Dr Andrea Lazzati

Dr Benedicte Gaborit

Dr Daigo Au

Dr Maria Sawaki

Pini D'

MSc Zaineb Mezdari

PhD Maria Pini
email: maria.pini@inserm.fr

MD Gabor Czibik

MD Julien Ternacle

MD Costin Radu

MD Yanyan Zhang

MD Serge Adnot

MD Corneliu Henegar

MD Geneviève A Derumeaux
email: genevieve.derumeaux@inserm.fr

MD Daigo Sawaki

MD Suzain Naushad

Lucille Lamendour

Dogus Altintas

PhD Marielle Breau

MSc Hao Liang

MSc Maissa Halfaoui

Mathieu Surenaud

Déborah Rousseau

Takehiko Yoshimitsu

Fawzia Louache

Philippe Gual

PhD Laura Braud

MD Raquel Mercedes

PhD Cécile Martel

PhD Nelly Buron

PhD Geneviève Marcelin

PhD Annie Borgne-Sanchez

PhD Roberta Foresti

PhD Roberto Motterlini

PhD Dogus Murat Altintas

MSc Juliette Bréhat

MD Elisabeth Riant

MD Thomas D'humières

PhD Cecile Martin

BSc Azania Abatan

MRCP Rizwan Sarwar

MSc Ophélie Marion

PhD Nadine Suffee

PhD Didier Morin

MD Stéphane Hatem

MRCP, DPhil Arash Yavari

MD, PhD ‡ Geneviève Derumeaux

Role of adipose tissue macrophages in the crosstalk between visceral adipose tissue and heart during high fat diet

Keywords: obesity, exercise, adipose tissue senescence, preadipocytes, bioenergetics, ATP, xanthine oxidase

Cette thèse, est l'aboutissement d'un long travail qui n'a été possible que grâce au concours de plusieurs personnes à qui je souhaite témoigner toute ma reconnaissance.

Pour commencer je tiens à remercier les membres du jury pour avoir accepté de consacrer du temps à évaluer mes travaux de recherche malgré leurs emplois du temps très chargés.

Ensuite, je remercie bien évidement le directeur de l'IMRB : Mr. Jorge BOCZKOWSKI et le directeur de l'UPEC : Mr. Jean-Luc DUBOIS-RANDÉ ainsi que toutes les personnes qui contribuent quotidiennement au bon fonctionnement de l'institut et de l'université pour nous permettre de travailler dans un cadre propice à nos activités de recherche scientifique.

Je remercie particulièrement le personnel des plateformes, surtout les membres de l'EP3, en particulier Adrien, Florine et Alice et le personnel de la plateforme d'imagerie : Xavier, Christelle et Wilfried, Merci à tous pour votre gentillesse et votre professionnalisme.

Merci également à tous ceux que je n'ai pas cité mais qui m'ont aidé d'une manière ou d'une autre.

Pour finir, je tiens à remercier chaleureusement tous les membres de ma Famille :

A mes chers parents, à qui je dois tout, merci d'avoir toujours été là pour moi, merci pour votre soutien et votre amour inconditionnel

خيرا يجازيكم و فيكم يبارك و منكم يتقبل هللا

A mes frères, merci pour votre aide et votre soutien, merci pour vos encouragements et merci d'être toujours là pour moi.

A mes oncles, merci pour votre soutien et vos encouragements, merci d'être toujours près de moi malgré les distances qui nous séparent.

A ma soeur Yousra, je ne pourrais jamais te remercier assez pour toute l'aide que tu m'as apportée, pour ton soutien et tes encouragements, pour m'avoir poussée à surmonter les difficultés et pour avoir été mon coach dans cette course de l'écriture du manuscrit : en comptant les pages écrites au fur et à mesure pour m'encourager, en exigeant des rapports détaillés de mon avancement, tout en prenant soin de contrôler mes heures de réveil et de coucher !! ce rythme militaire aura fini par payer, Merci Yousra, t'es un amour! Je te souhaite tout le bien que tu mérites.

A mes cousines les « BenLag », qui n'ont jamais arrêté de m'encourager et de me soutenir tout au long de mes études et dont la compagnie me fait tellement de bien.

A mes beaux-parents, qui m'ont toujours encouragée, aidée, et poussée à aller toujours plus loin, merci à vous, merci pour tout.

A mes chers enfants, Balkys et Oways, que je remercie du fond du coeur de faire partie de ma vie, d'être ma source de motivation, d'être ma raison de me battre, d'être ma source de bonheur, de joie et d'amour…Merci pour votre patience et votre soutien.

Et pour finir… A Moi !! Parce que j'ai franchi des montagnes pour en arriver là… -Establish the chronological order of events leading to visceral AT senescence in obesity -Identify the AT cells mainly affected by cellular senescence -Explore the role of senescent AT macrophages in the crosstalk between AT and heart.

-Compare different strategies in order to mitigate visceral AT senescence

We showed that AT senescence is an early and primary event that could orchestrate subsequent maladaptive changes in AT and heart function and remodeling. Besides, our results demonstrated the importance of early macrophage senescence in diet-induced and age-related cardiometabolic dysfunction and may shed new light on the search for developing better therapeutic options, including senolytic therapies, in the context of obesity in order to prevent cardiac and metabolic disorders

Résumé

L'obésité a augmenté de façon spectaculaire ces dernières années à travers le monde et ceci est devenu un problème majeur de santé publique. En effet, cela augmente le risque de décès, principalement des suites de maladies cardio-vasculaires. La sédentarité, ainsi qu'une alimentation riche en énergie, sont des facteurs connus pour favoriser le stress oxydatif et altérer la structure ainsi que la fonction du tissu adipeux (TA). Le TA viscéral, en particulier, a été identifié par notre équipe et par d'autres comme étant responsable des dommages causés aux organes distants par l'intermédiaire d'un sécrétome pro-fibrotique et pro-inflammatoire.

De façon intéressante, la sécrétion de ces facteurs coïncide avec l'accumulation de cellules sénescentes au niveau du TA au cours du vieillissement naturel mais également dans le contexte de l'obésité. La sénescence cellulaire est définie par l'arrêt irréversible du cycle cellulaire et est déclenchée par différents facteurs parmi lesquels on retrouve le raccourcissement des télomères ou encore le stress oxydatif. Son impact au niveau fonctionnel est démontré par les observations selon lesquelles l'élimination sélective des cellules sénescentes prévient l'insulino-résistance et retarde l'apparition des pathologies liées au vieillissement. Cependant, les mécanismes de l'initiation et de l'évolution de la sénescence au cours du temps dans le TA sont encore mal compris, de même que les cellules affectées et les principaux acteurs restent inconnus. Les principaux objectifs de mon projet de thèse consistaient donc à :

-Etablir l'ordre chronologique des événements conduisant à la sénescence prématurée du TA viscéral, et donc à son altération, dans le contexte de l'obésité, -Identifier les principales cellules affectées par la senescence, -Explorer le rôle des macrophages sénescents du TA viscéral dans le dialogue entre TA et coeur, -Comparer différentes stratégies visant à atténuer les altérations métaboliques et cardiaques liées à l'obésité.

Notre travail a démontré que la sénescence du TA est l'un des premiers évènements apparaissant lors de l'obésité et que cette senescence avait un rôle majeur dans les altérations métaboliques et cardiaques liées à cette pathologie. De plus, nos résultats ont mis en évidence l'implication des macrophages du TA et leur rôle crucial dans les complications mentionnées, apportant ainsi de nouveaux horizons et de nouveaux éléments dans la recherche et le développement de nouvelles thérapies pour traiter l'obésité et ses comorbidités.

Introduction

The world faces an obesity epidemic afflicting the entire cross-section of society, with increased risk for type 2 diabetes mellitus (T2DM), dyslipidaemia and cardiovascular disease.

Physical inactivity and high energy content in diet are known to alter adipose tissue structure and function. Visceral adipose tissue (VAT) is considered paramount to obesity diseasome and our team has contributed to identify a key role of this organ in promoting cardiac alterations and remodeling through a profibrotic and proinflammatory secretome. Interestingly, these secreted factors were coincident with the accumulation of senescent cells in adipose tissue during both natural aging and obesity. Besides, some studies demonstrated that selective elimination of senescent cells prevents insulin resistance and delays the onset of age-related pathologies.

In the following section, we will summarize the current knowledge about obesity diseasome and clinical implications, before going more in depth by describing adipose tissue physiology and physiopathology with a special focus on obesity-induced adipose tissue remodeling and dysfunction and finally give an overview of therapeutic approaches in obesity-associated metabolic complications.

SECTION 1

OBESITY

Chapter 1: Definition and Epidemiology

Obesity is the consequence of accumulation of fat mass following an imbalance between calorie intake and expenditure. In adult patients, body mass index (BMI) is commonly used to measure the severity of the overweightness. Although it provides no information about the percentage of fat mass in the body, BMI proved useful in clinical evaluation of patients with BMI ≥ 25 kg/m² considered overweight and patients with BMI ≥ 30 kg/m² defined as obese.

Over the past decades, the incidence of obesity has dramatically increased worldwide, especially in Western societies, and the developing world. The worldwide prevalence of obesity nearly tripled between 1975 and 2016 and obesity is now officially recognized as a chronic disease and global epidemic by the World Health Organization. Specifically, in 2016, more than 1.9 billion adults (39% of the world's adult population) were overweight, and 650 million (13% of the world's adult population) were obese.

Obesity is associated with increased risk for type 2 diabetes mellitus (T2DM), dyslipidemia, hypertension and cardiovascular disease and is associated with elevated all-cause mortality compared to normal weight. 1,2 It was estimated that in 2015, approximately 1 in every 11 adults has T2DM globally, and about 75% of patients with diabetes mellitus live in developing countries 3 (figure 1).

Figure 1: Estimated total number of adults (20-79 years) living with diabetes mellitus, highlighting the top three countries or territories for number of adults with diabetes mellitus (20-79 years) in 2015. (From Zheng & al., Nat Rev Endocrinol 2018) 4 T2DM is characterized by an impairment in insulin action (particularly in insulin-sensitive tissues: liver, muscle and adipose tissue become insulin-resistant), which pancreatic β-cells are not able to compensate by increasing insulin secretion. This imbalance results in abnormal blood levels of glucose. 5 Associated complications include but are not limited to macrovascular complications leading to cardiovascular disease (CVD) and microvascular complications affecting lower limb, kidney, retina and nervous system. 4 Global Burden of Disease Study 2013 identified diabetes mellitus (all forms) as the ninth major cause of reduced life expectancy. 5 In 2010, it was estimated that diabetes mellitus caused 3.96 million deaths in adults aged 20-79 years during that year (6.8% of global mortality). 6 This estimate was raised to 5 million deaths due to diabetes mellitus and its complications during 2015 in an International Diabetes Federation (IDF) report.

From an economic perspective, about 12% of the global health expenditure was spent on treatment of T2DM and its related complications in 2015. 3

Chapter 2: Clinical research and limitations

To address the dual epidemics of obesity and diabetes mellitus, many observational studies and randomized clinical trials showed the importance of prevention by maintaining a healthy body weight with a focus on maintaining energy balance through regular physical activity and healthy diet. 7,8 Major clinical trials have demonstrated that intensive lifestyle interventions can lower the incidence of diabetes mellitus by 58% compared with control groups and are more effective than pharmacological approaches. 9 Further studies indicated that not all adipose tissue pads are equal, but different anatomical depots show their own specific characteristics. The significance of varying body fat distribution is best illustrated by different clinical outcomes. Accumulation of visceral adipose tissue (VAT) corrodes health more than subcutaneous fat (SAT) does. 1,10 Indeed the inability of subcutaneous adipose tissue to store surplus triglycerides may lead to the development of ectopic fat depots in different organs such as the liver, the heart, the muscle and the pancreas.

This ectopic lipid deposition is of growing interest, since it is an important contributor to the development of obesity comorbidities 11 and may result from a dysfunctional subcutaneous adipose tissue not being able to appropriately store the lipid overload, which is redirected to cells of internal organs, leading to steatosis and organ dysfunction. Regional body-fat distribution is one of the key variables that explains the metabolic heterogeneity of obesity and its associated cardiovascular risks. This differential repartition of adipose tissue accumulation is due to many factors including genetics, environment, gender and age, [12][13][14][15] (figure 2)

Figure 2: Heterogeneity in WAT repartition and factors involved. (From Guglielmi, V. & al., Eat Weight Disord 2018). 10 Observations that a proportion of obese patients have a significantly lower risk for cardiometabolic abnormalities led to the concept of metabolically healthy obesity (MHO). This condition is characterized by lower amounts of ectopic fat (visceral and hepatic), together with normal glucose and lipid metabolism parameters, preserved insulin sensitivity and no hypertension compared to unhealthy obese patients. 16 Such an observation raises therefore the crucial need to characterize the body fat distribution to evaluate the percentage of "ectopic adipose tissue" in the adiposity index. This can be performed in routine clinic evaluation by different techniques of bioelectrical impedance analysis, or quantitative magnetic resonance for measuring total body water, fat, and lean tissue, as well as imaging techniques to further define ectopic fat depots. 17 . 1H-magnetic resonance spectroscopy (MRS) is a reliable noninvasive quantitative method to assess the pancreatic triglyceride content in vivo, which has been validated in rodents and in humans. 18,19 Another important factor to be evaluated in obese patients is the remodeling of adipose tissue as progression toward obese state is often accompanied by pericellular collagen deposition resulting in decreased extracellular matrix (ECM) flexibility. 20 Fibrosis within the adipose tissue compartment is one of the factors that may influence the development of obesityrelated comorbidities, 21 and this parameter can also be measured using the techniques mentioned above. However, the metabolic dysfunction associated with fibrosis may also depend on which fat pads are affected.

Obesity and comorbidities were furthermore studied from an interesting and newly developed point of view which consists in visualizing the potential interrelationships among human diseases and set up a network in which connections are based on common genetic or functional origins. This aims to understand the complex intertwined factors that may be part of an individual disease in order to analyse all the disorders and diseases of an organism as a whole. 22 According to this concept, a representation of obesity and T2DM "diseasome" was proposed, 23 (figure 3). One 2013) 23 Basic research is particularly helpful in deciphering mechanistic aspects underlying the dysregulation of metabolism, insulin resistance and inflammation that is often not possible through human observational studies. By contrast, suitable adaptations of human conditions to animal models allow intervention studies that can greatly facilitate mechanistic, hence drugable facets of pathology. For instance, basic research has extended our understanding about the pathophysiological role of adipose tissue in obesity-related cardiometabolic diseases. Further to the identification of epicardial adipose tissue as being profibrotic for the adjacent myocardium, 24 our group has uncovered the important role of remote visceral adipose tissue in promoting cardiac fibrosis in the context of obesity and aging in a mouse model. 25,26 Chapter 3: Animal models

The great degree of anatomical and physiological similarities in humans and mammals encouraged to investigate mechanisms and assess novel therapies in animal models before applying to humans. To further underscore the importance of in vivo models, assessment of physiological functions and systemic interactions between organs requires a whole organism that is not available via in vitro studies. Several human diseases affect also animal species such as allergies, cancer, epilepsy, myopathies, hypertension, and Type I diabetes. The mechanisms are also very similar in most of the cases and 90% of the veterinary drugs used to treat animals are identical or very similar to those used to treat humans. 27 Moreover, a number of major discoveries in basic science and medicine have been possible thanks to observations and testing on animals. A relevant example is the treatment of Type I diabetes by insulin that was established in the dog by Banting and McLeod, who received the Nobel Prize in 1921 for their discovery. 28 However, not all results obtained in animals are confirmed in humans. Several reasons were proposed to explain these failed translations. For example, some studies use inappropriate models or neglect the differences between the model used and humans when the percentage of homology is very high. Also, over 95% of genes are homologous between mice and humans, 29 yet, this does not exclude physiological discrepancies due to differences in the members of gene families, gene redundancies and nuanced interspecies differences in the regulation of expression. Accordingly, these aspects are increasingly better described and understood. 27 Several animal models are available for the study of obesity, which are classified into monogenic and polygenic models. The monogenic models are characterized by mutations in single genes having important roles in metabolism. For example, leptin gene in ob/ob (obese) mice, leptin receptor in db/db (diabetic) mice, tub (tubby) mice, Zucker fatty rats (ZFR), and carboxypeptidase E gene in fat/fat mice. The polygenetic models of obesity are lines of mice or other animals differing in body weight gain and fat deposition dynamic. One of the more intensively studied models of polygenic obesity is the C57BL/6J mice submitted to high fat diet (HFD), also known as model of diet-induced obesity (DIO). 30,31 SECTION 2 ADIPOSE TISSUE:

PHYSIOLOGICAL STRUCTURE & FUNCTION Chapter 1: Diversity of adipose tissue depots in humans and mice

Adipose tissue in humans and mice is divided into two major subtypes: white adipose tissue (WAT) and brown adipose tissue (BAT).

BAT is localized mostly within the supraclavicular region in humans 32 and within the interscapular region in mice but also within the supraclavicular region like in humans. 33 Brown adipocytes contain multilocular lipid droplets and a high number of mitochondria expressing the mitochondrial uncoupling protein 1 (UCP1). Their primary function is to dissipate energy in the form of heat (instead of producing ATP), 34 a thermogenic function that may have a role in the regulation of energy balance. 35 WAT is the major adipose tissue in mammals and is mainly composed of large adipocytes with a single lipid droplet and much fewer mitochondria than brown adipocytes. However, inducible brown adipocytes, also known as beige adipocytes, can be found inside WAT. Beige adipocytes are phenotypically distinct from both white and brown adipocytes but functionally closer to brown type. Their accumulation in WAT is termed browning and is induced by cold exposure or exercise. 36 It has been suggested that browning process could have beneficial effects on metabolism. Hence, the potential therapeutic role of browning in obesity and aging has gained a lot of attention. 37,38 There are two anatomically and functionally distinct subtypes of WAT: visceral white adipose tissue (VAT) and subcutaneous white adipose tissue (SAT). VAT in humans consists in omental, mesenteric, retroperitoneal, gonadal and pericardial WAT 39 whilst SAT is located in upper (deep and superficial abdomen) and lower (gluteofemoral) body regions. 40 In mice, VAT is composed of perigonadal, perirenal and mesenteric WAT, and SAT is located all around leg muscles in the inguinal region (figure 4). On average, SAT represents more than 80% of total body fat in humans and act as a protective barrier against external mechanical stress, dermal inflammatory processes and heat dispersion. 40 Clinically, it has been found to have beneficial effects on metabolism. 41 By contrast, VAT represents 10-20% of total body fat in men and 5-10% in women 10 and is commonly associated with metabolic disorders, such as diabetes and cardiovascular disease. 42 In addition to major WAT pads, small tissue-associated or -infiltrating adipose tissue, also known as ectopic fat is distributed across the body, 43 (figure 4).

In subsequent sections, WAT will be in focus, and "adipose tissue" or "AT" refers to WAT. Chapter 2: Adipose tissue cell types and their functions

I) Adipocytes

Mature adipocytes are the parenchymal cells of adipose tissue. They have a key function in regulating lipid metabolism and maintaining normal energy homeostasis by controlling fatty acid (FA) turnover through lipogenesis and lipolysis. As such, mature adipocytes have an important role in the regulation of circulating levels of FA and glucose.

Circulating FAs are derived from the combination of dietary fat intake and endogenous de novo synthesis. Fatty acids serve as substrates for energy production and precursors for the synthesis of lipids involved in the membranes (triglycerides) and the cellular signaling (e.g.

prostaglandines and leukotrienes). Despite the importance of FAs in physiological processes, their oversupply results in toxic cellular effects. This lipotoxicity is characterized by disruption of biological membrane integrity, alteration of cellular acid-base homeostasis and generation of harmful bioactive lipids. These adverse effects, in turn, induce endoplasmic reticulum stress, mitochondrial dysfunction, inflammation and cell death. [START_REF] Alcendor | Sirt1 regulates aging and resistance to oxidative stress in the heart[END_REF][START_REF] Tocchi | Mitochondrial dysfunction in cardiac aging[END_REF] To avoid toxicity of free FAs (FFAs), adipocytes are able to detoxify non-esterified FAs by esterification with glycerol to produce inert triacylglycerols (TAGs), a process called lipogenesis.

1.1) Lipogenesis

The canonical stimulant of lipogenesis is insulin. Prior to lipogenesis per se insulin increases glucose uptake through glucose transporter GLUT4 into adipocytes and induces glycolysis. The combined action of glycolysis and pyruvate dehydrogenase activity provides glycerol-3phosphate (the backbone of TAGs) and acetyl-CoA (the FFA precursor), respectively. FFAs, as previously mentioned, can also be derived from dietary fat intake. In that case, lipoprotein lipase (LPL), secreted by adipocytes and localized within capillaries lumen is a key enzyme that hydrolyzes TGs contained in chylomicrons and very low-density lipoproteins (VLDL) to provide FFAs, [START_REF] Ravaglia | Plasma amino acid concentrations in patients with amnestic mild cognitive impairment or Alzheimer disease[END_REF] (figure 5).

1.2) Lipolysis

During fasting or sympathetic activity, lipogenesis is inhibited, whilst lipolysis is induced. These states are typically characterized by low insulin concentrations as well as activation of the βadrenoreceptor signaling by catecholamines released from sympathetic nerves stimulated by low blood glucose levels. Consequences of these upstream events are increased TAG breakdown followed by the release of glycerol and FFA from adipocytes. At least three different enzymes are involved in this process:

-Adipose triglyceride lipase (ATGL) catalyzes the initial step of lipolysis by converting TAGs to diacylglycerols (DAGs).

-Hormone-sensitive lipase (HSL) is mainly responsible for the hydrolysis of DAGs to monoacylglycerols (MAGs) and finally, -MAG lipase (MGL) hydrolyzes MAGs.

ATGL and HSL are responsible for more than 90% of TG hydrolysis subject to β-adrenergic stimulation, activation of adenylate cyclase and PKA, [START_REF] Ravaglia | Plasma amino acid concentrations in patients with amnestic mild cognitive impairment or Alzheimer disease[END_REF] (figure 6). (From Bódis, K. & al., Eur J Clin Invest. 2018) [START_REF] Ravaglia | Plasma amino acid concentrations in patients with amnestic mild cognitive impairment or Alzheimer disease[END_REF] Adipocytes express 4 types of adrenergic receptors: beta1, beta2, beta3 and alpha2. Beta receptors are mostly expressed in VAT, and catecholamine-induced lipolytic response is higher in VAT. Alpha2-receptors (anti-lipolytic effect) are highly expressed in SAT. On the other hand,

VAT is less sensitive to insulin as opposed to SAT so insulin-stimulated lipogenesis is preferentially occurring in the SAT. 10,39,[START_REF] Ravaglia | Plasma amino acid concentrations in patients with amnestic mild cognitive impairment or Alzheimer disease[END_REF] These characteristic differences may explain why SAT is a metabolically healthier fat pad compared to VAT.

1.3) Secretory activity

The discovery of leptin in 1994 [START_REF] Denicola | Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis[END_REF] and adiponectin one year later [START_REF] Wu | Geroncogenesis: metabolic changes during aging as a driver of tumorigenesis[END_REF] led to a widespread recognition of adipose tissue as an endocrine organ. Other adipose tissue-secreted signaling mediators, known as adipokines, have been identified since then, and were involved in the communication linking adipose tissue with other organs of the body with a role in maintaining systemic homeostasis in physiological conditions or participating in organ remodeling or failure in pathological processes such as aging and obesity (see Section 3, chapter 4, part V).

Leptin, a 16 kDa protein hormone, is an adipokine secreted by adipocytes, acting to regulate food intake and energy expenditure. This occurs by communicating information about the degree of adiposity to the brain. Specifically, increasing adiposity enhances plasma leptin levels, which then reduces food intake by inhibiting secretion of neuropeptide Y and Agoutirelated protein in the hypothalamus, whilst simultaneously boosts energy expenditure. [START_REF] Deelen | A metabolic profile of all-cause mortality risk identified in an observational study of 44,168 individuals[END_REF]51 Much of our understanding about leptin's role in metabolic regulation comes from observations made in leptin deficient ob/ob mice and congenital leptin deficient humans.

Importantly, both affected mice and humans show severe obesity, together with glucose intolerance and insulin resistance, whilst symptoms were reversed by leptin administration. 52,53 Leptin exerts its effect on target tissues by binding to its receptor Ob-R.

This receptor is found in the brain but has also been identified in peripheral tissues such as adipose tissue, skeletal muscle, liver, pancreatic beta cells, blood mononuclear cells, lymphocytes, and endothelial cells. Through its insulin-like effects on glucose metabolism leptin is considered as potential treatment to attenuate hyperglycemia in type 1 diabetes in mice, rats, and lipodystrophy syndromes in humans. [54][55][56] Expression and secretion of leptin is subject to complex regulation with a plethora of factors involved. For example, leptin is upregulated by insulin, glucocorticoids, TNFα, estrogens, and CCAAT/enhancer-binding protein-α and downregulated by β3-adrenergic activity, androgens, free fatty acids, growth hormone, and peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists. 57 Leptin is not the only adipokine that adipocytes secrete. As a matter of fact, after the discovery of leptin, a number of other adipokines have been identified, the most important of which is adiponectin. As opposed to leptin levels, plasma levels of adiponectin are elevated in lean human subjects and reduced in with obesity and type 2 diabetes. 58,59 Adiponectin secretion is upregulated via PPAR-γ agonists such as the anti-diabetic drugs thiazolidinediones (TZDs) and this contributes to improve insulin sensitivity as extensively demonstrated in animal and human subjects treated with TZDs. 60 Interestingly, adiponectin as much as leptin are predominantly produced in SAT rather than VAT.

Beyond adipokines, adipose tissue is a highly dynamic secretory organ that employs a plethora of lipids, metabolites, noncoding RNAs, and extracellular vesicles (EVs) to relay information to other organs of the body. EVs -encompassing microparticles, exosomes, and apoptotic bodies -are emerging as a novel mode of communication between cells and have been recently implicated in physiological and pathological processes. 61 For instance in metabolic syndrome, they were shown to promote liver fibrosis by up-regulating tissue inhibitor of matrix metalloproteinase (TIMP)-1 and integrin ανβ-5, whilst downregulating some matrix metalloproteinases (MMP-7 and MMP-9). 62 Furthermore, EV isolated from obese adipose tissue of mice induced an M1-like macrophage polarization with secretion of proinflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, and led to the development of insulin resistance when injected in vivo to WT mice. 63 (figure 7).

II) Adipose-derived stem cells (ADSCs) and preadipocytes

Adipocyte progenitors are mostly localized in perivascular areas where adipogenesis starts. 64,65 Adipogenesis is the process that results in the formation of mature adipocytes. It is divided into two major stages: the determination phase -or commitment -and terminal differentiation. The determination phase is the conversion of the ADSC into a preadipocyte without any change in morphology but with a loss of multipotency. A fundamental property of preadipocytes is their ability to constantly proliferate and differentiate into mature adipocytes, allowing adipose tissue to have plasticity and capacity for expansion. 65 Terminal differentiation refers to the mechanisms that a committed preadipocyte undergoes transformation into a mature adipocyte, starting with growth arrest, followed by mitotic clonal expansion before final differentiation. 64 Adipogenesis is mainly regulated by preadipocytes themselves. Underlying regulation involves several pathways in a complex organization and at different levels. WNT signaling pathway for example promotes adipogenesis through upregulation of WNT5B gene or inhibits it by upregulating WNT10 gene. Hedgehog signaling pathway suppresses preadipocyte differentiation by downregulating peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2) expression. Transforming growth factor β (TGFβ) and preadipocyte factor-1 (Pref-1/ Dlk1) are other inhibitors of the adipogenic transformation of preadipocytes, whilst bone morphogenic proteins (BMPs) promote the commitment of pluripotent stem cells to the adipogenic lineage. This complex regulation ensures control of preadipocyte homeostasis within adipose tissue. 64,[66][67][68][69][70][71] Moreover, several other parameters modulate adipogenesis such as extracellular matrix (ECM) composition and stiffness, 72 the secretome of adipocytes and SVF cells, 73 epigenetics, 74 miRNAs, 75 androgenic hormones, 76 etc…

In addition to responsiveness to extrinsic factors, intrinsic cell properties are known to shape preadipocyte dynamics. Tchkonia & al. reported the existence of two different preadipocyte subtypes in humans, which differ in their capability for replication and differentiation. 77 Taken together, the differences observed between fat pads come down to inherent differences in a highly complex regulatory circuit.

During differentiation, as the cells progress from immature stem cells to committed preadipocytes and eventually to mature adipocytes, the cell surface markers change accordingly, with CD34 being the most frequently used preadipocyte cell marker. 65 However, a large variety of cell surface markers expressed in ADSCs and committed preadipocytes has been reported 64,65,78,79 (Table 1). The origin and fate of preadipocytes are not entirely clear; several lines of evidence support mesenchymal origin but hematopoietic origin is not altogether excluded. 80 Preadipocytes are mainly giving rise to mature adipocytes but when put in adequate environment they are able to differentiate into other cell types, like for example macrophages. This property was demonstrated in an elegant set of experiments by Charriere & al: when injected into the peritoneal cavity or co-cultured with macrophages, preadipocytes were rapidly and efficiently converted into macrophages. 81 Other studies showed that macrophages also can transform into preadipocytes when co-cultured with ADSCs and mature adipocytes, further showing a significant plasticity between these cells. 73

III) Endothelial cells and their progenitors

Mature adipocytes are not the only cells found in adipose tissue, in normal state, they represent one-third of the cells in total ; the remainder is a combination of a heterogenous collection of cells known as the stromal vascular fraction (SVF), which includes mesenchymal stem cells (MSCs), preadipocytes, fibroblasts, endothelial cells along with their progenitors, and immune cells. [82][83][84][85][86] Endothelial cells (ECs), pericytes and endothelial progenitor cells (EPCs) are found in the adipose tissue as a part of the SVF. ECs constitute the vessels and are surrounded by pericytes. EPCs are circulating and attracted by angiogenic factors produced by cells under hypoxic conditions. Adipose tissue expansion requires vessel remodeling and growth. The chain of events is as follows: tissue expansion creates a hypoxic microenvironment, which induces lactate production by adipocytes and preadipocytes, IL-8

secretion by macrophages and pro-angiogenic factors including VEGF, TGF-β, angiopoietins, leptin and others by adipocytes and other adipose tissue cells. 87 The combination of these factors facilitates EPCs recruitment and differentiation to form new vessels in a process called angiogenesis. ECs and EPCs are identified by their specific marker CD31 and studies showed that they may represent 25% of total SVF cells. 88,89

IV) Adipose tissue immune cells

Immune cells, also known as leucocytes, are normally found in adipose tissue, interspersed with adipocytes. They are the local sentinels of the immune system.

Leucocytes originate from bone morrow and/or lymphoid organs and found in blood/lymphatic circulation system, with some of them becoming tissue residents. They are divided into 3 subtypes: granulocytes, lymphocytes, and monocytes. (MMe) or "oxidized" (Mox) macrophages, which are both associated with insulin resistance, 92- 96 (figure 9).

Granulocytes

Introduction

During obesity, as a consequence of calorie overload, adipose tissue undergoes progressive expansion and remodeling leading to functional alterations with adverse effects on metabolism locally and systemically. 97 In physiological condition, hyperglycaemia following food intake stimulates insulin secretion in pancreatic β-cells. Insulin stimulates then glucose uptake and lipogenesis in adipocytes through PI3K/Akt signalling pathway and adipose tissue homeostasis is preserved. [START_REF] Ravaglia | Plasma amino acid concentrations in patients with amnestic mild cognitive impairment or Alzheimer disease[END_REF] In the context of calorie overload, adipose tissue maintains whole body energy homeostasis through hyperplasia (cell number increase ie. increased adipogenesis) and hypertrophy (cell size increase). 98 In obesity, excessive lipogenesis, together with impaired adipogenesis leads to adipocyte hypertrophy, lipotoxicity, cell dysfunction and death. [START_REF] Ravaglia | Plasma amino acid concentrations in patients with amnestic mild cognitive impairment or Alzheimer disease[END_REF]99 Adipocyte death by necroptosis has been shown to dramatically increase in obese mice and humans. 100,101 Adipocyte hypertrophy and death results in excessive leptin, cytokine and chemokine secretion leading to immune cell recruitment and formation of crown-like structures (CLS)i.e. macrophages and other immune cells surrounding dead or dying adipocytes. 100 Unlike acute inflammation happening in physiological condition, inflammation in obese AT becomes persistent and systemic over the time, which is known as chronic low-grade inflammation. 91

Chapter 1: Obesity and insulin resistance

I) Insulin signaling pathway in adipocytes

Insulin is an endocrine peptide hormone that binds intramembrane receptors in target cells to orchestrate an integrated anabolic response to disposable nutrients.

Although insulin receptors are expressed in many cell types, the role of insulin in glucose homeostasis is mainly characterized by insulin's direct effects on skeletal muscle, liver, and white adipocytes. 102 In skeletal muscle, insulin increases glucose uptake and glycogen synthesis. 103,104 In liver, it promotes glycogen synthesis, increases lipogenic and decreases gluconeogenic gene expression. [105][106][107][108] In WAT, our focus here, insulin inhibits lipolysis, induces lipogenesis, and enhances glucose uptake. The antilipolytic effect of insulin is mediated via insulin-dependent activation of adiposespecific phospholipase A2 (AdPLA2) which inhibits adenylate cyclase and also via Akt activated mammalian target of rapamycin (mTORC1) which inhibits β-adrenoreceptor-induced ATGL transcription. [START_REF] Ravaglia | Plasma amino acid concentrations in patients with amnestic mild cognitive impairment or Alzheimer disease[END_REF] On the other hand, mTORC1 induces lipogenesis through activation of sterol regulatory element-binding protein (SREBP1c), [START_REF] Ravaglia | Plasma amino acid concentrations in patients with amnestic mild cognitive impairment or Alzheimer disease[END_REF] (figure 11).

Insulin also promotes adipogenesis through the transcription factor peroxisome proliferatoractivated receptor-γ (PPARγ). 110

Stimulation of glucose transport into adipocytes is another main function of insulin, despite the fact that it only accounts for a small fraction of whole-body glucose disposal. 111 This process is mediated by PKB/Akt which promotes Glucose transporter 4 (GLUT4) translocation into cell membrane, (figure 11). Internalized glucose provides a source of glycerol-3phosphate to which fatty acids can be esterified, and insulin enhances lipoprotein lipase activity in adipose tissue endothelium. At the level of the whole organism, indirect effects of insulin via tissue cross-talk are also important and sometimes predominate over direct effects. Among several examples, a particularly well-studied one is the insulin-adipocyte-hepatocyte axis and its control of hepatic glucose production where insulin suppresses hepatic gluconeogenesis by inhibiting lipolysis in WAT. 114,115

II) Mechanism of insulin resistance in adipocytes

When higher circulating insulin levels are needed to achieve the integrated glucose-lowering response described above, we can talk of insulin resistance. Hyperglyceamia and hyperinsulinemia are both features of insulin resistance as we can see in T2D patients.

Glucose and lipid overconsumption, usually seen in obesity create a toxic environment for organs and are responsible for the alterations in both insulin target tissues and pancreatic βcells. The defective insulin action at target cells is due to receptor defects together with decreased surface insulin receptor (INSR) content, but also to post-receptor defects, i.e impaired signal transduction. In adipocytes, as in muscle and liver, a decrease in tyrosine kinase activity in T2D patients was shown. 116,117 Glucose transport alterations in adipocytes resulting from INSR dysfunction aggravate the situation even though WAT is not a quantitatively significant site of insulin-stimulated glucose disposal, accounting for <5% of an oral glucose load in humans. 118,119 Yet adipose-specific deletion of GLUT4 in mice resulted in liver and skeletal muscle insulin resistance without any change in body weight 120 , highlighting once again the importance of indirect effect of insulin through cross-talk between organs and the role of adipokines such as, in this case, retinol binding protein 4 (RBP4) and nicotinamide N-methyltransferase, 121,122 (figure 12).

The impact of adipocyte insulin resistance on lipid metabolism is of immense pathophysiological importance, considering that AT is the major source of plasma FFA, and that lipolysis is a critical and very fast regulator of hepatic gluconeogenesis which regulates fasting glycemia, [123][124][125] (figure 12). However, whether insulin resistance to suppression of adipose lipolysis involves primarily increased lipolysis per se, decreased re-esterification of FA, or both is not clear, although some studies move toward lipid storage defects rather than lipolysis defects. 126,127 Interestingly, unlike liver and skeletal muscle, the specific molecular defects related to adipocyte insulin resistance are largely uncharacterized. Studies have mostly focused on autocrine and paracrine effects of inflammatory cytokines and their impact on insulin signalling, 128-130 despite the fact that some studies have shown that insulin resistance was evident after few days of HFD feeding and before significant inflammation develops,

suggesting that non-inflammatory mechanisms are probably responsible for the initial dysfunction leading to adipocyte insulin resistance. (TLRs), a family of transmembrane proteins that play an important role in the innate immune system. 133,134 Inflammatory response can also be activated in case of tissue injuries. Regardless of the nature of the evoking stimulus inflammation trends towards resolution and homeostasis.

