to the PVHant. A limitation of our study could be that we used POMC antibodies, instead of antibodies directed to the peptide released from POMC cleavage: α-MSH. Indeed, food intake decrease is actually associated with a release of α-MSH and not POMC. However, amylin knockout mice also exhibit a similar phenotype, with an excess of AgRP fiber development and a decrease of α-MSH fiber development into the PVHant [START_REF] Lutz | Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus[END_REF], suggesting that the opposing effect we observed might not be due to the immunolabeling of POMC (a precursor). Another possibility is that our POMC results could reflect an effect of the use of tamoxifen per se. Indeed, we showed that the neonatal administration of tamoxifen to wildtype pups decreased the amount of POMC neurons in the ARH. Furthermore, as previously discussed, neonatal androgen exposure decreases hypothalamic POMC expression, POMC neuronal count and POMC axons within the ARH, without affecting the AgRP neuronal population [START_REF] Nohara | Early-Life Exposure to Testosterone Programs the Hypothalamic Melanocortin System[END_REF]. These data suggest that sex hormones preferentially influence the POMC neuronal circuit during the neonatal period. To overcome the problems encountered by the use of tamoxifen, we plan to repeat AgRP and POMC brain immunolabeling in mice with a constitutive induction of IGN (Vily-Petit et al. 2020). These mice already overexpress IGN at birth and do not require tamoxifen administration.

Furthermore, to determine whether neonatal IGN is a requisite for the development of ARH AgRP and POMC feeding circuits, we could perform the same experiments on mice with a conditional deletion of IGN, a murine model that has already been developed by our laboratory.

Since IGN seems to exert a neurodevelopmental role, it would be pertinent to determine whether the neonatal peak of IGN is regulated physiologically or nutritionally. As our laboratory demonstrated that the digestion of dietary protein stimulates IGN (Duraffourd et al. 2012), we speculate that the neonatal IGN peak might be regulated by the rich protein content of milk. To test whether the neonatal peak of IGN is nutritionally regulated, we could start by measuring IGN during the neonatal period in fasted pups. If the IGN peak still takes place, this would suggest that it is not nutritionally regulated.

Furthermore, it could be interesting to study whether the neonatal induction of IGN could restore the insufficient development of ARH projections observed in offspring of undernourished dams [START_REF] Delahaye | Maternal Perinatal Undernutrition Drastically Reduces Postnatal Leptin Surge and Affects the Development of Arcuate Nucleus Proopiomelanocortin Neurons in Neonatal Male Rat Pups[END_REF][START_REF] Yura | Role of Premature Leptin Surge in Obesity Resulting from Intrauterine Undernutrition[END_REF]. A same rationale can also be applied to the soluble fibers contained in breast milk. Indeed, mother milk contains fibers that can be modulated by food [START_REF] Bourgot | Maternal Short-Chain Fructooligosaccharide Supplementation Influences Intestinal Immune System Maturation in Piglets[END_REF][START_REF] Bouret | Development of Hypothalamic Circuits That Control Food Intake and Energy Balance[END_REF][START_REF] Oozeer | Intestinal Microbiology in Early Life: Specific Prebiotics Can Have Similar Functionalities as Human-Milk Oligosaccharides[END_REF]). These fibers could thus also be used by the neonates to provide substrates for the neonatal surge of IGN.

Determining the molecular mechanism underlying the neurodevelopmental effect of neonatal IGN

Our results showed that hypothalamic STAT3 phosphorylation levels at P9 in I overexp G6pc P1 were equivalent to those of control littermates. This was surprising for us as mouse pups with a constitutive deletion of IGN have diminished hypothalamic STAT3 phosphorylation (unpublished data), suggesting that IGN does stimulate hypothalamic STAT3 phosphorylation during the neonatal period. Nevertheless, a limitation of this experiment is that we measured STAT3 phosphorylation in whole hypothalami, which could fail to highlight slight variations of STAT3 phosphorylation specifically in the ARH.

Therefore, we plan to conduct STAT3 phosphorylation immunolabeling in brain slices of I overexp G6pc P1 pups and control littermates at P9 and P15 (coinciding with the time of ARH axonal outgrowth) (Bouret 2004a). Another potential candidate mediating the effect of IGN on ARH neurodevelopment is the calcitonin gene-related peptide (CGRP). Indeed, CGRP is needed for the decrease of food intake induced by IGN [START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF], and has been shown

to bind the ARH [START_REF] Sexton | Localization of Binding Sites for Calcitonin Gene-Related Peptide in Rat Brain by in Vitro Autoradiography[END_REF]). RAMP1 and RAMP3, which are components of one of the receptors of CGRP (AMY1) [START_REF] Hay | Update on the Pharmacology of Calcitonin/CGRP Family of Peptides: IUPHAR Review 25: Pharmacology of the Calcitonin/CGRP Family[END_REF], have been shown to mediate the neurotrophic role of amylin in ARH axonal outgrowth [START_REF] Lutz | Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus[END_REF]. Therefore, CGRP could bind to the same receptor and induce the same intracellular pathway than amylin and also exert a trophic role on ARH neurons [START_REF] Lutz | Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus[END_REF]

Main results

In this work, we also showed that inducing IGN specifically during the first two postnatal weeks protected mice from developing increased adiposity, increased circulating leptin levels and a diminished amount of hypothalamic STAT3 phosphorylation when fed a high-fat/high-sucrose diet during adulthood. All of these parameters are hallmarks of obesity and leptin resistance [START_REF] Bouhajja | Affinity Kinetics of Leptin-Reactive Immunoglobulins Are Associated with Plasma Leptin and Markers of Obesity and Diabetes[END_REF][START_REF] Izquierdo | Leptin, Obesity, and Leptin Resistance: Where Are We 25 Years Later?[END_REF][START_REF] Jung | Molecular Mechanisms of Central Leptin Resistance in Obesity[END_REF][START_REF] Kettaneh | High Plasma Leptin Predicts an Increase in Subcutaneous Adiposity in Children and Adults[END_REF]. Therefore, we propose that inducing IGN during the neonatal period could be beneficial for long-term metabolic health.

Potential mechanisms explaining the long-term beneficial metabolic effects of neonatal IGN

An excessive development of ARH axonal projections has been associated with metabolic problems during adulthood [START_REF] Chang | Maternal High-Fat Diet and Fetal Programming: Increased Proliferation of Hypothalamic Peptide-Producing Neurons That Increase Risk for Overeating and Obesity[END_REF][START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF]. Therefore, the protective metabolic effects observed in adult I overexp G6pc P1 mice could not be directly and solely correlated to the increase of AgRP projections into the PVHant. One possible explanation for the protective metabolic effects observed in I overexp G6pc P1 mice could be an improvement of hypothalamic leptin signaling throughout life. In this thesis, I showed that the hypothalamic STAT3 phosphorylation/circulating leptin ratio is increased both during the neonatal period and during adulthood in I overexp G6pc P1 mice. We plan to reinforce these data and further analyze leptin sensitivity in I overexp G6pc P1 mice in two ways. First, during the neonatal period, 4-hour fasted I overexp G6pc P1 and control pups will receive an intra-peritoneal (IP) injection of leptin, and their hypothalami will be collected after 30 minutes to quantify STAT3 phosphorylation. If leptin-induced STAT3 phosphorylation increases in hypothalami of I overexp G6pc P1 pups in comparison to control littermates, this would reflect higher hypothalamic leptin sensitivity. Second, during adulthood, we will measure food intake and hypothalamic STAT3 phosphorylation in I overexp G6pc P1 and control mice having received an IP injection of leptin. Having an enhanced hypothalamic leptin sensitivity could indeed protect mice from developing excessive diet-induced obesity in the long-term (de Git et al.

2018).

On another note, the fact that IGN protected long-term adiposity in mice only when it was induced during the first two weeks of life raises the possibility that neonatal IGN could have beneficially programmed other tissues that pursue their maturation during the neonatal period, such as the adipose tissue [START_REF] Han | The Spatiotemporal Development of Adipose Tissue[END_REF] [START_REF] Agnoux | Perinatal Protein Restriction Affects Milk Free Amino Acid and Fatty Acid Profile in Lactating Rats: Potential Role on Pup Growth and Metabolic Status[END_REF], which is the major precursor of IGN [START_REF] Croset | Rat Small Intestine Is an Insulin-Sensitive Gluconeogenic Organ[END_REF]. Therefore, it could be interesting to study the potential relationship between perinatal protein intake, neonatal IGN, and the neonatal development of adipose tissue.

Given our data on the metabolism of mice with an induction of IGN since P12, we could question whether the induction of IGN per se (independently of nutrients) in adults is actually beneficial. Indeed, my results showed that inducing IGN in adults (at least since P12)

does not exert any beneficial effects on metabolism (among the parameters analyzed).

However, we previously demonstrated that the induction of IGN by a diet rich in proteins or soluble fibers is a causal link in their metabolic benefits [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF][START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF]Duraffourd et al. 2012;[START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF][START_REF] Penhoat | Protein-Induced Satiety Is Abolished in the Absence of Intestinal Gluconeogenesis[END_REF]. We think that the explanation for this dissonance could be the supply of substrates for IGN. Indeed, proteinand soluble fiber-enriched diets supply the substrates necessary to induce IGN, in addition to directly or indirectly stimulating the expression of gut gluconeogenic enzymes. Altogether, our results strongly suggest that, in adult mice, both G6Pase induction and substrate availability are important for obtaining a strong induction of the portal glucose signal, leading to beneficial effects on metabolism (for example, increased satiety and improved glucose tolerance). However, inducing intestinal G6Pase per se in the first two weeks of life is sufficient to promote a healthy metabolism in the adult, whatever the diet.

Altogether, the second part of this thesis suggests that IGN exerts a neurodevelopmental role during the early postnatal period, and that its induction in this specific time-window could be beneficial for long-term adiposity when fed a high-fat/highsucrose diet. Therefore, this thesis opens a door for exploring the potential therapeutic role of inducing IGN during the early postnatal period in cases of perinatal malnutrition that could lead to the adult-onset of metabolic complications like obesity.

Conclusion

This thesis clearly demonstrates the pivotal and therefore decisive role of the early postnatal period on life-long health of mice. On the one hand, our work emphasizes the vulnerability of the neonatal period because injecting a single dose of 20 µg of tamoxifen on postnatal day 1 strikingly altered life-long female metabolism and male bone weight.