In the context of obesity and metabolic syndrome, local and systemic inflammation is observed, but the process is quite different, as the resolution part is absent, and inflammation becomes chronic and progressively harmful. This process is known as chronic low-grade inflammation. 133,134 Evidence that obesity causes inflammation started emerging in the 1990s. Hotamisligil et al. showed that genetically obese rodents had increased expression of the TNF-α gene in WAT and that the neutralization of TNF-α protein by anti-TNF-α antibodies decreased the resistance of these animals to insulin action, hence, establishing the link between obesity, inflammation and insulin resistance. 135 During obesity, saturated fatty acids, particularly lauric acid and palmitic acid present in high concentration in AT stimulate inflammatory response through the TLR4 signaling pathway. 136

TLR4 is expressed in innate immune cells, including macrophages, but also in other cell types such as adipocytes. Once the FA is bound to TLR4, it activates the kinase proteins JNK-1 and IKK-β, which in turn activate the transcription factors nuclear factor kappa B (NF-κB) and the activating protein (AP-1). Both proteins translocate to the nucleus and activate the transcription of many genes encoding pro-inflammatory cytokines including TNF-α , COX-2, and IL-6 137,138 and chemokines such as MCP-1. 139 In adipocytes, JNK-1 and IKK-β are also capable of phosphorylating IRS-1 at the serine 307 residue, hence reducing IRS-1 interaction with the insulin receptor beta subunit, which results in decreased signal transduction and insulin resistance, 140,141 (Figure 13). Initial obesity-associated inflammation is generated as a stress response by the adipocytes themselves, and by the subsequent necroptotic cell death probably induced by adipocyte hypertrophy. 100 Then, progressively immune cells become more potent sources of inflammation inside AT. 91,142 Within the expanding AT, the recruitment of cells of the innate immune system such as macrophages, dendritic cells, neutrophils, and mast cells increases. 91,143 These cells play in fact a key role in the initiation of inflammatory response and RBP4 was shown to have a crucial role in regulating and activating this process. 144 Recruited macrophages in turn activates adaptive immune cells and both B and T cells accumulate in the tissue (figure 14). (From Bharath, L. P. & al., Compr Physiol. 2017) 91 Macrophages correspond to about 40% of total WAT cells in obese human and mice as compared to only 18% in lean controls, and their accumulation was shown to be directly proportional to adiposity in both models. 145,146 The increase in ATM number is partly due to recruitment from monocyte trafficking and partly due to local proliferation of recruited macrophages. 92 ATM cell populations are highly plastic during obesity and tend to switch toward pro-inflammatory phenotype (M1, MMe) with secretion of cytokines and chemokines such as IL-6, TNF-α, IL-12 and MCP-1, whereas lean AT is dominated by M2 macrophages and production of anti-inflammatory cytokines such as IL-10, IL-1Ra, IL-4 and IL-13. 147 A cross-talk between macrophages and adipocytes via cytokines and adipokines production and secretion contributes also significantly to the increasing inflammation and dysfunction, 134 (figure 15). Obesity have been shown to affect also the distribution of lymphocytes in AT. B and T cells accumulate, except for Treg population which decreases. 143,156 Treg cells have an anti-inflammatory effect, directly, through their TCR receptor or indirectly, through cytokine production. 157,158 B and T cells (CD4+ and CD8+) participate to sustain inflammation and promote insulin resistance, and their depletion in mice showed protection from metabolic disturbance in response to HFD feeding, despite important weight gain. 159,160 Chapter 3: Obesity and adipose tissue fibrosis

Fibrosis is defined as an excessive accumulation of extracellular matrix (ECM) components, which results from an imbalance between excess synthesis of fibrillar components such as collagens and fibronectin, and an impairment in degradation of these proteins. ECM production is part of a regenerative step occurring in response to tissue injury and inflammation, however, if the damage persists, like in the context of obesity and low-grade inflammation, central cellular actors remain activated, and continuously produce fibrillar molecules that accumulate in the tissue as fibrotic deposition. 20 Thus, while ECM synthesis and tissue remodeling are initially a physiological response with beneficial effect, excessive accumulation of fibrosis in the absence of resolution of inflammation have deleterious effect on tissue homeostasis and function. 20,161 Fibrosis has been extensively studied in the liver, as we will describe later, whereas adipose tissue fibrosis has been recently identified in obesity and associated with metabolic disorders. Now it is considered as a hallmark of metabolically dysfunctional AT (figure 17). The components involved in the formation of adipose ECM are mostly collagens and fibronectin with collagen type I being the major ECM framework, hence playing a key role in the structure and function of mesenchymal tissues. 20 Collagen type VI is another major ECM protein found in AT, which contribute to ECM stability. Mature collagen VI is composed of three subunits: α1, α2, and α3, which are required for stable formation of the protein and are regulated both at transcript and posttranslational levels. 20 Obese mice lacking collagen VI showed reduced AT fibrosis and improved metabolic parameters. 162 During the onset of obesity, adipose tissue expansion and adipocyte hypertrophy is faster than neovascularization and, rapidly, adipocytes reach the diffusional limit of oxygen. This hypoxic environment causes an increase in the level of hypoxia-inducible factor 1α (HIF-1α) and the activation of HIF target genes including collagen type I, alpha 1 (COL1A1), COL3A1, and lysyl oxidase, an enzyme that mediates cross-linking of collagens, [163][164][165] (figure 18). Inhibition of HIF-1α activity both pharmacologically and genetically resulted in significant metabolic improvements and reduction of adipose tissue fibrosis and immune cell infiltration. 166 Hypoxia also stimulates cytokine and chemokine release from AT macrophages and inhibits preadipocyte differentiation by down regulating PPARγ in these cells. 167 In parallel, TGF-β, whose levels correlate with adiposity in humans and rodents, also inhibits adipogenesis by upregulating connective tissue growth factor (CTGF) in adipocytes. 168 In AT, ECM components are mostly produced by stromal vascular cells, among which preadipocytes and macrophages are central players. Both express and secrete collagens, but more importantly, they tightly regulate fibrogenesis process under both physiological and pathological conditions, 169,170 (figure 18).

In vitro studies demonstrated that, when they are in contact with activated macrophages, preadipocytes display a fibroblastic-like phenotype and produce high level of ECM molecules such as collagen I, tenascin-C, fibronectin and its receptor α5-integrin, and activin A. 169,171 Marcelin et al. identified a special subset of progenitor cells: the CD9 high PDGFRα+ progenitors that become profibrotic in obese patients and mice and responsible for AT fibrosis with a loss of tissue expandability and increased insulin resistance. 172 Macrophages regulate fibrogenesis process also by releasing chemokines that attract fibroblasts and other inflammatory cells. On the other hand, macrophages produce soluble mediators including TGF-β1 and platelet-derived growth factor (PDGF) which activate fibroblasts and control ECM dynamics by regulating the matrix metalloproteinases (MMPs, a family of endopeptidases that cleave collagenous proteins) and their inhibitors (TIMPs). 173 Activated fibroblasts differentiate into α-smooth muscle actin (SMA) expressing myofibroblasts to produce collagens, elastins, and proteoglycans. Obesity-induced AT fibrosis may induce rigid extracellular environment that restrains adipocyte expansion and then triggers adipocyte cell death and inflammation. In this context, AT growth is limited, and excess lipids are stored in ectopic tissues (figure 19). This was further confirmed by observations made in collagen VI knockout obese mice that showed increased flexibility of the ECM and bigger adipocytes compared to obese control mice, together with reduced cell death and inflammation and subsequently improved metabolic situation. 165 Giving the important role of AT fibrosis in obesity-associated metabolic dysfunction, targeting this parameter seems to be of interest in the context of clinical research. Indeed, studies have shown that collagen levels are reduced by adiponectin and by treatment with PPARγ agonists in mouse AT. 162 However, in patients, AT fibrosis is still persistent two years after surgicalinduced body weight reduction.

I) Definition of cellular senescence

Cellular senescence was originally identified by Hayflick and Moorhead, in 1965, as a state of "permanent" cell cycle arrest resulting from the limited proliferative capacity of human fibroblasts in culture condition (replicative cellular senescence). 179,180 This process was later associated with telomere shortening or dysfunction which triggers DNA damage response (DDR) and subsequent cell cycle arrest. [181][182][183][184] However, senescence can also be induced in response to other stress conditions such as oncogene activation, oxidative stress, or metabolic stress before any telomere shortening. This is defined as premature cellular senescence. 185 Thus, senescence is rather defined as a state of stable cell cycle arrest in response to various stresses. 182,184,186 Cells lose their ability to proliferate, ensuring they do not become cancerous. Senescent cells are usually characterized by a flattened morphology, a higher activity of β-galactosidase, cell cycle arrest, they are metabolically active, and they produce a wide range of cytokines, chemokines, matrix-remodeling proteases and growth factors.

Collectively, these secreted factors are known as senescent-associated secretory phenotype (SASP). 187 Recent data suggest that the SASP is not a unique entity, but functionally distinct senescent states depending on the stress-inducing condition, the cell type, and the time that the cells were maintained in senescence. 188 Important distinctions include senescence with or without persistent DNA damage that would lead to the activation of distinct signalling pathways.

II) Replicative cellular senescence

Replicative cellular senescence is induced by telomere shortening. Telomeres are regions of repetitive nucleotide sequences at each end of a chromosome (5'-TTAGGG-3' for vertebrates).

Each round of DNA replication reduces the telomere length because the telomerase cannot continue their duplication all the way to the end of a chromosome, so after each duplication the end of the chromosome is shortened and once the critical length is reached, cell division is stopped, [189][190][191] (figure 20). Critically short, uncapped telomeres induce DDR which triggers senescence. 192 In vivo, replicative senescence is a physiological process accumulating with

III) Premature cellular senescence

As previously mentioned, senescence can be activated through telomere-independent mechanisms and independently of the age (figure 21). Oncogene-induced senescence (OIS) appears in response to the hyperactivation of oncogenes such as Ras or Raf, or the inactivation of tumor suppressors like PTEN. [195][196][197] The oncogenic stress leads to hyperproliferation with increased DNA replication and accumulation of incomplete replication intermediates. This activates DDR which induces senescence. 195,[198][199][200] Oxidative stress, with the generation of reactive oxygen species (ROS), leads to the accumulation of DNA single-strand breaks, activation of DDR and finally emergence of premature senescence. 185,[201][202][203] Metabolic stress is another factor inducing premature senescence. Excessive calorie intake was shown, for instance, to promote oxidative stress and senescence in the adipose tissue of mice. 176,204 High glucose levels were also shown to be responsible for in vitro senescence induction in other cell types such as endothelial cells 205 and proximal tubule cells 206 . IV) Biomarkers of senescence

1.2) Morphological and metabolic changes

Senescent cells are characterized by a specific morphology, they are enlarged and flattened when compared to their dividing cell counterparts. They have several vacuoles and sometimes more than one nucleus. In addition, nuclear membrane integrity is disrupted due to a downregulation of Lamin B1 gene expression, a protein that plays an important role in nuclear structure. 208,209 Furthermore, accumulation of dysfunctional mitochondria and increased levels of reactive oxygen species (ROS) are observed in senescent cells. 210 Lysosomal activity in those cells is also altered, resulting in increased levels of β-galactosidase activity at pH 6.0.

Senescence-associated β-galactosidase (SA-β-gal) is now a common and widely adopted biomarker of cellular senescence. 211

1.3) Chromatin reorganization and changes in gene expression

Chromatin is composed of DNA wrapped around nucleosomes that are formed from histones and associated proteins that bind DNA or the histones. The canonical histones are highly synthesized in S phase to package the newly replicated DNA. Non-canonical histone variants are endowed with specific functional properties determined by their non-canonical protein sequences and their constitutive expression in contrast with the replication-dependent expression of canonical histones.

Chromatin reorganization and formation of senescence-associated heterochromatin foci (SAHF) are another feature of senescent cells (figure 23). These modifications in chromatin likely contribute to differential genome expression and the maintenance of the senescent state. 195,212 SAHFs were originally described in oncogene-induced senescence (OIS) and DNA damage-induced senescence. 213 Those structures are enriched for repressive epigenetic marks, including lysine 9 di-or-tri-methylated histone H3 (H3K9me2/3), heterochromatin protein 1 (HP1), and macroH2A, whereas euchromatic marks are absent. 213,214 SAHFs structures were shown to play an important role in silencing genes that promote proliferation, like E2F-target genes. 213 Certain histone variants and modifications do not just coincide with senescence, but they seem to have a functional role in inducing senescence. Specifically, a recently discovered Nterminal proteolytic product of histone H3.3 represses the expression of proliferation genes. 215 Moreover, macroH2A1 has been implicated in the feedback regulation of SASP gene expression during RASval12-induced senescence. 216 The epigenetic angle in regulating senescence is further underscored by observations suggesting that histone methyltransferase MLL1 is required for the expression of SASP components in oncogene-induced senescence. 217 Most recently, the Mann group identified H2A.J, a novel histone variant, differing from the canonical H2A by only five amino acids with an emerging functional role in senescence, aging and cancer. 218 Activation of DDR pathways is involved in both the induction and maintenance of senescent state. DDR kinases including ATM, ATR, CHK1, and CHK2, and other proteins like p53 and CDC25 are the main effectors of this pathway, 198 (figure 22). Another indicator of DNA damage is the phosphorylated form of H2A.X, a variant of the H2A protein family, which is required for checkpoint-mediated cell cycle arrest and DNA repair following double-stranded DNA breaks. 220

1.5) Senescent-associated secretory phenotype (SASP)

Senescent cells are characterized by pro-inflammatory SASP that mediates non-cell autonomous effects of senescence. This secretome is comprised of a complex mixture of cytokines, chemokines, growth factors, and proteases, among others. 191,221 However, the precise composition of this secretome depends on many factors such as cell type, micro-environment, or senescence-inducing stimulus, and its regulation is achieved at different levels, from transcriptional regulation to autocrine feedback loops. 187,222 Interestingly, persistent DDR appears to be critical for the development of the SASP. 187 SASP has both beneficial and deleterious effects, for example, it mediates immune cell recruitment to senescent cells and/or cancer cells in order to facilitate their clearance, which prevents tumor development. 223,224 Paradoxically, however, SASP have been shown to promote cancer cell progression via secretion of pro-angiogenic factors and other proteins involved in extracellular matrix remodeling. 222

1.6) Concluding remarks

The numerous features that characterize senescent cells make them easy to identify using specific markers (figure 24).

However, the described mechanisms of senescence are not necessarily dependent on one another and hence a senescent cell might not display all the biomarkers that we mentioned.

For instance, it has been shown that expression of CDK inhibitors p16 INK4A and p21 CIP1 is not always associated with DNA damage, and in this case, this senescent phenotype lacks a proinflammatory SASP. 182,187,[225][226][227] Moreover, most of the cells expressing markers of DNA damage in vivo, especially in a non-pathological context, are not necessarily senescent and might just be responding to a transient repairable damage. Another example is the p53-and p16 INK4A -independent induction of senescence by chromatin-dependent alteration of S-phase progression that occurs without DNA damage and checkpoint activation, but leads to cell cycle arrest. 228

V) Adipose tissue senescence in aging and obesity

Senescent cells accumulate in AT with aging 178 and obesity 193,204 and studies showed that visceral AT is more prone to senescence compared to subcutaneous AT. 229 Senescent cells have been shown to be involved in the development and progression of numerous diseases including obesity and type 2 diabetes. 176,177 Senescent AT also interferes with normal physiological functions of remote tissues and organs via SASP, inducing pathologies such as cardiovascular disease (CVD) and kidney disease. 177,230,231 In particular, our team has identified a profibrotic SASP that induces cardiac remodeling and dysfunction. 26 Hence, cell senescence is known to promote metabolic and other kinds of diseases, however, the potential role of these metabolic diseases as drivers of cell senescence and accelerators of biological aging was also proposed and extensively studied. [232][233][234][235][236] Senescent AT is characterized by several types of dysfunction. Alteration of AT renewal and defective adipogenesis were for instance linked to accumulation of AT senescent cells in aging and obesity. 237 Changes toward inflammatory status in senescent AT of both aged and obese humans and mice, 238,239 and aberrant adipocytokine production have been observed. Indeed, increased expression of plasminogen activator inhibitor-1 (PAI-1) and interleukin-6 (IL-6), 240 elevated levels of IL-1β, 241,242 TNFα, 204 and secretion of C-C motif chemokine ligand 2 (CCL2)

and C-X-C motif chemokine ligand 1 (CXCL1) accompanied with activation and migration of macrophages and neutrophils, 243 have been described.

Accumulation of senescent cells and chronic inflammation in AT in both obesity and aging give rise to insulin resistance, 244,245 and activated p53 has been proposed to be a central player given that transgenic overexpression of activated p53 results in senescence and insulin resistance, whereas its inactivation results in opposite effects in diabetic animal models. 178,204 Other senescent molecules such as p16 INK4a and p21 CIP1 were associated with insulin resistance. 246,247 However, it still remains unclear how activated p53, p16 INK4a and p21 CIP1 lead to insulin resistance in AT and whether other parameters such as inflammation are required in this context.

Although senescent AT in obesity and aging show multiple similarities, several studies demonstrated that pathways leading to senescence in these two states are different. 248,249 For instance, Zhuohao Liu et al. showed that during aging, unrestrained p53 activation in adipocytes elicits AT senescence through downregulation of murine double minute 2 (MDM2) expression, however, this protein was found to be upregulated, together with p53 protein in obese AT. 250

VI) Adipose tissue cell-type specific senescence

AT is composed of various cell types including mature adipocytes, progenitor cells, endothelial cells and immune cells. The composition as well as senescent status of those different cell populations change with aging and obesity (figure 25).

Mature adipocytes develop senescence in response to various oxidative stresses that appears with aging and obesity. P53 levels were higher in adipocytes differentiated from aged animals when compared with those differentiated from younger animals. 251 P53 levels were also found upregulated, together with p21 in adipocytes from diabetic subjects, 204 and DNA damage in adipocytes was described as an early event during the onset of obesity. 243,252 Senescent progenitor cells including preadipocytes are highly increased in obese and aged AT, 178,237 and this was shown to impair AT regeneration due to a decrease in adipogenic capacity of those cells on one hand, 253,254 and inhibition of adipogenesis in nearby progenitor cells through SASP molecules on the other hand. 237,255 Activin A, a member of the transforming growth factor superfamily which is regulated by p21 CIP1 is one of those molecules. 237,256 Adipogenesis was shown to be regulated by p16 INK4a as in the absence of this protein in progenitor cells, adipogenesis was enhanced. 257 Endothelial cells play an important role in AT by participating to lipid handling function of AT and to the regulation of adipogenesis. 258 An Impairment in endothelial function was observed in both aging and obesity, 258,259 and increased expression of the senescent marker γ-H2AX in AT endothelial cells of obese people was reported. 260 Moreover, p16 INK4a expression has been found upregulated in AT endothelial cells with aging. This increase in p16 INK4a expression was associated with a decrease in the gene expression of CD36 and adipocyte-fatty acid binding protein (A-FABP), proteins involved in lipid handling. 261 Immune cells are also affected by senescence. For instance, two populations of senescent T cells were recently identified in adipose tissue from obese and diabetic animals and humans, 262,263 and one of them was shown to increase inflammation and insulin resistance in visceral AT by producing osteopontin, a macrophage activating cytokine highly expressed in obese adipose tissue. 264 Senescent macrophages were also identified in adipose tissue of aged mice by using a mouse model with luciferase expression under the control p16 INK4a

promoter. 265 Another study showed upregulation of senescence-related genes in resident AT macrophages in response to activation of inflammasome pathway in aged mice which was linked to alterations in lipolysis in adipocytes. 266 Furthermore, Minamino et al. demonstrated that upregulation of p53 in adipocytes and macrophages induced senescence, insulin resistance and inflammatory cytokine production in mouse fat. Conversely, when they repressed p53 in adipocytes and macrophages under high-fat diet condition, using a dominant-negative approach, those effects were not observed anymore. 204 These data suggest that AT immune cell senescence play an important role in tissue dysfunction and associated metabolic disorders both in aging and obesity, however very few things are known about this and the mechanisms involved. The chronological order of events leading to and initiating AT senescence and dysfunction are unclear as well and need to be defined. diseases. 237,240,268,269 Few years later, Kirkland et al. (2015) developed the first chemical mixture of compounds that specifically target and eliminate senescent cells. 270 Dasatinib and Quercetin (D+Q) are used in combination to act as a senolytic drug which can kill cells by targeting the pro-survival pathways (as they are up-regulated in senescent cells) such as Bcl-2/ Bcl-Xl. This drug showed beneficial effects on aging-and obesity-induced metabolic disorders by improving insulin sensitivity, reducing inflammation and AT macrophage infiltration, and restoring AT integrity in mouse models. 271,272 These drugs are now being tested in clinical trials and have proved beneficial effects in diabetic kidney disease and pulmonary fibrosis. 273,274 Several other senolytics were later developed including fisetin, a quercetin analog targeting PI3K-Akt signaling pathway, 275,276 Bcl-2 inhibitor or heat shock protein 90 (HSP90) inhibitor, etc, some of which proved their efficiency and showed beneficial effects whereas some others require more investigations. 277,278 Targeting the SASP components instead of senescent cells resulted in great improvements of metabolic function, as well. Indeed, Kirkland et al. managed to improve insulin sensitivity, promote adipogenesis and reduce AT inflammation in aged mice by treating them with a pharmacological compound that inhibit pro-inflammatory SASP production. 237,279 Preventing the emergence of senescent cells by interfering with pathways that can induce senescence-associated replicative arrest are an interesting option but it carries the risk of promoting cancer, as it occurs when p16 INK4a , the retinoblastoma (Rb) protein, or p53 are diminished or inactivated. 280,281 Lifestyle interventions are another kind of treatment that proved efficiency in reducing senescence and preventing aging-and obesity-associated metabolic abnormalities. Caloric restriction for instance delays cellular senescence and increase life span. 282 Exercise and physical activity, whose beneficial effects have been shown to be mainly mediated by AT, 283,284 has both senolytic and senomorphic effects. Indeed, the number of p16 INK4a positive cells in AT of old women was shown to negatively correlate with physical activity, 285 and some other studies indicated reductions in AT pro-inflammatory cytokine expression in obese subjects. 286,287 These non-targeted approaches have the great advantage to physiologically improve metabolic parameters without any side-effects and with a more global action.

Chapter 5: Conclusion

As we explained and described in the previous sections, low grade inflammation, fibrosis and senescence are the main features characterizing the obese AT and those were extensively studied over the past few years. Obesity-induced AT inflammation was first described in 2003 by Stuart P. Weisberg et al. 288 Then the first descriptions of fibrosis as a feature of obese AT started in 2009. 162 At the same period, senescence, which up to then was only studied in the context of aging was, for the first time, associated with obesity, when Minamino et al.

identified a crucial role for AT p53 in the regulation of insulin resistance. 204 Since then, inflammation, fibrosis and senescence are extensively studied in obese individuals as well as in animal models of obesity. In particular, models of HFD-induced obesity are our main interest here, so we browsed the current state of knowledge in the literature and the models that were used i.e. durations of HFD (table 2). In exploring AT in C57BL6/Rj mice under HFD (60% of fat) little attention has been given to early events that may trigger deleterious secondary changes. More specifically these studies focused on the global changes in adipose tissue and description of senescence feature without detailing:

-the underlying mechanisms initiating the process of cellular senescence within adipose tissue -the specific role of adipose tissue cell populations -the time course of the occurrence of senescence after initiation of high fat diet -the confounding effect of age -the impact of a dysfunctional adipose tissue on remote organs such as the heart.

Our work aimed to contribute to these important unresolved questions.

Objectives of my thesis:

Obesity epidemic increases worldwide and is associated with major risk for chronic disease such as type 2 diabetes mellitus (T2DM), hypertension, and cardiovascular disease. 1,2 Despite a better understanding of underlying mechanisms, many questions remain to be answered since the role of adipose tissue senescence has recently emerged as a potential key contributor to the noxious effects of adiposity and development of a myriad of co-morbidities representing the "obesity diseasome".

Therefore, my thesis project aimed to study the senescence of visceral adipose tissue, as a key player in premature aging and more specifically in cardiac remodeling and dysfunction. 25,26,178,204 My work was organized around murine models of aging and high-fat diet-induced obesity in order to: 2) Evaluate the role of osteopontin on visceral adipose tissue remodeling and dysfunction in the context of aging, and in the cross-talk between adipose tissue and heart.

3) Understand the mechanisms initiating adipose tissue senescence by focusing on the role of mitochondria in the context of high-fat diet induced-obesity.

4) Contributing to the analysis of the mechanisms associated with cardiac aging.

67

Technical sheet n°1

Animal models

Objective & Rationale

In vivo models were used in order to assess physiological functions and systemic interactions between organs, that is not feasible via in vitro models because it requires a whole organism.

Ethics

All -Comparison between young and aged mice:

2-and 12-month-old WT male mice were used and were separated in 2 groups each. First group was put under CD or HFD for 5 weeks and second one was put under CD or HFD for 20 weeks. A total of 8 groups (4 young and 4 aged) composed of 4-5 mice each.

-AT and heart time course assessment:

2-month-old WT male mice were maintained on CD or HFD for 2, 5 or 10 weeks. A total of 6 groups composed of 5 to 7 animals each.

-Senolytic treatment:

5-month-old WT male mice were used. They were separated in 3 groups composed of 5 to 9

mice each with similar bodyweights: CD, HFD, or HFD + senolytic and the duration of diet was 10 weeks. The treatment started 5 weeks after beginning of diet, (see details of the treatment in the technical sheet n°5).

-Clodronate treatment:

2-month-old WT male mice were used. They were separated in 2 groups. First group was put under CD or HFD for 2 weeks and second one was put under CD or HFD for 10 weeks. Each group was separated in 3 sub-groups (CD, HFD Vehicle, HFD Clodronate). A total of 6 groups composed of 7 to 13 mice each. The group of mice undergoing 2 weeks of diet started the treatment together with the diet whereas the group of mice undergoing 10 weeks of diet started the treatment 8 weeks after the beginning of diet, (see details of the treatment in the technical sheet n°6).

2 nd study: Adipose tissue osteopontin promotes macrophage senescence C57/BL6JRj mice (referred as wild-type: WT) were purchased from Janvier Labs, France.

Osteopontin knockout mice (B6.129S6(Cg)-Spp1tm1Blh/J) were purchased from Jackson laboratory (ME, USA), which had been back-crossed with C57/BL6J mice more than 10 generations, and kept as homozygous (referred as OPN-/- (Derumeaux et al., 2008), starting 5 weeks after the initiation of HFD. Another group of HFD mice was treated with allopurinol (1mM in the drinking water) (Bravard et al., 2011), starting 5 weeks after the initiation of HFD, alone or in combination with exercise, up to 10 weeks of HFD. Body weight and status of glucose homeostasis (metabolic tests: ITT, GTT) were monitored at baseline before starting the exercise or the pharmacological intervention and at the end of the protocols, (see details of the treatment in the technical sheet n°8).

In a separate set of experiments, 3 months-old p16LUC heterozygote mice (obtained from N E Sharpless (Chapel Hill, NC, USA) and bred at CNRS-TAAM, France), harboring a knock-in of the luciferase gene into the Cdkn2a locus (Sorrentino et al., 2014), were exposed to CD and HFD for 10 weeks. Body weight gain and luminescence analysis were followed after 1, 2, 5 and 10 weeks of HFD, (see details of the treatment in the technical sheet n°9).

th study: Impaired phenylalanine catabolism is a key determinant of cardiac aging

Global p21-/-mice backcrossed to C57BL/6 background for at least 10 generations (Jackson Laboratory, USA) as well as wild-type (WT) littermates were used. For cardiac phenotyping, male WT and p21-/-mice were followed from the age of 2 to 15 months and myocardial structure and function were sequentially evaluated. Animals were euthanized and tissues harvested for histology and molecular biology at ages 2, 6, 10 and 15 months. For the drug treatment (Phe and BH4), 11-month-old WT mice (Janvier Labs, France) were used, (see details of the treatment in the technical sheet n°7).

In vivo Energy Metabolism Assessment

Objective

GTT measures the capacity of the organism to use glucose as energy substrate and eventual defects in peripheral disposal. ITT measures the global insulin sensitivity and points out potential insulin signalling defects.

Methods

Metabolic tolerance tests were carried out in mice starved for 5-6 hours. GTT was performed after intraperitoneal injection of glucose (0.8-1.5 mg/g body weight in distilled water) and glucose concentration was measured in blood samples obtained from tail vein at baseline, 15, 30, 60, 90, 120 minutes after injection with an automatic glucometer (Accu-Chek Performa, Roche, Germany). For ITT, insulin solution was injected intraperitoneally (0.3-0.5 mU/g body weight, Humulin, Lilly, France) with evaluation of blood glucose levels at baseline, 30, 60, 90, 120 minutes after injection (figure 26).

Data analysis

The values obtained were plotted using the software Prism-Graphpad to perform a graph

showing blood glucose values over the time (figure 26). The areas under the curves were then measured and compared between groups.

Technical sheet n°3

Echocardiographic Assessment

Definition/ Objective

In animal studies, echocardiography is a technique used to explore cardiac function of live animals in a non-invasive way. It gives the advantage to assess cardiac function during the course of the experimental protocol without interfering with it.

Principle

This procedure consists in analysing heart morphology and assessing wall motion and dimensions using a 2-dimensional transthoracic echocardiography in conscious mice.

Echocardiography transducer (or probe) is placed on the thorax , and images are taken through the chest wall.

Methods

Anesthesia: Mice were accustomed to handling before being subjected to transthoracic echocardiography in conscious state to avoid cardiac depressor effect of anesthetic agents.

Acquisition: In such a state heart rates were typically above 600 beats per minute (bpm). Data acquisition was performed by a single person for each cohort of animals and images were acquired from a parasternal position at the level of the papillary muscles using a 13-MHz linear-array transducer with a digital ultrasound system (Vivid 7, GE Medical System, Horton, Norway). Left ventricular diameters and ejection fraction, and anterior and posterior wall thicknesses were serially obtained from gray-scale M-mode acquisition. Peak systolic values of radial strain rate of the anterior and posterior wall were obtained using Tissue Doppler Imaging.

Haemodynamic Cardiac Assessment

Definition/ Objective

This procedure aims to assess in vivo haemodynamic of cardiac left ventricular function by measuring systolic and diastolic arterial pressure as well as blood pressure in the left ventricle including isovolumetric pressure development (dP/dtmax) and pressure decay (dP/dtmin).

Methods

In vivo haemodynamic measurements were performed just before mice were sacrificed following protocol as indicated before. 290 Briefly, haemodynamic evaluation was performed in mice placed on a homeothermic operating table in supine position under 1.5% isoflurane anesthesia with spontaneous breathing. A 1.4-Fr microcatheter (Millar Instruments, Houston, USA) was calibrated manually before each experiment, inserted via the right carotid artery into the aorta and subsequently advanced to the left ventricle. Data were collected after at least 10 min of baseline, using the lowest isoflurane concentration tolerated to ensure minimal cardiodepression during measurement of peak rates of isovolumetric pressure development (dP/dtmax) and pressure decay (dP/dtmin). The microcatheter was then withdrawn to the aorta for measurement of systolic and diastolic pressure. Data were analyzed using the IOX software (EMKA, France).

In vivo haemodynamic cardiac assesment was performed by Dr C. Radu

Technical sheet n°5

Senolytic Treatment

Definition

Senolytic drugs are chemical compounds that target and eliminate senescent cells by inducing apoptosis in those cells which are initially protected against cell death. Here we used Dasatinib and Qercetin (D+Q) drug which target the pro-survival pathways such as Bcl-2/ Bcl-Xl to selectively kill senescent cells over non-senescent ones. This drug showed beneficial effects on aging-and obesity-induced metabolic disorders by improving metabolic parameters and restoring AT integrity in mice models. 271,272 Objective Senolytic drugs are used in basic research for a better understanding of senescent cells by analysing the direct or undirect consequences of senescent cells elimination, and also in clinical trials to target the fundamental aging mechanisms that contributes to several disorders.

Methods

The drug was composed of a combination of two senolytic compounds: Dasatinib (SIGMA-CDS023389-25MG) and QUERCETIN(BIO-TECHNE 1125/100). The treatment was given to the mice through oral gavage at a concentration of 5 mg/kg for Dasatinib and 50 mg/kg for Quercetin per day (solution was prepared at 1 µg/µL for D and 10 µg/µL for Q in PEG 400 10%), for three consecutive days every 2 weeks as previously described.

Phenylalanine and BH4 Treatment

Definition

Phenylalanine (Phe) is an essential amino acid which is degraded into Tyrosine by the hepatic enzyme phenylalanine hydroxylase (Pah). BH4 is the essential cofactor for Pah.

Objective

These treatments were used to explore the role of Phe in driving cardiac aging and the impact of aging on liver Phe catabolism.

Methods

Phe was subcutaneously administered to the old mice in a dose of 200 mg/kg twice a day or vehicle (1x PBS) for one month. BH4 was intraperitoneally administered in a dose of 10 mg/kg/die 2x/die or vehicle (1x PBS with 10 mM sodium ascorbate and citric acid to pH4.5) over six weeks. General state of the mice (body weight & wellbeing) was closely monitored.

Z. Mezdari performed this experiment under the supervision of G. Czibik.

Exercise Training

Objective

This intervention is used to mimic physical exercise in humans and aims to explore and decipher the mechanisms associated with metabolic improvements in the context of HFDinduced obesity. Recent findings show that exercise initiated after long-term HFD reduces senescent phenotype markers in visceral adipose tissue. 283 In our protocol of short-term HFD, exercise was used to evaluate through which mechanism the amelioration of HFDinduced adipose tissue senescence could happen.

Methods

Technical sheet n°9

In vivo Luciferase Detection

Definition

Luciferase is an oxidative enzyme that produce bioluminescence by catalyzing the reaction between the luciferin substrates and other co-factors. The oxidized luciferin resulting from this reaction which is electronically excited releases photons when returning to the ground state. Luciferase bioluminescence, an established model in the field of cancer and aging 294,295 was used in our study to detect p16 INK4A protein in P16-LUC mice: a transgenic mouse model where luciferase is expressed under the control of p16 INK4A promoter.

Objective

This technic was used to detect p16 INK4A protein and determine the precise location of senescence and its evolution over time under HFD-induced obesity.

Methods

Luciferase activity was detected and analyzed with the collaboration of Dr Seguin, Paris Descartes University as previously described. 289 Mice were anesthetized with isoflurane, and then injected intraperitoneally with D-luciferin substrate (Caliper Life Sciences; 15 mg/ml in PBS), 10 ul/g body weight. Images were taken with the PhotonIMAGER system (BIOSPACE).

After substrate administration, peak luminescence was measured at 6-8 minutes. For simultaneous image capture of more than 3 animals, a wide-angle lens was used. To compare multiple images taken at the same exposure point, PhotoAcquisition Software for PhotonIMAGER (BIOSPACE) was used following substrate administration, and acquisition lasted for 10 minutes (from t=10min to t=20min after luciferin injection). An additional ROI was employed to normalize for background luminescence on each image for all analyses.

This experiment was performed by M. Pini and D. Sawaki.

Technical sheet n°10

Dissection: Blood and Tissue Harvesting

Objective

Dissection step is particularly important and critical because the way of collecting blood and harvesting tissues impacts the experiments outcome. So, this procedure must be done as carefully as possible to keep the sample integrity and at the same time, as quick as possible to avoid any protein degradation in the samples.

Methods

At the end of the indicated period of diet or treatment, mice were weighed, then anesthetized and blood was collected directly from inferior vena cava (figure 28). After blood collection, they were euthanized by cervical dislocation, followed by excision of organs starting with the heart. Heart was canulated and washed with PBS (figure 28). All samples were weighed before processing and then cut in two parts: one for histology and one molecular biology. Samples intended for histology were kept in formalin at 4°C for at least 48 hours before embedding.

For molecular biology, tissues were snap-frozen and stored at -80 °C, until they were powdered and aliquoted using mortar, pestle and liquid nitrogen. Plasma was prepared from whole blood samples by centrifugation at 5000g for 10 minutes at 4°C and supernatant was stored at -80°C.