However, on the other hand, our work also features a new developmental cue: IGN. Our laboratory has extensively reported the beneficial effects exerted by IGN in adult mice metabolism, but this is the first time we study whether IGN could modulate the neonatal development of mice. Given the presence of IGN in humans [START_REF] Battezzati | Nonhepatic Glucose Production in Humans[END_REF][START_REF] Hayes | Is Intestinal Gluconeogenesis a Key Factor in the Early Changes in Glucose Homeostasis Following Gastric Bypass?[END_REF][START_REF] Mithieux | Comment about Intestinal Gluconeogenesis after Gastric Bypass in Human in Relation with the Paper by Hayes et al[END_REF], our present work opens a door to exploring IGN as a therapeutic strategy in cases of perinatal malnutrition. Together with leptin, ghrelin and amylin, IGN is also a neurotrophic signal in the neonatal period, essential for a proper development of the hypothalamic circuitry, and as a metabolic signal in adulthood, involved in the regulation of food intake and energy homeostasis.

Homéostasie du glucose

L'homéostasie est définie comme un état d'équilibre relativement stable.

L'homéostasie du glucose, ou contrôle de la glycémie, est un besoin physiologique qui maintient la glycémie à environ 0,9-1 g/L, lorsqu'elle fonctionne de manière optimale.

L'apport nutritionnel en glucose varie tout au long de la vie d'un animal, allant d'un apport alimentaire excessif ou normal au jeûne et à la famine. Divers processus métaboliques permettant de s'adapter à ces différentes circonstances ont ainsi évolué. Pendant la période post-prandiale (c'est-à-dire, juste après un repas), l'afflux de substrats énergétiques conduit à une augmentation de la glycémie et à l'activation des voies métaboliques qui stockent le glucose sous forme de glycogène et de lipides. Au contraire, lorsque la nourriture se fait rare (comme pendant le jeûne), la glycémie est maintenue à 0,9-1 g/L en raison de l'activation des voies métaboliques impliquées dans la production endogène de glucose: la dégradation des réserves de glycogène hépatique (c'est-à-dire la glycogénolyse) et la production de novo de glucose (c'est-à-dire la néoglucogenèse) dans le foie [START_REF] Bernard | Acute Hyperinsulinemia and Its Reversal by Vagotomy after Lesions of the Ventromedial Hypothalamus in Anesthetized Rats*[END_REF], les reins et l'intestin [START_REF] Croset | Rat Small Intestine Is an Insulin-Sensitive Gluconeogenic Organ[END_REF][START_REF] Rajas | The Glucose-6 Phosphatase Gene Is Expressed in Human and Rat Small Intestine: Regulation of Expression in Fasted and Diabetic Rats[END_REF].

Pendant la période post-prandiale, la glycogenèse (la synthèse du glycogène) et la lipogenèse (la synthèse de novo des acides gras et des triglycérides) ont lieu. Les sites principaux des réserves de glycogène d'un organisme se trouvent dans le foie et dans le muscle squelettique. Les réserves de glycogène présentes dans le muscle squelettique ne fournissent du glucose que pour une utilisation locale car le muscle n'a pas l'enzyme obligatoire pour la production endogène de glucose, la glucose-6-phosphatase. Les réserves de glycogène dans le foie sont, en revanche, plus pertinentes quantitativement pour l'homéostasie du glucose car elles fournissent du glucose aux tissus qui en ont besoin grâce au processus de glycogénolyse pendant la période post-absorptive. La lipogenèse est principalement régulée par l'insuline, le glucose (qui l'induisent) et le glucagon (qui l'inhibe).

Les deux hormones régulent la lipogenèse en agissant aux niveaux transcriptionnel et posttraductionnel.

Entre deux repas, lorsque la nourriture ingérée a déjà été digérée, absorbée et stockée (moment défini comme la période post-absorptive, ou le début de la période de jeûne) l'organisme n'a plus d'apport exogène en glucose. Dans ce cas, la glycémie est maintenue par la production endogène de glucose (PEG) (Figure 3A). La PEG provient de la dégradation du glycogène hépatique (c'est-à-dire de la glycogénolyse) et de la production de glucose de novo à partir de substrats carbonés non glucidiques (c'est-à-dire la néoglucogenèse). Une enzyme clé, la glucose-6-phosphatase, catalyse la dernière étape de la glycogénolyse et de la néoglucogenèse (figure 3B).

Le glycogène hépatique est une ressource énergétique facilement disponible pour maintenir la glycémie dans un organisme. Cependant, à mesure que la période postabsorptive se poursuit et que le jeune se prolonge, les réserves de glycogène risquent de s'épuiser complètement. Dans ce scénario, la voie de la néoglucogenèse est fortement stimulée dans les trois tissus néoglucogéniques pour maintenir la glycémie à 0,9-1 g/L. La sous-unité catalytique de la glucose-6-phosphatase (codée par le gène G6pc) n'est présente que dans le foie, les reins et l'intestin [START_REF] Rajas | The Glucose-6 Phosphatase Gene Is Expressed in Human and Rat Small Intestine: Regulation of Expression in Fasted and Diabetic Rats[END_REF], ce qui en fait les seuls organes néoglucogéniques. Ces trois organes sont nécessaires au contrôle glycémique, mais chacun exerce un rôle différent et complémentaire dans l'homéostasie du glucose. En ce sens, le passage de la période post-absorptive à un jeûne prolongé se caractérise par une redistribution de la production de glucose entre la néoglucogenèse hépatique, rénale et intestinale (Soty et al. 2017).

Le rôle de la néoglucogenèse intestinale est particulièrement intéressant car cette fonction physiologique ne se limite pas au maintien de la glycémie lors d'un jeûne prolongé.

La néoglucogenèse intestinale peut être progressivement stimulée pendant la période postprandiale par des nutriments spécifiques, conduisant à une persistance de la production de glucose dans la veine porte pendant la période post-absorptive [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF][START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF]Duraffourd et al. 2012;[START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF]. Étant donné que l'intestin est situé en amont du système nerveux portal, cette production a la capacité de déclencher un signal intestincerveau. Ce signal initie un circuit neuronal qui joue un rôle majeur dans le maintien de la santé métabolique, qui sera décrit au chapitre 3 (Soty et al. 2017). principal producteur de ghréline est la cellule oxyntique gastrique. L'effet orexigénique de la ghréline est principalement transmis par le récepteur du sécrétagogue de l'hormone de croissance de type 1a (GHS-R type 1a). La ghréline induit la prise alimentaire en partie en liant le GHS-R de type 1a qui est présent dans les neurones AgRP/NPY de l'ARH [START_REF] Chen | Ghrelin Receptors Mediate Ghrelin-Induced Excitation of Agouti-Related Protein/Neuropeptide Y but Not pro-Opiomelanocortin Neurons[END_REF]).

La néoglucogenèse intestinale régule avantageusement la santé métabolique via un circuit intestin-cerveau

La néoglucogenèse intestinale entraîne la libération de glucose dans la veine porte déclenchant un circuit neuronal intestin-cerveau crucial pour la santé métabolique. Ce glucose est détecté par des senseurs SGLT3 présents dans la veine porte [START_REF] Delaere | The Role of Sodium-Coupled Glucose Co-Transporter 3 in the Satiety Effect of Portal Glucose Sensing[END_REF], qui cible l'hypothalamus via des afférents splanchniques [START_REF] Delaere | The Role of Sodium-Coupled Glucose Co-Transporter 3 in the Satiety Effect of Portal Glucose Sensing[END_REF]. Le glucose portal stimule la voie de signalisation STAT3 dans l'ARH de l'hypothalamus [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF], via un mécanisme dépendant du CGRP [START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]. La néoglucogenèse intestinale peut déclencher le signal glucose portal pendant la période post-absorptive dans certaines conditions, par exemple lorsqu'elle est induite par des nutriments spécifiques, comme une alimentation riche en protéines ou en fibres solubles (Figure 15), et pendant la période néonatale.

Les régimes enrichis en protéines ou en fibres solubles sont connus pour diminuer la faim et la prise alimentaire, et améliorer la santé métabolique. La digestion des protéines et des fibres pendant la période post-prandiale initie un signal intestin-cerveau, qui aboutit à l'induction des enzymes néoglucogéniques intestinales. En conséquence, l'activité néoglucogénique intestinale est pleinement augmentée et persiste pendant la période post-absorptive (Duraffourd et al. 2012;[START_REF] Mithieux | Portal Sensing of Intestinal Gluconeogenesis Is a Mechanistic Link in the Diminution of Food Intake Induced by Diet Protein[END_REF]. Notre laboratoire a montré que la néoglucogenèse intestinale est le lien mécanistique obligatoire des bénéfices métaboliques induits par les régimes enrichis en protéines ou en fibres solubles (Figure 15) [START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF], 2015[START_REF] Furness | Integrated Neural and Endocrine Control of Gastrointestinal Function[END_REF]Duraffourd et al. 2012).

Pendant longtemps, la communauté scientifique croyait que la néoglucogenèse néonatale n'était assurée que par le foie et les reins [START_REF] Girard | Adaptations of Glucose and Fatty Acid Metabolism during Perinatal Period and Suckling-Weaning Transition[END_REF]). Chatelain et ses collègues ont par la suite rapporté pour la première fois que la néoglucogenèse intestinale est aussi fortement induite pendant la période néonatale [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]. Plus précisément, les taux d'ARNm et de l'activité de la glucose-6-phosphatase atteignent un pic au jour 5 post-natal (P5) [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]. Le pic de néoglucogenèse intestinale survenant pendant la période néonatale chez le rat [START_REF] Chatelain | Development and Regulation of Glucose-6-Phosphatase Gene Expression in Rat Liver, Intestine, and Kidney: In Vivo and in Vitro Studies in Cultured Fetal Hepatocytes[END_REF]) a également été signalé chez le porc [START_REF] Darcy-Vrillon | Glucose, Galactose, and Glutamine Metabolism in Pig Isolated Enterocytes during Development[END_REF], la souris [START_REF] Ménard | Ultrastructural Localization of Intestinal Glucose-6-Phosphatase Activity during the Postnatal Development of the Mouse[END_REF][START_REF] Menard | Glucose-6-Phosphatase Activity in Mouse Small Intestine during Postnatal Development[END_REF] et des cobayes [START_REF] Anderson | Gluconeogenesis in Jejunal Mucosa of Guinea Pig[END_REF]) (Figure 16).

Le développement de modèles murins originaux dont le gène G6pc est spécifiquement invalidé ou surexprimé dans l'intestin a permis d'étudier le rôle de la néoglucogenèse intestinale indépendamment de la nutrition. Des souris dépourvues de néoglucogenèse intestinale (I.G6pc -/-) ont été obtenues via la stratégie de biotechnologie CRE/lox inductible par le tamoxifène [START_REF] Penhoat | Protein-Induced Satiety Is Abolished in the Absence of Intestinal Gluconeogenesis[END_REF]. Les souris I.G6pc -/-développent des altérations métaboliques sévères, telles qu'une intolérance au glucose et à l'insuline, une stéatose hépatique, une résistance à la leptine hypothalamique, et développent plus rapidement un phénotype de diabète de type 2 sous un régime riche en graisses et en saccharose [START_REF] Soty | A Gut-Brain Neural Circuit Controlled by Intestinal Gluconeogenesis Is Crucial in Metabolic Health[END_REF][START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]. Des souris surexprimant la néoglucogenèse intestinale (I. atteignant un pic entre P8 et P12 [START_REF] Ahima | Postnatal Leptin Surge and Regulation of Circadian Rhythm of Leptin by Feeding. Implications for Energy Homeostasis and Neuroendocrine Function[END_REF][START_REF] Devaskar | Developmental Changes in Ob Gene Expression and Circulating Leptin Peptide Concentrations[END_REF]. Ce pic exerce un effet neurotrophique sur les projections à partir de l'ARH des neurones POMC et AgRP via la phosphorylation de STAT3 et d'ERK (Bouret 2004b;[START_REF] Bouret | Distinct Roles for Specific Leptin Receptor Signals in the Development of Hypothalamic Feeding Circuits[END_REF]. Le rôle neurotrophique de la leptine est limité à la période néonatale (Bouret 2004b). Comme pour la leptine, les taux plasmatiques de ghréline chez la souris sont élevés pendant la période postnatale. Cependant, les niveaux plasmatiques de ghréline augmentent autour de P10 et atteignent les niveaux adultes vers P14 [START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF], période pendant laquelle les niveaux plasmatiques de leptine diminuent [START_REF] Ahima | Postnatal Leptin Surge and Regulation of Circadian Rhythm of Leptin by Feeding. Implications for Energy Homeostasis and Neuroendocrine Function[END_REF]. La ghréline stabilise la croissance axonale de l'ARH de la mi-vie postnatale au sevrage en interférant avec la signalisation de la leptine sur STAT3 [START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF].

Programmation développementale du syndrome métabolique

La prévalence croissante du diabète et de l'obésité dans le monde est alarmante et représente un défi majeur pour la santé publique. Des données épidémiologiques, des études cliniques et expérimentales montrent une association entre l'exposition périnatale à des stress nutritionnels et le développement de troubles métaboliques chez l'adulte [START_REF] Cottrell | Developmental Programming of Energy Balance and the Metabolic Syndrome[END_REF].

Les stress périnataux les plus rapportés et étudiés sont des déséquilibres nutritionnels, qui peuvent affecter le milieu hormonal périnatal de la progéniture.

L'exposition périnatale à la dénutrition ou à la suralimentation affecte la signalisation de la leptine et de la ghréline chez le souriceau, soit en modifiant les taux sanguins hormonaux, soit en modifiant la sensibilité centrale à ces hormones. D'une part, la dénutrition périnatale peut modifier le taux plasmatique néonatal de leptine, altérant ainsi les circuits hypothalamiques AgRP/NPY et POMC/CART et provoquant des troubles métaboliques à l'âge adulte [START_REF] Delahaye | Maternal Perinatal Undernutrition Drastically Reduces Postnatal Leptin Surge and Affects the Development of Arcuate Nucleus Proopiomelanocortin Neurons in Neonatal Male Rat Pups[END_REF][START_REF] Yura | Role of Premature Leptin Surge in Obesity Resulting from Intrauterine Undernutrition[END_REF]. D'autre part, la suralimentation périnatale peut également modifier les niveaux néonataux de leptine et de ghréline, et altérer les circuits hypothalamiques AgRP/NPY et POMC/CART [START_REF] Bouret | Hypothalamic Neural Projections Are Permanently Disrupted in Diet-Induced Obese Rats[END_REF][START_REF] Chen | Maternal and Postnatal Overnutrition Differentially Impact Appetite Regulators and Fuel Metabolism[END_REF][START_REF] Vogt | Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding[END_REF]. Le développement périnatal est donc une période cruciale de la vie dont dépend la santé métabolique d'un individu.

Mots clés :

 clés Néoglucogenèse intestinale, diabète, obesité, développement néonatal, hypothalamus. LIST OF ABBREVIATIONS ............................................................................................................................... 3.1. Impaired hypothalamic development ................................................................................ 104 3.2. Impaired development of other organs ................................................................................. 107 Pancreas ............................................................................................................................................. 107 The gastrointestinal tract ............................................................................................................ 108 Adipose tissue .................................................................................................................................. 109 AIM OF STUDY ................................................................................................................................................... 110 RESULTS .............................................................................................................................................................. 113 Tamoxifen treatment in the neonatal period affects glucose homeostasis in adult mice in a sex-dependent manner.............................................................................................................................. 114 Neonatal intestinal gluconeogenesis affects the development of hypothalamic feeding circuits and adult body composition .................................................................................................... 139 GENERAL DISCUSSION .................................................................................................................................. 170 1. Neonatal tamoxifen administration: highlighting the crucial role of sex hormones during the neonatal period on adult energy homeostasis? .................................................... 171 Main results ........................................................................................................................................... 171 The testosterone surge and sexual differentiation ................................................................ 171 2. Neonatal intestinal gluconeogenesis .......................................................................................... 174 2.1. Does intestinal gluconeogenesis (IGN) control the postnatal development of hypothalamic AgRP and POMC circuits? .................................................................................... 174 Main results ...................................................................................................................................... 174 An increase of AgRP and a decrease of POMC projections into the PVHant ............ 175 Determining the molecular mechanism underlying the neurodevelopmental effect of neonatal IGN ................................................................................................................................ 176 2.2. Is the effect of IGN on hypothalamic neuronal circuits associated with a long-term effect on mice metabolism? ............................................................................................................. 177 Main results ...................................................................................................................................... 177 Potential mechanisms explaining the long-term beneficial metabolic effects of neonatal IGN ..................................................................................................................................... 3. Conclusion ............................................................................................................................................. SCIENTIFIC COMMUNICATIONS ................................................................................................................ Résumé en français ......................................................................................................................................... REFERENCES .....................................................................................................................................................

Figure 1 .

 1 Figure 1. Glycogenesis. .....................................................................................................................................

Figure 2 .

 2 Figure 2. Metabolic pathways leading to triglyceride synthesis in the liver. .............................

Figure 3 .

 3 Figure 3. Endogenous glucose production and the glucose-6-phosphatase complex. ............

Figure 4 .

 4 Figure 4. Re-distribution of glucose production among the three gluconeogenic organs in various nutritional situations. .......................................................................................................................

Figure 5 .

 5 Figure 5. Gluconeogenic pathways in mammals. ...................................................................................

Figure 6 .

 6 Figure 6. A simplified schematic model of hypothalamic neuronal circuits regulating food intake. .....................................................................................................................................................................

Figure 7 .

 7 Figure 7. The median eminence. ..................................................................................................................

Figure 8 .

 8 Figure 8. Schematic model of the cyto-and chemoarchitecture of the mouse paraventricular nucleus of the hypothalamus (PVH). ..........................................................................................................

Figure 9 .

 9 Figure 9. Hormonal regulation of food intake and energy expenditure. ......................................

Figure 10 .

 10 Figure 10. Leptin signaling through LepRb. ............................................................................................

Figure 11 .

 11 Figure 11. Afferent innervation of the hepato-portal region. ...........................................................

Figure 12 .

 12 Figure12. Innervation of the gastro-intestinal tract by the autonomous nervous system. .

Figure 13 .

 13 Figure 13. Anatomy of the enteric nervous system. .............................................................................

Figure 14 .

 14 Figure 14. Schematic model of the neural circuit initiated by intestinal gluconeogenesis. ..

Figure 15 .

 15 Figure 15. Time course of the nutritional induction of intestinal gluconeogenesis.................

Figure 16 .

 16 Figure 16. Developmental changes in glucose-6-phosphatase (the mandatory enzyme of gluconeogenesis) mRNA levels in the liver, the kidney and the intestine. ..................................

Figure 17 .

 17 Figure 17. I.G6pc-/-mice. ...............................................................................................................................

Figure 18 .

 18 Figure 18. I.G6pcoverexp mice. ....................................................................................................................

Figure 19 .

 19 Figure 19. Neural tube formation during primary neurulation. ......................................................

Figure 20 .

 20 Figure 20. Major derivatives of neural crest subpopulations along the anterioposterior axis. ...................................................................................................................................................................................

Figure 21 .

 21 Figure 21. Development of the liver venous drainage. ........................................................................

Figure 22 .Figure 24 .

 2224 Figure 22. Hypothalamic development. ....................................................................................................Figure 23. Global trends of diabetes. From the IDF Diabetes Atlas, 9th edition, 2019. ........... 102

  will be substantially stimulated. Excess glucose molecules in the cytosol are transformed into pyruvate molecules through the glycolysis pathway. Pyruvate molecules then enter the mitochondria where they are transformed into Acetyl-CoA and then into Citrate by the Krebs cycle. Then, Citrate molecules are transported from the mitochondria back to the cytosol, where the ATP citrate lyase enzyme transforms it again into Acetyl-CoA, which is the substrate used to start the lipogenic pathway. In the cytosol, the major enzyme of lipogenesis, the Acetyl-CoA Carboxylase (ACC) carboxylates Acetyl-CoA, thereby forming Malonyl-CoA, which can be then transformed into a longer-chain fatty acid, Palmitate, by another major lipogenic enzyme, the Fatty Acid Synthase

Figure 2 .

 2 Figure 2. Metabolic pathways leading to triglyceride synthesis in the liver. Increased glucose levels induce Glucokinase (GK) and liver Pyruvate kinase (L-PK) for glycolysis; ATP citrate lyase, Acetyl-CoA carboxylase (ACC), and Fatty acid synthase (FAS) for lipogenesis; Fatty acid elongase 6 (ELOVL6) and Stearoyl-Coa desaturase-1 (SCD1) for fatty acid elongation and desaturation steps; and lastly Glycerol-3-phosphate acyltransferase (GPAT) and Diacylglycerol acyltransferase (DGAT) for triglyceride (TG) synthesis. F6P: fructose 6-phosphate; F1,6P2: fructose 1,6 diphosphate; G6P: glucose 6phosphate; PEP: phosphoenol pyruvate. Illustration made with Servier Medical Art.

Figure 4 .