Technical sheet n°11

Plasma Assay

Objective

To assess metabolic parameters and also identify the factors implicated in the crosstalk between the organs, we measured protein concentrations in plasma using Enzyme Linked

ImmunoSorbent Assay (ELISA) or Luminex bead-based multiplex assay.

Principle

ELISA assay is based on immuno-enzymatic detection of proteins in a liquid sample and their quantification by colorimetry. Luminex technology is also used to detect and quantify proteins in a liquid sample but it is a bead-based multiplex assay, so, as opposed to ELISA, it has the advantage to analyse several proteins at the same time using only a small amount of the sample.

Methods

For ELISA assay, we used the kit ELISA R&D Systems, (Minneapolis, MN, USA) (figure), and for

Luminex assay, we used the kit Luminex R&D Systems, (Minneapolis, MN, USA) (figure).

Plasma cytokines were measured following the manufacturer's instructions. Used specific kits are described in the supplemental table.

Data analysis

ELISA plates were analysed with an absorbance microplate reader TECAN infinite 200 and data obtained were processed with Magellan software. Luminex plates were processed and analysed with a Bio-Plex 200 device using xMAP technology of Luminex (Bio-Rad, USA).

Plasma assay was supervised and performed by M. Pini with the help of S. Naushad and M. Halfaoui.

Technical sheet n°12

Senescence-associated β-galactosidase staining

Definition

Senescent cells display an altered lysosomal activity resulting in increased levels of βgalactosidase activity at pH 6.0.

Objective

Senescence-associated β-galactosidase (SA-β-gal) is commonly used as a biomarker of cellular senescence, 211 and it was used here to assess senescence in freshly dissected hearts and fat pads.

Methods

Sections of freshly harvested hearts were incubated for 1 hour at 37°C in β-galactosidase staining solution containing 1 mg/ml X-Gal (Sigma), 40 mM citric acid, 150 mM NaCl, 2 mM MgCl2, 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide with the pH adjusted to 6.0. Stained sections were then scanned. For AT, small pieces (5 mm-square pieces) of freshly dissected tissues were directly subjected to staining solution (1 mg/ml Xgal, Sigma-Aldrich), and kept at 37°C for 8 to 12 hours. The images were taken by GR digital camera (RICOH, Japan).

Z. Mezdari participated to this experiment under the supervision of M. Pini and D. Sawaki.

Flow Cytometry

Definition

Flow cytometry is a technology that rapidly analyzes single cells or particles as they flow past single or multiple lasers while suspended in a buffered salt-based solution. Each particle is analyzed for visible light scatter and one or multiple fluorescence parameters.

Methods

Dissected adipose tissues were kept on ice and minced to 1-3 mm pieces in heparinized cold PBS. After snap vortexing, PBS was removed and tissues were digested with collagenase II solution (2mg/mL, Sigma-Aldrich) at 37°C water bath with gently shaking for 20 min. Digested tissue was then passed through 70 µm mesh and centrifuged to spin down stromal vascular fraction (SVF). After RBC lysis (ACK Lysing Buffer, Gibco, A1049201), the SVF cells were suspended in PBS supplemented 2% fetal-calf serum (FCS), and stained with appropriate antibodies for surface marker and isotype controls for 30 min at 4°C in the dark chamber. Used antibodies and fluorophores are listed in the supplemental table. Living and dead cell discrimination was performed with 7-AAD staining (Molecular Probes). For intracellular p16 staining, SVF cells stained with surface markers were then fixed and permeabilized with Cytofix/Cytoperm solution (BD Bioscience). The cells were then stained with Alexa 488 conjugated p16 antibody (2D9A12, Abcam) and corresponding isotype controls. Flow cytometry was performed with LSRII (BD Bioscience). Data was evaluated and analyzed with FlowJo (TreeStar).

Isolation and cell culture of Bone Marrow-Derived Macrophages

Objective

These cells are used as a model to study macrophage cells in vitro for a mecanistic approach that would be complementary of in vivo interventions and observations.

Methods

Mouse bone-marrow cells were isolated from femurs and tibias. After RBC lysis (LCK buffer, Sigma-Aldrich) and filtration through 70 µm mesh, bone-marrow cells were plated with macrophage culture media (MC media: RPMI 1640 medium (Gibco) supplemented with 5%FCS, penicillin/streptomycin (Gibco) and 30 ng/ml MCSF (R&D systems)). Five days after differentiation into macrophages, the cells were dissolved with Accutase (Sigma-Aldrich), counted and re-plated at 2 x10 5 /well density in 12-well non-coated plate.

We confirmed that over 95% of cells were diferentiated into CD11b/F4/80 double positive macrophages by this method.

BMDM immunofluorescent staining

BMDM were passed onto an OPN-coated 8-well Lab-Tek II chambered cover glass (Nalge Nunc) at 1.5 x10 4 /well density. After incubated as described above, the BMDM were washed with chilled PBS buffer and fixed with 2% paraformaldehyde solution for 10 min at room temperature and permeabilized with 0.05% Triton-X (Sigma-Aldrich). The cells were blocked with 2% BSA solution (Sigma-Aldrich) and subjected to p16 and F4/80 antibody treatment for overnight. Corresponding 2nd antibodies conjugated with fluorescent dyes were used to visualize with DAPI nucleus counter-staining. The images were taken using an Axioplan M2

Imaging microscope (Zeiss).

Phagocytosis assay

Isolated BMDM were used as effector cells and Human leukemia cell-line (HL60) was used as target cells. HL60 cells were labeled with pHrodo as previously described 298 with minor modification. In brief, the HL60 cells were washed twice with PBS buffer and incubated at room temperature for 30 min with 0.1 µg/ml pHrodo. The cells were washed with PBS buffer twice, and suspended in the RPMI medium containing 0.5% FCS, and used for engulfment. To 5.0 x 10 4 BMDM in 500 µl of RPMI medium containing 5% FCS in a 12-well plate, 2.0 x 10 5 pHrodo-labeled HL60 cells were added, and incubated at 37°C for 0, 30, 60, and 120 min. The cells were detached from the plate by incubating with 0.25% trypsin and 1mMEDTA, and collected by centrifugation. After stained with appropriate antibodies for surface marker and isotype controls, cells were further suspended in 500 µl of PBS buffer containing 2% FCS and analyzed by flow cytometry on the LSRII (BD Bioscience). Data was evaluated and analyzed with FlowJo (TreeStar). For time-lapse imaging, 1.5 x10 4 /well BMDM were passed onto an 8well Lab-Tek II chambered cover glass (Nalge Nunc), and incubated at 37°C for 0, 30, 60, and 120 min with 6 x 10 4 pHrodo-labeled HL60 cells in 250 µl of RPMI medium containing 5% FCS and observed by fluorescence microscopy.

Z. Mezdari participated and was trained to phagocytosis assay under the supervision of D. Sawaki and Y. Zhang.

Isolation and cell culture of

Preadipocytes

Methods

Preadipocytes were isolated from iWAT and eWAT stroma vascular fraction (SVF) and differentiated in an adipogenic culture media after two passages to eliminate nonpreadipocyte cell contamination. In detail, WAT was aseptically minced and incubated with collagenase Type II (1 mg/mL, Sigma-Aldrich) at 37°C for 45 min. After digestion, serumcontaining medium was added to the suspension and filtered through a 100 μm cell strainer.

Cell culture experiments on preadipocytes

Primary cell metabolic rate measurements

For bioenergetics assessment, the Mitochondrial Stress Test was performed on iWAT-and eWAT-differentiated adipocytes (20,000 cells/well) using the XF24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA). Oxygen consumption rate (OCR), indicative of mitochondrial activity, and extracellular acidification rate (ECAR), an index of glycolysis, were determined as described 299 .

ATP measurements

Differentiated primary preadipocytes and 3T3-L1 were lysed and intracellular ATP content measured by the ATP-lite assay kit (Perkin Elmer, Villebon-sur-Yvette, France) 300 .

Cell culture treatments

Primary preadipocytes were isolated from WAT of 5-month-old CD male mice. At differentiation, cells were incubated at different time points (for the time course: 30 min-3h-6h-24h or as specified in the figure legend) with adenosine triphosphate (ATP)-and uric acidloaded liposomes (200 μM, Sigma-Aldrich, France) as compared to two control applications: liposome-free (lipo-free) and liposome alone (CTL).

In addition, to assess the role of ATP as a senescence trigger, we performed interference experiments by co-administration of the xanthine oxidase (XO) an allopurinol inhibitor.

Preadipocytes were subjected for 6 hours to in vitro caloric overload in the form of either high glucose/palmitate (Metabolic media: MM) or ATP-loaded liposomes and co-administration of allopurinol (1mM).

Fully differentiated 3T3-L1 adipocytes, cultured in an atmosphere of 5% CO2 and 20% O2 at 37°C, were incubated for 24 hours with ATP or uric acid (200 μM, Sigma-Aldrich, France). ATP and the other molecules were encapsulated in liposomes (Sigma-Aldrich, France)

Superoxide level measurement

Cytosolic and mitochondrial ROS levels were measured in differentiated preadipocytes (7000/well) by quantification of CellROX (10 µM) and MitoSOX (5 µM) fluorescence intensity levels, respectively. Fluorescence readings were done by a microplate reader (Tecan, Männedorf, Switzerland).

This experiment was performed by M. Pini

Technical sheet n°18

Cell culture experiments on C2C12 and AML-12

C2C12

AML-12 cell line

AML-12 (ATCC® CRL-2254™) hepatocytes were maintained in DMEM/F-12 (1:1) culture media supplemented with 10% FBS, 10 µg/ml insulin, 5.5 µg/ml transferrin, 5 ng/ml selenium, 40 ng/ml dexamethasone and 1% penicillin/streptomycin. Cells were sub-cultivated from subconfluent flasks with a ratio from 1:4 to 1:8. For WB analysis, cells were plated on 6-well plates and harvested at 80-90% of confluence. AML-12 cells were treated with Nutlin3a (10 µM) against vehicle (DMSO) and transfected with indicated siRNA (Supplementary Table 2) at 80% confluence using Lipofectamine RNAiMAX Transfection Reagent (ThermoFischer Scientific 13778030) following the manufacturer's instructions. Transfection efficiency was constantly checked using BLOCK-IT Alexa Fluor Red Fluorescent Control (ThermoFischer Scientific 14750100). In other experiments AML-12 cells were treated with Nutlin3a without or with BH4 (10 µM). Cells were lysed for protein extraction, whilst conditioned media was used to determine tyrosine concentration.

This experiment was performed by G.Czibik

Technical sheet n°19

Histological Assessment

Objective

Histological assessment consists in analysing the structure and properties of biological tissues.

It is also used in order to get information about spatial location and/or evaluate the density of molecules and proteins of interest by histochemistry or immunohistochemistry technics.

Principle

Histochemical staining is based on biochemical reactions that can detect specific tissue components such as lipids, carbohydrates, or proteins. Immunohistochemical staining is based on immunochemical reaction used to detect a specific antigen thanks to a specific antibody.

Methods

Histochemical staining: Formalin-fixed organs were paraffin-embedded and sectioned into 7

µm thickness using a rotary microtome (Leica). The sections were deparaffinized using xylene and a graded series of ethanol dilutions. For general overview, adipocyte size measurements and counting of CLS sections were stained with hematoxylin and eosin (Sigma-Aldrich, France).

For determination of adipose tissue, liver and cardiac fibrosis, sections were stained with Picrosirius red using a commercial kit (Polysciences Europe GmbH, Germany) following the manufacturer's instructions.

Immunohistochemical staining:

Immunofluorescence was used to detect proteins of interest within their native environment, including co-localization with cell-specific markers. Briefly, paraffin-embedded sections were rehydrated, followed by heat-mediated antigen retrieval with citrate buffer pH6.0 (Dako). Sections were blocked with 30% goat serum and incubated overnight at +4 °C with primary antibodies (described in the supplemental table). The following day sections were washed, incubated with corresponding Alexa Fluor 488, 555 or 647 conjugated secondary antibodies (Invitrogen), washed again and mounted with Fluoroshield Mounting Medium With DAPI (Dako).

BODIPY fluorescent staining: Dissected adipose tissues were washed in PBS and cut into 3-5 mm pieces. Tissues were then incubated for 1h with BODIPY dye (Invitrogen D3835), a lipid marker, at a concentration of 1/1000, Isolectin GS-IB4 (Invitrogen I21411), a capillary marker, at a concentration of 1/100, and TO-PRO™-3 Iodide (642/661; Invitrogen T3605), marker of nuclei, at 1/1000 concentration. After PBS wash, tissues were put on the ibidi culture slide with 10% glycerol solution and observed under confocal microscope.

Data analysis

The pictures with hematoxyline & eosin and Sirius Red staining were captured using an

Technical sheet n°20

Evaluation of Gene Expression

Methods

Total RNA extraction : Total RNA was isolated from powdered snap-frozen tissues using the RNeasy mini kit (Qiagen) for adipose tissue, after a chloroform-mediated lipid removal step,

or RNeasy mini fibrous kit (Qiagen) for heart and liver, following the manufacturer's instructions. RNA concentration was then measured with NanoDrop ND-2000.

Reverse transcription: Because RNA is very unstable molecule, we use an enzyme called reverse transcriptase in order to convert the extracted RNA into complementary DNA (cDNA).

This step was performed using the High Capacity cDNA kit as instructed by the manufacturer (Applied Biosystems).

Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR):

This technique consists in amplifying a specific DNA fragment thanks to a DNA polymerase and a combination of two DNA primers that are complementary to the 3′ ends of each of the sense and anti-sense strands of the DNA target (also known as "forward" and "reverse" PCR primers respectively) .

A thermocycler machine (ref) was used for the amplification and amplicon products were detectable in real time by fluorescence.

For more precision and efficacy (as compared with Syber green method for example), we used Taqman probes (Applied Biosystems). This technology consists of a fluorophore covalently attached to the 5'-end of the oligonucleotide probe and a quencher at the 3'-end. The quencher inhibits any fluorescence signal as long as it stays close to the fluorophore. This probe anneals within the specific DNA region (single stranded DNA) and once the Taq polymerase extends the primer and synthesizes the nascent strand, its exonuclease activity degrades the probe and releases the fluorophore, which, relieved from the quenching effect, becomes fluorescent. Hence, the fluorescence detected in the thermocycler is directly proportional to the amount of DNA template present in the PCR. So, quantitative measurements of the accumulation of the product during the exponential stage provide an estimation of transcript levels.

Our qRT-PCR experiments were performed in fast multiplexed reactions using two different fluorophores: FAM for the gene of interest and VIC for the β-actin as endogenous control. We used TaqMan probes which were conjugated to a minor groove binder (MGB) moiety with increased binding affinity. All qRT-PCR reagents, including inventoried Taqman-MGB probes (described in the supplemental table) as well as qRT-PCR instrument were products of Applied Biosystems.

Data analysis

Relative

Technical sheet n°21

Evaluation of Protein Expression

Objective

Protein synthesis results from mRNA translation by ribosomes. Thus, protein expression should be reflecting gene expression. However, this is not always the case, given that protein expression can be regulated at different levels. So, when deciphering the mechanisms underlying potential changes observed in physiological, pathological or experimental condition, both gene and protein expression should be evaluated.

Methods

Total protein extraction: Pulverised tissue samples (100-150 mg of eWAT) were lysed with T-PER lysis buffer (ThermoFisher) supplemented with 0.05 mM phenylmethylsulphonyl fluoride (PMSF), 0,05 mM proteasome inhibitor MG-132 (Sigma-Aldrich), and protease/phosphatase inhibitor cocktail tablet (ThermoFisher) complete with sonication and passing through a 21 G needle. To pellet any debris, lysates were centrifuged at 12,000 g for 15 minutes at 4°C, and the middle fraction (between pellet and lipid layer) was transferred to fresh tubes.

Protein assay: Bradford assay (BioRad) was used to determine protein concentrations, sample concentrations were then equalised with lysis buffer and boiled with Laemmli buffer.

Western blot: Equal amounts of protein lysates were loaded onto precast denaturing polyacrylamide gels (Nupage 12% Bis Tris gel, Novex, Invitrogen). Proteins were separated by electrophoresis, then transferred onto polyvinylidene difluoride (PVDF) membrane (Invitrogen) using a transfer cell (Bio-Rad) in ice-cold transfer buffer (48 mM Tris base, 390 mM glycine, 0.1% SDS, 20% methanol (v/v)) at 125V for 2 hours. Membranes were blocked in 1x skimmed milk for 1 hour at room temperature, followed by overnight incubation with primary antibody (described in the supplemental table) diluted in blocking buffer at 4°C. The following morning membranes were washed, incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody in blocking buffer for one hour. Blots were developed with the aid of enhanced chemiluminescence (ECL) reagents (normal, Prime & Select) and digital images were obtained with a dedicated imaging system (Azure Biosystems).

Membranes were stripped of antibodies and reprobed with an antibody raised against β-actin conjugated to HRP as loading control. This procedure consisted in incubation for 15min in Guanidine-HCl-based stripping solution (6M Guanidine-HCl, 0.2% Nonidet P-40, 0.1M βmercaptoethanol, 20mM Tris-HCl, pH7.5 and 100 mM 2-mercaptoethanol) and at room temperature with gentle agitation, followed by extensive washing and blocking. 301

Data analysis

The images obtained were analysed using imageJ software. Intensity of signals corresponding

to the protein of interest were normalized on the intensity of signals corresponding to the βactin and values were plotted using Prism-Graphpad software.

Technical sheet n°22

Statistical Analysis

Data were acquired and analysed blind to age or treatment. Unpaired, two-tailed t-test was used to compare two groups. More than two groups were compared with one-way analysis of variance (ANOVA), with Bonferroni or Tukey's correction for multiple comparisons. Two-way ANOVA was used to compare groups with time-dependent changes, with Bonferroni or Tukey's correction for more than two groups. Data are presented as original images or mean ± SEM. Annotations: ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated. A p value of < 0.05 was considered significant and p value >0.05 was considered non-significant.

104

The result section is presented in two main sections:

➢ Assessment of adipose tissue senescence in the context of high fat diet-induced obesity and in the context of aging:

1) The first study is entitled "Visceral adipose tissue macrophage senescence promotes metabolic and cardiac disorders during the onset of HFD-induced obesity". It is presented as commented figures and the article is being finalised and prepared for submission in Diabetes journal. This study is the core of my thesis work. I conducted this project and performed most of the experiments, with the support and participation of my team.

2) The second study is entitled "Osteopontin promotes adipose tissue remodeling through macrophage senescence". It is presented as an article submitted to JCI Insight and is associated to a published letter to Circulation in which I was co-author. This aims at evaluating the role of osteopontin on visceral adipose tissue remodeling and dysfunction in the context of aging, and in the cross-talk between adipose tissue and heart. My participation to this study consisted in helping with organ isolation, preparation for FACS analysis, quantitative PCR analysis, and imaging experiments.

3) The third study is entitled "Adipose tissue senescence is mediated by increased ATP content after a short-term high fat diet exposure" and is presented as original article under revision in Aging Cell. My contribution to this study consisted in participating to the bibliographic searches, helping with in vivo procedures (body weight measurements, metabolic tests, exercise training), organ isolation, imaging experiments and data analysis.

➢ Analysis of the mechanisms associated with cardiac aging:

The fourth study is entitled "Dysregulated phenylalanine catabolism is a defining characteristic of cardiac aging". It is presented as original article under revision in Circulation. My contribution to this study consisted in participating to the in vivo procedures (animal handling, measurement of body weight, water and food intake, drug administration, metabolic tests), organ isolation and processing, preparation of sample aliquots for collaborators, quantitative PCR analysis, imaging experiments, data analysis and critically reading the manuscript with suggestions.

ETUDE 1 Résumé

Le tissu adipeux viscéral est un facteur de risque majeur des comorbidités associées à l'obésité tel que le diabète de type 2 et les maladies cardiovasculaires. Cependant, malgré les études effectuées sur cet organe dans le contexte de l'obésité, très peu de choses sont connues sur

Introduction

The world faces an obesity epidemic afflicting the entire cross-section of society, with increased risk for type 2 diabetes mellitus (T2DM), dyslipidaemia and cardiovascular disease. 302 As an inseparable part of chronic positive imbalance, surplus energy accumulates in adipose tissue (AT) as fat. Contrary to early beliefs, 303 AT is not an inert storage structure, but an organ with significant plasticity and secretory activity with potential impact on the function of remote organs. [START_REF] Deelen | A metabolic profile of all-cause mortality risk identified in an observational study of 44,168 individuals[END_REF]304 Moreover, not all adipose tissue pads are equal; accumulation of visceral adipose tissue (VAT) corrodes health more than subcutaneous fat does. 305 This suggests the existence of VAT-specific pathological processes spreading injury to other organs, such as the heart, as we recently demonstrated in the context of aging. 26 Observations in obese individuals and in high-fat diet (HFD)-induced obesity murine models, led to the discovery of chronic low-grade inflammation 306 and increased leptin secretion. [START_REF] Denicola | Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis[END_REF]288,[307][308][309] Moreover, adipocyte hypertrophy, tissue remodeling, 172 hypoxia, 310,311 and accelerated senescence 204,283,312 have been implicated in cardiometabolic dysregulation.

These studies used long dietary regimens (typically >15 weeks) without focusing on the initiation of VAT abnormalities.

Therefore, our study aimed at studying the time-course of abnormalities occurring early in VAT after initiation of high fat diet. We also aimed to compare young (2 months old) and aged (12 months old) wild type C57BL6-J mice, in order to identify the potential confounding factor of aging which may per se affect VAT and heart.

Here we present the comparison of VAT changes in young and old mice undergoing HFD regimen and we focus on the presentation of the analysis of the time-course in young mice to characterize the changes in VAT during this short exposure to HFD.

Similar analysis are scheduled in the group of old mice and work is in progress.

Results

I) Metabolic parameters in young and aged mice undergoing HFD

To explore the response of differently aged mice to caloric overload, C57BL6 (WT) male mice of 2 and 12 months of age were fed a high-fat diet (HFD; vs. chow as control diet) for the duration of 5 or 20 weeks (figure 1). After initiation of HFD, cross-sectional area of adipocytes increased as early as 5 weeks as compared with CD groups in both young and aged mice (figure 3A,B).

However, the number of crown-like structures (CLS) significantly increased only in young mice at a later stage of 20 weeks as compared with CD mice, whilst a similar trend was observed in old mice (figure 3C). Adipose tissue macrophages (ATMs) are important cellular components of the adipose tissue and they are concentrated in CLS. Interestingly, HFD significantly increased the total number of ATM (Mac 3) in young mice after a 20-weeks duration of HFD but not in old mice. This increase in ATM was with a higher number of ATMs, which were positive for p16 (figure 4). Next, systemic glycaemia was evaluated. HFD worsened glucose tolerance as early as 5 weeks with further aggravation at 20 weeks in all groups, but at a lesser extent in 12-month-old than in 2-month-old mice.

After 20 weeks of HFD, a more severe pattern of insulin resistance was observed in young versus old mice on HFD as compared with control chow diet groups, whilst ITT curves between 2-and 12-month-old mice on HFD were comparable after 5 weeks of HFD and worse than those of CD group (figure 5).

II) Time course of metabolic alterations in young mice submitted to HFD

In order to explore early sequence of structural, functional and molecular alterations in high fat diet-induced obesity, 2-month-old C57BL/6 male mice were fed chow (CD) or high-fat diet (HFD) and euthanized at 2, 5 and 10 weeks after the initiation of the diet (figure 6). Plasma levels of adipokines (leptin and adiponectin) and insulin showed a significant upregulation of leptin as early as 2weeks of HFD, whilst no significant change was observed for adiponectin. Insulin levels significantly increased after 10 weeks of HFD (figure 8).

III) Time course of VAT alterations and remodeling during the onset of obesity

Since VAT is commonly regarded as a source of major cardiometabolic risk in both mice, [313][314][315] and humans, [316][317][318] we analysed VAT remodeling time course during a 10-week HFD duration.

Adipocyte size progressively increased as early as 2 weeks after HFD initiation (figure 8A-B), followed by an increase of crown like structures at 5 weeks (figure 8C). As angiogenesis lagged behind the rate of adipocyte expansion, we also demonstrated a capillary rarefaction after 10 weeks of HFD (figure 10). 122

We then analysed the transcript levels of genes VAT involved in the regulation of energy metabolism: we observed an upregulation of leptin gene expression as early as 2weeks of HFD but no variation for adiponectin (figure 12A-B) and a downregulation of the genes coding for Insulin-like growth factor-1 (Igf-1) and for Glucose transporter-4 or GLUT-4 protein (Slc2a4) at Metabolic dysfunction detectable before systemic inflammation and major VAT remodeling prompted us to seek for other potential causes of impaired glucose homeostasis. Since adipose tissue senescence has been proposed to contribute to dysmetabolic state in obesity, 204,252,283 we assessed senescence markers in VAT and identified an increase in VAT senescent cells as early as 2 weeks of HFD, as shown by the upregulation of transcript and protein levels of senescence markers Cdnk2a (p16) and Cdnk1a (p21) (figure 13). These data indicative of cellular senescence were not associated with significant increase in DNA damage as demonstrated by γH2AX staining (figure 14). Taken together, our data show that VAT senescence is induced at a very early stage, through DNA damage-independent pathway, and precedes inflammation and fibrosis during VAT remodeling. This chronological order is also confirmed when we look at transcript levels of senescence-, inflammation-, and fibrosis-related genes (figure 15).

A B IV) Time course of cardiac alterations during HFD-induced obesity

Obesity is known to alter the cardiac structure and function. 25 In order to analyse the time course of cardiac remodeling and dysfunction, echocardiography was sequentially performed in both CD and HFD mice and hearts were examined for structural changes after 2, 5 and 10 weeks of HFD vs CD. As opposed to long-term HFD feeding (20 weeks), 25 10 weeks of HFD did not increase heart weight to tibia length, an index of cardiac hypertrophy (figure 16A). Left ventricular ejection fraction, an index of systolic function, remained unaltered (figure 16B). By contrast, systolic strain rate, a sensitive index of myocardial contractility obtained by deformation imaging identified a reduced contractility as early as 2 weeks of HFD, followed by progressive aggravation with duration of HFD (figure 16C). Interestingly, strain rate also declined in CD-fed mice with time, which is consistent with age-related decline (figure 16C). Cross-sectional area (CSA) of transversely sectioned cardiomyocytes did not increase by HFD until 10 weeks of HFD (figure 17A-B). Myocardial interstitial fibrosis, another important component of cardiac remodeling, also remained unchanged in the duration of observation with a trend to a slight increase after 10 weeks of HFD (figure 17C-D).

A B C

Collectively, we did not find major changes in cardiac structure within 10 weeks of HFD, but 2 weeks of HFD sufficed to compromise cardiac contractility.

V) Senolytic treatment as a therapeutic option to rescue metabolic and cardiac function

Our previous data showed that VAT senescence as compared to inflammation and fibrosis is the only parameter which is concordant with cardiometabolic alterations observed at 2 weeks of HFD. Hence, we used a senolytic treatment to specifically eliminate senescent cells. We used 5-months old mice for a 10-week HFD regimen followed by a 4-week Dasatinib + Quercetin (D+Q vs. Vehicle) treatment and compared them to a CD group (figure 18). While p16 senescent marker was increased by HFD, senolytic treatment decreased the number of p16 positive cells without any significant change in body weight or adiposity index between the two HFD groups (figure 19).

A B D C E F VI) Time course of AT cell-specific senescence during HFD-induced obesity

As the efficiency of global elimination of senescent cells was not convincing, we shifted toward more specific and targeted options. So, we determined which cell-type(s) were affected by HFD-induced senescence in VAT of 2-month old mice using immunofluorescent co-labelling.

We found an increased co-localization between p16 and macrophage marker CD68 in HFD at all time-points investigated (figure 21A). Similar observations were made with p21 marker (figure 21B). By contrast, senescence did not occur in CD3+ lymphocytes before 5-10 weeks of HFD (figure 21C,D) and in PDGFR+ preadipocytes before 10-week HFD (figure 21 E,F). Globally, in VAT, under HFD, p16 positive signal was not only found in macrophages while the majority of p21 positive signal was colocalised to CD68+ macrophages (figure 22). Taken together, these data showed that senescence induction in response to HFD was occurring in adipose tissue macrophages at an early stage preceding that of other cells.

A B

To further confirm that induction of senescence in adipose tissue macrophages occurs in response to caloric overload, we subjected in vitro bone marrow-derived macrophages (BMDMs) from 2-month-old mice to metabolic stimulation with high glucose + palmitate (metabolic media: MM) and observed an increase in both p16 and p21 protein expression (figure 23). These findings confirm that macrophages undergo senescence in response to caloric overload. Clodronate-liposome treatment reduced the number of adipose tissue macrophage (ATM)

(figure 25A-B). Interestingly, clodronate-liposome treatment was associated with a significant reduction in the number of p21+ cells (figure 25C), whilst the number of p16+ cells was not changed (figure 25D-E), consistent with the observation that the majority of p21 but not p16 colocalised to CD68+ macrophages (figure 22). However, a closer look at senescent macrophages, i.e. p16+ CD68+ and p21+ CD68+ clearly showed a reduction of both Interestingly, clearance of senescent macrophages was associated with a complete rescue of glucose metabolism as shown by glucose tolerance and insulin resistance tests (figure 26A-D) despite no significant change in body weight (figure 26E). Similarly, plasma adipokine levels were also normalized by clodronate treatment (figure 26F-G). Furthermore, cardiac function was compromised with vehicle, as with HFD alone, but

Clodronate-liposome treatment completely restored healthy cardiac function (figure 27).

A B C

C To explore whether clearance of senescent macrophages could remedy adverse consequences of longer caloric overload, the above experiment was repeated with 10 weeks of HFD and clodronate/vehicle treatment 2 weeks prior to sacrifice (figure 28). In sharp contrast with observations made in 2-week HFD, these mice showed no improvement of metabolic function (figure 29A-D) with no change in body weight between the 2 HFD groups (figure 29E). In Response:

We appreciate the comments and further questions raised by Wang and his colleagues in relation to our recent work, stating the importance of the visceral adipose tissue (VAT) and its fibrotic secretome in the promotion of age-related cardiac remodeling and dysfunction. 1 As they suggested, the effect of aging on VAT and its secretome is still poorly understood. Fat tissue is a nexus of pathways involved in longevity, metabolic dysfunction, and low-grade inflammation. Abnormal ectopic fat accumulation (or visceral adiposity), a common hallmark of aging and obesity, represents per se a risk factor for accelerated aging with the accumulation of senescent cells within adipose tissue. Recent data, including ours, suggest that chronological aging induces cumulative alterations in the VAT leading to the modification of its secretome toward a senescent-associated secretory phenotype, including a major increase in osteopontin (OPN) production. 1,2 We also recently demonstrated the predominant role of circulating profibrotic factors in comparison with the role of proinflammatory factors in triggering cardiac remodeling. Thus, an increased plasma level of fibrotic factor transforming growth factor β is released by VAT in a short-term high-fat diet model that induces cardiac alteration together with myocardial interstitial fibrosis as early as 5 weeks. 3 Furthermore, we demonstrated that a high-fat diet promotes immune cell senescence before the actual development of obesity. 4 Both aging and obesity induce immune cell senescence, which may result in a modification of their polarization and secretory profile. Our hypothesis is that adipose tissue macrophages (resident and recruited) undergo a senescent phenotype in the context of aging or a high-fat diet. These changes accelerate age-related tissue deterioration through paracrine mechanisms involving the secretion of senescent-associated secretory phenotype cytokines and initiate a vicious circle by promoting senescence in the local tissue microenvironment, eventually contributing to the deterioration of remote organs (heart).

Our observation that the OPN-knockout mouse or pharmacological inhibition of the OPN model ameliorated not only cardiac aging, but also age-related insulin resistance, indicates the crucial role of OPN in the induction of insulin resistance. It is of interest that OPN-knockout mice showed decreased insulin resistance despite their higher fat mass, suggesting that the senescence-associated remodeling of VAT, but not the VAT mass, is the culprit for insulin resistance progression. It is intriguing that remodeled myocardium (ventricle) itself shows a very low number of senescent cells despite an obvious age-related remodeling and dysfunction (D. Sawaki et al, unpublished data), which may indicate that influences from external organs such as VAT, and circulating factors, are more important. 1 Because heart failure with preserved ejection fraction is a multicomorbidity disease including aging and obesity as the biggest risk factors, 5 we are currently investigating the role of OPN in heart failure conditions such as metabolic stress or pressure over-

Introduction

In obesity, adipose tissue (AT) undergoes cellular senescence involving adipose tissue macrophage (ATM) activation, which enhances AT remodeling through proinflammatory signaling. (1-5) During the aging process, AT remodeling has also been described, (6)

Results

Aging promotes accumulation of senescent cells in visceral adipose tissue

With aging, senescent cells accumulate in VAT as demonstrated by higher luciferase activity in p16-luciferase mice (Fig. 1a) and increased senescence-associated beta-galactosidase (SA--Gal) activity within VAT (Fig. 1b). This was in line with the age-related up-regulation of transcript and protein levels of senescence markers (i.e. p16 and p21, Fig. 1c) in the 12-month VAT as compared with the 3-month VAT (Fig. 1d,e).

VAT senescence was associated with an increase in gene expression of matricellular proteins (Supplemental Fig. 1a) and pro-inflammatory markers (Supplemental Fig. 1b). This included increased expression levels of Galectin3 (Lgals3) and osteopontin (OPN; Spp1), a key component of SASP ( 14) that has been reported to induce a deleterious remodeling of the heart.( 9) Interestingly, upregulation of Spp1 (OPN gene) was one of the strongest discriminative genes associated with VAT aging, together with senescence (Cdkna1 and Cdkna2) and phagocytosis-related genes (Fig. 1f,g). Based on this finding, first we established senescent cells (p16-positive) mainly localizing in OPN-rich VAT areas (Fig. 1h) OPN also colocalized within macrophages (F4/80) and T-lymphocytes (CD3) (Fig. 1i, Supplemental Fig. 1c).

Of note, age-related VAT changes included an increase in body-weight, adipose tissue depot weight, adipocyte size, and interstitial fibrosis together with an impaired glucose tolerance/insulin resistance, higher adipokine levels and an alteration of glucose and fatty acid metabolism-related gene expression (Supplemental Fig. 1d-k).

Taken together, our data suggest that VAT senescence may be governed by tissue/local OPN accumulation.

Osteopontin is a critical regulator of adipose tissue and macrophage senescence Since OPN was highly expressed in aged VAT aging, we first aimed to dissect the role of OPN in inducing VAT cellular senescence by genetic deletion and pharmacological inhibition.

We found a decreased VAT senescence in12-month OPN-knockout (OPN-/-) mice as compared with wild-type littermates (WT) as evidenced by lower SA--Gal activity (Fig. 2a) and p16 protein levels (but not p21 and p53, Fig. 2b,c). Interestingly, this reduced VAT senescence in OPN-/-mice was associated with lower VAT fibrosis (Supplemental Fig. 2a) and improved glucose tolerance and insulin sensitivity (Supplemental Fig. 2b,c) despite higher body weight, larger adipose tissue mass and adipocyte size (Supplemental Fig. 2d-f).

Similarly, pharmacological OPN inhibition by Agelastatin A (AA)(9) induced a dramatic decrease of p16 expression in VAT (Fig. 2d) and in CD68+ ATM (Fig. 2e) in 12-month WT mice.

AA treatment also improved VAT structure (i.e. reduced adipocyte size), adipokine secretion profile and glucose homeostasis (Supplemental Fig. 2g-j). Taken together, these results show that OPN deficiency or inhibition improves VAT structure, metabolic function and reduces the percentage of senescent ATM.

To firmly establish the role of OPN in inducing senescence in macrophages, we next subjected bone marrow-derived macrophages (BMDM) from 3-month WT mice to in vitro OPN-matrix treatment (by dish coating, Fig. 2f). In naïve BMDM, OPN induced p16 transcript and protein expression in a dose-and time-dependent manner (Fig. 2g & Supplemental Fig. 2k).

Interestingly, this p16 induction was coupled with similarly increased Ccl2 expression but with decreased Il1b expression (Supplemental Fig. 2l). Exogenous OPN also promoted p16 expression levels in OPN-/-macrophages (Supplemental Fig. 2m). We then confirmed the role of OPN in vivo with OPN-enriched Matrigel implantation (compared with vehicle), which increased infiltration of macrophages and up-regulated p16 expression in recruited macrophages in young WT mice (Fig. 2h,i).

Collectively, our results indicate that OPN is an important extracellular matrix protein that potentiates VAT senescence and metabolic dysfunction during aging through VAT immune cell senescence.