 4 Figure 4. Re-distribution of glucose production among the three gluconeogenic organs in various nutritional situations.When animals are fed a standard starch-based diet, glucose production from the kidney represents about 15% to 20% of the endogenous glucose production (EGP) at the beginning of the post-absorptive period. Kidney glucose production increases to about 50% at 24 hours of fasting[START_REF] Pillot | Protein Feeding Promotes Redistribution of Endogenous Glucose Production to the Kidney and Potentiates Its Suppression by Insulin[END_REF]). Intestinal glucose production accounts for about 5% to 7% of EGP at the beginning of the post-absorptive period and increases to about 20 to 25% at 24 hours of fasting in rats[START_REF] Croset | Rat Small Intestine Is an Insulin-Sensitive Gluconeogenic Organ[END_REF][START_REF] Mithieux | Contribution of Intestine and Kidney to Glucose Fluxes in Different Nutritional States in Rat[END_REF]. Comparable augmentations of the contributions of the intestine and the kidney take place upon eating a protein-enriched diet, a situation in which the intestine accounts for about 20% and the kidney about 45% of EGP at the beginning of the post-absorptive period[START_REF] Pillot | Protein Feeding Promotes Redistribution of Endogenous Glucose Production to the Kidney and Potentiates Its Suppression by Insulin[END_REF]. A comparable contribution of the intestine to about 20% of EGP was measured upon a fiber-enriched diet[START_REF] Vadder | Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits[END_REF]. Kidney glucose production was not determined in this situation.(From Soty et al., 2017).

Figure 5 .

 5 Figure 5. Gluconeogenic pathways in mammals.Gluconeogenic substrates (glycerol, alanine, lactate, glutamine and propionate, in blue boxes) enter the gluconeogenesis pathway. Enzymes specific to gluconeogenesis are written in blue. Oxaloacetate is transported to the cytosol through the malate/aspartate shuttle. For comparative purposes, enzymes specific to glycolysis are written in yellow.

Figure 6 .

 6 Figure 6. A simplified schematic model of hypothalamic neuronal circuits regulating food intake. Full legend in next page.

Figure

  Figure6. A simplified schematic model of hypothalamic neuronal circuits regulating food intake. On the left side: non exhaustive illustration of the neuronal populations regulating food intake in each hypothalamic nucleus. On the right side: non exhaustive illustration of the receptors to peripheral signals expressed in each hypothalamic nucleus. Hypothalamic nuclei involved in the regulation of food intake communicate with each other, forming neuronal circuits. Among these nuclei, the arcuate nucleus (ARH) plays a central role. Indeed, it has both orexigenic AgRP/NPY and anorexigenic POMC/CART neurons, and it expresses receptors to many peripheral signals. The ARH sends axonal projections to all other nuclei regulating food intake. Importantly, the ARH is located near the median eminence (ME), a circumventricular organ with tanycytes lining the wall of the third ventricle (3V) and which projections are in contact with fenestrated capillaries that facilitate blood/brain exchanges. ARH: arcuate nucleus; VMH: ventromedial nucleus; DMH: dorsomedial nucleus; LH: lateral hypothalamic area; PVH: paraventricular nucleus; AgRP: agouti-related peptide; NPY: neuropeptide Y; POMC: pro-opiomelanocortin; CART: Cocaine and Amphetamine Regulated Transcript; MCH: Melanin-Concentrating Hormone; SF1: steroidogenic factor 1; BDNF: brain-derived neurotrophic factor; CRH: corticotrophin-releasing hormone; TRH: thyrotropin-releasing hormone; InsR: insulin receptor; LepRb: leptin receptor; GHSH: ghrelin receptor; GLP1R1: GLP-1 receptor; ApidoR1: adiponectin receptor; CCK1: CCK receptor; GLP-1: glucagon-like peptide. Adapted from the doctorate thesis manuscript of Dr. Fanny Langlet. Illustration partly made with Servier Medical Art

Figure 7 .

 7 Figure 7. The median eminence.(1) The structure of the median eminence is mainly made of specialized glial cells called tanycytes, which cell bodies line the wall of the third ventricle, and their projections are in contact with fenestrated capillaries from the hypophyseal portal blood system. (2) Cytoarchitecture of the rat mediobasal hypothalamus. Distribution of tanycytes (red, labeled for vimentin), blood vessels (white, labeled by the panendothelial antibody clone RECA-1), neurons (white, labeled for the neuronal marker HuC/D), astrocytes (green, labeled for GFAP), and all cell nuclei are stained blue with Hoechst. Frames in (A) and (B) are shown with all color channels in (C) and (D), respectively. Scale bar: (A) 100 µm. 3V: third ventricle; DMH: dorsomedial nucleus of the hypothalamus; VMH: ventromedial nucleus of the hypothalamus; GFAP: glial fibrillary acidic protein. (Panel 2 from Prevot et al., 2018).

Figure 8 .

 8 Figure 8. Schematic model of the cyto-and chemoarchitecture of the mouse paraventricular nucleus of the hypothalamus (PVH).The anterior PVH (on the left side) contains the majority of magnocellular neuroendocrine neurons that project to the neurohypophysis (also known as posterior pituitary, PP) and parvicellular neuroendocrine neurons that project to the median eminence for further transport to the adenohypophysis (also known as anterior pituitary, AP). The posterior PVH (on the right side) contains most of the descending preautonomic neurons. * indicates the anterior PVHpv, which along with the posterior PVHpv, sends projections to the adenohypophysis. The line was not drawn to avoid confusion. pv: periventricular part; am: anterior magnocellular part; mm: medial magnocellular part; pml: posterior magnocellular part, lateral zone; mpd: medial parvicellular part, dorsal zone; pmm: posterior magnocellular part, medial zone; dp: descending division, dorsal parvicellular part; mpv: medial parvicellular part, ventral zone; lp: descending division, lateral parvicellular part; OXY: oxytocin; VAS: vasopressin; CRH: corticotrophin-releasing hormone; TRH: thyrotropinreleasing hormone; SS: somatostatine; DMX: dorsal motor nucleus of the vagus nerve; CGS: central gray, spinal cord; IML: intermediolateral column, spinal cord.(From Biag et al. 2012).

Figure 9 .

 9 Figure 9. Hormonal regulation of food intake and energy expenditure.Non exhaustive list of hormones secreted by the adipose tissue and the gastrointestinal tract. These hormones regulate food intake by targeting several areas of the nervous system.

Figure 10 .

 10 Figure 10. Leptin signaling through LepRb. Description of the signaling pathways are in the main text.

Figure 11 .

 11 Figure 11. Afferent innervation of the hepato-portal region.Spinal afferent innervation is shown in red broken lines. Spinal afferents are connected to the dorsal root ganglia (DRG) at thoracic levels Th7 through Th13. Thickness of lines represents the relative abundance of innervations, and the diameter of dots represents sensory terminals in the various compartments. Sensory information is then relayed to the parabrachial nucleus (PBN), and to a lesser extent, to the nucleus of the tractus solitarius (NTS) in the dorsal vagal complex (DVC). dmnX: dorsal motor nucleus of the vagus; Amy: amygdala; PVN: paraventricular nucleus of the hypothalamus; LHA: lateral hypothalamic area(From Berthoud, 2004).

Figure 12 .

 12 Figure 12. Innervation of the gastro-intestinal tract by the autonomous nervous system.The sympathetic nervous sytem is illustrated in green lines, and the parasympathetic nervous system in red lines. Adapted from the doctorate thesis manuscript of Dr. Filipede Vadder.

Figure 13 .

 13 Figure13. Anatomy of the enteric nervous system. Location of the enteric nervous system represented by the myenteric plexus (also known as the Auerbach plexus), and the submucosa (SMP) plexus (also known as the Meissner plexus) (From Furness, 2012).

Figure 14 .

 14 Figure 14. Schematic model of the neural circuit initiated by intestinal gluconeogenesis.Intestinal gluconeogenesis leads to the production of glucose, which is released in the portal vein(1). This glucose is detected by SGLT3 sensors in the portal vein (2), and induces a nerve signal (the portal glucose signal) that subsequently activates brain regions responsible for regulating energy balance (the brainstem and the hypothalamus) and reward circuits(3). A central reflex arc then increases satiety, and leads to weight loss and improvements in blood sugar control and insulin sensitivity (4). For clarity, not all nerve connections have been shown in this illustration. PBN: parabrachial nucleus; DVC: dorsal vagal complex; SGLT3: glucose/sodium cotransporter 3. Adapted from the doctorate thesis manuscript of Dr. Flore Sinet.

Figure 15 .

 15 Figure 15. Time course of the nutritional induction of intestinal gluconeogenesis.Upper chain: during the postprandial period, peptides from dietary protein act as antagonists on the µ-opioid receptor (MOR) to signal the brain, which initiates the intestinal release of vasoactive intestinal peptide (VIP). The activation of the VIP receptor (VIPR) activates the adenylate cyclase and the subsequent production of cAMP, which then induces the expression of intestinal gluconeogenesis (IGN) genes in enterocytes. During the postabsorptive period, glutamate (Glu) and glutamine (Gln) (from digested protein or from the blood) are converted into glucose that signals the brain via SGLT3 sensors and portal glucose sensing. Lower chain: soluble fibers travel along the proximal intestine to reach the distal gut. They are then fermented by microbiota, producing the short-chain fatty acids propionate (Pro) and butyrate (But). On the one hand, propionate signals the brain by acting as agonist on the free fatty acid receptor 3 (FFAR3). The brain then induces IGN gene expression in both the proximal and the distal gut via neural VIP signaling. On the other hand, butyrate induces IGN gene expression in the distal gut by increasing cAMP. Propionate and succinate are then used as substrates of IGN, initiating portal glucose sensing. Later on (more slowly), insoluble fibers are fermented and may promote the same processes. These steps have been graphically distinguished for clarity, but many of them can overlap under physiological conditions of nutrition. PBN: parabrachial nucleus; NTS: nucleus of the solitary tract; PVN: paraventricular nucleus of the hypothalamus(Soty et al. 2017).

Figure 16 .

 16 Figure 16. Developmental changes in glucose-6-phosphatase (the mandatory enzyme of gluconeogenesis) mRNA levels in the liver, the kidney and the intestine. (A) Representative Northern blot of Glucose-6-Phosphatase mRNA levels in the liver, kidney and jejunum during the suckling period in rats. (B) Densitometric quantification of four different Northern blots. The results are expressed as percentage of Glucose-6-Phosphatase mRNA levels found in the liver of 48-hour starved adult rats. (From Chatelain et al., 1998).

Figure 17 .

 17 Figure 17. I.G6pc-/-mice.(A) Generation of transgenic mice lacking intestinal gluconeogenesis using the inducible CRE/lox system[START_REF] Penhoat | Protein-Induced Satiety Is Abolished in the Absence of Intestinal Gluconeogenesis[END_REF]. The exon 3 of the catalytic subunit of the glucose-6phosphatase (the G6pc gene) is flanked between two loxP sequences. The recombinase Cre is fused to a mutated version of the ligand-binding domain of the human estrogen receptor (ERT2), and placed under the control of the villin promoter (which expression is restricted to the intestinal epithelium), was used to achieve spatiotemporal control of gene inactivation. Following administration of tamoxifen (a selective estrogen receptor modulator), the Cre-ER T2 is able to enter the nucleus and induces the inactivation of the G6pc gene by excising the loxP sites, which leads to a truncated, non-functional version of the protein. (B) PCR analysis confirming the specific excision of the exon3 of the G6pc gene in the intestine, illustrated by the amplification of a 595 bp fragment in I-G6pc -/-mice. (C) Only a residual and non-specific intestinal glucose-6-phosphatase enzymatic activity is observed in I-G6pc -/-mice. Pr: promoter; WT: wild-type; bp: base pair; * p <0.05 Student's t test. From the doctorate thesis manuscript of Dr. FloreSinet.

  G6Pase enzymatic activity. Both parameters were unchanged in the liver and the kidneys[START_REF] Vily-Petit | Intestinal Gluconeogenesis Prevents Obesity-Linked Liver Steatosis and Non-Alcoholic Fatty Liver Disease[END_REF]) (Figure18).The metabolic phenotype: a protection from the onset of diabetes on a high-fat/highsucrose diet I.G6pc overexp mice are protected from developing obesity and diabetes when fed a high-fat/high-sucrose diet[START_REF] Vily-Petit | Intestinal Gluconeogenesis Prevents Obesity-Linked Liver Steatosis and Non-Alcoholic Fatty Liver Disease[END_REF]. When fed a high-fat/high-sucrose diet for 12 weeks, mice that overexpressed intestinal gluconeogenesis weighted less than wild-type mice and had better glucose tolerance, lower fasting glycaemia and insulinemia and an improved hepatic insulin sensitivity (Vily-Petit et al. 2020). I.G6pc overexp mice were also protected from developing hepatic steatosis after being fed a high-fat/high-sucrose diet for wild-type mice, which was associated with an inferior hepatic expression of genes implicated in the lipogenesis pathway and lipid uptake[START_REF] Vily-Petit | Intestinal Gluconeogenesis Prevents Obesity-Linked Liver Steatosis and Non-Alcoholic Fatty Liver Disease[END_REF]. Hepatic steatosis can deteriorate into non-alcoholic steatohepatitis (NASH), which is a condition in which the metabolism of the liver is immerse in inflammation and fibrosis[START_REF] Benedict | Non-Alcoholic Fatty Liver Disease: An Expanded Review[END_REF].

Figure 18 .

 18 Figure 18. I.G6pcoverexp mice. (A) Schematic representation of the generation of transgenic mice with a constitutive induction of the catalytic subunit of the glucose-6-phosphatase (the G6pc gene) specifically in the intestine, according to the CRE/lox biotechnology strategy. Briefly, a STOP cassette flanked between two loxP sequences and the complementary DNA of the G6pc gene were inserted in the Rosa26 locus, which exhibits a strong transcriptional activity but is blocked here by the stop cassette. The Cre recombinase is expressed under the villin promoter to induce an intestine-specific genetic modification. (B) Representative western blot analyses of the catalytic subunit (G6PC) of the glucose-6-phosphatase in the three gluconeogenesis organs: the liver, kidney and intestine. The graph shows the quantification of protein expression in I.G6pc overexp and wild-type (WT) mice. (C) Glucose-6-phosphatase (G6Pase) activity in the the liver, the kidney and the intestine, relative to protein concentration. (Vily-Petit et al. 2020).

(

  Figure19)[START_REF] Park | Induction and Segregation of the Vertebrate Cranial Placodes[END_REF]. During neurulation, the neural plate border gives rise to neural crest cells on both sides of the neural tube. Neural crest cells then go through an epithelial-to-mesenchymal transition and subsequently migrate along the antero-posterior axis of the developing embryo. Once they reach their final destination, cells differentiate into diverse cell types that can be grouped into four distinct subpopulations according to their new localization: cranial, vagal, trunk or sacral (Figure20)[START_REF] Rothstein | The Molecular Basis of Neural Crest Axial Identity[END_REF]. Neural crest cells differentiate into a multitude of cells such as neurons and glia from the peripheral and the enteric nervous systems, melanocytes, cartilage and bone, smooth muscle cells of the great vessels, and enterochromaffin cells of the adrenal gland[START_REF] Rothstein | The Molecular Basis of Neural Crest Axial Identity[END_REF]. Cranial placodes also differentiate into a multitude of cells, but unlike neural crest cells, they migrate shorter distances within the embryo and are generally restricted to the head region[START_REF] Park | Induction and Segregation of the Vertebrate Cranial Placodes[END_REF].

Figure 20 .

 20 Figure 20. Major derivatives of neural crest subpopulations along the anterioposterior axis. Differences in migratory patterns from subpopulations of neural crest cells, and their major derivatives. This illustration represents the Hamburger and Hamilton developmental stage 16 of a chick embryo. NC: neural crest. (Rothstein et al. 2018).

Figure 19 .

 19 Figure 19. Neural tube formation during primary neurulation. (A) The neural plate formation is the first step of neurulation. The neural plate border will later give rise to both neural crest cells and the placodes (the latter do not figure in the illustration). (B) Cells from the medial hinge point (MHP) stimulate the bending of the neural plate and the elevation of the neural folds. (C) Both sections of the neural plate border continue to approach as the neural plate continues to bend. (D) The tips of the neural folds fuse and form a closed neural tube. The closed tube separates from the surface ectoderm. The neural plate border gives rise to neural crest cells that will subsequently migrate. DLHP: cells from the dorsolateral hinge point. From discovery.lifemapsc.com.

E12. 5 ,

 5 Figure 21. Development of the liver venous drainage. (A) The umbilical veins in dark blue (RUV: right umbilical vein; LUV: left umbilical vein) carry oxygenated blood from the placenta and run on the lateral sides of the vitelline veins in light blue (RVV: right vitelline vein and LVV: left vitelline vein). (B) The liver bud develops at the foregut-midgut junction. (C) The vitelline veins in light blue enter and ramify within the septum transversum, and form the hepatic sinusoids. The two umbilical veins give rise to both intra-and extra-hepatic branches (RIHB: right intra-hepatic branch; REHB: right extrahepatic branch). (D) The extra-hepatic branches of the umbilical veins regress, represented by the gray color, along with the extra-hepatic portion of the left vitelline vein. (E) The right intra-hepatic vitelline vein becomes the portal vein (PV). CCV = common cardinal vein; SV: sinus venosus; STM: septum transversum; DUOD = duodenum; DV = ductus venosus; PHCVC = post-hepatic caudal vena cava. (Crawford et al. 2010).

Figure 22 .

 22 Figure 22. Hypothalamic development. (A) Major stages and (B) endocrine regulation of hypothalamic development[START_REF] Bouret | Organizational Actions of Metabolic Hormones[END_REF][START_REF] Bouret | Development of Hypothalamic Circuits That Control Food Intake and Energy Balance[END_REF].

  projections originating from the DMH or the VMH, axons from the ARH project to their targets relatively late. ARH axons connect to the DMH by P6, the PVH by P10, and the LH by P12. However, ARH axonal projections exhibit an adult-like distribution around P16(Bouret 2004a). Regarding the elongation of axons from AgRP/NPY neurons, both the intensity and the quantity of AgRP/NPY immune-reactive fibers increases from P0 to P21 in the PVH, and from P0 to P15 in the LH and the DMH[START_REF] Nilsson | Maturation of the Hypothalamic Arcuate Agouti-Related Protein System during Postnatal Development in the Mouse[END_REF].During the postnatal period, axonal outgrowth from POMC neurons is regulated by leptin signaling (see section 4.2 of this chapter). The diffusible axon guidance cues Netrin, Slit and Semaphorin, which are highly expressed in the PVH during development[START_REF] Xu | Expression of Robo/Slit and Semaphorin/Plexin/Neuropilin Family Members in the Developing Hypothalamic Paraventricular and Supraoptic Nuclei[END_REF], might be involved in POMC axonal development. Loss of Neuropilin 1 and 2 receptors in POMC neurons disrupts the development of axonal projections to the PVH, specifically, supporting a role for neuropilins/semaphorins in POMC axon guidance[START_REF] Coupe | Development of the Hypothalamic Melanocortin System[END_REF][START_REF] Van Der Klaauw | Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance[END_REF]. In parallel, morphological and functional data suggest that autophagy might also be involved in the development of POMC axonal projections. Neonates lacking the autophagy related 7 gene (Atg7) in POMC neurons display a reduced density of POMC-containing axonal projections to each of their target nuclei, including the PVH, the DMH and the LH and develop obesity, and impaired glucose homeostasis during adulthood[START_REF] Coupé | Loss of Autophagy in Pro-Opiomelanocortin Neurons Perturbs Axon Growth and Causes Metabolic Dysregulation[END_REF]

Figure 23 .

 23 Figure 23. Global trends of diabetes. From the IDF Diabetes Atlas, 9th edition, 2019.

Figure 24 .

 24 Figure 24. The perinatal environment influences the developmental programming of key organs regulating energy balance.This represents a possible mechanism leading to higher susceptibility to develop metabolic disorders during adulthood[START_REF] Bouret | Organizational Actions of Metabolic Hormones[END_REF].

Fat

  and lean mass were determined with Nuclear Magnetic Resonance spectroscopy (Minispec LF90 II; BRUKER Society, Wissembourg, France; ANR-11-EQPX-0035 PHENOCAN). Physical activity and energy expenditure were determined by indirect calorimetry (Phenomaster, TSE Systems GmbH). All procedures were performed by the ANIPHY platform, SFR Lyon-Est from Lyon 1 University.

  01 and ***p<0.001. Statistical details of experiments can be found in the figure legends. Statistical analyses were performed with GraphPad Prism 6 software.