Role of adipose tissue macrophages to drive visceral adipose tissue senescence during aging

To further establish the role of OPN in inducing ATM senescence and thus driving VAT remodeling and dysfunction, we focused on ATM phenotype in 3-and 12-month WT and OPN-/-mice. First, we found that p16 was highly expressed in ATM (CD68+) in 12-month WT as compared with 12-month OPN-/-mice whilst it was not expressed in young mice (Fig. 3a). FACS phenotyping with conventional surface markers (Supplemental Fig. 3a,b) (15, 16) confirmed that aging increased the percentage of Ly6c-CD206+ cells within the CD11b+F4/80+ cells in WT mice (Fig. 3b) but did not show any significant difference in VAT immune cell subpopulations between 12-month WT and OPN-/-mice (Fig. 3c). Interestingly, FACS analysis targeting p16 showed higher p16 protein levels in F4/80+ ATM in 12-month vs 3-month WT mice, and remarkably reduced p16 expression in ATM-derived 12-month OPN-/-mice (Fig. 3d), which was in line with our observation of lessened VAT senescence in 12-month OPN-/-mice (Fig. 2a-c). In parallel, T lymphocytes (CD3+) were expressing p16 with aging whilst OPN deficiency reduced it (Supplemental Fig. 3c).

As a proof of concept for a key role of ATM senescence to induce VAT senescence, we eliminated ATM with liposome-packaged clodronate in 12-month WT mice (Fig. 3e). We first observed depletion of ATM by clodronate (Fig. 3f and Supplemental Fig. 3d) together with reduced OPN (Fig. 3g) and p16 levels in VAT (Fig. 3h). This was associated with a dramatic improvement in VAT remodeling (decreased adipocyte size), adipokine secretion profile and glucose tolerance/insulin resistance (Fig. 3i-l).

Collectively our data reinforce the hypothesis of a key role of OPN-related ATM senescence to drive age-dependent VAT alterations. This led us to further characterize the link between OPN and the occurrence of senescence in ATM.

Impact of aging on adipose tissue macrophage function

Hence, we evaluated the effects of ATM senescence and the role of OPN on efferocytosis, an important macrophage function to eliminate cell debris and apoptotic cells and to maintain tissue integrity.( 17) First, staining of VAT showed an increased co-localization between TUNEL and CD45+ cells together with a shorter distance between TUNEL+/CD45+ cells in 12-month OPN-/-as compared to WT animals (Fig. 4a-c). However, there was a similar agedependent increase in TUNEL+ cells in WT and OPN-/-(Supplemental Fig. 4a), whilst the CD45+ cell percentage was higher in the aged OPN-/-as compared with aged WT mice (Supplemental Fig. 4b).

Since TUNEL positivity may not represent only apoptotic chromatin change but also DNA strand break and DNA repair process, (18, 19) we stained VAT for phosphorylated H2AX (H2AX) in both 3-and 12-month WT and OPN-/-mice and found a comparable age-dependent increase in H2AX+ cells between genotypes (Fig. 4d,e). In sharp contrast, however, H2AX+ cells mainly co-localized with CD45+ cells in OPN-/-VAT but not in WT, as observed with TUNEL+ cells (Fig. 4f). We then performed staining for cleaved-caspase 3, a central executioner of apoptosis, to evaluate the final step of apoptosis in the VAT (Fig. 4g).( 20) Cleaved-caspase 3 mostly co-localized with CD45+ cells in 12-month OPN-/-, but not in WT (Fig. 4h). The genotype-related difference in TUNEL, H2AX and cleaved-caspase 3 demonstrates an impairment in ATM phagocytic function to clear the age-related cellular damage accumulation in aged WT as opposed to OPN-/-mice. To confirm this in vivo observation, we performed an in vitro phagocytosis assay (Supplemental Fig. 4c-e), which displayed impaired phagocytic activity in BMDM derived from 12-month WT mice, which was prevented in BMDM derived from age-matched OPN-/-mice (Fig. 4i and Supplemental Fig. 4d,e). In line with these results, phagocytosis-related gene expression levels were higher in OPN-/-VAT (Fig. 4j) and in OPN-/-BMDM (Supplemental Fig. 4f) compared to WT. Conversely, OPN decreased BMDM phagocytic activity and related-gene expression levels compared to vehicle treatment (Supplemental Fig. 4g,h).

These results highlight that, coincident with the induction of senescence, OPN impairs a major ATM function, i.e. efferocytosis. By contrast, genetic OPN deletion restores ATM functionality whilst preventing ATM senescence.

OPN-/-BMDM transplantation rejuvenates visceral adipose tissue and restores metabolic function during aging

To further establish the role of OPN in VAT aging, we conducted bone marrow transplantation (BMT) from 4-month WT and OPN-/-donors to WT recipients after total body irradiation, and kept them until the age of 16-month (Fig. 5a). Blood leukocytes were reconstituted with donorderived cells (CD45.2+) over 95%, without affecting their number (Supplemental Fig. 5a-c).

After OPN-/-BMT, VAT SA--Gal staining slightly decreased as compared with WT BMT (Fig. 5b), together with decreased total p16-positive cells and p16-positive ATM (Fig. 5c,d), but without any change in p16+CD3+ cells (Supplemental Fig. 5d). OPN positive cells markedly decreased in OPN-/-BMT VAT (Fig. 5e), along with OPN gene expression (Fig. 5f). Blunted senescence and OPN expression in OPN-/-BMT VAT associated with normalized adipokine gene expression pattern, smaller adipocyte size, and improved glucose tolerance/insulin sensitivity (Fig. 5g-j). Finally, OPN-/-BMT reduced TUNEL+ cells (Fig. 5k) and increased colocalization of cleaved caspase-3+CD45+ cells in VAT (Fig. 5l), along with higher efferocytosisrelated gene expression (Fig. 5m).

Collectively, our results indicate that ATM-derived OPN exerts an important role in promoting ATM/VAT senescence and dysfunction.

Discussion

In this study, we showed that age-related VAT remodeling and senescence are strongly related to ATM senescence with impairment of major macrophage functions, such as efferocytosis.

Importantly, we uncovered a key role for the matricellular protein OPN to drive these processes.

VAT senescence during aging

Our data pointed out that VAT remodeling occurs during the aging process together with cellular senescence. Indeed, comparison of 3-month and 12-month mice highlighted that VAT aging begins in middle-aged mice, earlier than previously reported in the literature.( 21) VAT aging is associated with structural remodeling (i.e. enlargement of adipocytes, fibrotic deposition), metabolic dysfunction (i.e. impaired glucose tolerance, insulin sensitivity, and adipokine secretion) and senescence (SA--Gal activity and p16/p21/p53).

Interestingly, we demonstrated that matricellular protein and senescence marker genes were the more discriminative than proinflammatory genes to drive VAT aging, whilst efferocytosisrelated genes were strongly down-regulated. This observation focused our attention on how OPN and ATM govern VAT senescence.

First, our results highlighted the important role of OPN, a member of the matricellular protein family, during VAT aging.( 13) Whilst the role of many matricellular proteins including OPN has been shown in VAT remodeling in obesity, (10, 22-24) we previously reported that VAT was the major source of OPN during aging. (9) Our current data reinforce this age-related upregulation of OPN (Spp1) and galactin3 (Lgals3), both involved in the regulation of macrophage function. (25) Genetic deletion and pharmacological inhibition of OPN reduced VAT and ATM senescence, whilst restoring efferocytosis, as shown by in vitro and in vivo assays. By contrast, OPN induced a senescent-like phenotype in naïve macrophages with reduced phagocytic activity, indicating that OPN has a key role in modulating ATM senescence and function. Interestingly, ATM's endocytotic dysfunction might be considered as one of the primary events to mediate VAT dysfunction in both aging and obesity as recently reported. (15, 26) Another interesting finding is that genetic OPN deletion resulted in significantly larger adipocyte-size and VAT mass in spite of their better metabolic function. This indicates that the OPN/senescence-free developmental aspects are more important to the adipocyte function than the demerit of cell-size enlargement.

Importance of macrophage senescence in VAT aging

Thus, to dissect the interplay between OPN and ATM senescence, we explored how OPNinducible ATM senescence may spread senescence to the adjacent tissue ultimately leading to VAT remodeling and dysfunction during the aging process. Indeed, the specific consequences of ATM senescence have not yet been fully addressed whilst other adipose tissue cell components have been reported to undergo senescence during aging. (1-3, 6) Better understanding of pathological processes affecting ATM, the most abundant AT immune celltype in natural aging, (27) is particularly important given their key role in AT homeostasis and functional maintenance.( 28) By contrast, ATM have been referred to as a source of harmful effects under stress-conditions such as over-nutrition responsible for a chronic, low-grade proinflammatory state.( 29) First, we established that aging induced p16 up-regulation in ATM of WT mice, which was blunted by OPN deficiency. The impact of ATM senescence was further demonstrated by deletion of ATM by clodronate-liposome treatment that ameliorated VAT structural remodeling (i.e. adipocyte hypertrophy), and more broadly, metabolic function (i.e, glucose tolerance and adipokine secretion profile). This clodronate-mediated improvement paralleled with reduced ATM senescence (lessened p16+CD68+ cells), indicating that ATM is a major contributor to age-related VAT and systemic metabolic dysfunction.

Next we demonstrated a senescence-associated impairment of ATM function regulated by OPN both in vivo and in vitro. In particular, we studied efferocytosis, which is crucial in maintaining AT homeostasis by removing apoptotic cells and cell debris. Whilst aging was associated with an increase in TUNEL+ cells in both WT and OPN-/-VAT, only aged OPN-/mice but not WT manifested efficient efferocytosis as observed by higher engulfment of TUNEL+ or cleaved-caspase 3+ cells. In vitro functional assay further indicated that aged OPN-/-BMDM exerted preserved phagocytic activity with higher expression levels of key efferocytosis-related genes such as Mertk. (30, 31) The importance of our findings is underscored by recent studies pointing out that tissue microenvironment is the most important determinant for macrophage phenotype and functionality. (32) For instance, tissue macrophages are differently programmed for efferocytosis, depending on apoptotic signals. (33, 34) In this regard, during the aging process, adipose tissue extracellular OPN would be crucial to direct recruited or naïve tissue macrophages to senescent reprograming at the cost of phagocytic function.

Most of the recent publications mentioned favorable effects by systemic elimination of senescent cells with senolytic drugs. (5, 35-37) On the other hand, induction of senescence in some cell types is beneficial during aging and repair processes. (9, 38-40) Beside senolytic drugs, we have identified OPN as a novel target for ameliorating cellular senescence in specific cell types such as adipose tissue macrophages, complementing our previous observations in cardiac fibroblasts.( 9) Collectively, our results showed that elimination of senescent ATM ameliorated AT remodeling and dysfunction, suggesting that macrophage-specific and senescence-targeted therapeutics (such as macrophage-directed pharmacological OPN inhibition) could be a viable strategy to control AT senescence and subsequent systemic metabolic alterations resulting from aging.

Methods

Mice: C57/BL6JRj mice (referred as wild-type: WT) were purchased from Janvier Labs, France.

Osteopontine knockout mice (B6.129S6(Cg)-Spp1 tm1Blh /J) were purchased from Jackson laboratory (ME, USA), which had been back-crossed with C57/BL6J mice more than 10 generations, and kept as homozygous (referred as OPN-/-). p16-luciferase mice (Strain Code 01XBT--B6.Cg-Cdkn2a tm3.1Nesh Tyr c-2J/Nci) were obtained from the NCI mouse repository via Dr Norman Sharpless (University of North Carolina).( 21 In Vivo luciferase Detection Exited luciferase activity was detected and analyzed under collaboration with Dr Seguin, Paris Descartes University as previously described.( 21) Briefly, mice were anesthetized with isoflurane, and then injected intraperitoneally with D-luciferin substrate (Caliper Life Sciences; 15 mg/ml in PBS). Images were taken with the PhotonIMAGER system (BIOSPACE).

After substrate administration, peak luminescence was measured at 6-8 minutes. For simultaneous image capture of more than 3 animals, a wide-angle lens was used. In our aging studies, in which animal size varied, mice were injected with 10 μl/g body weight of substrate.

To compare multiple images taken at the same exposure point, PhotoAcquisition Software for PhotonIMAGER (BIOSPACE) was used following substrate administration, and acquisition lasted for 10 minutes (from t=10min to t=20min after luciferin injection). An additional ROI was employed to normalize for background luminescence on each image for all analyses.

Metabolic tolerance tests:

Glucose Tolerance Test (GTT): Mice were fasted for 6-7 hours before the study, and then given glucose solution intraperitoneally (1.5 mg/g body weight). Blood samples were collected from tail vein at baseline, 15, 30, 60, 90, 120 minutes after injection, and glucose concentration was measured with an automatic glucometer (Accu-Chek Performa, Roche, Germany).

Insulin Tolerance Test (ITT): Mice were fasted for 6-7 hours before study, and then given insulin solution intraperitoneally (0.5 mU/g of body weight, Umelin, Lilly, France). Blood samples were collected from tail vein at baseline, 30, 60, 90, 120 minutes after injection, and glucose concentration was measured as above.

In vivo Matrigel plug assay.

OPN-enriched in vivo matrigel plug assay was performed according to the manufacture's instruction and previous report. (41) The total of 6 male, 5-month old WT mice were used. In brief, total 500 μl of Matrigel,(cat#. 354234, Corning) was mixed with 100 l of mouse recombinant OPN (R&D) 50 g or PBS (as vehicle). Mixed solution was subcutaneously injected to groin sites of anesthetized mice. After 14 days, the Matrigel plug was removed, fixed in 4% formaldehyde with mesh-cassettes (Trajan), and further processed for immunohistochemistry.

In vivo macrophage depletion

The total of 17 male WT mice, 12-month old and weighed 30~36 g were used. Before in vivo macrophage depletion, mice were undergone baseline assessment of metabolic functions (GTT and ITT). The mice were then injected intraperitoneally with 70 mg/kg clodronate in liposomes or PBS control liposomes (ClodLip BV, Amsterdam, Netherlands) every 4 days for 4 weeks (70% dose used in reference).( 42) Their body weights were recorded at the same time. After final metabolic assessments, the mice were euthanized by neck-dislodgement. The organs and bloods were harvested and processed for further evaluations.

Osteopontin inhibition by small molecule

The total of 20 male WT mice, approximately 11-month old and weighed 28~35 g were used.

Agelastatin A (AA), small organic molecule known to inhibit OPN production in vivo, was synthesized and kindly provided by Professor Takehiko Yoshimitsu at Graduate School of Pharmaceutical Sciences, Okayama University, Okayama, Japan. After baseline assessment of metabolic functions (GTT and ITT), the mice were given 1.5 mg/kg of AA or vehicle only (2-Hydroxypropyl-β-cyclodextrinonly, Sigma-Aldrich) intraperitoneally every 4 days for 4 weeks.( 9)Their body weights were recorded at the same time. This protocol was developed with informative advices from Pro.Yoshimitsu. After final metabolic assessments, the mice were euthanized by neck-dislodgement. The organs and bloods were harvested and processed for further evaluations.

Mouse bone-marrow transplantation (BMT) Irradiated (9.5 Gy, whole-body), 8-week old male wild-type mice (CD45.1: B6.SJL-Ptprca Pepcb/BoyCrl, congenic strains, C57BL/6 background) were purchased from Charles-River Laboratory (France) as recipient. Bone-marrow cells were taken from femurs of donor mice (4month old male WT and OPN-/-mice as described above; CD45.2) and 1-day after irradiation, the 3-month old recipient mice received 2 millions of donor bone-marrow cells by intravenous tail injection. Mice were kept and aged in house until 16-month old. Body weight was recorded monthly, and peripheral blood reconstitution was analyzed by FACS at 2, 4, 5, 9 and 12-month after BMT. Used antibodies and fluorophores are listed in the supplemental table.

Plasma adipokine measurement

Collected whole blood samples were centrifuged and plasma fraction was separated, stored at -80 °C. Plasma levels of adipokines were assessed with a Magnetic Luminex screening assay kit (Adiponectin: LXSAMSM-1, Leptin: LXSAMSM-19, Bio-Techne, Abington, UK) following the manufacturer's instructions.

Tissue processing and Histology

Dissected organs were fixed with 4% formaldehyde solution (Sigma-Aldrich) immediately and subjected to paraffin-embedding after at least 1 week fixation. The sections were deparaffinized using xylene and a graded series of ethanol dilutions. Adipose tissue sections ( 5m thickness) were stained with hematoxyline & eosin (Sigma-Aldrich, France) for general appearance observation and adipocyte size measurement, and Sirius Red for tissue fibrosis measurement, respectively.

For immunofluorescent staining, deparaffinized and hydrated sections were subjected to heatmediated antigen retrieval (Citrate buffer pH6.0, Dako) and blocking with 30% goat serum.

Primary antibodies were applied with antibody diluent (Dako) over-night at 4°C in humidified chamber. Corresponding 2 nd antibodies conjugated with fluorescent dyes were used followed with DAPI nucleus counter-staining.

The pictures with hematoxyline & eosin and Sirius Red staining were captured using an Axioplan 2 Imaging microscope (Zeiss). Fluorescent images were taken using an Axioplan M2 Imaging microscope (Zeiss). For adipocyte size, at least 200-300 cells per sample were traced and quantified surface area. For interstitial fibrosis, 3-5 images per sample were measured.

Red area normalized by total surface area. All data were measured with the ImageJ software (NIH).

SA--Gal staining

Dissected VATs in 5 mm-square pieces were directly subjected to staining solution (1 mg/ml Xgal, Sigma-Aldrich), and kept at 37°C for 12 hours. The images were taken by GR digital camera (RICOH, Japan). Quantitative PCR Total RNA was extracted from 80mg powdered adipose tissue samples with RNA easy fibrous kit (Qiagen). RNA from cultured macrophages was extracted with RNA easy mini kit (Qiagen) as manufacturer's instruction. First strand DNA synthesis was synthesized from 0.5-1.0 g total RNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and a thermal cycler SimpliAmp (Applied Biosystems) according to manufacturer's instruction.

Quantitative PCR was performed and analyzed with TaqMan® Universal PCR Master Mix and StepOne™ Real-Time PCR System (Applied Biosystems). eta-actin was used as internal control. Used Taqman primers are described in the supplemental table.

Western Blot Analysis

Eighty-90 mg of adipose tissues was homogenized in "Platonic" urea lysis buffer containing 7 M urea, 10% glycerol (v/v), 10 mM Tris-HCl (pH6.8), 1% SDS, 1 mM dithiothreitol, supplemented with protease and phosphatase inhibitor cocktail tablets (Pierce/Thermofisher, 88265, France). After vortexing, lysates were sonicated, passed through a 21G needle at least 6 times, rotated for 30 minutes at 4°C and spun to obtain supernatant. Protein concentration was adjusted according to Bradford method (Bio-rad). Denatured total protein (20µg) was loaded to 10% SDS-PAGE. Separated proteins were transferred to PVDF membranes (Invitrogen), blocked in Pierce™ Clear Milk Blocking Buffer (Thermofisher, 37587, France) and probed for selected proteins with a specific primary antibody at 4°C overnight, followed by incubation with the corresponding HRP-conjugated secondary antibodies (Abcam).

Densitometric quantification was performed with ImageJ software, and normalized to -actin levels in each sample. Primary antibodies are described in the supplemental table.

Adipose tissue immune cell and macrophage flow cytometric analysis (FACS).

Dissected adipose tissues were kept on ice and minced to 1-3 mm pieces in heparinized cold PBS. After snap vortexing, PBS was removed and tissues were digested with collagenase II solution (Sigma-Aldrich) at 37°C water bath with gently shaking for 20 min. Digested tissue was then passed through 70 m mesh and centrifuged to spin down stromal vascular fraction (SVF). After RBC lysis (ACK Lysing Buffer, Gibco, A1049201), the SVF cells were suspended in PBS supplemented 2% fetal-calf serum (FCS), and stained with appropriate antibodies for surface marker and isotype controls for 30 min at 4°C in the dark chamber. Used antibodies and fluorophores are listed in the supplemental table. Living and dead cell discrimination was performed with 7-AAD staining (Molecular Probes). For intracellular p16 staining, SVF cells stained with surface markers were then fixed and permeabilized with Cytofix/Cytoperm solution (BD Bioscience). The cells were then stained with Alexa 488 conjugated p16 antibody (2D9A12, Abcam) and corresponding isotype controls. Flow cytometry was performed with LSRII (BD Bioscience). Data was evaluated and analyzed with FlowJo (TreeStar).

Generation of bone-marrow derived macrophages (BMDM)

Mice bone-marrow cell was isolated from femurs and tibias from 3-or 12-month old WT and OPN-/-mice. After RBC lysis (LCK buffer, Sigma-Aldrich) and passed through 70 m mesh, bone-marrow cells were plated with RPMI 1640 medium (Gibco) supplemented with 5%FCS, penicillin/streptomycin (Gibco) and 30 ng/ml MCSF (R&D systems). Five days after differentiation into macrophages, the cells were treated with Accutase (Sigma-Aldrich), counted and re-plated at 1.5 x10 5 /well in 12-well non-coated plate. The BMDMs were further subjected to OPN stimulation experiment or phagocytosis assay.

BMDM stimulation by osteopontin

Modulation of BMDM by OPN protein was evaluated as previously described with minor modification.( 43) Briefly, 12-well plates were coated with 20 or 40 g of mouse recombinant OPN (R&D systems) for 20 hours at 4°C and then stabilized with 0.5% polyvinylpyrrolidone (Sigma-Aldrich) for 1 hour at room temperature. BMDM (1.5 × 10 5 /well) were re-plated in RPMI 1640 medium supplemented with 5%FCS and 30 ng/ml MCSF. Twenty-four or 36 hours after passage, the cells were harvested for RNA extraction.

BMDM immunofluorescent staining BMDM were passed onto an OPN-coated 8-well Lab-Tek II chambered cover glass (Nalge Nunc) at 1.5 x10 4 /well density. After incubated as described above, the BMDM were washed with chilled PBS buffer and fixed with 2% paraformaldehyde solution for 10 min at room temperature and permeabilized with 0.05% Triton-X (Sigma-Aldrich). The cells were blocked with 2% BSA solution (Sigma-Aldrich) and subjected to p16 and F4/80 antibody treatment for overnight. Corresponding 2nd antibodies conjugated with fluorescent dyes were used to visualize with DAPI nucleus counter-staining. The images were taken using an Axioplan M2 Imaging microscope (Zeiss).

Phagocytosis assay

Human leukemia cell-line, HL60 cells were labeled with pHrodo; iFL Green STP Ester (amine reactive) (Thermofisher) as previously described ( 44) with minor modification. In brief, the HL60 cells were washed twice with PBS buffer and incubated at room temperature for 30 min with 0.1 g/ml pHrodo. The cells were washed with PBS buffer twice, and suspended in the RPMI medium containing 0.5% FCS, and used for engulfment. To 5.0 x 10 4 BMDM in 500 µl of RPMI medium containing 5% FCS in a 12-well plate, 2.0 x 10 5 pHrodo-labeled HL60 cells were added, and incubated at 37°C for 0, 30, 60, and 120 min. The cells were detached from the plate by incubating with 0.25% trypsin and 1mM EDTA, and collected by centrifugation. The cells were labelled with surface marker of CD11b and F4/80, further suspended in 500 l of PBS buffer containing 2% FCS and analyzed by flow cytometry on the LSRII (BD Bioscience).

Data was evaluated and analyzed with FlowJo (TreeStar). For time-lapse imaging, 1.5 x10 4 /well BMDM were passed onto an 8-well Lab-Tek II chambered cover glass (Nalge Nunc), and incubated at 37°C for 0, 30, 60, and 120 min with 6 x 10 4 pHrodo-labeled HL60 cells in 250 l of RPMI medium containing 5% FCS and observed by fluorescence microscopy.

Discriminative gene analysis

In addition to exploring individual changes involving various molecular targets, we aimed to evaluate also the contextual importance of the changes affecting major themes that characterize the adipose tissue functional signature under the effect of aging. To this purpose we measured the VAT expression profile of a panel of 43 genes selected to illustrate 5 major themes characterizing the functional signature of the adipose tissue: efferocytosis, matricellular proteins, senescence, inflammation, adipose tissue functions and secretome. A random-forest supervised learning algorithm was applied iteratively to evaluate and rank the relative discriminative value (i.e. feature importance) associated with changes in these gene expression profiles induced by aging. These computations were performed using the R environment for statistical computing and the algorithmic implementation available in the "randomForest" R package (version 4.6-12). To assess the relative discriminative value associated with each measured expression profile we averaged the feature importance provided by the random forest algorithm over 10,000 successive iterations performed for each of the analyzed situations. The individual discriminative value of each transcriptional profile was expressed as percentages of the total discriminative power of the panel of selected transcriptional descriptors. The resulting values were plotted after grouping them by functional category and ranking the available categories in the decreasing order of their strongest transcriptional descriptor, to illustrate the contextual importance of the functional changes induced by VAT aging

Statistics

All data are presented as mean+SEM. Difference between two groups was analyzed by unpaired t-test. Comparisons between multiple groups were done using one-or two-way ANOVA followed by Tukey or Bonferroni test. These analysis were performed in GraphPad Prism 5.0. for Win (GraphPad Software, La Jolla, California, USA). All t-tests were two-tailed. Antibodies used for immunohistochemistry and western-blot analysis

INTRODUCTION

White adipose tissue (WAT) senescence has emerged as an important contributor to the development of comorbidities associated with obesity (Ahima, 2009; Tchkonia et al., 2010) and aging (Xu et al., 2018). Our group has recently highlighted the impact of WAT senescence on cardiac disorders in the context of aging (Sawaki et al., 2018) and obesity (Ternacle et al., 2017), strengthening evidence that WAT can develop a senescence-associated secretory phenotype (SASP) that entails the release of various profibrotic and pro-inflammatory proteins (Campisi, 2005; Coppe et al., 2008; Munoz-Espin and Serrano, 2014) and contribute to spreading the senescence to remote organs (Tchkonia et al., 2010). Release of bioactive lipids by WAT may additionally affect the senescence phenotype (Das, 2019). Cellular senescence is a fundamental process that drives aging and its consequences, such as frailty and the development of chronic diseases, leading to multi-morbidity and increasing mortality worldwide (McHugh and Gil, 2018). Senescence involves an irreversible arrest of the cell cycle, followed by increased expression of SASP and altered metabolic activity. The accumulation of senescent cells in multiple tissues is central to the pathogenesis of age-related diseases.

Therefore, senolytic strategies targeting fundamental aging processes (Baker et al., 2016; Baker et al., 2011; Xu et al., 2018) and metabolic dysfunction (Palmer et al., 2019) are now extensively investigated. Importantly, exercise and physical activity were shown to attenuate progression of age-related disorders (Derumeaux et al., 2008), and also obesity and HFD-induced cardiometabolic disorders (Schafer et al., 2016).

Despite intensive investigation on the role of WAT in metabolic diseases, the timeline and the underlying mechanisms of WAT senescence have not been fully explored in the context of obesity. A better understanding of these processes could help developing effective strategies targeting senescent cells and preventing obesity-related tissue deterioration. Mitochondrial dysfunction and impairment of adenosine triphosphate (ATP) production are critical in the pathogenesis of obesity-associated diseases and mitochondria are also fundamental in the regulation of fatty acid metabolism, energy expenditure and disposal of reactive oxygen species (ROS) (Lefranc et al., 2019; Lowell and Shulman, 2005). Notably, ATP released by malfunctioning mitochondria to extracellular space during cell death or from senescent cells is recognized as part of damage-associated molecular patterns (DAMP), molecules that induce inflammatory response and are critically involved in the pathogenesis of several diseases (Basisty et al., 2020; Nakahira et al., 2015). Therefore, the potential role of mitochondrial alterations in obesity-induced WAT senescence and their modulation by physical exercise remains to be elucidated. Here we hypothesized that energetic abundance in caloric overload leads to overproduction of ATP, contributing to the induction of senescence.

We used a short-term high fat diet (HFD, 1 to 10 weeks) versus a chow diet (CD) exposure in adult mice (5 months old) to follow the time-course of senescence induction in WAT and the impact on glucose homeostasis. We then used physical exercise as a senostatic strategy to investigate whether metabolic alterations were reversed together with WAT senescence in the context of ongoing HFD. Since mitochondria are at the nexus of both metabolic and aging associated disorders (Correia-Melo et al., 2016; Tavallaie et al., 2020), we investigated mitochondrial function in WAT and primary adipocytes.

We demonstrate here that WAT senescence: 1) occurred as early as 2 weeks of HFD in both subcutaneous (inguinal, iWAT) and visceral (epididymal, eWAT) WAT preceding the development of chronic systemic inflammation and WAT fibrosis; 2) was associated with increased WAT glycolytic and mitochondrial activity leading to increased local ATP.

We identified the role of ATP as a causal mechanism of WAT senescence by demonstrating that liposome-delivered ATP induced preadipocyte senescence in vitro, whilst inhibition of ATP degradation by allopurinol amplified WAT senescence in both in vitro and in vivo experiments. Furthermore, short-term exercise decreased preadipocyte ATP content in iWAT but not in eWAT and, concomitantly prevented HFD-induced WAT senescence.

RESULTS

WAT cellular senescence occurs shortly after initiation of HFD

In order to assess the kinetic of WAT senescence after the initiation of HFD, we followed the animals during a 10-week time course and focused the analysis on eWAT. In parallel with an increase of body weight, fat mass and leptin expression (Fig. S1A), WAT senescence occurred as early as 2 weeks after the initiation of HFD, as demonstrated by a gradual increase in SA-β-galactosidase (SA-β-Gal) activity (Fig. 1A), progressive upregulation of p16 (cyclin dependent inhibitor 2A, Cdkn2a) and p21 (cyclin dependent inhibitor 1A, Cdkn1a) (Fig. 1B) and higher proportion of p16-and p21-positive cells in the adipocyte precursor pool (PDGFRα+ cells) (Fig. 1C). This resulted in increased WAT senescence as assessed in vivo by a 10-week HFD intervention in p16 LUC /+ mice, demonstrating increased bioluminescence in the abdominal cavity area, in relation to the activation of p16INK4a (Fig. 1D).

Since obesity may compromise mitochondrial homeostasis (Lefranc et al., 2019), we next investigated mitochondrial function to assess its contribution to cellular senescence.

During the first five weeks following HFD initiation, we found no evidence of mitochondrial dysfunction based on the assessment of mitochondrial genes (cytochrome C and Tfam), citrate synthase activity and activity of respiratory complexes, such as complex IV (cytochrome C oxidase) and complex V (ATP synthase) (Fig. S1B-C). In addition, we found no evidence for oxidative DNA damage (anti-8-oxoguanine staining), despite a modest increase in mitochondrial ROS (MitoSOX), but not cytosolic ROS (CellROX), in both iWAT and eWAT HFD-derived preadipocytes compared with CD preadipocytes (Fig. S1D).

We then assessed the beneficial effect on WAT remodeling of an exercise intervention (daily swimming) initiated after 5 weeks of HFD in comparison with sedentary HFD and CD animals (Fig. S1E). To avoid the confounding effect of body weight loss with exercise, we selected mice with similar body weight (Fig. S1F) and fat mass (Fig 1E) in each regimen group.

As expected, HFD progressively increased body weight (Fig. S1F), adiposity (Fig 1E ) and adipocyte size in both iWAT and eWAT (Fig. S1G-H). Exercise reduced adipocyte size in both WAT depots and iWAT weight (Fig. S1G-H and Table S1), without altering the food intake (CD-SED 2.8±1 g/day vs. CD-EX 3.4±0.6 g/day, p=NS and HFD-SED 3.7±1.0 g/day vs. HFD-EX 3.7±1.2 g/day, p=NS). Although 10-weeks HFD did not induce WAT fibrosis compared to CD (Fig. S1I-K), it increased the expression of key profibrotic factors such as transforming growth factor β1 (Tgfb1), fibronectin (Fn1) and tissue inhibitor of metalloproteinase 1 (Timp1) in eWAT but not in iWAT (Fig. S1L). Exercise significantly reduced the expression of these profibrotic factors in eWAT of HFD animals.

Also, HFD increased leptin and decreased adiponectin levels in both plasma and tissue (Fig. S1M-N). Despite similar body weight, exercise decreased plasma leptin levels in HFD mice and increased plasma adiponectin levels in CD but not in HFD mice. Additionally, HFD impaired glucose tolerance (GTT) and insulin sensitivity (ITT) (Fig. S1O-P) whilst exercise improved them in HFD mice.

In parallel, the adipogenic function declined early in HFD mice as demonstrated by the downregulation of markers of lipogenesis (fatty acid synthase, Fasn) and lipolysis (perilipin 1, Plin1) in both iWAT and eWAT (Fig. S1Q). HFD also impaired glucose metabolism as shown by a decreased expression of insulin-sensitive glucose transporter (solute carrier family 2 member 4, Slc2a4) and hormone insulin-like growth factor 1 (Igf1) mRNA in both WAT depots (Fig. S1R).

We next focused on the impact of exercise on WAT senescence. Whilst SA-β-Gal activity was increased after 10 weeks of HFD in both WAT and preadipocytes, it was fully normalized by exercise (Fig. 1F-I).

HFD induces cellular senescence in various types of WAT cells, out of systemic inflammation.

Having demonstrated a HFD-induced SA-β-Gal activity in WAT, we then explored additional markers of cellular senescence. First, HFD increased p21 expression in both iWAT and eWAT when p16 was upregulated only in eWAT (Fig. 2A). Exercise decreased only p21 in the iWAT depot (Fig. 2A). Then we assessed p16 and p21 expression in preadipocytes after verifying that senescence was not influenced by culture conditions prior to differentiation (Fig. S2A). HFD upregulated p16 and p21 expression in eWAT-but not in iWAT-derived preadipocytes, whilst exercise normalized it (Fig. 2B). Furthermore, a 10-week HFD increased p16+/ PDGFRα+ by 6-fold in iWAT and 9-fold in eWAT (Fig. 2C). Similarly p21+/ PDGFRα+ increased by 4-fold in iWAT and 5.5-fold in eWAT (Fig. 2D). Interestingly, exercise blunted senescence in both WAT depots (Fig. 2C-D). In addition, WAT senescence was associated with adipocyte hypertrophy (Fig. S2B). HFD also increased the number of crown-like structures (CLS, 15fold in eWAT vs. 3-fold in iWAT) -immune cells surrounding dying adipocytes (Fig. S2C).

Both CD45+ and CD45-cells exhibited a senescent phenotype, as demonstrated by an increase in the S139 phosphorylated form of H2A.X expression -a marker of double-stranded DNA break and senescence (Vergoni et al., 2016) -in HFD, but was ameliorated by exercise (Fig. S2C-D). Furthermore, HFD increased p16+ co-localization with both iWAT and eWAT Mac3+ macrophages whilst exercise normalized it (Fig. S2E). By contrast, p21+ co-localization with macrophages increased only in eWAT and was not normalized with exercise (Fig. S2F). A similar pattern was observed with p16 staining in eWAT lymphocytes (Fig. S2G).

Remarkably, WAT senescence occurred without systemic inflammation as plasmatic levels of proinflammatory factors [C-C-Chemokine-Ligand (CCL2), tumor necrosis factor α (TNFα), interleukin 6 (IL6), interleukin 10 (IL10)] were undetectable. Despite upregulation of

Mcp1/Ccl2 in eWAT but not iWAT (Fig. S2H), HFD did not significantly increase proinflammatory factors in eWAT secretome (Fig. S2I).

To better discriminate the individual importance of the factors involved in the HFD-driven alteration of WAT, we applied a supervised predictive model to a panel of transcriptional descriptors selected to illustrate adipocyte function, senescence, inflammation and tissue remodeling in iWAT (Fig. 2E) and eWAT (Fig. 2F). Interestingly, the best predictors of HFDinduced changes in eWAT were leptin and the chemotaxis marker Ccl2, followed closely by the senescence marker p21, while leptin increase was the main change occurring in iWAT.

Altogether, these data reveal that, WAT cellular senescence induced by HFD affects multiple cell populations of adipogenic and immune origins in both depots, is rescuable by exercise and precedes systemic inflammation.

HFD-induced WAT senescence is associated with increased adipocyte bioenergetics.