  Consistently, ERα are widely expressed in the hypothalamus during the perinatal time frame of brain circuit development[START_REF] Tena-Sempere | Neonatal Imprinting and Regulation of Estrogen Receptor Alpha and Beta MRNA Expression by Estrogen in the Pituitary and Hypothalamus of the Male Rat[END_REF]) and a neonatal tamoxifen treatment can alter cell survival in the hippocampus[START_REF] Nobakht | Effects of Tamoxifen on Morphological and Ultrastructural Aspects of Developing Hippocampus of Rat[END_REF], affect brain progestin receptors[START_REF] Gonzales | Anatomically-Specific Actions of Oestrogen Receptor in the Developing Female Rat Brain: Effects of Oestradiol and Selective Oestrogen Receptor Modulators on Progestin Receptor Expression[END_REF], kisspeptin neurons in the arcuate nucleus of the hypothalamus[START_REF] Ichimura | Neonatal Exposure to SERMs Disrupts Neuroendocrine Development and Postnatal Reproductive Function through Alteration of Hypothalamic Kisspeptin Neurons in Female Rats[END_REF][START_REF] Parandin | Oestrogenic Action of Neonatal Tamoxifen on the Hypothalamus and Reproductive System in Female Mice[END_REF] Mahdavi-Shahri 2017) and precursors of neurons secreting the gonadotropin releasing hormone[START_REF] Agça | Modulation of Estrogen Receptors during Development Inhibits Neurogenesis of Precursors to GnRH-1 Neurones: In Vitro Studies with Explants of Ovine Olfactory Placode[END_REF]. In agreement with the role of sex steroids on neonatal programming(Tramunt et al. 2020), it could be possible that the neonatal tamoxifen exposure interferes with the ER-driven development of hypothalamic nuclei controlling energy balance and programs the metabolism of female mice into greater adiposity and metabolic disorders.

Figure 1 :

 1 Figure 1: Efficient recombination of floxed alleles following the use of tamoxifen at postnatal day 1. (A) Scheme of the floxed G6pc1 allele (G6pc1 lox ) and the exon 3-deleted G6pc1 allele (G6pc1 del ) from an original mouse model we developed (Penhoat et al. 2011). Exons are indicated by black rectangles and the 5' and 3' untranslated sequences are represented by white rectangles. LoxP sequences are represented as triangles. (B) Genomic

Figure 2 :

 2 Figure 2: Neonatal tamoxifen administration affects energy homeostasis in a sexdependent manner. (A) Body weight, (B) cumulated food intake, (C) body composition and (D) body composition normalized to body weight of NTT (green circles) and vehicle-treated (black circles) male mice (mean ± SEM, n=8-9). (E) Body weight, (F) cumulated food intake, (G) body composition and (H) body composition normalized to body weight of NTT (blue triangles) and vehicle-treated (black triangles) female mice (mean ± SEM, n=5-8). (I) liver weight, (J) liver weight normalized to body weight in female mice. (K) Scd1, Elovl6, Fasn, Acaca, Srebp1c, Chrebp: hepatic relative mRNA levels of genes coding proteins involved in

Figure 3 :

 3 Figure 3: Modest improvement of glucose homeostasis in male mice treated with tamoxifen in the neonatal period, after one and two months on a HF/HS diet. (A) Insulin tolerance, (B) glucose tolerance, with the corresponding A.U.C.s, and (C) 6-hour fasting glycaemia of NTT (green circles) and vehicle-treated (black circles) male mice after one month on a HF/HS diet (mean ± SEM, n=8-9). (D) Insulin tolerance, (E) glucose tolerance, with the corresponding A.U.C.s, and (F) 6-hour fasting glycaemia and (G) insulinemia of NTT (green circles) and vehicle-treated (black circles) male mice after two months on a HF/HS diet (mean ± SEM, n=8-9). HF/HS: high-fat/high-sucrose; A.U.C.: Area under the curve. Statistical analyses performed: T-tests for A.U.C.s, glycaemia and insulinemia. Two-way ANOVA, followed by multiple comparisons and SIDAK test for tolerance tests. *p<0.05.

Figure 4 :

 4 Figure 4: Impaired glucose homeostasis in female mice treated with tamoxifen in the neonatal period, after one and two months on a HF/HS diet. (A) Insulin tolerance, (B) glucose tolerance with the corresponding A.U.C.s, and (C) 6-hour fasting glycaemia of NTT (blue triangles) and vehicle-treated (black triangles) female mice after one month on a HF/HS diet (mean ± SEM, n= 5-8). (D) Comparison of the A.U.C.s of the glucose tolerance for female mice after one (yellow triangles) and two months (orange triangles) on a HF/HS diet (mean ± SEM,n=5-8). (E) Insulin tolerance, (F) glucose tolerance with the corresponding A.U.C.s, and (G) 6-hour fasting glycaemia and (H) insulinemia of NTT (blue triangles) and vehicle-treated (black triangles) female mice after two months on a HF/HS diet (mean ± SEM, n=5-8). HF/HS: high-fat/high-sucrose; A.U.C.: Area under the curve. Statistical analyses performed: T-tests for A.U.C.s, glycaemia and insulinemia. Two-way ANOVA, followed by multiple comparisons and SIDAK test for tolerance tests and panel D. *p<0.05; **p<0.01; ***p<0.001.

Figure 5 :

 5 Figure 5: Metabolic parameters of NTT and vehicle-treated female mice treated with tamoxifen in the neonatal period and fed a standard diet throughout life. (A) Weight, (B) body composition normalized to body weight, (C) 6-hour fasting glycaemia and (D) glucose tolerance with the corresponding A.U.C. of NTT (blue open triangles) and vehicletreated (black open triangles) female mice fed a standard diet (mean ± SEM, n=4). NTT: Neonatal tamoxifen treatment; A.U.C.: area under the curve. Statistical analyses performed: T-tests for weight, A.U.C. and glycaemia. Two-way ANOVA, followed by multiple comparisons and SIDAK test for body composition and glucose tolerance. *p<0.05; **p<0.01.

Figure 6 :

 6 Figure 6: Decreased energy expenditure and physical activity in female mice treated with tamoxifen in the neonatal period. (A) Energy expenditure normalized to body lean mass and (B) spontaneous physical activity during 24 hours of NTT (green circles) and vehicle-treated (black circles) male mice fed a HF/HS diet during adulthood (mean ±SEM, n=8-9). (C) Energy expenditure normalized to body lean mass and (D) spontaneous physical activity during 24 hours of NTT (blue triangles) and vehicle-treated (black triangles) female mice fed a HF/HS diet during adulthood (mean ± SEM, n=5). (E) Energy expenditure normalized to body lean mass during 24 hours of NTT (blue open triangles) and vehicletreated female mice (black open triangles) fed a standard diet during adulthood (mean ± SEM, n=4). NTT: Neonatal tamoxifen treatment; HF/HS: high-fat/high-sucrose. T-tests were performed for statistical analyses. *p<0.05; **p<0.01.

Figure 7 :

 7 Figure 7: Decreased tibia weight in male mice treated with tamoxifen in the neonatal period. (A) Tibia length and (B) tibia weight of NTT (green circles) and vehicle-treated male mice (black circles) fed a HF/HS diet during adulthood (mean ± SEM, n=6-7). (C) Body weight gain, (D) tibia length and (E) tibia weight of NTT (green open circles) and vehicle-treated (black open circles) male mice fed a standard diet during adulthood (mean ± SEM, n=4-5). NTT: Neonatal tamoxifen treatment; HF/HS: high-fat/high-sucrose. Statistical analyses performed: T-tests for tibia length and weight. Two-way ANOVA, followed by multiple comparisons and SIDAK test for body weight gain. *p<0.05; **p<0.01; ***p<0.001.

Figure 1 .

 1 Figure 1. Efficient recombination of floxed alleles following the use of tamoxifen at postnatal day 1.

Figure 2 .

 2 Figure 2. Neonatal tamoxifen administration affects energy homeostasis in a sex-dependent manner.

Figure 3 .

 3 Figure 3. Modest improvement of glucose homeostasis in male mice treated with tamoxifen in the neonatal period, after one and two months on a HF/HS diet.

Figure 4 .Figure 5 .

 45 Figure 4. Impaired glucose homeostasis in female mice treated with tamoxifen in the neonatal period, after one and two months on a HF/HS diet.

Figure 6 :

 6 Figure 6: Decreased energy expenditure and physical activity in female mice treated with tamoxifen in the neonatal period.

Figure 7 :

 7 Figure 7: Decreased tibia weight in male mice treated with tamoxifen in the neonatal period.

  Abbreviations: IGN, intestinal gluconeogenesis; AgRP: agouti-related peptide; POMC: proopiomelanocortin; ARH: arcuate nucleus; PVHant: paraventricular nucleus anterior; PVHpost: paraventricular nucleus posterior; DMH: dorsomedial nucleus; LH: lateral hypothalamic area; 3V: 3 rd ventricle; STAT3: transcription factor signal transducer and activator of transcription 3; CGRP: calcitonin gene-related peptide; HF/HS, high-fat/highsucrose.

  Epidemiological data, clinical studies and experimental research have indicated that a deleterious nutritional environment during the perinatal period may exacerbate the susceptibility of an individual to develop metabolic diseases later in life[START_REF] Cottrell | Developmental Programming of Energy Balance and the Metabolic Syndrome[END_REF][START_REF] Moullé | Effects of Nutrient Intake during Pregnancy and Lactation on the Endocrine Pancreas of the Offspring[END_REF].Key organs that regulate energy balance develop during gestation and pursue their maturation during the first postnatal weeks in rodents. In this respect, an inadequate perinatal environment can alter the development of the hypothalamus, which is a central regulator of energy balance[START_REF] Sainsbury | Hypothalamic Regulation of Energy Homeostasis[END_REF][START_REF] Schneeberger | Hypothalamic and Brainstem Neuronal Circuits Controlling Homeostatic Energy Balance[END_REF]. The hypothalamus contains several nuclei, from which the arcuate nucleus (ARH) stands out as a central integrator of peripheral hormonal and nutritional cues. The ARH contains orexigenic neurons containing neuropeptide Y and the agouti-related peptide (NPY/AgRP neurons) and anorexigenic neurons containing the cocaine amphetaminerelated transcript and pro-opiomelanocortin (POMC/CART neurons)[START_REF] Cone | Anatomy and Regulation of the Central Melanocortin System[END_REF] Wu et al. 2009;[START_REF] Wu | GABAergic Signaling by AgRP Neurons Prevents Anorexia via a Melanocortin-Independent Mechanism[END_REF]. These neurons transmit peptidergic signals through axons that project into other hypothalamic nuclei implicated in energy balance regulation such as the paraventricular nucleus (PVH), the dorsomedial nucleus (DMH) and the lateral nucleus (LH)[START_REF] Berthoud | Multiple Neural Systems Controlling Food Intake and Body Weight[END_REF][START_REF] Cone | Anatomy and Regulation of the Central Melanocortin System[END_REF].