We next assessed whether a 10-week HFD intervention compromised mitochondrial homeostasis and whether exercise exerted beneficial effects on WAT bioenergetics (Stanford et al., 2015). Mitochondrial biogenesis and oxidative metabolism in WAT were impaired by HFD (Fig. S3A-B). The expression of the master regulators of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α) and its downstream genes, were downregulated by HFD in both WAT depots and preadipocytes (Fig. S3A). HFD also downregulated genes related to fatty acid oxidation, such as peroxisome proliferator-activated receptor alpha (Pparα) and related genes in both WAT and preadipocytes (Fig. S3B). HFD increased iWAT and eWAT mitochondrial content as assessed by immunofluorescence staining of a mitochondria-specific marker, Tom20 (Fig. 3A) with unaltered citrate synthase activity in whole WAT (not shown) and in mature adipocytes (Fig. 3B). Exercise normalized mitochondrial content, blunted HFD-induced mitochondrial biogenesis alterations and upregulated genes related to WAT browning (Pgc1α, Prdm16, and Ucp1) in iWAT (Fig. 3A-B; Fig. S3A-B). We further assessed mitochondrial expression profile in eWAT and found the HFD-induced downregulation of several genes related to mitochondrial homeostasis, beta-oxidation, morphogenesis (fission/fusion, autophagy and apoptosis), as illustrated by a heatmap representation of their expression profiles (Fig. S3C). These changes were associated with the downregulation of mitofusin2 (Mfn2) in both WAT (Fig. S3D).

Next, we analyzed mitochondrial oxygen consumption (OCR) and glycolytic activity (extracellular acidification rate, ECAR) in real time by the Seahorse technology in preadipocytes (Fig. S3E-F). HFD increased mitochondrial and non-mitochondrial OCR in preadipocytes derived from both WAT depots (Fig. 3C). In addition, HFD enhanced glycolysis (ECAR) in both WAT depots, while exercise restored it only in iWAT (Fig. 3D).

Interestingly, ATP-linked oxygen consumption was increased by HFD in preadipocytes from both iWAT and eWAT, suggesting a higher respiratory chain and ATP synthase activity (Fig. 3E). Accordingly we found a higher ATP content in preadipocytes derived from both WAT depots in HFD sedentary mice, whilst exercise reduced ATP content in iWAT but not eWAT, drawing our attention to potential differences in the purine catabolism pathway between the two fat depots (Fig. 3 F,G). Thus, we explored the first and only unidirectional purine catabolic enzyme, xanthine oxidase (XO), which catalyzes the oxidation of hypoxanthine to xanthine, and of xanthine to uric acid. Interestingly, XO protein level was considerably higher in iWAT than in eWAT, without any impact of diet-or exercise-intervention (Fig. 3H). This finding was further confirmed by the increased XO staining of primary iWAT-versus eWAT-derived preadipocytes (Fig. 3I). As a consequence, plasmatic uric acid, the end-degradation product of ATP, slightly increased with HFD, but was normalized by exercise (Fig. S3G). Interestingly HFD did not increase uric acid content in both WAT depots (Fig. 3J). HFD also increased the expression of the purinergic receptor P2X, ligand-gated ion channel, 7 (P2rx7), a modulator of cell damage and ATP release, in eWAT but not in iWAT (Fig. 3K).

These results, highlighting the increase in HFD-related ATP content in both WAT depots and the normalization by exercise only in iWAT, are summarized in Figure 4 together with the impact on senescence.

Role of ATP in WAT senescence.

To evaluate the specific role of intracellular ATP in WAT senescence, we assessed SA-β-gal activity, p16 and p21 expression in iWAT-and eWAT-derived primary adipocytes treated with ATP-loaded liposomes. First, we demonstrated the difference between iWAT and eWAT after ATP-loaded liposome delivery and showed that ATP peaked at 30 minutes after treatment in iWAT-derived preadipocytes (Fig. S5A), followed at 6 hours by a decline in ATP content concomitant with an increase in uric acid. In contrast, in eWAT-derived preadipocytes, ATP levels remained elevated over 24 hours. This is consistent with the difference in XO protein expression in the respective WAT depots (Fig. 3H-I), whilst purinergic receptor expression increased in both iWAT-and eWAT-derived preadipocytes in a similar manner at 6 hours of ATP stimulation (Fig. S5C). Interestingly, ATP-loaded liposomes induced p21 expression at 6 hours after treatment in both iWAT and eWAT, and p16 expression at 6 hours in eWAT and delayed at 24 hours in iWAT (Fig. 5A-B). Thus, we evaluated ATP-inducible senescence compared to either liposome free, liposome control or uric acid-loaded liposome after 6-hour treatment (Fig. 5C-F). Senescence occurred only after delivery of ATP, but not uric acid, in comparison with liposome-free and control conditions (Fig. 5C-F). Of note, ATP-induced senescence was higher in eWAT than in iWAT (1.5 fold vs 1.2 fold, p=0.0011).

Furthermore, to prove the role of ATP accumulation in initiating WAT senescence in the context of HFD, we inhibited ATP catabolism by allopurinol, a XO inhibitor, in both ATP-and in high glucose/palmitate-(metabolic media: MM, to mimic in vitro caloric overload) treated preadipocytes (Fig. 5C-D). Notably, MM increased SA-β-gal positivity only in eWAT, but not in iWAT, aligned with the differences in XO levels between the two fat depots (Fig. 5C-D).

Preventing the degradation of ATP with allopurinol aggravated senescence in ATP-and MMtreated iWAT preadipocytes. In eWAT allopurinol increased SA-β-gal positivity only in ATPbut not MM-treated preadipocytes (Fig. 5C-D). Furthermore, allopurinol increased p21 expression in both iWAT and eWAT ATP-treated preadipocytes, and p16 only in eWAT (Fig. 5E-F). Similar results were obtained in treating 3T3-L1 adipocytes with ATP-and uric acidloaded liposomes, lending support to the critical role of ATP, but not uric acid, in inducing WAT senescence (Fig. S5D-E).

To further establish ATP as a trigger of WAT senescence, we inhibited the degradation of ATP by allopurinol administration after 5-week HFD in vivo, alone or in combination with exercise (Fig. S5F). The effectiveness of allopurinol treatment was assessed by a 15% decrease in uric acid plasma levels together with a similar trend in eWAT lysates (Fig. S5G). Furthermore, allopurinol increased p21+ adipocyte precursors in iWAT, but not eWAT, of HFD-SED mice and reduced the beneficial effect of exercise mainly in iWAT (Fig. 5G). Allopurinol similarly increased p16+ adipocyte precursors and reduced exercise rescue in both HFD iWAT and eWAT. Allopurinol, as opposed to exercise, did not affect body weight, adiposity, SA-β-gal activity, insulin sensitivity (ITT) and glucose tolerance (GTT) (Fig. S5H-K).

Altogether these results indicate a key role of ATP, but not uric acid, in the induction of WAT senescence in a 10-week HFD intervention. Remarkably, addition of allopurinol reduced ATP degradation and amplified the pro-senescent effect of ATP in caloric overload in vitro and in vivo.

DISCUSSION

Our data highlight the accumulation of senescent cells within both visceral and subcutaneous WAT as a very early event occurring two weeks after HFD initiation in parallel with metabolic alterations but independent of systemic inflammation or WAT fibrosis. Furthermore, we identify HFD-induced ATP overproduction by increased glycolysis and oxidative phosphorylation, as a putative mechanism underlying WAT senescence. Exercise prevents metabolic alterations independently of major body weight and adiposity reduction and ameliorates senescence mainly in iWAT. This finding is consistent with the high expression of xanthine oxydase in iWAT, which allows ATP degradation and explains the full rescue of senescence during exercise in this fat depot.

WAT senescence occurs early after HFD initiation

In the context of caloric overload, the energy storage in WAT reaches a threshold that initiates a cascade of events leading ultimately to systemic inflammation and pathological consequences, including increased insulin resistance and remote organ damage (Minamino et al., 2009; Palmer et al., 2019; Ternacle et al., 2017). Whilst most of the literature evaluates long-term HFD, we focused our investigation on 10-week HFD in adult mice. Although we did not observe WAT fibrosis, nor local or systemic inflammation, HFD induced glucose intolerance, insulin resistance and leptin production consistent with increased fat mass and adipocyte hypertrophy together with organization of immune cells in crown-like structures (Murano et al., 2008).

From a detailed kinetic during the 10-week HFD exposure, our main observation is that cellular senescence represents one of the earliest events occurring in WAT, two weeks after the initiation of HFD as demonstrated by increase in SA-β-gal activity, senescence markers (Cdkn1a and Cdkn2a) and p16 and p21 positive staining. The increased positive staining was associated with adipocyte hypertrophy, as recently reported in other cell types (Anderson et al., 2019; Biran et al., 2017; Neurohr et al., 2019). Furthermore, we observed an increase in in vivo p16INK4a expression at 10-week HFD.

Senescence occurred in the different WAT cell populations, including mature adipocytes, preadipocytes, adipocyte progenitors and immune cells. Interestingly, the senescence phenotype was maintained in differentiated HFD-derived preadipocytes, reflecting the in vivo characteristics of parental tissue (Hausman et al., 2008). This finding is important considering the impact of WAT senescence on systemic disorders and lifespan (Baker et al., 2016).

Of note, we did not observe any tissue induction of classical pro-inflammatory SASP factors (CCL2, TNFa, IL6, IL10, PAI1) (Coppe et al., 2008; Munoz-Espin and Serrano, 2014; Saker et al., 2016) as opposed to longer (i.e. 30 vs 10 weeks in our model) HFD exposure in older (12-13 vs 7 months) animals (Schafer et al., 2016). Instead, we observed an upregulation of several SASP-associated chemokines ( Mcp1) and profibrotic cytokines (Tgfb1, Fn1, Timp1) in eWAT but not iWAT. Indeed, MCP-1 is secreted by senescent cells and has recently been correlated with biological age in mammals (Yousefzadeh et al., 2018), whilst TGFβ promotes matrix remodeling in remote organs (Sawaki et al., 2018). Our current data and recent observations (Sawaki et al., 2018; Ternacle et al., 2017) support the premise that senescent WAT is an important source of profibrotic cytokines, known to alter tissue homeostasis prior to the induction of a pro-inflammatory secretome. This profibrotic secretome contributes to organ remodeling, physical dysfunction and premature aging (Munoz-Espin and Serrano, 2014; Sawaki et al., 2018; van Deursen, 2014).

Increased ATP content initiates WAT senescence without mitochondria dysfunction

Mitochondria are important for senescence and the SASP production (Correia-Melo et al., 2016; Passos et al., 2010). In the context of high calorie intake mitochondria increase ROS generation as a toxic by-product of oxidative phosphorylation. Our data are consistent with this observation since the 10-week HFD regimen resulted in marginally increased mitochondrial ROS production in WAT exerting a potential trophic effect (Lee et al., 2009). Importantly, ROS mildly increased after WAT senescence occurred in the HFD kinetic. Additionally, mitochondria play a critical role in maintaining cellular activities by generating energy in the form of ATP. In HFD sedentary mice, our data show an elevated ATP content in both iWAT and eWAT preadipocytes, but exercise normalized it only in iWAT. Thus, we explored the possibility of increased purine catabolism in iWAT. First, we observed substantially higher expression of xanthine oxidase, the enzyme controlling purine degradation, in iWAT than in eWAT. Moreover, XO uses molecular O2 as an essential cofactor, which is provided by exercise hyperemia that improves blood flow in the neighboring iWAT. The combination of high XO expression, HFD-increased levels of substrate for purine degradation and O2 cofactor is consistent with the difference between iWAT and eWAT senescence prevention by exercise.

We also show a HFD-induced expression of P2rx7, a modulator of cell senescence (Cho et al., 2014) increased in obesity, only in eWAT.

Since high concentrations of ATP and its end-degradation products have been shown to be endogenous "danger-associated molecular patterns", DAMPs (Basisty et al., 2020; Nakahira et al., 2015), we further demonstrated that ATP treatment induced senescence in both primary preadipocyte and 3T3-L1 adipocyte culture, whilst uric acid did not. Indeed ATP, but not uric acid-loaded liposomes rapidly induced an increase in SA-β-gal activity and an upregulation of p16 and p21.

Exercise exerts a "senostatic" action preventing WAT senescence during HFD

Our study extends an important observation showing that exercise prevents adipose tissue senescence in HFD (Schafer et al., 2016) and adds mechanistic clues to a triggering role for ATP. The significance of our present findings is further highlighted by the lack of confounding factors known to interfere with senescence, such as physiological age (Sawaki et al., 2018) and weight loss (Ma et al., 2020), in our study design.

Exercise promotes mitochondrial biogenesis and improves function in multiple organs including the heart (Judge and Leeuwenburgh, 2007). In the current experimental stage of HFD, we show that exercise reduced global WAT respiration, mitochondrial ATP-linked oxygen consumption and glycolysis, ultimately resulting in reduced ATP accumulation in iWAT but not in eWAT. Moreover, exercise improved insulin sensitivity in obesity by modulating the excessive energy generation. These findings are in line with the normalization of HFD-induced increase in mitochondrial mass, a pattern closely reflecting that observed in other models of senescence (Korolchuk et al., 2017; Stab et al., 2016). As ATP acts as a danger signal on immune cells (Faas et al., 2017), our data show that exercise-related ATP decrease reduced p16 and p21 expression in WAT macrophages, which may improve the clearance of senescent cells.

Our data are in line with a recent observation showing that restraining mitochondrial ATP synthesis by a respiratory chain complex IV moderate inhibitor might alleviate age-associated disorders (Tavallaie et al., 2020).

Clinical impact

Given the worldwide obesity epidemics, there is an increasing interest in public health programs encouraging healthy eating behavior and physical activity. Aerobic training is known to have a beneficial impact at different life stages on several aspects of human health, such as cognitive function, cardiovascular, endocrine and immune responses. Importantly, the benefit of exercise has been demonstrated in pathological conditions, including diabetes, cancer, and cardiovascular disorders (Myers, 2003). Here we add mechanistic insights to the role of exercise in preventing both cellular senescence and metabolic alteration in the context of obesity, reinforcing the concept that adipose tissue dysfunction affects glucose homeostasis (Pini et al., 2016). Indeed, we demonstrate that exercise improves WAT cellular senescence, adipokine dysregulation and adipocyte bioenergetics independent of weight loss, favoring the recovery of glucose homeostasis in obesity. Therefore, exercise through the control of local ATP seems an interesting approach to prevent the induction of cellular senescence and its potential deleterious consequences.

In summary exercise is an excellent senostatic strategy without any side effect, for preventing obesity-associated senescence in WAT.

EXPERIMENTAL PROCEDURES

Animals

Five months old C57/BL6JRj mice (WT, Janvier Labs, France) were subjected to chow diet (CD) and HFD (60% calories from fat, D12492, irradiated; Research Diets Inc., New Brunswick, NJ) for 1, 2 and 5 weeks to assess the kinetics of epididymal white adipose tissue (WAT) senescence.

In a separate set of experiments, CD and HFD mice were followed up to 10 weeks. After 5 weeks on the respective diets, mice were randomly assigned to: CD-sedentary (CD-SED), CDexercise (CD-EX), or HFD-sedentary (HFD-SED), HFD-exercise (HFD-EX); n = 15/group (Fig. S1E). The exercise consisted of two swimming sessions/day for 5 days/week during 4

weeks (Derumeaux et al., 2008). Mice were 7 months old at necropsy. To avoid any bias related to differences in body weight, the whole investigation was conducted in mice with matched body weights and adiposity levels, as measured at the end of the protocol (n=8/group).

In a separate set of experiments, 3 months-old p16 LUC heterozygote mice (obtained from N E Sharpless (Chapel Hill, NC, USA) and bred at CNRS-TAAM, France), harboring a knock-in of the luciferase gene into the Cdkn2a locus (Sorrentino et al., 2014), were exposed to CD and HFD for 10 weeks. This model aimed to explore in vivo the HFD-induced transcription of p16INK4a, a marker of senescence.

In a separate set of experiments, one group of HFD mice was treated with allopurinol (1mM in the drinking water) (Bravard et al., 2011), starting 5 weeks after the initiation of HFD, alone or in combination with exercise, up to 10 weeks of HFD (Fig. S5F).

All experiments were performed in accordance with the guidelines of our Institutional Animal Care and Use Committee (IACUC). The protocol was approved by the IACUC of the French National Institute of Health and Medical Research, U955, Creteil, France (ComEth 15-001).

In vivo procedures

During follow-up animals underwent weekly body weight (BW) measurements. Daily food consumption was monitored over one week in all groups. Insulin (ITT) and glucose (GTT)

tolerance tests were performed at baseline, before exercise and at the end of exercise intervention (Sawaki et al. 2018).

Plasma and tissue collection and measurements

On the day of the sacrifice, mice were not fasted and CD and HFD exercise groups had the last swimming session 24 hours before. Blood was collected and processed as described (Sawaki et al. 2018). Subcutaneous (inguinal, iWAT), visceral (epididymal, eWAT) and perirenal (pWAT)

WAT samples were collected, weighed and immediately processed for further analysis. Plasma leptin and adiponectin were quantified by ELISA (R&D Systems, Minneapolis, MN, USA).

Conditioned media (eWAT secretome) and plasma were analyzed for a selected panel of proinflammatory mediators (Campisi, 2005; Coppe et al., 2008; Munoz-Espin and Serrano, 2014; Saker et al., 2016), using a Luminex Bio-Plex cytokine assay kit (Bio-Rad, USA). Plasma and WAT uric acid was quantified by a colorimetric assay kit (BioVision, Milpitas, CA, USA).

For WAT measurements, we used the sample deproteinization preparation kit (BioVision, Milpitas, CA, USA).

Histological assessment

Sections of iWAT and eWAT were stained with hematoxylin & eosin for adipocyte size measurements. Sirius Red and immunofluorescent staining procedures were performed as described (Sawaki et al., 2018). Antibodies are reported in the Table S2.

Primary cell culture

Preadipocytes were isolated from iWAT and eWAT stroma vascular fraction (SVF) and differentiated in an adipogenic culture media after two passages to eliminate non-preadipocyte cell contamination. In detail, WAT was aseptically minced and incubated with collagenase Type II (1 mg/mL, Sigma) at 37°C for 45 min. After digestion, serum-containing medium was added to the suspension and filtered through a 100 μm cell strainer. SVF cells were centrifuged for 10 min at 350 g and suspended in pre-warmed adipogenic culture media, consisting of DMEM high glucose with Glutamax (Gibco) supplemented with 10% new born calf serum (Thermofisher), 2.4 nM human insulin (Sigma) and 1% antibiotic solution (Pen-Strep solution, Sigma). To induce differentiation, SVF cells were seeded near confluence and medium was replenished every day to allow selection by adhesion during the first 3 days and then changed every 2 days. SVF cells were cultured in an atmosphere of 5% CO2 and 20% O2 at 37°C. Based on preliminary experiments, day 7 was the time of differentiation used for all experiments.

SA-β-galactosidase activity

For senescence assays, portions of iWAT and eWAT depots freshly dissected and respectively differentiated preadipocytes were incubated with staining solution (1 mg/ml Ultrapure X-gal, Sigma-Aldrich, France) (Itahana et al., 2007; Sawaki et al., 2018). For β-galactosidase activity in tissue, a score of relative intensity of the signal (0=absent, 1=low, 2=medium, 3=high, 4=very high) was used.

Primary cell metabolic rate measurements

For bioenergetics assessment, the Mitochondrial Stress Test was performed on iWAT-and eWAT-differentiated adipocytes (20,000 cells/well) using the XF24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA). Oxygen consumption rate (OCR), indicative of mitochondrial activity, and extracellular acidification rate (ECAR), an index of glycolysis, were determined as described (Foresti et al., 2015).

ATP measurements

Differentiated primary preadipocytes and 3T3-L1 were lysed and intracellular ATP content measured by the ATP-lite assay kit (Perkin Elmer, Villebon-sur-Yvette, France) (Braud et al., 2018).

Cell culture treatments

Primary preadipocytes were isolated from WAT of 5-month-old CD male mice. At differentiation, cells were incubated at different time points (for the time course: 30 min-3h-6h-24h or as specified in the figure legend) with adenosine triphosphate (ATP)-and uric acidloaded liposomes (200 μM, Sigma-Aldrich, France) as compared to two control applications: liposome-free (lipo-free) and liposome alone (CTL).

In addition, to assess the role of ATP as a senescence trigger, we performed interference experiments by co-administration of the xanthine oxidase (XO) an allopurinol inhibitor.

Preadipocytes were subjected for 6 hours to in vitro caloric overload in the form of either high glucose/palmitate (Metabolic media: MM) or ATP-loaded liposomes and co-administration of allopurinol (1mM).

Fully differentiated 3T3-L1 adipocytes, cultured in an atmosphere of 5% CO2 and 20% O2 at 37°C, were incubated for 24 hours with ATP or uric acid (200 μM, Sigma-Aldrich, France).

ATP and the other molecules were encapsulated in liposomes (Sigma-Aldrich, France) (Braud et al. 2018).

Superoxide level measurement

Cytosolic and mitochondrial ROS levels were measured in differentiated preadipocytes (7000/well) by quantification of CellROX (10 µM) and MitoSOX (5 µM) fluorescence intensity levels, respectively. Fluorescence readings were done by a microplate reader (Tecan, Männedorf, Switzerland).

Quantitative Real-Time PCR

Total RNA was isolated from total iWAT and eWAT, the differentiated preadipocytes and 3T3-L1 adipocytes using RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA).

Quantification was performed with Qubit RNA HS Assay Kit (Life Technologies, Thermo Fisher Scientific, MA, USA). First strand DNA synthesis was realized using 0.25-0.5 ng total RNA and qPCR was performed and analyzed as described (Sawaki et al. 2018). Taqman assays are reported in the Table S2.

RNA profiling

PAMM-087ZA RT 2 Profiler TM PCR Array (Qiagen, Valencia, CA, USA) was performed on 600 ng of total RNA from eWAT. Real-time PCR was then performed using Brillant II SYBR Green QPCR Master Mix (Agilent) and Mx3005 Pro thermocycler (Agilent).

Statistical analyses

All statistical analyses were carried out using GraphPad Prism 5 (La Jolla, CA) and the R environment for statistical computing. All data are reported as mean±SEM. Comparisons between multiple groups were done using one-or two-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered as significant. All images were processed with the ImageJ (NIH, https://imagej.nih.gov/ij/). To evaluate the importance of the changes affecting major biological annotations that characterize the WAT functional signature under the effect of HFD, we measured the iWAT and eWAT expression profile of a panel of 22 genes illustrating adipocyte function, inflammation, tissue remodeling and senescence. A random-forest supervised learning algorithm ("randomForest" R package, version 4.6-12) was applied iteratively to evaluate and rank the relative discriminative power associated with changes in these gene expression profiles induced by HFD in iWAT and eWAT. To assess the relative discriminative power associated with each measured expression profile we averaged the feature importance provided by the random forest algorithm over 10,000 successive iterations performed for each of the analyzed situations. The individual discriminative power of each transcriptional profile was expressed as percentages of the total discriminative power of the panel of selected transcriptional descriptors. The resulting values were plotted after grouping them by functional category and ranking the available categories in the decreasing order of their strongest transcriptional descriptor, to illustrate the contextual importance of the functional changes induced by diet in the two WAT depots. Separately, the expression of a panel of several mitochondria genes was quantified by using RT2 ProfilerTM PCR Array. A linear regression model was used to test for differential expression in relation to diet. Significant transcriptional changes were then annotated in relation to three main themes characterizing mitochondrial function (i.e. morphogenesis, homeostasis and beta-oxidation) and illustrated graphically using a heat-map representation.

Tavallaie, M., Voshtani, R., Deng, X., Qiao, Y., Jiang, F., Collman, J.P., and Fu, L. (2020). Moderation of mitochondrial respiration mitigates metabolic syndrome of aging. Proc Natl Acad Sci U S A. Tchkonia, T., Morbeck, D.E., Von Zglinicki, T., Van Deursen, J., Lustgarten, J., Scrable, H., Khosla, S., Jensen, M.D., and Kirkland, J.L. ( 2010). Fat tissue, aging, and cellular senescence. Aging Cell 9, 667-684.

FIGURE LEGENDS

Figure 1. Short-term high fat diet regimen induces adipose tissue senescence. Detailed kinetics of epidydimal adipose tissue (eWAT) senescence after initiation of HFD at 1, 2, 5 and 10 weeks compared with chow diet (CD) group (n=5/group). A: β-galactosidase (SA-β-gal) activity in eWAT. B: Gene expression for senescence markers, p16 (Cdkn2a) and p21 (Cdkn1a), in eWAT C: Representative images of 5-week HFD and quantification by immunofluorescence staining in eWAT of co-localization with p16 (green) or p21 (green) and marker of adipocyte precursor platelet-derived growth factor receptor alpha, PDGFRα (red).

Sections were counterstained with DAPI (blue). Double positive cells are expressed as the mean number positive cells over adipocytes (n=3-4/group, 5 pictures per mouse; magnification x200, scale bar = 50 μm). D: Whole-body luminescence for individual p16 LUC transgenic male mice are depicted at 10-week HFD. Bioluminescence (BLI) is expressed in arbitrary unit (AU) and calculated as BLI = BLI ROI area of interest (in Ph/s/sr) / BLI ROI LED (internal control -LED-in ph/s/sr), where Ph/s/sr is photon per second per steradian (CD n = 2, HFD n = 3). E: Fat mass (adiposity index) of four groups of mice: CD-sedentary (CD-SED), CD-exercise (CD-EX), HFD-sedentary (HFD-SED), HFD-exercise (HFD-EX); n=8/group. Adiposity index calculated by the ratio between the sum of three WAT weights and body weight per mouse. F-G: Representative images of SA-β-gal in iWAT and eWAT and quantification of relative intensity of signal (0=absent, 1=low, 2=medium, 3=high, 4=very high). H-I: Representative images and quantification of SA-β-gal in preadipocytes derived from iWAT and eWAT (n=4 mice/group, 8 pictures/mouse; magnification x200, scale bar = 50 μm). For gene expression, results are expressed as fold mRNA change relative to CD-SED values set to 1; n = 5/group. All data are presented as mean ± SEM. For the kinetics after HFD initiation (panels A-B-C), statistical significance was evaluated by one-or two-way ANOVA followed by Bonferroni correction. *P < 0.05 vs. CD; π < 0.05 vs. 1-week HFD. For all other experiments, statistical significance was evaluated by a one-or two-way ANOVA followed by Bonferroni correction. *P < 0.05; **P < 0.01; ***P < 0.001 for differences due to diet regimen within sedentary and exercise groups (* = diet effect) and e P < 0.05; ee P <0.01; eee P < 0.001 for differences between sedentary and exercise groups fed the same diet ( e = exercise effect).

E-F:

The discriminative power of four major functional themes illustrating the HFD impact on WAT transcriptional profile. Transcriptional changes of a panel of selected genes were evaluated separately in iWAT and eWAT. Their discriminative power was estimated by iterative testing in a supervised predictive model and is expressed as percentage of their total discriminative power. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 for differences due to diet regimen within sedentary and exercise groups (* = diet effect) and e P < 0.05; ee P <0.01; eee P < 0.001 for differences between sedentary and exercise groups fed the same diet ( e = exercise effect). J: Uric acid concentration in iWAT and eWAT tissue lysates, n = 6-12/group). K: Gene expression analysis in iWAT and eWAT for purinergic receptor P2X, ligand-gated ion channel, 7 (P2rx7). For gene expression, results are expressed as fold mRNA change relative to CD-SED values set to 1; n = 6/group. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 for differences due to diet regimen within sedentary and exercise groups (* = diet effect) and e P < 0.05; ee P <0.01; eee P < 0.001 for differences between sedentary and exercise groups fed the same diet ( e = exercise effect). The table displays the differences between iWAT and eWAT together with the effect of regimen and exercise in terms of adipose tissue function, senescence markers and ATP and uric acid contents. preadipocytes stimulated for 6h with uric acid, high glucose/palmitate (metabolic media: MM) or ATP-loaded liposomes alone or in combination with allopurinol (5 pictures per condition; magnification x400, scale bar = 25 μm). E-F: Gene expression analysis for senescence (Cdkn1a, Cdkn2a) in chow diet (CD) iWAT-and eWAT-derived preadipocytes stimulated for 6h with uric acid-or ATP-loaded liposomes alone or in combination with allopurinol. For gene expression, results are expressed as fold mRNA change relative to Lipo-free values set to 1; n = 3/group. G: Representative images and quantification by immunofluorescence staining in iWAT and eWAT of co-localization with p21/CDKN1A (green) and PDGFRα (red). Sections were counterstained with DAPI (blue). Double positive cells are expressed as the mean number of positive cells over adipocytes (n=3-4/group, 5 pictures per mouse; magnification x200, scale bar = 50 μm). All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 for differences with reference control (liposome alone in vitro and control diet in vivo), e P < 0.05 for differences between sedentary and exercise groups fed the same diet ( e = exercise effect) and # P < 0.05; ## P <0.01 for the effect of allopurinol treatment between groups. Toutes ces données apportent la preuve de l'effet délétère et pathogène de l'augmentation des taux plasmatiques de Phe sur le coeur et offrent de nouvelles cibles thérapeutiques pour le traitement des altérations cardiaques associées au vieillissement.

Background: Aging myocardium undergoes progressive cardiac hypertrophy and interstitial fibrosis with diastolic and systolic dysfunction. Recent metabolomics studies shed light on amino acids in aging.

The present study aims to dissect how aging leads to elevated plasma levels of the essential amino acid phenylalanine (Phe) and how Phe may promote age-related cardiac dysfunction.

Methods:

We studied cardiac structure and function, together with Phe catabolism in WT and p21 -/- mice aged between 2 and 24 months, the latter known to be protected from cellular senescence. To explore Phe's effects on cellular senescence and ectopic Phe catabolism we treated primary adult rat or human AC-16 cardiomyocytes with Phe. To establish a role for Phe in driving cardiac aging, wild-type (WT) male mice were treated twice a day with Phe (200 mg/kg) for a month. Finally, we treated aged WT mice with tetrahydrobiopterin (BH4; 10 mg/kg), the essential cofactor for Phe-degrading enzyme phenylalanine hydroxylase (Pah) or restricted dietary Phe intake. The impact of aging on liver Phe catabolism was explored in vitro in AML-12 hepatocytes subjected to Phe or Nutlin3a (a p53 activator)induced senescence with or without p21 siRNA or BH4, with Pah protein and tyrosine levels determined.

Results: Natural aging induced progressive increase in plasma Phe levels concomitant with cardiac dysfunction, whilst p21 deletion delayed these changes. Phe treatment induced cardiac deterioration in young WT mice, strikingly similar to that occurring with age, whilst it triggered cellular senescence, redox and epigenetic changes in vitro. Pharmacological restoration of Phe catabolism with BH4 or dietary Phe restriction abrogated the rise in plasma Phe and reversed senescent cardiac alterations in aged WT mice to a great extent. Our findings obtained from aged mice and human samples indicated a key role for hepatic Phe catabolic decline in elevated plasma Phe levels compromising cardiac integrity.

Conclusions:

Our findings establish a pathogenic role for increased Phe levels in cardiac aging and highlight Phe/PAH modulation as a potential therapeutic target for age-associated cardiac impairment.

Aging leads to increased cellular senescence in organs, thus disrupting tissue structure and function through a senescence-associated secretory phenotype consisting of pro-inflammatory cytokines, extracellular matrix-degrading proteins and profibrotic factors. 1 Aging is therefore a major risk factor for many life-threatening disorders including cardiovascular diseases, mainly heart failure. 2 A recent report has shown that during aging cardiomyocytes acquire a senescent-like phenotype that contributes to the age-related myocardial dysfunction. 3 Indeed, aging myocardium undergoes adverse structural alterations characterized by progressive cardiomyocyte hypertrophy, interstitial fibrosis and inflammation, ultimately leading to diastolic and systolic dysfunction. [4][5][6] Apart from traditional cardiovascular risk factors (e.g. hyperlipidaemia, systemic hypertension and obesity), recognized to accelerate cardiac aging, 7 specific mechanisms have been explored, 8 among others mitochondrial oxidative stress, 3,9 and myocardial activation of phosphoinositide 3-kinase (PI3K). 10 In addition, our group has recently pointed out that cardiac aging is a multisystem disease resulting from complex and intertwined interactions with other organs, in particular visceral adipose tissue that exerts remote adverse effects by releasing profibrotic factors, such as osteopontin and TGFβ. 6 In parallel, metabolomics shed light on the link between amino acids and aging/heart failure. In particular, plasma levels of phenylalanine (Phe) in human subjects increase with age, [11][12][13][14] and inversely correlate with leukocyte telomere length, 15 a marker of aging. 16 In addition, increased serum Phe levels have been proposed as one of the metabolic fingerprints of heart failure. 17,18 Collectively, these findings suggest that Phe may induce systemic senescence and cardiac dysfunction. Levels of Phe, an essential amino acid, are regulated through degradation into tyrosine catalyzed by the tetrahydrobiopterin (BH4)dependent rate-limiting enzyme phenylalanine hydroxylase (Pah) physiologically restricted to liver and kidney. 19 Tyrosine (Tyr) is the precursor of catecholamines, which have been shown to be increased in the aging process 20 and in heart failure. How aging leads to elevated Phe levels and how abnormal Phe catabolism promotes cardiac senescence are currently unknown.

In the present study we demonstrate that Phe administration induced firstly a remarkable age-mimicking cardiac deterioration in young mice and secondly cellular senescence in vitro. Hepatic Phe catabolism declined with age in a p21-dependent manner, whilst p21 deficiency prevented age-related cardiac dysfunction. Substantiating a role for impaired Phe catabolism in cardiac aging, the Pah cofactor BH4 or dietary Phe restriction abrogated the age-related rise in plasma Phe and reversed age-associated cardiac alterations. Here we identify Phe/PAH modulation as a potential therapeutic strategy for promoting cardiac health and preventing age-related cardiomyopathy.

Methods

The data, analytic methods, and study materials are available to other researchers, on reasonable request, for the purpose of reproducing the results or replicating the procedures presented here.

Animal husbandry

All

Cardiac phenotyping

WT and p21 -/-male mice were followed from the age of 2 to 24 months of age. These mice were sequentially evaluated for myocardial structure and function (conscious echocardiography & in vivo hemodynamic measurements). Animals were euthanized and tissues harvested for histology, molecular biology or metabolic readouts (at ages 2, 6, 10 15 and 24 months). Other WT mice received in vivo Phe (as subcutaneous injections in a dose of 200 mg/kg 2x/die or 1x PBS as vehicle) or BH4 (as intraperitoneal injections in a dose of 10 mg/kg/die 2x/die or vehicle consisting of 1x PBS with 10 mM sodium ascorbate and citric acid to pH4.5) treatment. Dietary Phe restriction was achieved using a custom-designed Phe-deficient diet (composition: Supplementary Table 1) supplemented with 20-25% Phe of control diet in the drinking water. General state of the mice (body weight & wellbeing) was closely monitored. All treatments were completed as scheduled without incidents.

Data analysis and statistics

Mice were randomly assigned to experimental groups and data were acquired and analyzed blind to genotype, age or treatment. Statistical analyses were performed using GraphPad Prism Software (version 6). Unpaired, two-tailed t-test was used to compare two groups, whilst more than two groups were compared using one-way analysis of variance (ANOVA), with Bonferroni post-hoc test for multiple comparisons. Two-way ANOVA was used to compare groups with time-dependent evolution of readouts, with Bonferroni post-hoc test for more than two groups. Data are presented as mean ± standard error of the mean or individual values. Annotations used: *p < 0.05, **p < 0.01, ***p < 0.001 compared to groups indicated. A p value of < 0.05 was considered significant.

Details of standard molecular biology, cell culture, histology, biochemistry and in vivo

experiments are described in the Supplementary Methods.

Results

p21 deficiency delays cardiac aging

As a molecular signature of aging first we explored senescence in aging hearts, using common markers of senescence in C57BL6 (WT) male mice between 2 and 24 months of age. We found that whilst p53 was not expressed before 2 years of age and p16 (cyclin-dependent kinase inhibitor 2a) not at all, p21 (cyclin-dependent kinase inhibitor 1a) was progressively expressed from 10 months of age (Fig. 1A-C).

These results encouraged us to study p21 -/-mice with age-matched WT littermates as controls. Genomic instability did not manifest in hearts of either genotype before 2 years of age (8-oxo-guanine, but not phosphorylated γ-H2A.X; Supplementary Fig. 1A). 4-hydrononenal (4-HNE), a marker of oxidative stress, was increasingly expressed with age in WT hearts, which was considerably delayed in p21 -/-mice (Supplementary Fig. 1B).

Structurally, heart weight to tibia length (HW/TL) was increased at 10 and 15 months of age, 5,6 but returned to young levels at 24 months of age in WT mice. Interestingly, HW/TL in p21 -/-mice did not significantly change over the observation period and was smaller than in WT mice at 10-15 months of age (Fig. 1D). Myocardial interstitial fibrosis and vimentin-positive area (cardiac fibroblasts) progressively increased with age in WT mice, which was significantly retarded in hearts of p21 -/-mice, with the first increase occurring at 24 months of age (Fig. 1E-F & Supplementary Fig. 1C). We found no evidence for cardiac amyloidosis in aged WT hearts (Supplementary Fig. 1D).

WT mice exhibited hallmarks of myocardial functional aging, including progressive reductions in relaxation (dP/dtmin) and contractility (dP/dtmax, systolic strain rate) recorded at comparable heart rates (Supplementary Fig. 1E-F), which was significantly delayed by p21 deficiency (Fig. 1G-I). Left ventricular ejection fraction was preserved during the observation period in both genotypes. (Fig. 1J). 5 Finally, urged by the widely reported observations of age-dependent increase in blood Phe levels in humans [11][12][13][14] we measured plasma Phe levels. Consistent with human studies, we found an age-dependent increase in WT animals (at 15 and 24 months), again significantly delayed in p21 -/-mice (first increased at 24 months; Fig. 1K). Collectively, p21 deficiency delayed age-related structural and functional myocardial damage together with a postponed rise in plasma Phe levels.

Ectopic Phe catabolism in the aging myocardium is delayed with p21 deficiency.

The constellation of myocardial structural/functional decline and increase in plasma Phe concentrations delayed in p21 -/-mice compared to age-matched WT littermates motivated us to explore Phe-related effects in aged hearts. First, we studied myocardial expression of Phe transporters. At 15 months of agethe last age in our series when p21 deficiency allowed full protection against age-related structural and functional decline (Fig. 1)transcript levels of Slc43a2 alone showed an age-dependent upregulation in WT hearts, which was prevented in hearts of p21 -/-mice (Fig. 2A). At this age, however, almost all Phe transporters showed higher myocardial expression in WT mice compared to hearts of p21 -/-mice (Fig. 2A). Given that Pah is the rate-limiting enzyme of Phe catabolism possibly leading to fumarate and the non-enzymatic, irreversible, fumarate-induced posttranslational modification known as succination 21,22 we explored ectopic Phe catabolism in the myocardium (Fig. 2B). Intriguingly, we found an age-dependent myocardial induction of Pah, fumarate and 2-SC (2-succinylcysteine, the structural basis for succination; Fig. 2C-D), delayed by p21 deficiency. In line with the local demand to supply BH4 for Pah activity, GTP cyclohydrolase 1 (Gch1), the rate-limiting enzyme in de novo BH4 biosynthesis (Fig. 2B), was upregulated in WT hearts at 15 months of age but not in hearts of p21 -/-mice.

The succination-sensitive Nrf2-antioxidant pathway 21 followed this pattern (Supplementary Fig. 3H-I).

Consistent with Pah-mediated production of fumarate, Pah and 2-SC co-localized in elderly murine and human hearts alike (Fig. 2E-F). In light of fumarate's energetic role as a Krebs-cycle intermediate, importantly we found no sign for compromised energetics up to 15 months of age; by contrast, mitochondrial complex V activity was impaired at 24 months of age in both genotypes (Fig. 2G, data not shown and Supplementary Fig. 2E). In search of further downstream consequences of ectopic Phe catabolism in the aging heart, we studied levels of H3K9me3, a recently proposed senescence marker. 23 Levels of this transcriptionally repressive histone code are regulated by KDM4 histone demethylases, whose cofactor 2-oxoglutarate (2-OG) is subject to competitive inhibition by increased fumarate levels (Fig. 2H). 24 Accordingly, H3K9me3 was increasingly expressed in WT myocardium starting at 10 months of age and this was delayed in hearts of p21 -/-mice (first induced at 24 months of age; Fig. 2I).

Taken together, our data demonstrate an age-associated induction of myocardial Phe catabolism with redox and epigenetic consequences, which was delayed by p21 deficiency.

In vitro phenylalanine treatment mimics changes characteristic of cardiac aging.

To identify affected cells within the myocardium, first we stained hearts of 24-month-old WT mice with p21 against cell type-specific markers. p21 co-localized with troponin I (cardiomyocytes) and vimentin (cardiac fibroblasts), but not CD31 (endothelial cells; Fig. 3A). To establish a role for Phe in cellular senescence in vitro, we treated primary adult rat cardiomyocytes with Phe. Phe mimicked in vivo findings in aged WT hearts in two important aspects: it selectively induced p21 (but not p16 and phospho-p53 or p-p53; Fig. 3B) and p21 expression was extranuclear (Fig. 3C). BH4 antagonized Phedependent p21 induction, suggesting that it may catalyze Phe catabolism (Fig. 3B). In support, we found that Phe treatment greatly elevated intracellular Phe levels (by ~35-fold), partially reversed by cotreatment with BH4 (Fig. 3D). Moreover, Phe treatment alone increased levels of Phe end-catabolite fumarate, whose levels BH4 did not further increase (Fig. 3E), probably due to the reactive nature of fumarate. These results establish cardiomyocytes as a powerful Phe-buffer when challenged with elevated Phe levels.

With regard to the p53-independent p21 induction by Phe, we hypothesized that Phe interferes with p21 protein degradation, another important way of p21 regulation. 25 To address this, we treated primary adult rat cardiomyocytes with Phe or vehicle without or with proteosome inhibitor MG132. Notably, Phe not only induced p21 protein, but also inversely reduced levels of a ~55 kDa unidentified band (Fig. 3F). MG132 alone increased the density of both bands (especially at ~55 kDa), but adding Phe enhanced band intensity only at 21 kDa, whilst reducing it at ~55 kDa (Fig. 3F). These findings suggested Phe inhibiting p21 ubiquitination, where the ~55 kDa band may correspond to an ubiquitinated form of p21 (one ubiquitin unit is 8.6 kDa, so native p21 with the maximal 4 lysil-ubiquitin units equals to 55.4, which is in close vicinity of the size of the unidentified band). To explore this, we immunoprecipitated p21 in MG132-treated human AC-16 cardiomyocytes and indeed found its ubiquitination prevented by Phe to a great extent (Fig. 3G).

Moreover, in Phe-treated cardiomyocytes there were increased cytosolic superoxide and suppressed reduced glutathione levels (GSH) rescued by the addition of BH4, with no changes in mitochondrial superoxide levels (Fig. 3H & Supplementary Fig. 3A-B). Notably, in Phe-treated cardiomyocytes BH4 greatly induced succination and succination-sensitive Nrf2 signaling (Fig. 3H-I & Supplementary Fig. 3C). Moreover, GSH levels after Phe/BH4 co-administration were dependent on the pentose phosphate pathway (PPP), as the PPP inhibitor dehydroepiandrosterone (DHEA) impaired the rescue (Supplementary Fig. 3D). This is consistent with the Phe/BH4-dependent upregulation of Nrf2 target G6pd, the rate-limiting enzyme of PPP (Fig. 3I). Apart from redox signaling Phe induced H3K9me3 expression, which was further enhanced by BH4 (Supplementary Fig. 3C).

Next, we explored Pah induction. In hearts of 24-month-old WT mice Pah was expressed by cardiomyocytes, cardiac fibroblasts and endothelial cells alike, however, the predominant expression came from cardiomyocytes (Supplementary Fig. 3E). For this reason we explored Pah expression in cardiomyocytes. Phe alone or in combination with BH4 failed to induce Pah protein (Supplementary Fig. 3F). Substances such as H2O2 (known to induce Pah in melanocytes) 26 or HDAC inhibitor sodium butyrate (to facilitate open chromatin and Pah transcription as reported for cardiac aging) 27 alone did not induce Pah levels in cultured cardiomyocytes, only if they were combined with Phe (Supplementary Fig. 3G-H).

Finally, since both p21 and Pah were expressed in vimentin+ cardiac fibroblasts of 24-month-old WT hearts mice, we treated murine neonatal cardiac fibroblasts with Phe. We found that Phe treatment induced Acta2, a marker of fibroblast activation, as well as p21, Mmp9, Gdf15 and Tgfb2, the latter two found induced in cardiac fibroblasts of 30-month-old WT mice 3 (Fig. 3J & Supplementary Fig. 3I).

Taken together, in vitro Phe treatment recapitulated multiple aspects of cardiac aging, including induction of senescence, ectopic Phe catabolism, succination, epigenetic and redox changes.

Experimental hyperphenylalaninemia promotes cardiac aging in vivo.

To replicate human age-related hyperphenylalaninemia [11][12][13][14] experimentally, we treated WT mice with Phe vs. vehicle (VEH). Capitalizing on our observations in the time-course experiments (Fig. 12) we subjected two groups of mice to a 1-month treatment, corresponding to the absence (5-month-old) or presence (11-month-old) of age-related myocardial structural, functional and molecular alterations (Fig. 4A).

Mice tolerated Phe treatment well without significant weight loss (Supplementary Fig. 4A). Plasma Phe levels increased with both age and Phe treatment, suggesting limited Phe catabolic reserve (Fig. 4B).

Phe treatment in young mice mimicked myocardial structural changes observed in vehicle-treated old mice, i.e. cardiac hypertrophy and interstitial fibrosis (Fig. 4C-G), but no amyloid depositions (Supplementary Fig. 4B). Phe administration to young mice strikingly recapitulated age-associated cardiac dysfunction, with impaired relaxation (dP/dtmin) and contractility (dP/dtmax & systolic strain rate) but preserved LV ejection fraction (Fig. 4H-K & Supplementary Fig. 4C-D). 5,6 Age and Phe comparably upregulated myocardial p21, Pah, Gch1, 2-SC, H3K9me3 and 4-HNE without altering myocardial energetics (Fig. 4L & Supplementary Fig. 4E-K). Like in natural aging p21 and Pah were expressed by both cardiomyocytes and cardiac fibroblasts with in vivo Phe treatment (Supplementary Fig. 4L-M).

Collectively, in vivo Phe treatment is sufficient to induce cardiac aging in young mice, whilst its failure to aggravate adverse myocardial changes in elderly mice suggests Phe's involvement in cardiac aging.

BH4 treatment rescues age-related cardiac dysfunction.

Since Phe induces cardiac aging, we hypothesized that augmenting Phe catabolism could improve cardiac function in older mice. To address this, we administered BH4 to 11-month-old WT mice for 6 weeks to enhance Pah activity and thereby reduce plasma Phe levels (Fig. 5A & Supplementary Fig. 5A).

BH4 neither influenced arterial pressures nor myocardial nitric oxide synthase (NOS) activity, a group of enzymes using BH4 as a cofactor (Fig. 5B-C). 26,27 By contrast, BH4 treatment suppressed ageassociated myocardial expression of p21, Pah, Gch1 & 2-SC (Fig. 5D & Supplementary Fig. 5B) and robustly reduced interstitial fibrosis and cardiac hypertrophy (Fig. 5E-H). In vivo BH4 treatment restored myocardial relaxation and contractility towards that of young mice (Fig. 5I-K). Thus, BH4 administration can rejuvenate aged myocardium.

BH4 rescues age-related hepatic Pah decline.

Had BH4 acted in the heart directly, we would have expected increased myocardial NOS activity and Phe catabolism. In the absence of such changes, we investigated the contribution of tissues physiologically expressing Pah, namely the kidney and liver. Whilst aging had no impact on renal Pah or Gch1 expression, we found markedly reduced hepatic Pah and Gch1 protein levels in old WT mice, the latter prevented by p21 deficiency (Supplementary Fig. 6A-B & Fig. 6A). From here we proceeded to determine levels of Phe transporters. Interestingly, quantitative RT-PCR analysis identified three distinct expression patterns amongst Phe transporters: 1. Expression declined in an age-dependent manner, independent of p21 (Slc7a5 & Slc16a10); 2. No change occurred with age and loss of p21 (Slc43a2) and 3. Age-dependent upregulation in WT livers was prevented by p21 deficiency (Slc1a5 & Slc7a8; Fig. 6B). Subsequently, we confirmed protein induction of Slc7a8 in 15-month-old WT livers absent in age-matched p21-/-livers (Fig. 6C). Collectively, these findings indicate that livers from p21-/-mice were immune to some Phe-related changes occurring with age.

Based on Slc7a8's higher affinity for Phe than its paralogue Slc7a5's, 28 we hypothesized that an agerelated Phe transporter shift may increase hepatic uptake of Phe with functional consequences. To obtain direct evidence in vivo, first we stained livers of vehicle-or Phe-treated WT mice, finding induction of Slc7a8, which associated with suppressed Pah expression (Fig. 6D). With Phe transport increased and Phe catabolism impaired, we reasoned that there should be more Phe in the liver. Indeed, we found that compared to 6-month-old vehicle-treated mice, Phe treatment substantially increased hepatic Phe content, comparable to that observed in older mice in 12-month-old WT mice untreated with Phe (Fig. 6E). These results indicate that Phe treatment and age both increase hepatic Phe content.

Next we explored if this excess Phe induces p21. We found an age-dependent induction of hepatic p21 expression (but not p-p53 and p16; Fig. 6F & Supplementary Fig. 6C). To address this further we treated murine AML-12 hepatocytes with Phe and found a dose-dependent p21 induction (Fig. 6G), whilst Pah expression declined (Fig. 6H).

Since aging induced liver senescence, we hypothesized that senescence undermines hepatic Phe catabolism. We therefore treated AML-12 cells with the p53 activator Nutlin3a to indirectly induce p21.

Nutlin3a induced hepatocyte senescence, downregulated Pah and lowered tyrosine production, changes reversed by siRNA-mediated p21 knockdown (Fig. 6I-J & Supplementary Fig. 6D-F). Moreover, BH4 administration rescued Pah and tyrosine levels from Nutlin3a-induced reduction in vitro (Supplementary Fig. 6G-I) and resulted in re-expression of hepatic Pah with reduced liver Phe content and increased hepatic Pah activity in vivo (Fig. 6K-M). 29,30 Finally, we found that in murine livers Pah was not only expressed at orders of magnitude greater levels than other BH4-dependent enzymes, but was also the only one that showed an age-dependent downregulation (Supplementary Fig. 6J). Analysis of a transcriptomic dataset from non-diseased human liver biopsies confirmed a progressive reduction in PAH expression with age (Fig. 6N). Taken together, advanced age undermines hepatic Phe catabolism that is reversible with BH4 treatment.

Dietary Phe restriction rejuvenates old hearts.

To reinforce that lowering Phe levels rejuvenates the aged myocardium, we designed a diet deficient of Phe differing only in the absence of Phe from its control diet (Supplementary Table 1). A pilot study showed that mice on Phe-deficient diet lost weight compared to either Phe-containing control diet or chow, probably due to interference with protein turnover. To circumvent this confounding effect, we re-supplemented 20-25% Phe of control diet in drinking water that stopped weight loss. Then we subjected 23-month-old WT mice to Phe-deficient diet with 20-25% Phe supplementation (Phe-) or control diet (Phe+) for 4 weeks and evaluated effects.

Food intake, body weight, adiposity index and glucose tolerance was not influenced by Phe-diet (Supplementary Fig. 7A-D). At the end of Phe-diet plasma Phe levels were reduced towards values observed around 2-6 months of age or 12.5 months with BH4 treatment (Fig. 7A vs. Fig. 1K, Fig. 4B & Fig. 5A). Importantly, myocardial p21 expression and readouts of ectopic Phe catabolism were reversed to a great extent by Phe-diet (Fig. 7B). Moreover, hearts of Phe-mice displayed substantially reduced interstitial fibrosis (Sirius red: by ~65%, vimentin+ area: by ~50%; Fig. 7C-E) and improvement of contractility and relaxation (dP/dtmax and dP/dtmin by ~30%, strain rate in conscious mice by 60%; Fig. 7G-I) at comparable heart rates and LV ejection fraction (Supplementary Fig. 7E-G). The strength of dietary Phe limitation is further illustrated by rendering 2-year-old hearts functionally and structurally comparable to much younger mice (Supplementary Fig. 7H-K). These results demonstrate that elevated Phe levels represent an important determinant in cardiac aging, which is reversible by therapeutic interventions.

Discussion

Our study provides new mechanistic insights into cardiac aging with important implications. Firstly, we demonstrate that aging increases plasma Phe levels and that elevated plasma Phe levels induce cardiac senescence and dysfunction, a phenotype inducible with Phe treatment in young mice. Secondly, we show that age-related increase in p21 suppresses hepatic Pah function, the latter crucial in controlling systemic Phe levels. Thirdly, p21 deficiency delays structural, functional and molecular alterations in the aging myocardium whilst Pah cofactor BH4 or dietary Phe restriction reverses them. Collectively, our findings may provide diagnostic clues for cardiac aging and open new therapeutic avenues to rejuvenate aged myocardium, an important challenge in the face of high cardiovascular burden in the growing population of elderly individuals. 31

Hyperphenylalaninemia promotes cardiac aging

Notably, our murine experiments recapitulated elevated plasma Phe levels observed in elderly human subjects. [11][12][13][14] Subsequently, we found that Phe excess induced myocardial p21, hence cardiac senescence. Furthermore, increased plasma levels of the essential amino acid Phe pointed to its impaired global catabolism, an observation particularly interesting in light of myocardial induction of Pah, Gch1 and 2-SC that we found in aged hearts confirming progressive induction of myocardial Pah transcripts by aging. 10,27 This strongly suggests that in hyperphenylalaninemia myocardium acts to maintain Phe homeostasis through induction of its catabolic machinery. In the course of this adaptation, however, cardiac Phe catabolism may itself promote myocardial aging through altered intracellular signaling by protein succination, 22 as we have demonstrated in aging human and murine myocardium. Equally, but not exclusively, increased Phe uptake with limited myocardial Phe catabolic capacity may result in the accumulation of toxic metabolites, such as phenylpyruvate, phenyllactate and phenylacetate. 32 Moreover, increased ectopic Phe catabolism into tyrosine may fuel catecholamine biosynthesis in tyrosine hydroxylase-expressing cells, such as certain neurons and chromaffin cells of the adrenal medulla. 20 Thus, elevated Phe levels have an adverse impact on cardiac health, as evidenced by structural and functional myocardial impairment with a signature of senescence and Phe catabolism.

BH4 treatment rejuvenates the aging myocardium by restoring hepatic Pah activity

Our findings support a paradigm of age-related hepatic hypocatabolism of Phe underlying age-related rise in plasma Phe and driving cardiac aging. This is in line with downregulation of Pah, one of the six BH4-dependent enzymes with the highest hepatic expression. Pah's vital dependence on its natural cofactor, BH4, is illustrated by hyperphenylalaninemia being a prominent feature of enzymatic deficiencies in de novo BH4 biosynthesis or the recycling pathway. 33 Importantly, we restored healthy cardiac structure and function in naturally aged mice through normalizing plasma Phe levels by BH4, consistent with revived hepatic Pah activity. 29,30 In line with this, we found reduced Pah and tyrosine levels in AML12 hepatocytes with pharmacologically stimulated senescence that could be rescued by both p21 knockdown and BH4. In theory, other BH4dependent enzymes, i.e. nitric oxide synthases (NOS) could play a role in the cardiac improvement observed with BH4 treatment. Improved vascular NOS coupling and subsequently reduced left ventricular afterload [34][35][36] is unlikely since systemic blood pressure remained unaltered by intraperitoneal BH4 delivery. Moreover, BH4 treatment did not increase myocardial NOS activity. Lack of such changes is consistent with the reported failure of exogenously delivered BH4 to reach efficient systemic concentrations 34 due to predelicted BH4 uptake of natural Phe-expressing organs: the liver and kidney. 37 Restored indices of myocardial Phe catabolism by BH4 correspond to BH4 uptake by the liver fuelling its Pah activity taking the burden of Phe catabolism off the heart. This is consistent with the lack of reduction in cardiac damage induced by pressure overload in cardiomyocyte-restricted Gch1 transgenic mice 38 as opposed to oral BH4 administration. 39 Finally, the salubrious myocardial effects of dietary Phe restriction underscores the importance of lowering systemic Phe levels to rejuvenate old hearts.

Collectively our data show that pharmacological restoration of hepatic Pah activity by BH4 may rejuvenate age-associated cardiac changes and have potential application in age-related cardiac disease, such as heart failure.

Age-dependent p21 compromises hepatic Pah activity

Whilst Phe induced p21 in hepatocytes and cardiomyocytes in vitro, aging upregulated p21 expression in both liver and heart, hallmarking senescence of these organs. Moreover, we uncover the absence of age-related rise in plasma Phe levels and cardiac decline in p21 -/-mice indicating that there are particular age-related gain-of-function(s) attributable to p21. Accordingly, in cultured hepatocytes we established a Phe-dependent p21 induction; experimental p21 induction on the other hand was sufficient to compromise Pah expression and activity in hepatocytes, which could be rescued by BH4 co-treatment. This constitutes a vicious cycle: Phe induces hepatic p21, p21 compromises hepatic Pah expression/activity, which raises systemic Phe levels and the cycle starts all over again. Interestingly, neither Phe-nor p21-mediated life-shortening is unprecedented; whilst dietary addition of Phe reduces lifespan in C. elegans 40 and ants, 41 p21 deficiency increases it in mice. 42 As for our findings, we identified one novel function for p21, which is the suppression of hepatic Pah expression/activity, through which p21 is a key node for the rise in plasma Phe levels with age.

Cardiac aging: a quiescent organ and cellular senescence?

Cellular senescence of the heart is intriguing due to the postmitotic status of cardiomyocytes (~30% of all heart cells occupying 80% of the heart's volume) 43 that do not fall under Hayflick's concept of senescence, the proliferative inability in otherwise division-competent cells. 44 Aged cardiomyocytes acquire a senescent-like phenotype characterised by persistent DNA damage at telomere regions that occur independent of cell division and telomere length. 3 This length-independent telomere damage in cardiomyocytes activates senescent pathways (p21) and raises secretion of pro-fibrotic andhypertrophic factors. 3 Autocrine/paracrine effects of this secretome include proliferation of cardiac fibroblasts and their activation towards myofibroblasts. In addition to this intramyocardial cascade, we uncovered a role for visceral adipose tissue, adding an endocrine enhancement to cardiac aging by releasing a profibrotic secretome (e.g. Tgfb1, osteopontin and leptin). 6 Therefore, cardiac aging seems to result from complex interactions as part of an integrated multi-disorder state.

The present study extends this organism-level approach to cardiac aging by studying circulating metabolic signals. Since metabolic screens showed increased plasma Phe levels occurring in both natural aging and heart failure, we specifically studied the role of plasma Phe in cardiac aging. 11, 13-15, 17, 18, 27 Our findings in age-related and experimentally induced hyperphenylalaninemia confirmed a causal relationship between elevated plasma Phe levels and cardiac decline.

A new piece in the puzzle: the natural course of cardiac aging

Murine investigations into cardiac aging traditionally explore animals with at least 18 months of age.

These studies among others identified dysfunctional sirtuin/NAD + biology, [START_REF] Alcendor | Sirt1 regulates aging and resistance to oxidative stress in the heart[END_REF] mitochondrial oxidative stress, 9 abnormal PI3K/Akt signaling, misfolded proteins, 10 telomere dysfunction, DNA damage 3 and compromised mitochondrial energetics [START_REF] Tocchi | Mitochondrial dysfunction in cardiac aging[END_REF] in aged hearts. Our findings synergize with the literature extending our understanding of the natural history of cardiac aging. For instance, in WT hearts at 24 months of age we observed elevated complex III activity with suppressed ATP synthase activity pointing to the former as a potential mitochondrial source of oxidative stress. 9 The early onset of age-related functional decline is most evident by the time-limited competitiveness of elite athletes, which typically predates heart disease. Accordingly, during our time-course we found clinically detectable impairments in myocardial structure and function as early as 10 months of age. Up to and including the age of 15 months we found no sign of myocardial DNA damage, impaired mitochondrial complex activities, alterations in NAD + /NADH levels (re: energetics and sirtuins). On the contrary, we observed an emergence of myocardial p21 expression, ectopic Phe catabolism with increased succination, redox imbalance and epigenetic changes starting at 10 months of age. These structural, functional and molecular alterations associated with elevated plasma Phe levels, were inducible with Phe treatment in vitro and in young WT mice in vivo, as well as were (depending on age at least partially) reversible with interventions controlling systemic Phe levels (in vivo BH4 treatment and dietary Phe restriction). These results demonstrate that dysregulated Phe catabolism is an earlyonset, persistent component of cardiac aging, preceding mechanisms described to date, and importantly, it is amenable to therapeutic correction.

Conclusions

Our findings unlock a novel approach to rejuvenate the aged myocardium by pharmacological restoration of aging-induced Phe catabolic decline. The potential of targeting Phe is highlighted by related abnormalities starting at middle age and persisting throughout life. Our observations may be extended to other age-related disorders, such as dementia, [START_REF] Ravaglia | Plasma amino acid concentrations in patients with amnestic mild cognitive impairment or Alzheimer disease[END_REF] susceptibility to cancer, [START_REF] Denicola | Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis[END_REF][START_REF] Wu | Geroncogenesis: metabolic changes during aging as a driver of tumorigenesis[END_REF] and all-cause mortality risk [START_REF] Deelen | A metabolic profile of all-cause mortality risk identified in an observational study of 44,168 individuals[END_REF] that could benefit from a strategy of counteracting the adverse effects of a declining PAH activity.

Methods

The data, analytic methods, and study materials are available to other researchers, on reasonable request, for the purpose of reproducing the results or replicating the procedures presented here.

Animal husbandry

Procedures

Cardiac phenotyping

Male WT and p21 -/-mice were followed from the age of 2 to 24 months of age. These mice were sequentially evaluated for myocardial structure and function as detailed later. Animals were euthanized and tissues harvested for histology and molecular biology (at ages 2, 6, 10 15 and 24 months).

In vivo drug treatment and phenylalanine-deficient diet

Phe in a dose of 200 mg/kg twice a day or vehicle (1x PBS) was subcutaneously administered to 11 month-old WT mice (Janvier Labs, France) for one month. BH4 in a dose of 10 mg/kg/die 2x/die or vehicle (1x PBS with 10 mM sodium ascorbate and citric acid to pH4.5) was intraperitoneally administered to 11 month-old WT mice over six weeks. General state of the mice (body weight & wellbeing) was closely monitored. In both cases drug treatment was completed as scheduled without incidents.

As an alternative approach to limit Phe levels in 24-month--old WT mice, we designed a special diet differing only in the absence of Phe from the corresponding control diet (composition of this diet is detailed in Supplementary Table 1). During a pilot study we observed that mice on Phe-deficient diet tended to lose weight compared to either Phe-containing control diet or chow, which presumably occurred due to Phe insufficiency-related impairment of protein synthesis. To circumvent this problem, using calculations based on food intake of control diet mice and water intake of mice on Phe-deficient diet we re-supplemented 20-25% Phe of control diet mice in drinking water that stopped weight loss.

Then we subjected 23-month-old WT mice to Phe-deficient diet with 20-25% Phe supplementation (Phe-) or control diet (Phe+) for 4 weeks and evaluated plasma Phe levels and myocardial effects.

Echocardiography in conscious mice

Mice were trained to be grasped for transthoracic echocardiography that was performed in non-sedated mice to avoid the cardiac depressant effect of anesthetic agents, as previously reported. 1 Heart rates were typically above 600 beats per minute (bpm). Data acquisition for a single cohort was performed by a single operator (ER). Images were acquired from a parasternal position at the level of the papillary muscles using a 13-MHz linear-array transducer with a digital ultrasound system (Vivid 7, GE Medical System, Horton, Norway). Left ventricular dimensions and ejection fraction, anterior and posterior wall thicknesses were serially obtained from M-mode acquisition. Relative LV wall thickness (RWT) was defined as the sum of septal and posterior wall thickness over LV end-diastolic diameter, and LV mass was determined using the uncorrected cube assumption formula (LV mass = 1.05x((AWTd+LVEDD+PWTd) 3 -(LVEDD) 3 )). Peak systolic values of radial strain rate of the anterior and posterior wall were obtained using Tissue Doppler Imaging (TDI) as previously described. 2 TDI loops were acquired from parasternal view with a careful alignment with the radial component of the deformation 3 at a mean frame rate of 514 fps and a depth of 1 cm. The Nyquist velocity limit was set at 12cm/s. Radial strain rate analysis was performed offline using the EchoPac Software (GE Medical Systems) blindly by a single operator (GD). Peak systolic of radial strain rate was computed from a region of interest positioned in the mid anterior wall and was measured over an axial distance of 0.6 mm. The temporal smoothing filters were turned off for all measurements. Because of the inevitable respiratory variability, we averaged peak systolic of radial strain rate on 8 consecutive cardiac cycles.

Invasive in vivo hemodynamic assessment of left ventricular function

In vivo hemodynamic measurements were performed just before mice of indicated ages were sacrificed. 1 Hemodynamic evaluation was performed in mice placed on a homeothermic operating table in supine position under 1.5% isoflurane anesthesia with spontaneous breathing. A 1.4-Fr microcatheter (Millar Instruments, Houston, USA) was calibrated manually before each experiment, inserted via the right carotid artery into the aorta and subsequently advanced to the left ventricle. Data were collected after at least 10 min of baseline, using the lowest isoflurane concentration tolerated to ensure minimal cardiodepression during measurement of peak rates of isovolumetric pressure development (dP/dtmax) and pressure decay (dP/dtmin). The microcatheter was then withdrawn to the aorta for measurement of systolic and diastolic pressure. Data were analyzed using the IOX software (EMKA, France).

Glucose Tolerance Test (GTT)

24-month-old WT mice on Phe-, Phe+ and chow diets were fasted for 7 hours and intraperitoneally injected with a glucose solution at a dose of 1.5 g/kg body weight. Blood samples were obtained from tail vein at baseline, 15, 30, 60, 90, 120 minutes after injection, and glucose concentration was measured with an automated glucometer (Accu-Chek Performa, Roche, Germany).

Tissue harvesting and processing

All mice were weighed, euthanized by cervical dislocation, followed by rapid excision of the organs of interest. The heart was cannulated through the aorta for perfusion with ice-cold 1x PBS, then blotted and weighed. The heart was cut in half perpendicular to its axis: the apical two-third was snap-frozen in liquid nitrogen, whilst the basic one-third was recannulated through the aorta, perfused with 10% formalin for histology. Hearts were kept in formalin at 4°C for at least 24-48 hours before embedding.

Snap-frozen tissues were kept at -80 °C, until they were powdered using a mortar and pestle cooled with liquid nitrogen and collected as aliquots. Livers and kidneys were processed in a similar manner.

Human heart biopsies

Human heart biopsies (right atrial appendage) were obtained from patients undergoing elective coronary artery bypass grafting. Biopsies were obtained after approval of the ethical committee (Comité de Protection des Personnes Ile-de France VI) of Pitié-Salpêtrière Hospital, Paris and informed consent was acquired from each patient prior to the procedure. 4

Human liver data

The available human liver transcriptomic data was generated in 33 non-diseased, beating heart liver donors with Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. 5 The annotations and normalized data were downloaded from NCBI Gene Expression Omnibus (accession number GSE107039). BioMart19 6 was used to map the microarray probesets to the human assembly in Ensembl release 96, revealing that there were 3 probesets that mapped to PAH. Although expression of only one probeset significantly correlated negatively with age, the expression of the other two demonstrated a similar trend. The data from these 3 probesets was reduced to a single variable by performing principal components analysis with IBM SPSS Statistics v25. The first principal component, which had a mean of 0 and standard deviation of 1, was regressed as a dependent variable against donor age in GraphPad Prism v8.

RNA work and quantitative RT-PCR

Total RNA from cells or powdered tissue was extracted using the RNeasy mini kit or RNeasy mini fibrous tissue kit (QIAGEN), respectively, as previously described. 7 RNA yield was measured with Nanodrop ND-1000, a >1.9 260/280 ratio was accepted. For reverse transcription the High Capacity cDNA kit was used according to the manufacturer's instructions (Applied Biosystems). Quantitative reverse transcriptase-polymerase chain reaction was used to quantify relative levels of genes of interest.

Briefly, amplification reactions were carried out using Fast Universal Master Mix on a StepOnePlus system (Applied Biosystems) in a multiplexed design (FAM: gene of interest; VIC: β-actin as endogenous control) in a reaction volume of 10 μl. All Taqman oligos used were inventoried Taqman-MGB oligos from Applied Biosystems. Relative expression was quantified using the ΔCT method with the formula RQ=2 -ΔΔCT (User Bulletin #2; Applied Biosystems). 7

Protein extraction and Western blotting

Cells were washed briefly with ice-cold 1x PBS, scraped in 400 μL T-PER lysis buffer (ThermoFisher) supplemented with 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and protease/phosphatase inhibitor cocktail tablet (ThermoFisher). Pulverised tissue samples (20-30 mg) were lysed in the same buffer accelerated by sonication and passing through a 21 G needle. To pellet any debris, lysates were centrifuged at 13,000 g for 10 minutes at 4°C, with the supernatant transferred to fresh tubes. Protein concentrations were determined with Bradford assay (Biorad) and lysates were diluted to equal concentration with lysis buffer. Protein lysates were then mixed with Laemmli buffer, vortexed and heated to 95°C for 5 min. Equal amounts of protein lysates in parallel with protein molecular weight marker (ThermoFisher) were loaded onto pre-cast polyacrylamide gels (Nupage 10 or 12% Bis Tris gel, Novex, Invitrogen) and separated by electrophoresis at 200V for 1 hour. Proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Invitrogen) using an electrophoretic transfer cell (Mini Trans-Blot, Bio-Rad) in ice-cold transfer buffer (48 mM Tris base, 390 mM glycine, 0.1% SDS, 20% methanol (v/v)) at 300 mA for 2 hours. Membranes were blocked in 1x skimmed milk (ThermoFisher) for 1 hour at room temperature, followed by overnight incubation with primary antibody diluted in blocking buffer at 4°C. The antibodies and concentrations used are shown in Supplementary Table 1.

Next day membranes were washed, followed by 60 minutes incubation in the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:2000-10000 in blocking buffer (all Abcam). Blots were developed using enhanced chemiluminescence (ECL) reagents (normal, Prime & Select) and blocking, sections were incubated overnight at +4 °C with primary antibodies raised against the following targets: 4-HNE (Millipore; goat, 1:200), Pah (Abcam; rabbit, 1:200) or 2-SC (Cambridge Biosciences; rabbit, 1:100; also see Supplementary Table 1). Next day sections were washed, then incubated with the corresponding HRP-conjugated secondary antibodies (Abcam; 1:200) for 30 minutes at room temperature, washed again, followed by incubation with 3,3'-diaminobenzidine (DAB; Sigma) under visual observation until the signal appeared. Then slides were either counterstained with haematoxylin or not, dehydrated and mounted.

Immunofluorescence was used for higher sensitivity and co-localization of proteins of interest. Briefly, paraffin-embedded sections were rehydrated, followed by antigen retrieval with citrate-assisted heat.

Sections were then blocked with 30% goat serum (using antibody dilution solution with background reducing components; Dako) or Bloxall™ artificial blocking agent (Dako) and incubated overnight at +4 °C with primary antibodies raised against target proteins (see Supplementary Table 1 for details).

The following day sections were washed in 1x TBST, incubated with a mixture of the corresponding Alexa Fluor 555-labelled secondary antibodies (Invitrogen) and Alexa-488-conjugated Phalloidin (to counterstain cardiomyocytes) for 30 minutes at room temperature, and washed again. In case of colabeling Alexa Fluor 555-labelled and Alexa Fluor 647-labelled secondary antibodies were used (Invitrogen), occasionally in combination with the avidin/biotin enhancement system (Dako). Specific protocols are available upon request. Slides were mounted with fluorescent mounting media with DAPI (4',6-diamidino-2-phenylindole; Abcam).

Isolation and culture of primary cells

Left ventricular cardiomyocytes were isolated from male Sprague Dawley rats (9 weeks, 300-350g) as described previously. 9 Briefly, rats were anesthetized with ketamine and xylazine (100 and 10 mg/kg, respectively) and received heparin (100 IU/kg). Hearts were excised and retrogradely perfused with an oxygenated (95% CO2, 5% O2) perfusion buffer (NaCl 113 mM, KCl 4.7 mM, KH2PO4 0.6 mM, Na2HPO4 0.6 mM, MgSO4-7H2O 1,2 mM, NaHCO3 12 mM, KHCO3 10 mM, HEPES 10 mM, Taurine 30 mM, phenol red 0.032 mM, D-glucose 5.5 mM, 2,3butanedionemonoxime 10 mM, pH 7.4) for 2 minutes to wash out the blood from the coronary arteries. Then, hearts were perfused with digestion buffer (perfusion buffer supplemented with 0.1 mg/mL liberase, 0.14 mg/mL trypsin-EDTA and 12.5 µM Ca 2+ ) for 10-12 minutes.

The hearts were then placed in stopping buffer (perfusion buffer supplemented with 10% New Born Calf Serum (NBCS) and 12.5 µM Ca 2+ ). Atria and the right ventricle were removed.