3. 2 .

 2 Modification of hypothalamic fibers in mice with a neonatal induction of intestinal gluconeogenesis Hypothalamic feeding circuits that project from the arcuate nucleus (ARH) to other hypothalamic nuclei involved in energy balance regulation develop mainly during the first two weeks of life in rodents, and show an adult-like organization already around P16 (Bouret 2004a). To determine whether neonatal IGN plays a role in the development of hypothalamic feeding circuits, we immunolabeled AgRP and POMC fibers in brain slices of mouse pups aged of 20 days. I overexp G6pc P1 pups exhibited about 35% more of AgRP immunoreactivity in the anterior part of the hypothalamic paraventricular nucleus (PVHant) in comparison to control littermates (Figure 2A,B). However, we measured equivalent AgRP immunoreactivity in the posterior part of the paraventricular nucleus (PVHpost), the lateral hypothalamic nucleus (LH) and the dorsomedial nucleus (DMH) of both I overexp G6pc P1 and control littermates (Figure 2A,B). Unexpectedly, P20 I overexp G6pc P1 pups exhibited a 20%

Figure 2 .

 2 Figure 2. The neonatal induction of IGN influences the development of AgRP and POMC fibers. (A) Schematic representation of hypothalamic nuclei that were quantified (with their corresponding bregma). Confocal images and quantification of (B) AgRP-immunoreactive and (C) POMC-immunoreactive fibers (mean ± SEM; n= 5-6). 3V: 3 rd ventricle; PVHant: anterior part of the paraventricular nucleus; PVHpost: posterior part of the paraventricular nucleus; LH: lateral hypothalamic area; DMH: dorsomedial nucleus. Two-way ANOVA, followed by multiple comparisons and SIDAK test were performed as statistical analyses. *p<0.05; **p<0.01.

Figure 3 .

 3 Figure 3. The neonatal induction of IGN protects the metabolism of adult mice fed a HF/HS diet. Analyses were performed in adult mice fed a HF/HS diet since the 6 th week of life (mean ± SEM; n= 6-7). (A) Weight gain. (B) Body fat mass normalized by total body weight. (C) Body lean mass normalized by total body weight. (D) Plasma leptin levels. (E) Hypothalamic STAT3 phosphorylation levels. Two-way ANOVA, followed by multiple comparisons and SIDAK test were performed as statistical analyses for weight gain. T-tests were performed for body composition and hypothalamic measurements. *p<0.05; **p<0.01; ***p<0.001.

Figure 4 .

 4 Figure 4. The P12 induction of IGN fails to affect the development of AgRP and POMC fibers. (A) Schematic representation of hypothalamic nuclei that were quantified (with their corresponding bregma). Confocal images and quantification of (B) AgRP-immunoreactive and (C) POMC-immunoreactive fibers (mean ± SEM; n= 5-6). 3V: 3 rd ventricle; PVHant: anterior part of the paraventricular nucleus; PVHpost: posterior part of the paraventricular nucleus; LH: lateral hypothalamic area; DMH: dorsomedial nucleus. Two-way ANOVA, followed by multiple comparisons and SIDAK test were performed as statistical analyses.

Figure 5 .

 5 Figure 5. The P12 induction of IGN fails to protect the metabolism of adult mice fed a HF/HS diet. Analyses were performed in adult mice fed a HF/HS diet since the 6 th week of life (mean ± SEM, n= 6-7). (A) Weight gain. (B) Body fat mass normalized by total body weight. (C) Body lean mass normalized by total body weight. (D) Plasma leptin levels. (E) Hypothalamic STAT3 phosphorylation levels. Two-way ANOVA, followed by multiple comparisons and

Figure 6 .

 6 Figure 6. Enhanced hypothalamic leptin signaling in I overexp G6pc P1 mouse pups. (A) Relative hypothalamic AgRP protein expression. (B) Relative hypothalamic POMC protein expression. (C) Relative hypothalamic STAT3 phosphorylation levels. (D) Plasma leptin levels. (E) Ratio of hypothalamic STAT3 phosphorylation levels to plasma leptin levels during the neonatal period. Stain-fee gels were used for the quantification of whole protein content in western blot analyses. (mean ± SEM, n= 5-7). Two-way ANOVA, followed by multiple comparisons and SIDAK test were performed as statistical analyses. *p<0.05.

Figure 1 .

 1 Figure 1. Murine model with an inducible induction of intestinal gluconeogenesis (IGN).

Figure 2 .

 2 Figure 2. The neonatal induction of IGN influences the development of AgRP and POMC fibers.

Figure 3 .

 3 Figure 3. The neonatal induction of IGN protects the metabolism of adult mice fed a HF/HS diet.

Figure 4 .

 4 Figure 4. The P12 induction of IGN fails to affect the development of AgRP and POMC fibers.

Figure 5 .

 5 Figure 5. The P12 induction of IGN fails to protect the metabolism of adult mice fed a HF/HS diet.

Figure 6 .

 6 Figure 6. Enhanced hypothalamic leptin signaling in I overexp G6pc P1 mouse pups.

2 .

 2 Contrôle central de la prise alimentaire L'homéostasie du glucose dépend des deux piliers de l'équilibre énergétique: la prise alimentaire et la dépense énergétique. La régulation de ces deux piliers nécessite une communication constante entre le système endocrinien et le système nerveux. Le système endocrinien signale au cerveau l'état énergétique d'un individu, et le cerveau contrôle alors la sécrétion hormonale de différents tissus afin de réguler l'équilibre énergétique. Deux des centres de régulation les plus importants sont l'hypothalamus et le tronc cérébral. L'hypothalamus est situé à la base du cerveau et, en tant que centre d'intégration majeur du corps, relie le système nerveux au système endocrinien par la glande pituitaire. Des expériences utilisant des lésions physiques de noyaux hypothalamiques spécifiques et des études utilisant des stratégies de ciblage génique conditionnel, spécifiques aux neurones, ont mis en évidence que la régulation de l'homéostasie énergétique engage un réseau neuronal interconnecté dans l'hypothalamus impliquant des noyaux spécifiques: le noyau arqué (ARH), le noyau ventromédial (VMH), le noyau dorsomédial (DMH), la zone hypothalamique latérale (LH) et le noyau paraventriculaire (PVH) (Figure 6). L'ARH est au centre de ce circuit neuronal. Il est situé juste au-dessus d'un organe circumventriculaire appelé éminence médiane. Cette localisation stratégique permet à l'ARH d'intégrer les signaux sanguins de faim et de satiété qui reflètent les réserves d'énergie du corps, puis d'envoyer des signaux neuro-peptidergiques aux noyaux intra-hypothalamiques, au tronc cérébral et au système cortico-limbique, conduisant finalement à la régulation de l'équilibre énergétique[START_REF] Berthoud | Multiple Neural Systems Controlling Food Intake and Body Weight[END_REF]. L'ARH contient plusieurs populations neuronales selon les neuro-peptides exprimés (Figure6). Deux populations neuronales sont considérées comme particulièrement importantes pour le contrôle de l'équilibre énergétique. La première population neuronale est généralement définie comme coexprimant les peptides pro-opiomelanocortin (POMC) et cocaine-amphetamine-related transcript (CART). La dépolarisation des neurones POMC/CART conduit à la libération de peptides comme l'α-MSH, qui agissent comme agonistes sur les récepteurs de mélanocortine 3 et 4 (MC3R et MC4R) situés dans les neurones en aval, conduisant à terme à une diminution de la prise alimentaire et à une augmentation de la dépense énergétique[START_REF] Cone | Anatomy and Regulation of the Central Melanocortin System[END_REF]. La deuxième population neuronale co-exprime le peptide Agouti-related peptide (AgRP), le neuropeptide-Y (NPY) et le GABA. L'activation des neurones hypothalamiques AgRP/NPY favorise l'alimentation et leur inhibition conduit à la famine(Wu et al. 2009;[START_REF] Wu | GABAergic Signaling by AgRP Neurons Prevents Anorexia via a Melanocortin-Independent Mechanism[END_REF]. La régulation de la prise alimentaire et de la dépense énergétique nécessite l'activité coordonnée et, la plupart du temps, opposée des populations neuronales POMC/CART et AgRP/NPY. Parmi tous les autres noyaux hypothalamiques, le PVH est particulièrement intéressant car il contient plusieurs populations neuronales qui modulent le système par la leptine et est profondément impliquée dans les actions développementales de la leptine[START_REF] Kwon | Leptin Signalling Pathways in Hypothalamic Neurons[END_REF][START_REF] Münzberg | Role of Signal Transducer and Activator of Transcription 3 in Regulation of Hypothalamic Proopiomelanocortin Gene Expression by Leptin[END_REF]. La leptine conduit à la phosphorylation de STAT3, qui se dimérise ensuite et se déplace vers le noyau où il diminue l'expression d'AgRP et augmente l'expression de POMC dans chaque population neuronale respective (Pour une revue, voir[START_REF] Kwon | Leptin Signalling Pathways in Hypothalamic Neurons[END_REF]. La stimulation des neurones POMC/CART et l'inhibition des neurones AgRP/NPY permettent la signalisation via MC3R et MC4R dans des neurones de second ordre, conduisant finalement à une diminution de la prise alimentaire. En opposition directe à la leptine, la ghréline est décrite comme la seule hormone signalant la faim. Le

4 .

 4 G6pc overexp ) ont été obtenues via la stratégie de biotechnologie CRE/lox constitutive (Vily-Petit et al. 2020). Les souris I.G6pc overexp sont protégées du développement de l'obésité, de la stéatose hépatique et du diabète de type 2 lorsqu'elles sont nourries avec un régime riche en graisses et en saccharose (Vily-Petit et al. 2020). Les résultats obtenus avec ces modèles murins transgéniques ont validé les résultats obtenus avec des régimes spécifiques concernant les effets bénéfiques de la néoglucogenèse intestinale. Aspects développementaux de l'axe intestin-cerveau chez les individus en bonne santé La veine porte et les neurones des ganglions nodosiques et de la racine dorsale (correspondant respectivement aux afférences vagales et splanchniques) impliqués dans l'axe intestin-cerveau stimulé par la néoglucogenèse intestinale se développent déjà au cours de la vie foetale.. Plusieurs études suggèrent effectivement que la veine porte pourrait déjà contenir des terminaisons vagales et splanchniques au début de la période postnatale et qu'elle est déjà innervée au moins depuis P5 chez le rat. La croissance axonale de l'hypothalamus a lieu au cours des trois premières semaines post-natales chez les rongeurs. Les axones de l'ARH se connectent au DMH à P6, au PVH à P10 et au LH à P12. Ces projections présentent la distribution retrouvée chez l'adulte à partir de P18 (Bouret 2004a). Le développement de ces circuits neuronaux hypothalamiques est un processus régulé par une signalisation intracellulaire initiée par plusieurs hormones. La leptine et la ghréline, qui régulent l'alimentation et la dépense énergétique à l'âge adulte, exercent en fait un rôle développemental pendant la période périnatale. Les taux plasmatiques de leptine chez les souris augmentent pendant la période postnatale,

6 .