The left ventricle was dissected into small fragments, then submitted to successive aspirationreflux with a pipette. Digested ventricles were filtered through a 250 μm cell filter. After a 10-minute incubation at 37°C, supernatants were discarded and cells wer e resuspended with calcium buffer (perfusion buffer supplemented with 5% NBCS and 12.5 µM Ca 2+ ). Subsequently, extracellular calcium was added incrementally up to 1 mM. Finally, cells were suspended in culture medium M199 (supplemented with 1% Insulin Tran sferrin Selenium -ITS) and seeded onto wells pre-coated with 10 µg/mL laminin (for immunoblotting, intracellular Phe or fumarate measurements: 2x10 5 cells in 6-well plates; for RNA/qRT-PCR: 10 5 cells in 12-well plates; for fluorescence: 2x10 4 cells in 8-well Labtek II chamber slides, ThermoFisher).

Cells were allowed to attach for 2 hours before starting any treatment. Duration of treatments was 4 hours at a final concentration of L-phenylalanine: 5 mM; 10 BH4: 10 µM; MG132: 1 µM).

At the end of treatments the majority of cells were rod -shaped, had cross-striations and no visible vesicles on their surface, whilst for microscopic detection all cells studied were such.

Treatments were performed in the M199 1% ITS media.

Primary cardiac fibroblasts were isolated from mouse neonates as described previously. 4 Briefly, neonatal C57BL6J mice (Janvier) were euthanized by decapitation, and ventricles were minced in icecold balanced salt solution. Subsequently, minced ventricles were incubated in a balanced salt solution containing trypsin for 24 hours at 4°C, and then further digested with 300 U/mL of collagenase type 2 for 30 to 45 minutes at 37°C with gentle agitation. Cell suspensions were filtered with a 70 μm poresize mesh and passed onto dishes with Medium 199 (ThermoFisher), supplemented with 10% fetal calf serum. Thirty minutes later, floating cells were removed, and the attached cardiac fibroblasts were maintained in culture until passage 3. For Phe treatment cells were seeded onto LabTek II chamber slides at a density of 70% of confluence. Having allowed to attach, cells were treated with 0.1, 0.5, 1, 2.5 and 5 mM Phe or vehicle (1x PBS) for 24 hours.

Culture of cell-lines

AC-16 cardiomyocytes (immortalized human ventricular myocytes) were maintained in DMEM/F12 (supplemented with 12.5% foetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin) in a humidified incubator at 37 °C with 5% CO2. 11 Treatments were performed in 1x PBS Mg+/Ca+ (supplemented with 17,5 mM glucose and 0,5 mM pyruvate) for 24 hours (with a final concentration of L-phenylalanine: 5 mM; BH4: 10 µM; MG132: 1 µM; dehydroepiandrosterone (DHEA): 10 µM; H2O2: 10 µM; sodium butyrate (SB): 5 µM as indicated). For microscopic evaluation, 10 4 cells were seeded onto 8-well LabTek II chamber slides. At the end of treatment cells were fixed with 4% paraformaldehyde at 37 °C for 10 minutes before permeabilization (0.1% Triton-X100 in 1x PBS for 1m minutes), blocking and primary antibody. For evaluation of redox status, 4x10 4 cells were seeded onto 24-well plates, received treatment as indicated and were processed for fluorescent probes as instructed (see later in further detail). Processing of AC-16 cells for co-immunoprecipitation experiments is described under a separate subchapter above. AML-12 cells (ATCC® CRL-2254™) were maintained in DMEM/F-12 (1:1) culture media supplemented with 10% FBS, 10 µg/ml insulin, 5.5 µg/ml transferrin, 5 ng/ml selenium, 40 ng/ml dexamethasone and 1% penicillin/streptomycin. Cells were sub-cultivated from subconfluent flasks with a ratio from 1:4 to 1:8. For WB analysis, cells were plated on 6-well plates and harvested at 80-90% of confluence. AML-12 cells were treated with Nutlin3a (10 µM) against vehicle (DMSO) and transfected with indicated siRNA (Supplementary Table 2) at 80% confluence using Lipofectamine RNAiMAX Transfection Reagent (ThermoFischer Scientific 13778030) following the manufacturer's instructions. Transfection efficiency was constantly checked using BLOCK-IT Alexa Fluor Red Fluorescent Control (ThermoFischer Scientific 14750100). In other experiments AML-12 cells were treated with Nutlin3a without or with BH4 (10 µM). Cells were lysed for protein extraction, whilst conditioned media was used to determine tyrosine concentration.

All cell culture experiments were performed in biological triplicates at the least.

Myocardial nitric oxide synthase (NOS) activity

Myocardial tissue was probed for NOS activity using a commercial NOS activity kit according to the manufacturer's instructions (BioVision). Briefly, myocardial tissue aliquots were lysed in ice-cold NOS Assay Buffer supplemented with Pierce protease/phosphatase inhibitor cocktail and centrifuged to clear the protein lysates from insoluble cell debris. Protein lysates were then incubated with a reaction mix containing NOS cofactors, NOS substrate and nitrite reductase at 37 °C for 60 minutes, followed by addition of NOS Assay Buffer. This was followed by three 10-minute incubations at room temperature with enhancer, probe and NaOH added, respectively. Fluorometric measurements took place with excitation at 360 nm and emission at 450 nm. NOS recombined protein acted as positive control. Sample values were determined against a standard curve and normalized to protein concentration (Bradford assay).

Mitochondrial complex and citrate synthase activity

Mitochondrial enzymatic activities were measured real-time in 96-well microplates by spectrofluorimetry (Tecan Spark) and normalized to protein concentration (BCA assay).

Briefly, pulverized heart samples were homogenized in Extraction Buffer (20 mM Tris-HCl, 250 mM sucrose, 2 mM EGTA, 40 mM KCl and 1 mg/mL BSA, pH 7.2) with 1 % Percoll and 100 µg/mL digitonin. After centrifugation at 10 000 g at 4 °C, pellets were washed in Extraction Buffer, dried and kept at -80°C until further analysis. Complex II/succinate dehydrogenase activity was measured in 10 mM KH2PO4 buffer (pH 7.2) with addition of 0.2 mM lauryl maltoside, 5 mM succinate, 25 µM oxidized decylubiquinone and 40 µM DCPIP (Dichloroindophenol sodium) at OD 600 nm with malonate (5 mM) as reference inhibitor. Complex III/coenzyme Qcytochrome c reductase activity was dettermined in 10 mM KH2PO4 buffer (pH 7.2) after addition of 0.2 mM lauryl maltoside, 60 µM oxidized cytochrome c, 300 µM KCN and 12.5 µM reduced decylubiquinone at OD 550 nm with antimycin A (1 µM) as reference inhibitor. Complex IV/cytochrome c oxidase activity was measured in 10 mM KH2PO4 (pH 7.2) after addition of 0.2 mM lauryl maltoside, 60 µM reduced cytochrome c at OD 550 nm) with KCN (300 µM) as reference inhibitor. Complex V/ATP synthase activity was determined at OD 340 nm in a reaction buffer (25 mM KH2PO4 (pH 8) with 0.2 mM lauryl maltoside, 2 mM ATP, 25 mM phosphoenolpyruvate, 7.5 U pyruvate kinase, 12.5 U lactate dehydrogenase, 500 µM NADH, 1 µM antimycin A 250 mM sucrose, 30 mM KCl, 0.2 mM EGTA, 5 mM MgCl2, 2 µM P1,P5-Di(adenosine-5′) pentaphosphate pentasodium salt (Ap5A), 0.1% BSA added, with oligomycin A (10 µM) as reference inhibitor. Citrate synthase activity was measured at OD 415 nm in a reaction mix containing 10 mM Tris, (pH 8). 150 μM 5,5'-Dithio-bis 2-nitrobenzoic acid, 300 μM acetyl-coA and 1 mM oxaloacetate. All reagents were purchased from Sigma Aldrich (France).

Determination of superoxide and metabolite levels

Levels of reduced glutathione (GSH; ThermoFisher), cytosolic (CellROX; ThermoFisher) and mitochondrial (MitoSOX; ThermoFisher) superoxide, phenylalanine (BioVision), tyrosine (BioVision) and fumarate (BioVision) levels as well as NAD + /NADH ratios were evaluated in cells, hearts, livers or plasma, with respective agents/kits following the manufacturer's recommendations. Specifically, cellular GSH levels were estimated with the aid of ThiolTracker™ Violet fluorescent probe (final concentration: 20 µM; ThermoFisher), whilst cytosolic and mitochondrial superoxide levels with CellROX and MitoSOX probes, respectively (CellROX final concentration: 5 µM; MitoSOX final concentration: 2 µM). Plasma, liver or cardiomyocyte phenylalanine levels were determined using an enzyme-coupled, fluorometric method (BioVision). Briefly, samples were deproteinized via trichloroacetate precipitation, neutralized, tyrosinase-treated (to remove tyrosine that may interfere) and diluted to fit in the standard curve. Fluorescence reading took place at 587 nm after excitation at 535 nm. Levels of tyrosine in liver, or conditioned media from treated cells were determined after deproteinization using 10 kDa cut-off columns with an enzyme-coupled, colorimetric method with reading at 491 nm (BioVision). Cellular and myocardial fumarate levels were determined using an enzyme-coupled colorimetric kit with reading at 450 nm (BioVision). Myocardial NAD + /NADH ratios were calculated based on direct measurements of NAD + and NADH levels of the same samples using an enzyme-coupled colorimetric kit with reading at 450 nm (BioVision).

Data analysis and statistics

Supplementary Table 1. Composition of the custom-designed Phe-deficient and control diets. Thus, our aim is to dissect the respective role of two senescence "stressors" on visceral adipose tissue, i.e. increase in nutrients and aging, together with their combination. Indeed we demonstrate the time course of senescence cells affecting specifically different adipose tissue cells with impact on the SASP and the remote effects on target organs such as the heart. This work is still in progress and has major implications in the prevention of deleterious consequences of adipose tissue senescence, requiring tailored "senolytic" therapy before translation to the clinical setting.

These observations are summarized in supplemental table 2 with comparison of metabolic parameters, adipose tissue remodeling, circulating cytokines and impact on heart between the 3 groups of age (2, 5 and 12 months old animals). For instance, the respective occurrence of cellular senescence as assessed by staining of senescence markers (p16 & p21) on the different cell types in visceral adipose tissue show a different profile according to the age, as it is shown bellow, for example, for preadipocytes.

senescence (especially p16) and macrophage marker F4/80 in VAT of aged WT mice. Since OPN's is known to impact on macrophage polarisation and function, 327 we explored the relationship among OPN, ATM and VAT senescence further.

➢ Importance of macrophage senescence in VAT aging

First, to dissect the interplay between OPN and ATM senescence, we explored how OPN may induce senescence in naïve WT BMDM. As expected, recombinant OPN induced a senescentlike phenotype in naïve macrophages with reduced phagocytic activity. Interestingly, recent reports suggest that ATM's endocytotic dysfunction may be one of the primary events to mediate VAT dysfunction in both aging and obesity. 328,329 Next, we speculated that OPN-inducible ATM senescence may compromise inherent macrophage functions and spread senescence to the adjacent tissue. Accordingly, we found that genetic deletion and pharmacological inhibition of OPN reduced both VAT and ATM senescence, whilst restoring efferocytosis, as shown by in vitro and in vivo assays.

In addition to ameliorated macrophage function, an interesting collateral finding was that genetic OPN deletion resulted in significantly larger adipocytes and gross VAT mass.

Intriguingly, OPN/senescence-free adiposity associated with better metabolic function, lending support to the concept that VAT quality is both critical and improvable.

Even though genetic deletion and pharmacological inhibition of OPN prevented or reversed functional ATM and VAT senescence, we used a secondary approach to specifically target macrophages. In the absence of available conditional OPN knockout mice, we chose to eliminate macrophages by clodronate-liposome treatment (an approach we also used in dietinduced obesity in Study 3). By deleting senescent and dysfunctional ATM (lessened p16+CD68+ cells) in elderly mice clodronate-liposome treatment ameliorated VAT structural remodeling (i.e., adipocyte hypertrophy), and more broadly, metabolic function (i.e. glucose tolerance and adipokine secretion profile). These findings strongly indicate that ATM is a major contributor to age-related VAT and systemic metabolic dysfunction.

Next, we dug further into the OPN-dependent senescence-associated impairment of ATM function. In particular, we studied efferocytosis (a key macrophage function found downregulated as a function of age by our discriminative gene expression analysis) both in vivo and in vitro. Efferocytosis is crucial in maintaining AT homeostasis by removing apoptotic cells and cell debris. Whilst aging was associated with an increase in TUNEL+ cells in WT and OPN-/-VAT alike, only aged OPN-/-mice manifested efficient efferocytosis evidenced by higher rate of engulfed TUNEL+ or cleaved-caspase 3+ cells. In vitro functional assay further supported healthier efferocytosis by OPN deficiency: compared to age-matched WT BMDM, aged OPN-/-BMDM exerted preserved phagocytic activity with higher expression levels of key efferocytosis-related genes such as Mertk. 330,331 The importance of our findings is underscored by recent studies finding tissue microenvironment to be the most important determinant for macrophage functionality, 332 whilst changes in apoptotic signals program tissue macrophages differently for efferocytosis. [333][334][335] In aging adipose tissue extracellular OPN seems to direct recruited or naïve tissue macrophages to senescent reprogramming at the cost of phagocytic function.

Several recent publications observed favourable effects by systemic elimination of senescent cells with senolytic drugs. 267,[336][337][338] On the other hand, induction of senescence in some cell types responsible for damage is beneficial during aging and repair processes, 280,320,339,340 not to mention in preventing cancer. Here we have identified OPN as a novel target for ameliorating cellular senescence in specific cell types such as adipose tissue macrophages, complementing our previous observations in cardiac fibroblasts. 320 Collectively, our results demonstrated that elimination of senescent ATM ameliorated AT remodeling and dysfunction. These observations suggest that macrophage-specific and senescence-targeted therapeutics (such as macrophage-directed pharmacological OPN inhibition) could be a viable strategy to control AT senescence with altered secretome and systemic metabolic decline in aging.

➢ Role of macrophages identified by studying time-dependent effects of HFD in young

WT mice

In caloric overload, the capacity of WAT's energy storage reaches a threshold that turns on a cascade ultimately resulting in pathological consequences, including systemic inflammation, increased insulin resistance and remote organ damage. 204,272,341 Whilst most research groups address the possibility of macrophage involvement we fed mice HFD for 2 weeks and treated them with clodronate-liposome, as we did in old mice (Study 3). These liposomes are known to be selectively phagocytosed by macrophages and internalised clodronate subsequently induces apoptosis. 350 Most importantly, we observed that clearance of macrophages restored normoglycemia. Although the intricate nature of macrophage dysfunction remains a matter of debate, these findings are consistent with the reversal of dysmetabolic state either with clodronate-mediated removal of macrophages 351,352 or genetically induced apoptosis of senescent cells in p16-ATTAC mice 353 on long-term HFD (>12 weeks). More importantly, these results may inform about impaired glucose homeostasis critically depending on the state of macrophages per se. The nonessential nature of chronic low-grade inflammation 349,354 or fibrotic remodeling of VAT 348 at this early stage may be reconciled by altered immune cells unable to preserve functional tissue, possibly rendering VAT (as well as probably other organs)

to further injury when persistently challenged.

In the past years, several studies converged to the concept that accumulation of senescent cells during aging or after metabolic challenge such as diabetes or obesity contributes to the overall decline in organ function and lifespan. Previous studies using p16INKATTAC mice and senolytic drugs proved the merits of removal of all senescent cells to improve organ function and longevity. Thus, these observations prompted us to test the association of Dasatinib plus Quercetin to prevent HFD-related cardiac and metabolic disorders. Interestingly senolytic treatment was not efficient to prevent the cardiac functional alteration induced by HFD, despite a moderate improvement in metabolic function as assessed by GTT and ITT. Such an observation is in line with our previous data demonstrating that cellular senescence is not only harmful but has beneficial roles in cardiac aging since the senescence induction in cardiac fibroblasts is pivotal in blunting fibrosis during aging and in improving therefore cardiac function. These data provide therefore arguments to develop cell type-specific senolytic approaches with the hypothesis that eliminating selectively pathogenic senescent cells will benefit cardiac and metabolic function.

Indeed, cardiac integrity is known to be negatively affected by HFD. 355 In hearts of HFD-fed mice we observed a slight, nonetheless significant time-dependent increase (i.e. not before 10 weeks of HFD) in cardiomyocyte cross-sectional area, but no increase in myocardial interstitial fibrosis. HFD compromised contractility prior to any detectable structural changes.

Importantly, the deficit in contractility by HFD was undone via clodronate-mediated clearance of macrophages. These observations support a vital role for macrophage dysfunction in early, HFD-induced cardiac impairment.

Despite the spectacular rescue effect achieved with pharmacological clearance of macrophages, global macrophage depletion cannot be a therapeutic aim, as life without macrophages would have dramatic effects. In an attempt to develop potentially translatable therapeutic options, first it is important to characterize these senescent macrophages deeper to understand the intricate mechanisms that shifted them into a senescent state. In addition to determining how they become senescent, it is imperative to learn how they induce metabolic and cardiac alterations.

Answers to the first question will set the course for the pathways involved that could be specifically targeted by carefully chosen senolytic or senostatic agents. The viability of this approach is suggested by recent studies showing distinct molecular vulnerabilities of senescent cells. 270,[275][276][277][278] Our data pave the way to adapt the strategy to screen potential senolytic drug candidates targeting specifically senescent tissue macrophages in order to increase the therapeutic efficiency with minimised side-effects as opposed to a global senolytic approach used in our present study. Specific delivery to target cells, i.e., macrophages may also be improved by encapsulating chosen senolytics into liposomes to be selectively phagocytosed by macrophages or even ATMs should the targeting strategy allows it.

Deciphering the mechanisms by which senescent macrophages induce metabolic and cardiac alterations need to be based on extensive analysis of the secretome of these cells. This ongoing work that I aim to contribute, will involve proteomic/metabolomic/lipidomics screening of local secretomes and plasma representing systemic circulation that reaches remote organs affected. Besides, a future area of interest is to identify circulating extracellular vesicles recently implicated in obesity. 356 Paracrine effects will be explored using co-culturing of macrophages with potentially affected cells (preadipocytes, fibroblasts, etc). 134,[169][170][171] In conclusion, our findings allow the speculation that macrophage senescence by HFD is one of the first pathological events, occurring well before local or systemic inflammation as well as major tissue remodeling take place. Its key pathogenic role is underscored by restored cardiometabolic status after in vivo pharmacological clearance of macrophages. Exploratory studies will shed further light on early macrophage senescence and dysfunction in VAT and possibly other organs that may take us a step closer to improved therapeutic targets in T2DM.

➢ Adipose tissue senescence is mediated by increased ATP content after a short-term high fat diet exposure

After the identification of the early and key role of macrophages in adipose tissue senescence, we aimed to explore mechanisms potentially underlying the accumulation of senescent cells within both visceral and subcutaneous WAT in response to experimental caloric overload.

Here we found that induction of senescence in either WAT depots was a very early event occurring two weeks after HFD initiation in parallel with metabolic alterations but independent of systemic inflammation or WAT fibrosis. Furthermore, we identified HFDinduced excessive ATP production by increased glycolysis and oxidative phosphorylation, as a putative mechanism underlying WAT senescence. Forced exercise in the form of daily swimming restored impaired systemic glucose homeostasis irrespective of reductions in body weight and adiposity. With regard to WAT senescence, we found that exercise disproportionately affected various fat pads, ameliorating senescence in subcutaneous AT but not in visceral AT. These fat-specific differences seemed to be inherent as demonstrated in tissues and primary preadipocytes derived from subcutaneous and visceral AT and were related to xanthine oxidase-mediated ATP disposal.

At the end of 10 weeks of HFD starting in male WT mice of 5 months of age, we did not observe WAT fibrosis, nor local or systemic inflammation, but glucose intolerance, insulin resistance and leptin production manifested consistent with increased fat mass and adipocyte hypertrophy along with organization of immune cells into crown-like structures. 357 Specifically, we found unaltered tissue levels of classical pro-inflammatory SASP factors (CCL2, TNFa, IL6, IL10, PAI1) 181,222,358 as opposed to longer (i.e. 30 vs. 10 weeks in our model) HFD exposure in older (12-13 vs. 7 months) animals. 283 Instead, we observed an upregulation of several SASP-related chemokines (Mcp1) and profibrotic cytokines (Tgfb1, Fn1, Timp1) in eWAT but not iWAT. Interestingly, of the affected factors MCP-1 is secreted by senescent cells and has recently been correlated with biological age in mammals, 359 whilst TGFβ promotes matrix remodeling in remote organs 360 . Our recent observations 341,360 lend support to the ability of senescent WAT to secrete great quantities of profibrotic cytokines that alter tissue homeostasis prior to the development of a pro-inflammatory secretome. This profibrotic secretome has been shown to contribute to organ remodeling, dysfunction, and premature aging. 181,188,360 Accordingly, we observed that cellular senescence as one of the earliest events occurring in WAT, two weeks after the initiation of HFD as demonstrated by increase in SA-βgal activity, and expression of senescence markers p16 and p21.

Increased ATP content initiates WAT senescence

Caloric overload is buffered by the ability of WAT to store surplus energy as fat. For a long time, WAT has been considered as a passive storage unit as a result of anabolic metabolism, whilst little is known about catabolic processes during excessive energy intake. As evidence for increased catabolism occurring in concert with enhanced anabolism in HFD-challenged adipocytes, we found elevated ATP levels in both subcutaneous and visceral adipose tissuederived preadipocytes, which was normalized by exercise only in subcutaneous WAT. This is consistent with marginally increased mitochondrial ROS production in WAT of HFD-fed mice, probably as a by-product of exaggerated oxidative phosphorylation.

As adipocytes are prototypic low-maintenance cells in terms of energy utilization, it is curious what happens to excess ATP and what changes it may induce. Firstly, there is a feedback inhibition by ATP and NADH at multiple upstream levels, which would slow down and eventually stop further catabolism. Secondly, we reasoned that should the adipocytes be able to turn on mechanisms to reduce ATP levels, they would be expected to do so. One such possibility is to enhance purine catabolism as a means to dispose excessive ATP. In parallel with increased responsiveness of subcutaneous WAT to exercise-induced ATP reduction, we observed substantially higher expression of xanthine oxidase (XO) -the rate-limiting enzyme controlling purine degradation -, in subcutaneous than in visceral WAT. Moreover, XO uses molecular O2 as an essential cofactor, which is provided by exercise hyperaemia that improves blood flow in the neighbouring subcutaneous WAT. The combination of high XO expression, HFD-derived increased substrate supply for purine degradation and O2 cofactor is consistent with the difference between subcutaneous and visceral WAT in exercise preventing/reversing senescence. We further demonstrated that ATP, but not uric acid-loaded liposomes induced senescence in primary preadipocytes derived from subcutaneous WAT and this was dependent on XO activity, as its inhibitor allopurinol provoked senescence when cells were challenged with ATP-loaded liposomes or increased glucose/fatty acid content in the media.

➢ Exercise exerts a "senostatic" action preventing WAT senescence during HFD Our findings confirm previous reports of exercise preventing adipose tissue senescence in HFD 283 and add mechanistic clues to a triggering role for ATP. The significance of our present results is further highlighted by the lack of confounding factors known to interfere with senescence, such as age 360 and weight loss, 361 in our study design.

Exercise promotes mitochondrial biogenesis and improves function in multiple organs including the heart. 362 Curiously, we found that exercise reduced global WAT respiration, mitochondrial ATP-linked oxygen consumption and glycolysis, ultimately resulting in reduced ATP accumulation in subcutaneous but not in visceral WAT. These observations extend our findings about increased purine degradation in calorically challenged subcutaneous WATderived preadipocytes and suggest that subcutaneous WAT is able to limit ATP production and activate ATP disposal in times of caloric overload, whilst visceral WAT cannot. Moreover, exercise improved insulin sensitivity in obesity in concert with modulating the excessive energy generation. These findings are in line with the normalization of HFD-induced increase in mitochondrial mass, a pattern closely reflecting that observed in other models of senescence. 363,364 As ATP acts as a danger signal on immune cells, 365 our data show that exercise-related ATP reductions moderate p16 and p21 expression in WAT macrophages, which may improve the clearance of senescent cells. Our data are in line with a recent observation showing that restraining mitochondrial ATP synthesis with a relatively weak respiratory chain complex IV inhibitor may alleviate age-associated disorders. 366

Clinical impact

Given the worldwide obesity epidemics, there is an increasing interest in public health programs encouraging healthy eating behaviour and physical activity. Aerobic training has a beneficial impact at different life stages on several aspects of human health, such as cognitive function, cardiovascular, endocrine, and immune responses. Importantly, the benefit of exercise has been demonstrated in pathological conditions, including diabetes, cancer, and cardiovascular disorders 367 . Here we add mechanistic insights to the role of exercise in preventing both cellular senescence and impaired glucose homeostasis in diet-induced obesity, reinforcing the concept that adipose tissue dysfunction affects glucose homeostasis. 368 As such, exercise ameliorates WAT cellular senescence, adipokine dysregulation and adipocyte bioenergetics independent of weight loss, favouring the recovery of glucose homeostasis in obesity. Therefore, exercise through the control of local ATP seems an interesting approach to prevent the induction of cellular senescence and its potential deleterious consequences. In summary, exercise is an excellent "senostatic" strategy for preventing obesity-associated senescence in WAT devoid of side effects together with mitigating cardiac-associated abnormalities.

Since my team is interested in studying the cross-talk between the adipose tissue and the heart in different settings mimicking clinical situations of aging (obesity, chronological aging), all our murine models are phenotyped on both metabolic and myocardial changes.

Therefore, I have participated in a study aiming to decipher mechanisms leading to cardiac aging during chronological aging.

Dysregulated Phe catabolism in natural aging

This study followed our interest in finding extracardiac cues to cardiac deterioration associated with natural aging (coined henceforth as "cardiac aging"). Results provide new mechanistic insights into cardiac aging with important clinical implications. Firstly, we demonstrate that aging increases plasma Phe levels in mice and that elevated plasma Phe

Hyperphenylalaninemia promotes cardiac aging

Notably, our murine experiments recapitulated elevated plasma Phe levels observed in elderly human subjects. [370][371][372][373] Subsequently, we found that Phe excess induced myocardial p21, hence cardiac senescence. Furthermore, increased plasma levels of the essential amino acid Phe pointed to its impaired global catabolism, an observation particularly interesting in light of myocardial induction of Pah, Gch1 and 2-SC that we found in aged hearts, confirming progressive induction of myocardial Pah transcripts by aging. 374,375 This strongly suggests that in hyperphenylalaninemia myocardium acts to maintain Phe homeostasis through induction of its catabolic machinery. In the course of this adaptation, however, cardiac Phe catabolism may itself promote myocardial aging through altered intracellular signalling by protein succination, 376 as we have demonstrated in aging human and murine myocardium. Equally, but not exclusively, increased Phe uptake with limited myocardial Phe catabolic capacity may result in the accumulation of toxic metabolites, such as phenylpyruvate, phenyllactate and phenylacetate. 377 Moreover, increased ectopic Phe catabolism into tyrosine may fuel catecholamine biosynthesis in tyrosine hydroxylase-expressing cells, such as certain neurons and chromaffin cells of the adrenal medulla. 378 Thus, elevated Phe levels have an adverse impact on cardiac health, as evidenced by structural and functional myocardial impairment with a signature of senescence and Phe catabolism.

BH4 treatment rejuvenates the aging myocardium by restoring hepatic Pah activity

Our findings support a paradigm of age-related hepatic hypocatabolism of Phe underlying agerelated rise in plasma Phe and driving cardiac aging. This is in line with downregulation of Pah, one of the six BH4-dependent enzymes with the highest hepatic expression. Pah's vital dependence on its natural cofactor, BH4, is illustrated by hyperphenylalaninemia being a prominent feature of enzymatic deficiencies in de novo BH4 biosynthesis or the recycling pathway. 379 Importantly, we restored healthy cardiac structure and function in naturally aged mice through normalizing plasma Phe levels by BH4, consistent with revived hepatic Pah activity. 380,381 In line with this, we found reduced Pah and tyrosine levels in AML12 hepatocytes with pharmacologically stimulated senescence that could be rescued by both p21 knockdown and BH4. In theory, other BH4-dependent enzymes, i.e., nitric oxide synthases (NOS) could play a role in the cardiac improvement observed with BH4 treatment. Improved vascular NOS coupling and subsequently reduced left ventricular afterload [382][383][384] is unlikely, however, since systemic blood pressure remained unaltered by intraperitoneal BH4 delivery. Moreover, BH4 treatment did not increase myocardial NOS activity. Lack of such changes is consistent with the reported failure of exogenously delivered BH4 to reach efficient systemic concentrations 382 due to predelicted BH4 uptake of natural Phe-expressing organs: the liver and kidney. 385 Restored indices of myocardial Phe catabolism by BH4 correspond to BH4 uptake by the liver fuelling its local Pah activity taking the burden of Phe catabolism off the heart. This is consistent with the lack of reduction in cardiac damage induced by pressure overload in cardiomyocyte-restricted Gch1 transgenic mice 386 as opposed to oral BH4 administration. 387 Finally, the salubrious myocardial effects of dietary Phe restriction underscores the importance of lowering systemic Phe levels to rejuvenate old hearts.

Collectively our data show that pharmacological restoration of hepatic Pah activity by BH4 may rejuvenate age-associated cardiac changes and have potential application in age-related cardiac disease, such as heart failure.

Age-dependent p21 compromises hepatic Pah activity

Whilst Phe induced p21 in hepatocytes and cardiomyocytes in vitro, aging upregulated p21 expression in both liver and heart, hallmarking senescence of these organs. Moreover, we uncovered a significant delay in age-related rise in plasma Phe levels and cardiac decline in p21 -/-mice indicating that there are particular age-related gain-of-function(s) attributable to p21. Accordingly, in cultured hepatocytes we established a Phe-dependent p21 induction; experimental p21 induction on the other hand was sufficient to compromise Pah expression and activity in hepatocytes, which could be rescued by BH4 co-treatment. This constitutes a vicious cycle: Phe induces hepatic p21, p21 compromises hepatic Pah expression/activity, which raises systemic Phe levels and the cycle starts all over again. Interestingly, neither Phenor p21-mediated life-shortening is unprecedented; whilst dietary addition of Phe reduces lifespan in C. elegans 388 and ants, 389 p21 deficiency increases it in mice. 390 As for our findings, we identified one novel function for p21, which is the suppression of hepatic Pah expression/activity, through which p21 is a key node for the rise in plasma Phe levels with age.

Cardiac aging: a quiescent organ and cellular senescence?

Cellular senescence of the heart is intriguing due to the postmitotic status of cardiomyocytes (~30% of all heart cells occupying 80% of the heart's volume) 391 that do not fall under Leonard

Hayflick's concept of senescence, the proliferative inability in otherwise division-competent cells. 179 Aged cardiomyocytes acquire a senescent-like phenotype characterised by persistent DNA damage at telomere regions that occur independent of cell division and telomere length. 392 This length-independent telomere damage in cardiomyocytes activates senescent pathways (p21) and raises secretion of pro-fibrotic and -hypertrophic factors. 392 Autocrine/paracrine effects of this secretome include proliferation of cardiac fibroblasts and their activation towards myofibroblasts. In addition to this intramyocardial cascade, we uncovered a role for visceral adipose tissue, adding an endocrine enhancement to cardiac aging by releasing a profibrotic secretome (e.g., Tgfb1, osteopontin and leptin). 320 Therefore, cardiac aging seems to result from complex interactions as part of an integrated multi-disorder state.

The present study extends this organism-level approach to cardiac aging by studying circulating metabolic signals. Since metabolic screens showed increased plasma Phe levels occurring in both natural aging and heart failure, we specifically studied the role of plasma Phe in cardiac aging. [371][372][373]375,[393][394][395] Our findings in age-related and experimentally induced hyperphenylalaninemia confirmed a causal relationship between elevated plasma Phe levels and cardiac decline.

A new piece in the puzzle: the natural course of cardiac aging

Murine investigations into cardiac aging traditionally explore animals with at least 18 months of age. These studies among others identified dysfunctional sirtuin/NAD + biology, 396 mitochondrial oxidative stress, 397 abnormal PI3K/Akt signaling, misfolded proteins, 374 telomere dysfunction, DNA damage 392 and compromised mitochondrial energetics 398 in aged hearts. Our findings synergize with the literature extending our understanding of the natural history of cardiac aging. For instance, in WT hearts at 24 months of age we observed elevated complex III activity with suppressed ATP synthase activity pointing to the former as a potential mitochondrial source of oxidative stress. 397 The early onset of age-related functional decline is most evident by the time-limited competitiveness of elite athletes, which typically predates heart disease. Accordingly, during our time-course we found clinically detectable impairments in myocardial structure and function as early as 10 months of age. Up to and including the age of 15 months we found no sign of myocardial DNA damage, impaired mitochondrial complex activities, alterations in NAD + /NADH levels (re: energetics and sirtuins). On the contrary, we observed an emergence of myocardial p21 expression, ectopic Phe catabolism with increased succination, redox imbalance and epigenetic changes starting at 10 months of age. These structural, functional and molecular alterations associated with elevated plasma Phe levels, were inducible with Phe treatment in vitro and in young WT mice in vivo, as well as were (depending on age at least partially) reversible with interventions controlling systemic Phe levels (in vivo BH4 treatment and dietary Phe restriction). These results demonstrate that dysregulated Phe catabolism is an early-onset persistent component of cardiac aging, preceding mechanisms described to date, and importantly, it is amenable to therapeutic correction.

Conclusions

Our findings unlock a novel approach to rejuvenate the aged myocardium by pharmacological restoration of aging-induced Phe catabolic decline. The potential of targeting Phe is highlighted by related abnormalities starting at middle age and persisting throughout life. Our observations may be extended to other age-related disorders, such as dementia, 399 susceptibility to cancer, 400,401 and all-cause mortality risk 402 that could benefit from a strategy of counteracting the adverse effects of a declining PAH activity.

ATP production turned out to be a good strategy for preventing obesity-associated senescence in AT and cardiac-associated abnormalities.

Since my team is interested in studying the cross-talk between the adipose tissue and the heart in different settings mimicking clinical situations of aging (obesity, chronological aging), all our murine models are phenotyped on both metabolic and myocardial changes.

Therefore, I have participated in a study aiming to decipher mechanisms leading to cardiac aging during chronological aging (Study 4). This study followed our interest in finding extracardiac cues to cardiac deterioration associated with natural aging (coined henceforth as "cardiac aging") and was based on the observation that there is an age-dependent increase in blood Phe levels. From here experimentation established Phe-related adverse cardiac effects in natural aging and impaired hepatic Pah activity as an underlying cause.

These findings unlock a novel approach to rejuvenate the aged myocardium by pharmacological restoration of age-induced Phe catabolic decline and may also be extended to other age-related disorders, such as dementia, susceptibility to cancer, and all-cause mortality risk that could benefit from a strategy of counteracting the adverse effects of a declining PAH activity.

Altogether our data indicate the noxious impact of high fat diet on adipose tissue that may act as a toxic organ releasing profibrotic and proinflammatory cytokines spreading senescence to remote organs with remodeling and dysfunction consequences as evidenced in the heart.

This PhD study underlines the need to integrate environmental factors in order to target the complex intertwined conditions participating to the organism aging and the related obesityand age-diseasome. Indeed, the accumulation of senescent cells leads to premature loss of tissue renewal, inducing tissue remodeling and organ dysfunction. Premature aging may be the consequence of the senescent cell accumulation but is also driven by interconnected biological mechanisms such as epigenetic alterations, genomic instability, telomere attrition, deregulated nutrient sensing, mitochondrial dysfunction, stem cell exhaustion, and altered intercellular communication.

Our data suggest that epigenetic mechanisms are of importance in the control of "inflammaging" and deserve additional work that we aim to contribute in the next future.

Indeed, epigenetic pathways are known to sense the environment and to reprogram the cell identity including endothelial and immune cells that may contribute to age-related organ dysfunction.

Graphical abstract: Adipose Tissue Remodeling as a central player in cardiometabolic disorders in the context of high-fat diet-induced obesity and aging.