 6 Objectif de notre étude Dans cette étude, notre objectif principal était de déterminer le rôle de la néoglucogenèse intestinale sur le développement postnatal des circuits alimentaires hypothalamiques chez la souris. Pour y parvenir, nous avons cherché à surexprimer la néoglucogenèse intestinale au début de la période postnatale. Pour cela, nous avons utilisé un nouveau modèle murin avec une surexpression inductible (dépendante du tamoxifène) du gène G6pc spécifiquement dans l'intestin. Cependant, ce nouveau modèle murin nécessite une administration de tamoxifène à la naissance. Par conséquent, nous avons déchiffré les effets associés à une administration néonatale de tamoxifène en soi avant de déterminer le rôle de la néoglucogenèse intestinale sur le développement néonatal des circuits d'alimentation hypothalamiques. 7. Premier article: le traitement au tamoxifène pendant la période néonatale affecte l'homéostasie du glucose chez les souris adultes de manière dépendante du sexe Le tamoxifène est un modulateur sélectif du récepteur des oestrogènes utilisé pour activer la recombinase CRE ERT2 (Metzger and Chambon 2001), permettant un contrôle tissulaire et temporel de la mutagenèse somatique induites chez les souris transgéniques. Les études intégrant le développement et le métabolisme nécessitent une modification génétique induite par l'administration de tamoxifène depuis la période néonatale. Dans cette étude, nous avons étudié les effets d'une administration néonatale de tamoxifène sur l'homéostasie énergétique chez des souris adultes mâles et femelles C57BL/6J. À cette fin, des souriceaux ont reçu une injection unique de 20 µg de tamoxifène un jour après la naissance. Nous avons mesuré le poids, la composition corporelle, la tolérance au glucose et à l'insuline, le métabolisme de base et la longueur et le poids du tibia chez des souris adultes traitées au tamoxifène pendant la période néonatale. Nos résultats montrent que l'administration néonatale de tamoxifène a exercé des effets dépendants du sexe sur l'homéostasie énergétique. Les souris femelles traitées avec du tamoxifène pendant la période néonatale ont montré une accélération de l'apparition de l'obésité et du diabète par rapport aux souris contrôles littermates, lorsqu'elles ont été soumises à un régime riche en graisses et en sucrose. Ces souris ont présenté une augmentation de plus de 60% de la masse grasse associée à une augmentation de la prise alimentaire, une diminution de l'activité physique et une diminution des dépenses énergétiques, une intolérance au glucose et à l'insuline, ainsi qu'une hyperglycémie et une hyperinsulinémie à jeun. Au contraire, les souris mâles traitées au tamoxifène pendant la période néonatale ont présenté une amélioration modeste de la tolérance au glucose et à l'insuline et une diminution de la masse maigre tout au long de la vie en raison d'une diminution de la densité osseuse. Dans l'ensemble, nos données suggèrent que l'administration néonatale de tamoxifène exerce une influence marquée et dépendante du sexe sur l'homéostasie énergétique des adultes. Nous recommandons donc que l'administration néonatale de tamoxifène pour le développement de modèles murins soit utilisée avec prudence dans des études sur l'homéostasie énergétique. 8. Deuxième article: La néoglucogenèse intestinale contrôle le développement neuronal hypothalamique AgRP au début de la période néonatale Pour déterminer le rôle de la néoglucogenèse intestinale sur le développement postnatal des circuits alimentaires hypothalamiques chez la souris, nous avons surexprimé le gène G6pc spécifiquement dans l'intestin dès P1 chez la souris. Nous avons analysé les circuits hypothalamiques AgRP et POMC et étudié leur composition corporelle. En parallèle, pour déterminer si ces effets sont restreints au pic postnatal de la néoglucogenèse intestinale, nous avons effectué les mêmes analyses chez des souris avec une surexpression intestinale de G6pc juste après le pic postnatal de néoglucogenèse intestinale (P12). Nos résultats montrent que l'induction de la néoglucogenèse intestinale à P1 a augmenté la densité des projections axonales AgRP vers le PVH chez des souris âgées de 20 jours. Cet effet était limité aux deux premières semaines postnatales car l'induction de la néoglucogenèse intestinale à P12 n'a pas eu d'effet sur la densité des projections axonales AgRP vers le PVH. Les souris avec une induction de la néoglucogenèse intestinale depuis P1 présentaient moins de masse grasse et une masse maigre accrue après deux mois de régime riche en graisses et en sucrose. Cet effet métabolique n'a pas été retrouvé lorsque la néoglucogenèse intestinale a été induite dès P12. Nos données suggèrent que la néoglucogenèse intestinale exerce un effet neurotrophique sur les projections axonales AgRP vers le PVH au cours des deux premières semaines de vie. L'induction de la néoglucogenèse intestinale au début de la période postnatale pourrait protéger la santé métabolique à long terme.

6. A simplified schematic model of hypothalamic neuronal circuits regulating food intake. On

	vascular endothelial cells that form the blood-brain barrier
	the left side: non exhaustive illustration of the neuronal populations
	regulating food intake in each hypothalamic nucleus. On the right side: non exhaustive
	illustration of the receptors to peripheral signals expressed in each hypothalamic nucleus.
	Hypothalamic nuclei involved in the regulation of food intake communicate with each other,
	forming neuronal circuits. Among these nuclei, the arcuate nucleus (ARH) plays a central
	role. Indeed, it has both orexigenic AgRP/NPY and anorexigenic POMC/CART neurons, and
	it expresses receptors to many peripheral signals. The ARH sends axonal projections to all
	other nuclei regulating food intake. Importantly, the ARH is located near the median
	eminence (ME), a circumventricular organ with tanycytes lining the wall of the third
	ventricle (3V) and which projections are in contact with fenestrated capillaries that facilitate
	blood/brain exchanges. ARH: arcuate nucleus; VMH: ventromedial nucleus;
	DMH: dorsomedial nucleus; LH: lateral hypothalamic area; PVH: paraventricular nucleus;
	AgRP: agouti-related peptide; NPY: neuropeptide Y; POMC: pro-opiomelanocortin;
	CART: Cocaine and Amphetamine Regulated Transcript; MCH: Melanin-Concentrating
	Hormone; SF1: steroidogenic factor 1; BDNF: brain-derived neurotrophic factor; CRH:
	corticotrophin-releasing hormone; TRH: thyrotropin-releasing hormone; InsR: insulin
	receptor; LepRb: leptin receptor; GHSH: ghrelin receptor; GLP1R1: GLP-1 receptor; ApidoR1:
	adiponectin receptor; CCK1: CCK receptor; GLP-1: glucagon-like peptide. Adapted from the
	doctorate thesis manuscript of Dr. Fanny Langlet. Illustration partly made with Servier Medical
	Art

Table 1 : Primer sequences used for gene expression analyses.

 1

	Targeted gene	Primer sequences
	Acetyl-CoA carboxylase alpha 1 (Acaca)	Fw: TGGCAGACCACTATGTTCCA
		Rev: GAGCAGTTCTGGGAGTTTCG
	Acyl-CoA Oxidase 1 (Acox1)	Fw: TGCCAAATTCCCTCATCTTC
		Rev: CTTGGATGGTAGTCCGGAGA
	Carbohydrate-responsive element-	Fw: GAAGCCACCCTATAGCTCCC
	binding protein, CHREBP (Chrebp)	Rev: CTGGGGACCTAAACAGGAGC
	Carnitine palmitoyltransferase1 (Cpt1a)	Fw: ACCTCCATGGCTCAGACAG
		Rev: AGCAGAGGCTCAAGCTGTTCA
	Cytochrome P450 (Cyp4a10)	Fw: TCCAGCAGTTCCCATCACCT
		Rev: TTGCTTCCCCAGAACCATCT
	Cytochrome P450 (Cyp4a14)	Fw: TCAGTCTATTTCTGCTGTTC
		Rev: GAGCTCCTTGTCCTTCAGATGGT
	Fatty acid elongase 6 (Elovl6)	Fw: ACAATGGACCTGTCAGCAAA
		Rev: GTACCAGTGCAGGAAGATCAGT
	Fatty acid synthase, FAS (Fasn)	Fw: TTCCAAGACGAAAATGATGC
		Rev: AATTGTGGGATCAGGAGAGC
	Peroxisome proliferator-activated	Fw: AGTTCACGCATGTGAAGGCTG
	receptor alpha PPARα (Ppara)

  . To test if CGRP is the missing link in the neurotrophic role of IGN on AgRP projections to the PVHant, two experiments could be performed. The first one is immunolabeling AgRP and POMC fibers on brains of I overexp G6pc P1 pups receiving an intracerebroventricular injection of a CGRP antagonist, such as CGRP 8-37[START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF]. The second experiment consists of incubating ARH explants with CGRP to measure ARH axonal outgrowth[START_REF] Soty | Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose[END_REF][START_REF] Steculorum | Neonatal Ghrelin Programs Development of Hypothalamic Feeding Circuits[END_REF].

	2.2. Is the effect of IGN on hypothalamic neuronal circuits associated with a long-term
	effect on mice metabolism?

ACKNOWLEDGEMENTS

I would first like to thank the members of my thesis jury. Thank you Pr. Fabien Van Coppenolle for having accepted to be the president of my thesis jury. Thank you very much Dr. Daniela Cota and Dr. Patricia Parnet for having accepted to take the time to read, evaluate and judge my thesis manuscript. I can only hope that you will find my thesis subject

Acknowledgments

We would like to thank Valerie Oréa and Delphine Guyonnet from the ANIPHY Platform, SFR Lyon-Est (Lyon 1 University, Rockefeller Campus) for assessing body composition, physical activity and energy expenditure, and the members of the animal facility ALECS.

Acknowledgments

We would like to thank Valerie Oréa and Delphine Guyonnet from the ANIPHY Platform (Lyon 1 University, Rockefeller Campus) for assessing body composition.

Disclosure statement:

The authors have nothing to disclose.

Author contributions

Declaration of interests

The authors declare no competing interests.

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