  List of abbreviations ................................................................................................................................ Abstract ................................................................................................................................................. Résumé .................................................................................................................................................. INTRODUCTION .......................................................................................................................... CONTEXT & LITERATURE REVIEW ................................................................................................ Section 1: OBESITY…………………………………………………………………………..……………………………………………….17

Chapter 1 : 24 Chapter 1 :

 1241 Definition and Epidemiology ........................................................................................... Chapter 2: Clinical research and limitations ...................................................................................... Chapter 3: Animal models ................................................................................................................ Section 2: ADIPOSE TISSUE: PHYSIOLOGICAL STRUCTURE & FUNCTION…………………………….……………..Diversity of adipose tissue depots in humans and mice ................................................ Chapter 2: Adipose tissue cell types and their functions ................................................................. I) Adipocytes ............................................................................................................................. II) Adipose-derived stem cells (ADSCs) and preadipocytes ....................................................... III) Endothelial cells and their progenitors ................................................................................. IV) Adipose tissue immune cells ................................................................................................. Section 3: ADIPOSE TISSUE: PATHOPHYSIOLOGICAL STRUCTURE & FUNCTION…………………….………….37 Introduction ....................................................................................................................................... Chapter 1: Obesity and insulin resistance ........................................................................................ I) Insulin signaling pathway in adipocytes ................................................................................ II) Mecanism of insulin resistance in adipocytes ....................................................................... Chapter 2: Obesity and low-grade inflammation .............................................................................. Chapter 3: Obesity and adipose tissue fibrosis ................................................................................. Chapter 4: Obesity and adipose tissue senescence .......................................................................... I) Definition of cellular senescence .......................................................................................... II) Replicative cellular senescence ............................................................................................. III) Premature cellular senescence ......................................................................................... IV) Biomarkers of senescence ................................................................................................. V) Adipose tissue senescence in aging and obesity ................................................................... VI) Adipose tissue cell-type specific senescence .................................................................... VII) Adipose tissue senescence as a therapeutic target for the treatment of aging-and obesity-related metabolic disorders ............................................................................................. Chapter 5: Conclusion ....................................................................................................................... OBJECTIVES ................................................................................................................................ METHODS .................................................................................................................................. RESULTS .................................................................................................................................... Study 1………………..…………………………………………………………………………..…………………………………………….105 Study 2………………..…………………………………………………………………………..…………………………………………….137 Study 3………………..…………………………………………………………………………..…………………………………………….184 Study 4………………..…………………………………………………………………………..…………………………………………….228 DISCUSSION .............................................................................................................................. CONLUSION & PERSPECTIVES ..................................................................................................... SUPPLEMENTAL TABLES ............................................................................................................. BIBLIOGRAPHY .......................................................................................................................... However, time course of AT senescence and mechanisms underlying its initiation are yet unclear, as well as the precise identity of the cells affected within the AT. Therefore, my Doctoral project aimed to:

Figure 3 :

 3 Figure 3: Type 2 diabetes related functional linkage network (T2DFN). (From Rende, D. & al., Plos

Figure 4 :

 4 Figure 4: Anatomy of major fat depots in rodents (A) and Humans (B), (From Tchkonia, T. & al., Cell Metabolism 2013)44

Figure 5 :

 5 Figure 5: Regulation of lipogenesis within adipocytes. CM: chylomicron; VLDL: very low-density lipoproteins; LPL: Lipoprotein lipase; G3P: glycerol-3-phosphate. (modified from exsymol.com: https://www.exsymol.com/en/dossier/lipogenese/ )

Figure 6 :

 6 Figure 6: Regulation of lipolysis within adipocytes. Black arrows indicate pathways of biochemical substrates and products. Blue arrows indicate joined pathways of biochemical substrates resulting in the respective product. Open arrows in red show the direction of change in the insulin-resistant state (From Bódis, K. & al., Eur J Clin Invest. 2018) 47

Figure 7 :

 7 Figure 7: Effects of extracellular vesicles (EVs) from adipocytes, hepatocytes, and skeletal muscle. (From Martínez MC, Andriantsitohaina R. Circ Res. 2017) 61

  are composed of neutrophils, basophils, and eosinophils. In adipose tissue, they are very few but still important as they are the first cells to migrate to sites of acute infection and injury and start immune response by inducing recruitment of further immune cells.90 Lymphocytes belong to one of three subpopulations: Natural Killer (NK) lymphocytes expressing CD16 cell surface marker (CD16+), B lymphocytes (CD19+) and T lymphocytes (CD3+). NK cells have two major functions. The first function is to eliminate tumours or infected cells through cytotoxic activity, whilst secondly NK cells additionally stimulate immune response by modulating other immune cells via cytokine production. B lymphocytes are activated in later stage during immune response as they trigger the adaptive phase. Their main role is to produce antibodies after being activated either directly by the antigens or by T lymphocytes.91 The process starts when B-cell link an antigen through specific membrane receptors known as B cell receptors (BCRs). Then, in a process common to all antigenpresenting cells (APCs), they display the antigen complexed with MHC class II molecules on their surface and T-cells recognize these complexes thanks to their T-cell receptors (TCRs). As a result, T-cells get activated and produce cytokines including IL-2, IL-4 and IL-5, which will in turn activate B-cells and allow them to produce antibodies (figure8). T lymphocytes are divided in 3 subpopulations: T cytotoxic lymphocytes (CD8+), T helper lymphocytes and regulatory T-cells (Treg) which are both CD4+. T cytotoxic lymphocytes have the same functions as NK cells except that they act in a more targeted way: they specifically kill cells with an antigen they recognized before. T helper lymphocytes participate in the elimination of target cells indirectly through modulation of other immune cells. Treg cells play an important role in modulating the immune response toward the end of it in order to make sure inflammation stops as soon as the danger is gone.91

Figure 8 :

 8 Figure 8: T-cell-dependant B-cell activation (Modified from preprotech.com: https://www.peprotech.com/de/immunology-research-booklet-2 )

Figure 9 :

 9 Figure 9: Adipose tissue macrophages (ATMs) classification. AAMs: alternatively activated macrophages; MME: metabolically activated macrophages; Mox: oxidized macrophages. (From Russo, L. & al., Immunology 2018) 92

  Once insulin protein binds to the extracellular α subunits of the receptor, it induces phosphorylation of tyrosine residues in the intracytoplasmic β subunits. This leads to phosphorylation of Insulin Receptor Subtrate (IRS-1) which, in adipocytes, induces Phosphatidyl Inositol 3 Kinase (PI3K) and protein kinase B (PKB/Akt) recruitment and activation, 102,109 (figure10).

Figure 10 :

 10 Figure 10: Insulin receptor (INSR) activation and the associated signal transduction. (From

Figure 11 :

 11 Figure 11: Insulin signaling pathway in adipocytes. Black arrows indicate pathways of biochemical substrates and products and unfilled thick arrows indicate the direction of change in insulin-resistant state. (From Bódis, K. & al., Eur J Clin Invest. 2018) 47

Figure 12 :Chapter 2 :

 122 Figure 12: Physiological impact of insulin resistance: an integrated perspective. (From Petersen, M.C. & al., Physiol Rev. 2018) 102

Figure 13 :

 13 Figure 13: Molecular mechanism of the effects of saturated (16:0) fatty acids on the TLR4 and NF-κB pathways. (Modified from Marcelo Macedo Rogero & al., Nutrients 2018) 134

Figure 14 :

 14 Figure 14: Immune cells infiltrates and inflammation in obese adipose tissue.

Figure 15 :

 15 Figure 15: Cross-talk between adipocytes and macrophages in lean or obese adipose tissue.(From Marcelo MacedoRogero & al., Nutrients 2018) 134

Figure 16 :

 16 Figure 16: Pictures of immunofluorescent staining showing the morphological difference between adipogenic/angiogenic cell clusters (B, E : 8 weeks-old diabetic mouse) and crown-like structures (C, F : 30 weeks-old mouse). Blue color is BODIPY (lipids), red color is CD68 (macrophages), and green color is Hoechst (nuclei). (From Nishimura, S. & al., Diabetes. 2007) 150

Figure 17 :

 17 Figure 17: Quantification of total fibrillar collagens by picrosirius red staining in subcutaneous and visceral AT in obese versus lean subjects. Data are represented as mean ± SEM.(FromSun, K & al., Cell Metab 2013) 20

Figure 19 :Chapter 4 :

 194 Figure 19: Obesity and adipose tissue dysfunction.(FromBuechler, C. & al., World J Diabetes 2015) 165

  age and measurement of telomere length in peripheral blood mononuclear cells (PBMC) is a commonly used technique and established marker of organismal aging in humans.193

Figure 20 :

 20 Figure 20: Schematic representation of replicative cellular senescence. (From Blackburn, E. & al., Science 2015) 194

Figure 21 :

 21 Figure 21: A general overview of premature cellular senescence. (From Liu, Z. & al., Clin Sci 2020) 177

Figure 22 :

 22 Figure 22: Mechanisms of cell cycle arrest (From https://www.cellsignal.com)

Figure 23 :

 23 Figure 23: Global and focal chromatin remodeling in senescence.(FromLee, S. & al., Nat Cell Biol 2019) 219

Figure 24 :

 24 Figure 24: Common biomarkers of senescence (From https://www.cellsignal.com)

Figure 25 :

 25 Figure 25: Causes and consequences of cellular senescence in different AT cell types. (From Liu, Z.& al., Clin Sci 2020) 177

1 )

 1 Compare the impact of high-fat diet-induced obesity on adipose tissue function and remodeling in young and aged mice: a. To identify potential age-dependent response, b. To establishing the chronological order of events leading to visceral adipose tissue alterations and senescence in high fat diet young mice, c. To explore the role of senescent adipose tissue macrophages in metabolic and cardiac dysfunction, d. To compare different strategies in order to mitigate visceral adipose tissue senescence.

Z.

  Mezdari contributed to the follow-up and the treatment of the different animal cohorts involved in the four studies presented in this thesis document together with M. Pini, D. Sawaki, G. Czibik and Y. Zhang.

Figure 26 :

 26 Figure 26: Representative curves of blood glucose concentrations obtained after Glucose tolerance test (GTT) and Insulin tolerance test (ITT)

Figure 27 :

 27 Figure 27: Pictures of the echocardiographic machine (left) and a conscious mouse undergoing echocardiography (right)

  271

Z.

  Mezdari participated to this experiment under the supervision of M.Pini and D. Sawaki

Mice underwent 4

 4 weeks of physical training together with CD or HFD that they started 5 weeks before. Exercise training consisted in 2 daily swimming sessions in a water tank maintained at 30°C to 32°C separated by a 6-hour break. Swimming sessions were supervised to avoid floating and/or clinging of individual animals and increased by 10 minutes each day until 90 minutes sessions were reached(Derumeaux et al., 2008). 293 Z. Mezdari performed this experiment under the supervision of M Pini.

Figure 28 :

 28 Figure 28: Dissection pictures: Abdominal excision (left), blood collection (middle), and canulation of heart (right)

Figure 29 :

 29 Figure 29: Flow cytometry: Description and principles (from Pranab Day, 2018). 296

Z.

  Mezdari participated to isolation and cell culture of BMDM under the supervision of D. Sawaki with the support of. Y. Zhang and M. Pini Technical sheet n°15Cell culture experiments on BMDMBMDM stimulation by osteopontinModulation of BMDM by OPN protein was evaluated as previously described with minor modification.297 Briefly, 12-well plates were coated with 20 or 40 µg of mouse recombinant OPN (R&D systems) for 20 hours at 4°C and then stabilized with 0.5% polyvinylpyrrolidone (Sigma-Aldrich) for 1 hour at room temperature. BMDM (1.5 × 10 5 /well) were re-plated in RPMI 1640 medium supplemented with 5%FCS and 30 ng/ml MCSF. Twenty-four or 36 hours after passage, the cells were harvested for RNA extraction.

  SVF cells were centrifuged for 10 min at 350 g and suspended in pre-warmed adipogenic culture media, consisting of DMEM high glucose with Glutamax (Gibco) supplemented with 10% new born calf serum (Thermofisher), 2.4 nM human insulin (Sigma) and 1% antibiotic solution (Pen-Strep solution, Sigma). To induce differentiation, SVF cells were seeded near confluence and medium was replenished every day to allow selection by adhesion during the first 3 days and then changed every 2 days. SVF cells were cultured in an atmosphere of 5% CO2 and 20% O2 at 37°C. Based on preliminary experiments, day 7 was the time of differentiation used for all experiments. This experiment was performed by M. Pini 91 Technical sheet n°17

  cell line C2C12 (ATCC® CRL-1772™) myoblasts maintained in DMEM supplemented with L-glutamine (2 mM), penicillin/streptomycin (1%) and 5% fetal bovine serum (FBS) were seeded either at a density of 5x10 4 cells (<50%) per well in 24-well plates or 10 4 cells in 4-well chamber slides. At 80% confluence, cells were treated with L-phenylalanine (1-10 mM), BH4 (10 µM), 4-CPA (1.5 mM) and/or N-acetylcysteine (500 µM) vs. vehicle overnight as indicated. Cells were analyzed for oxidative stress with MitoSOX™ (mitochondrial superoxide; 5 µM, ThermoFisher Scientific), CellROX™ (cytosolic superoxide; 5 µM, ThermoFisher Scientific), gene or protein expression, or various metabolites (see below).

Axioplan 2

 2 Imaging microscope (Zeiss). Immunohistochemical staining images were obtained with an Axioimager M2 fluorescent microscope (Zeiss). Confocal microscope (Zeiss LSM 510 Meta) was used for BODIPY fluorescent staining. All quantifications were done blindly, using the ImageJ software Histological assessement was performed by Z. Mezdari, Dr. M. Pini, Dr. D. Sawaki, Dr. G. Czibik, S. Naushad, M. Halfaoui, H. Liang, and Dr. D. Altintas.

Figure 30 :

 30 Figure 30: (A) Principle of the TaqMan technology (from https://en.wikipedia.org/wiki/TaqMan) ; (B)Representative qPCR amplification curve (from https://bitesizebio.com/24581/what-is-a-ct-value/ )

Figure 31 :

 31 Figure 31: Representation of the different steps of Western blot ( from https://www.novusbio.com/application/western-blotting)

  les évènements primaires qui sont à l'initiation des altérations du tissu adipeux observées chez les individus obèses. Nous avons donc focalisé notre attention sur les mécanismes initiateurs du remodelage du tissu adipeux et de la sénescence du TA viscéral en établissant une cinétique des événements en analysant chaque type cellulaire et en particulier les macrophages de cet organe du fait de leur rôle reconnu dans les altérations métaboliques et cardiaques. Nous avons utilisé un modèle murin pour comparer l'effet d'un régime riche en graisses chez des souris jeunes et des souris âgées puis dans un second temps observé chez les souris jeunes les modifications du tissu adipeux viscéral et du coeur à différentes durées de ce régime. Enfin, nous avons traité ces souris par sénolytiques (association Dasatinib/Quercétine) puis par clodronate afin d'éliminer spécifiquement les macrophages. Les résultats obtenus ont montré que les souris jeunes répondent de manière plus rapide et plus intense au stress métabolique comparé aux souris âgées. Nous avons également pu observer que la sénescence du tissu adipeux était un élément précoce au cours du régime riche en graisses et qu'elle affectait en priorité les macrophages chez les souris jeunes, en apparaissant avant l'inflammation et la fibrose qui caractérisent habituellement le tissu adipeux dans le cadre de l'obèsité. De plus, la survenue de sénescence coïncide avec l'apparition de troubles métaboliques et cardiaques. Enfin, le rôle crucial des macrophages a été confirmé par une complète restauration des paramètres métaboliques et cardiaques à la suite de leur élimination par clodronate alors que le traitement sénolytique ne normalisait pas ces paramètres. Ces résultats montrent donc l'importance et le rôle clé que jouent les macrophages sénescents dans les complications métaboliques et cardiaques lors de l'obésité et apportent des arguments pour le développement de traitements ciblant spécifiquement les macrophages.

Figure 1 :Figure 2 :

 12 Figure 1: Schematic of treatment protocol. Young (2m) or aged (12m) mice fed CD or HFD either for 5 weeks or for 20 weeks, a total of 8 groups (n=4-5/group).

Figure 3 :

 3 Figure 3: A, Representative images of VAT stained with Hematoxilin and Eosin (H&E), magnification: 100x, scale-bar: 100 µm. B, Quantification of adipocyte cross-sectional area (CSA). C, number of crown like structures (CLS) ; (n=4-5/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant,*p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 4 :

 4 Figure 4: A, Representative images of VAT co-stained with p16 (senescence marker), Mac3 (macrophage marker), and DAPI (nuclei), magnification: 400x, scale-bar: 25 µm. B, Quantification of Mac3 positive cells. C, Quantification of p16 and Mac3 double positive cells; (n=4-5/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 5 :

 5 Figure 5: A-F, Temporal plot and area-under-curve (AUC) analysis of Glucose tolerance test (GTT; A-C) and insulin tolerance test (ITT; D-F); (n=4-5/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 6 :Figure 7 :

 67 Figure 6: Schematic of treatment protocol. 2-months-old mice fed CD or HFD either for 2 weeks or 5 weeks or 10 weeks, a total of 6 groups (n=5-10/group).

Figure 8 :

 8 Figure 8: A-C, Plasma levels of Leptin (A), Adiponectin (B), and Insulin (C); (n=5/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 8 :

 8 Figure 8: A, Representative images of VAT stained with Hematoxilin and Eosin (H&E), magnification: 100x, scale-bar: 100 µm. B, Quantification of adipocyte cross-sectional area (CSA). C, number of crown like structures (CLS) ; (n=15-20 images/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 9 :

 9 Figure 9: A-B, Representative images of CD3 (lymphocyte marker) immunostaining in VAT (A) and quantification of CD3 positive cells (B). Representative images of CD68 (macrophage marker) immunostaining in VAT (C) and quantification of CD68 positive cells (D). Magnification: 200x, scalebar: 50 µm; (n=9-12 images/group). E-H, Quantitative RT-PCR analysis of inflammation-related genes in VAT;(n=5-10/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's posthoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 10 :Figure 11 :

 1011 Figure 10: A, Representative images of VAT co-stained with BODIPY (lipid marker) and Isolectin B4 (IB4; capillary marker). Magnification: 400x, scale-bar: 50 µm. B, Quantification of capillary density normalized to cell size; (n=8-15 images/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test;ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 12 :

 12 Figure 12: A-D, Quantitative RT-PCR analysis of metabolism-related genes in VAT; (n=5/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

  123

Figure 13 :

 13 Figure 13: A-B, Representative images of P16 (senescent marker) immunostaining in VAT (A) and quantification of P16 positive cells (B); (n=15 images/group). C-D, Representative images of P21 (senescent marker) immunostaining in VAT (C) and quantification of P21 positive cells (D); (n=7-8 images/group). Magnification: 200x, scale-bar: 50 µm. E-F, Quantitative RT-PCR analysis of p16 (E)

Figure 14 :

 14 Figure 14: A-B, Representative images of γH2AX (DNA damage marker) and DAPI (nuclei marker) co-immunostaining in VAT (A) and quantification of γH2AX positive nuclei (B); (n=12 images/group). Magnification: 400x, scale-bar: 25 µm. Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 15 :

 15 Figure 15: Summary of quantitative RT-PCR analysis of senescence-, fibrosis-, inflammation-related gene in VAT; Data are presented as mean ± SEM (n=5/group).

Figure 16 :

 16 Figure 16: A-C, Evaluation of cardiac function; Heart weight to tibia length (HW/TL; A); Left ventricular ejection fraction (LVEF; B); Systolic strain rate (C); (n=5-7/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test;ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 17 :

 17 Figure 17: A-B, Representative images of hearts stained with wheat-germ agglutinin (WGA: plasma membrane marker; A), and quantification of cardiomyocyte cross-sectional area (CSA; B). Representative images of hearts stained with Picrosirius red (collagen marker; C) and quantification of interstitial fibrosis normalized to cell number (D); (n=10-16 images/group). magnification: 200x, scalebar: 50 µm. Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 18 :

 18 Figure 18: Schematic of treatment protocol. 5-months-old mice fed CD or HFD for 10 weeks and treated with or without senolytics (D+Q) for the HFD ones, a total of 3 groups (n=5-11/group).

Figure 19 :

 19 Figure 19: A-B, Representative images of P16 (senescence marker) and CD68 (macrophage marker) co-immunostaining in VAT, white arrows show colocalisation (A) and quantification of P16 and CD68 double positive cells (B); (n=12-17 images/group). Magnification: 200x, scale-bar: 50 µm. C, Body weight (n=5-11/group). D, Adiposity index (n=5-11/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test;ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 20 :

 20 Figure 20: A-D, Temporal plot and area-under-curve (AUC) analysis of Glucose tolerance test (GTT; A, B) and insulin tolerance test (ITT; C, D). E, Heart weight to tibia length (HW/TL). F, Systolic strain rate; (n=5-11/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 21 :

 21 Figure 21: A, Representative images of P16 (senescence marker) and CD68 (macrophage marker) co-immunostaining in VAT and quantification of P16 and CD68 double positive cells. B, Representative images of P21 (senescence marker) and CD68 (macrophage marker) co-immunostaining in VAT and quantification of P21 and CD68 double positive cells. C-D, Representative images of P16 or P21 (senescence marker) and CD3 (lymphocyte marker) co-immunostaining in VAT and quantification of P16 or P21 and CD3 double positive cells. E-F, Representative images of P16 or P21 (senescence marker) and PDGFRα (preadipocyte marker) co-immunostaining in VAT and quantification of P16 or P21 and PDGFRα double positive cells. (n=7-15 images/group). Magnification: 200x. Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

Figure 22 :

 22 Figure 22: A, Percentage of p16 positive cells per cell type. B, Percentage of p21 positive cells per cell type. Data are presented as mean ± SEM (n=7-15/group).

Figure 23 :

 23 Figure 23: A, Schematic of treatment protocol: Bone Marrow-Derived macrophages (BMDM) cells treated with metabolic media. B-C, Representative images of P16 (B) and P21 (C) immunostaining in BMDM

Figure 24 :

 24 Figure 24: Schematic of treatment protocol. 2-months-old mice fed CD or HFD for 2 weeks and treated with or without clodronate for the HFD ones, a total of 3 groups (n=5-11/group).

Figure 25 :

 25 Figure 25: A-C, Representative images of P21 (senescence marker) and CD68 (macrophage marker) co-immunostaining in VAT, white arrows show colocalisation (A) and quantification of CD68 positive cells (B), and quantification of P21 positive cells (C); D-E, Representative images of P16 (senescence marker) and CD68 (macrophage marker) co-immunostaining in VAT, white arrows show colocalisation (D) and quantification of P16 positive cells (E). F, Quantification of P21 and CD68 double positive cells. G, Quantification of P16 and CD68 double positive cells. H, Quantification of adipocyte crosssectional area (CSA). N=10-11 images/group. Magnification: 200x, scale-bar: 50 µm. Data are

Figure 26 :

 26 Figure 26: A-D, Temporal plot and area-under-curve (AUC) analysis of Glucose tolerance test (GTT;A, B) and insulin tolerance test (ITT; C, D); (n=5-10/group). E-F, Quantitative RT-PCR analysis of metabolism-related genes in VAT; (n=7/group). Data are presented as mean ± SEM analysed withANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 27 :

 27 Figure 27: A-C, Evaluation of cardiac function; Heart weight to tibia length (HW/TL; A); Left ventricular ejection fraction (LVEF; B); Systolic strain rate (C); (n=7-9/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

Figure 28 :

 28 Figure 28: Schematic of treatment protocol. 2-months-old mice fed CD or HFD for 10 weeks and treated with or without clodronate for the HFD ones, a total of 3 groups (n=8-12/group).

Figure 29 : 2 RésuméFebruary

 292 Figure 29: A-D, Temporal plot and area-under-curve (AUC) analysis of Glucose tolerance test (GTT; A, B) and insulin tolerance test (ITT; C, D). E, Body weight. N=8-12/group. Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

  but the underlying mechanisms are not yet fully understood, and particularly the specific role of ATM in aggravating such AT remodeling remains to be clarified. Aging increases ectopic AT accumulation and more specifically, visceral AT (VAT) depots.(7) Age-associated VAT remodeling includes adipocyte hypertrophy, deposition of extracellular matrix, and accumulation of immune cells.(8) Furthermore, our group has identified a major role of VAT in inducing remote tissue remodeling through the release of profibrotic factors, including transforming growth factor (TGF and osteopontin (OPN), both components of the senescenceassociated secretory phenotype (SASP) amongst a variety of growth factors, proinflammatory cytokines/chemokines as well as adipokines.(9) We recently established VAT as the major source of OPN during aging, extending knowledge on the reported role of OPN to participate in the AT pro-inflammatory status.(9-12) OPN is a matricellular protein involved in intra-and extra-cellular signaling mediating cell-to-cell interaction, immune cell function, inflammation, and tissue remodeling.(13) Notably, our group has demonstrated that OPN modulates the function of fibroblasts and contributes to age-related cardiac disorders.(9) However, the link between age-related OPN production, VAT senescence and impairment of macrophage function is still elusive. Here we show that during the aging process, ATM undergo a senescence phenotype, which impairs ATM function and contributes to age-related VAT remodeling and dysfunction. These senescent macrophages show global decline of cell motility, focal adhesion and endocytosis. Thus senescent ATM are inefficient in clearing apoptotic VAT cells. Conversely, depleting senescent ATM blunts VAT senescence and restores healthy VAT structure and function. Importantly, we uncover a major role of OPN in inducing ATM senescence by demonstrating that the loss of OPN attenuates ATM senescence, prevents age-related ATM dysfunction and finally rejuvenates VAT. Reciprocally, extracellular OPN drives naïve macrophages toward a senescent phenotype and impairs ATM phagocytic activity. Taken together, our data indicate that age-related OPN accumulation in VAT governs ATM senescence, which is a major contributor to VAT remodeling.

  ) All mice were housed in a specific pathogen free platform at a constant temperature (22°c), with a 12-hour light-dark cycle and unrestricted access to a chow diet (CD, A0310, Safe Diets, France) and water. Chow diet composition corresponded to 13.5% of fat, 61.3% of carbohydrate and 25.2% of protein. During follow-up, animals underwent a monthly body-weight (BW) evaluations.

  deoxynucleotidyl transferase-dUTP nick end labeling) staining was performed according to the manufacture's instruction (ApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit, Millipore) with minor modifications. Briefly, deparaffinized and hydrated sections were subjected proteinase K treatment (20 mg/mL, Qiagen), and incubated with TdT enzyme for 1 hour. After stopping the reaction, digoxigenin-conjugated fluorescein was applied to visualize the signals. CD45 immunohistochemistry and DAPI counter-staining was performed to show the TUNEL positive cell localization.

P

  -value <0.05 was considered significant (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Study approval All animal experiments were approved by the Institutional Animal Care and Use Committee of the French National Institute of Health and Medical Research (INSERM)-Unit 955, Créteil, France (ComEth 15-001) and Institut Gustave Roussy's Institutional Animal Care and Use Committee (Villejuif, France). Author contributions D.S., M.P. and Y.Z. designed, conducted most of the experiments and data analysis, and prepared the manuscript; Z.M., S.N., H.L. and M.H. helped with organ isolation and imaging experiments; L.L. and D.A. performed gene and protein expression analysis; M.B. assisted with mouse work and live imaging experiments; M.S. performed plasma analysis; D.R., P.G. and T.Y. donated materials and provided helpful suggestions; F.L. and S.A. provided helpful suggestions and contributed to the manuscript; C.H. performed gene discriminative analysis; and G.C. and G.D. supervised this work and helped to write the manuscript.

Figures & legends Fig. 1

 legends1 Figures & legends

Fig. 2

 2 Fig. 2 OPN is a critical regulator for VAT senescence. a Representative SA--Gal staining (bluegreen) of epididymal VAT from 12-month old WT and OPN-/-mice. b Representative Westernblot analysis using lysates from 12-month old WT and OPN-/-mice VAT. -actin was used as loading control. c Representative immunostaining of VAT (p16: /p21/p53: green, wheat germ agglutinin; WGA: red, DAPI: blue), arrow-heads indicate green-positive cells. Bar graphs

Fig. 4

 4 Fig. 4 Role of OPN in age-related ATM dysfunction. a Representative immunostaining of VAT from 3-and 12-month WT and OPN-/-mice (TUNEL: green, CD45: red, DAPI: blue). Green arrow-heads indicate TUNEL single-positive cells, and white arrow-heads indicate TUNEL and CD45 double-positive cells. Scale bar=50 m. b Quantification of co-localization of TUNEL and CD45 positive cell % to total cell number. n=3 mice/group. c Quantification of distance

Fig. 5

 5 Fig. 5 OPN-deficient bone marrow-derived macrophage attenuates age-related AT senescence, remodeling and dysfunction. a Schematic protocol of bone-marrow transplantation (BMT) from WT or OPN-/-donor to WT recipient to evaluate the effect of OPN-deficient BMDM. b

Figure 2 .

 2 Figure 2. HFD induces cellular senescence in various types of WAT cells, indipendent of systemic inflammation. A-B:Gene expression for p16 and p21 in iWAT and eWAT (A) and in iWAT-and eWAT-derived preadipocytes (B); n=3-6/group. C: Representative images and quantification by immunofluorescence staining in iWAT and eWAT of co-localization with p16 (green) and PDGFRα (red). Sections were counterstained with DAPI (blue). Double positive cells are expressed as the mean number of positive cells over adipocytes (n=4 mice/group, 5 pictures per mouse; arrow, original magnification x200, scale bar = 50 μm). D: Representative images and quantification by immunofluorescence staining in iWAT and eWAT of colocalization with p21 (green) and PDGFRα (red). Sections were counterstained with DAPI (blue). Double positive cells are expressed as the mean number of positive cells over adipocytes (n=4 mice/group, 5 pictures per mouse; arrow, original magnification x200, scale bar = 50 μm). E-F: The discriminative power of four major functional themes illustrating the HFD impact on WAT transcriptional profile. Transcriptional changes of a panel of selected genes were

Figure 3 .

 3 Figure 3. Adipose tissue senescence is associated with increased adipocyte bioenergetics. A: Representative images of CD-SED and HFD-SED mice and quantification by immunofluorescence staining in iWAT and eWAT of translocase of outer mitochondrial membrane 20 (Tom20) over adipocytes (n=5 mice/group, 4 pictures per mouse; arrow, original magnification x200, scale bar = 50 μm). B: Citrate synthase activity in iWAT and eWAT; n = 5/group. C: Changes in mitochondrial and non-mitochondrial oxygen consumption (OCR) after bioenergetic analysis of iWAT-and eWAT-derived preadipocytes isolated from CD and HFD mice, sedentary (SED) or exercise (EX) groups. Data are mean ± SEM of four independent experiments with five technical repeats. D: Changes in extracellular acidification rate (ECAR), an index of glycolysis. Glycolysis is determined through measurements of the surrounding media before injection of compounds. Data are mean ± SEM of four independent experiments with five technical repeats. E: Changes in ATP-linked oxygen consumption. Data are mean ± SEM of four independent experiments with five technical repeats. F: Purine degradation scheme. G: Intracellular ATP content in iWAT-and eWAT-derived preadipocytes; n = 6-8 wells/group. H: Representative images and quantification by immunofluorescence staining in iWAT and eWAT of xanthine oxidase (XO, green). Positive cell expressed as the mean number of positive cells over adipocytes (n=3 mice/group, 3 pictures per mouse; magnification x200, scale bar = 50 μm). I: Representative images of immunofluorescence XO staining in iWAT-and eWAT-derived preadipocytes (magnification x400, scale bar = 25 μm).J: Uric acid concentration in iWAT and eWAT tissue lysates, n = 6-12/group). K: Gene expression analysis in iWAT and eWAT for purinergic receptor P2X, ligand-gated ion channel, 7 (P2rx7). For gene expression, results are expressed as fold mRNA change relative to CD-SED values set to 1; n = 6/group. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 for differences due to diet regimen within sedentary and exercise groups (* = diet effect) and e P < 0.05; ee P <0.01; eee P < 0.001 for differences between sedentary and exercise groups fed the same diet ( e = exercise effect).

Figure 4 .

 4 Figure 4. A "heat map-like" table.The table displays the differences between iWAT and eWAT together with the effect of regimen and exercise in terms of adipose tissue function, senescence markers and ATP and uric acid contents.

Figure 5 .

 5 Figure 5. Role of ATP in adipose tissue senescence. A-B: Gene expression analysis for senescence (Cdkn1a, Cdkn2a)in chow diet (CD) iWAT-and eWAT-derived preadipocytes after 7 days of differentiation and treatment with ATP-loaded liposomes compared with two control applications: liposome-free (lipo-free, red line) and liposome alone (CTL); n = 4-6/group. C-D: Representative images of SA-β-gal in chow diet (CD) iWAT-and eWAT-derived

  Fig. 1

  animal work was approved by the Institutional Animal Care and Use Committee of the French National Institute of Health and Medical Research (INSERM)-Unit 955, Créteil, France (ComEth 15-001). Global p21 -/-mice backcrossed to C57BL/6 background for at least 10 generations (Jackson Laboratory, USA) as well as wild-type (WT) littermates or C57BL/6 mice of indicated ages (Janvier Labs, France) were kept in individually ventilated cages in a high-health facility with 12-hour lightdark cycle, controlled temperature (20-22°C) and humidity. Water and chow were provided ad libitum.

Figure 1 .

 1 Figures & Legends

Figure 2 .

 2 Figure 2. Development of ectopic phenylalanine (Phe) catabolism in the aging myocardium and its delay with p21 deficiency. A, Myocardial transcript levels of Phe transporters in 2 and 15 monthold WT and 15 month-old p21 -/-mice (n=5-8/group). B, Schematic to illustrate simplified Phe catabolism (phenylalanine hydroxylase: Pah; tyrosine: Tyr; fumarate: FU; 2-succinylcysteine: 2-SC; GTP cyclohydrolase 1: Gch1; tetra/dihydrobiopterin: BH4/BH2). C, Myocardial immunofluorescence for Pah and 2-SC in WT and p21 -/-mice at indicated ages (n=4/condition). D, Cardiac fumarate levels in indicated conditions (n=5-10/condition) E-F, Representative Pah/2-SC immunofluorescent colabeling in murine (E; n=3/condition) and human (F) hearts at indicated ages and genotypes. G Mitochondrial complex activities (normalized to citrate synthase activity; n=8-9/group; in hearts of 2, 15 and 24 month-old WT mice. H, Fumarate interferes with the activity of histone demethylase 4 (KDM4) resulting in higher levels of H3K9me3 (2-oxoglutarate: 2-OG). I, Myocardial immunofluorescence for H3K9me3 in WT and p21 -/-mice at indicated ages (n=4/condition). Data are presented as original images (C, E-F, magnification: 200x, scale-bar: 50 µm; I, magnification: 400x,

Figure 3 .

 3 Figure 3. Effects of phenylalanine treatment in vitro. A, Myocardial p21 expression against markers of cardiomyocytes (troponin I), fibroblasts (vimentin) and endothelial cells (CD31) in 24-month-old WT mice. B, Immunoblot of indicated markers of senescence in primary adult rat cardiomyocytes treated with Phe (5 mM) and/or BH4 (10 µM; n=3/treatment). C, Immunofluorescence of p21 after Phe treatment. D-E, Intracellular Phe (D) and fumarate (E) levels in primary adult rat cardiomyocytes treated with Phe and/or BH4 (n=6/treatment). F, Immunoblot of indicated markers of senescence in primary adult rat cardiomyocytes treated with Phe and/or proteosome inhibitor MG132 (MG: 10 µM; n=3/treatment). G, Co-immunoprecipitation of p21 and reprobing for ubiquitin in human AC-16 cells after indicated treatments. H, Representative images of cytosolic superoxide (CellROX), reduced glutathione (GSH, ThiolTracker), 2-SC and Nrf2 in primary adult rat cardiomyocytes treated with Phe (5 mM) and/or BH4 (10 µM; n=3/treatment). I, qRT-PCR analysis of Nrf2 target genes in vehicle and Phe+BH4 treated primary adult rat cardiomyocytes (n=4/treatment). J, Representative images of alpha smooth muscle actin (Acta2) expression in cardiac fibroblasts isolated from WT murine neonates treated with indicated concentrations of Phe (n=3/condition). For all microscopic images, magnification: 400x, scale-bar: 25 µm, except for GSH: magnification: 200x, scale-bar: 50 µm. Data are presented as original images (A-C, F-H, J) or mean ± SEM analyzed with two-tailed unpaired t-test (I) or one-way ANOVA with Bonferroni post-hoc test (D-E); ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001 as indicated.

Figure 4 .

 4 Figure 4. Experimental hyperphenylalaninemia promotes cardiac aging in vivo. A, Schematic of treatment and evaluation protocol. B, Plasma Phe levels in vehicle-or Phe-treated WT mice of 6 or 12 months of age (n=8-11/group). C, Heart weight to tibial length (HW/TL) for the same groups (n=8-11/group). D, Representative images of hearts stained with wheat-germ agglutinin (WGA), sirius red or vimentin for the same groups (n=4/group). E-G, Quantification of cardiomyocyte cross-sectional area (CSA; E; n=4/group), interstitial fibrosis (Sirius red, F; n=4/group) and vimentin-positive areas (G; n=4/group) in hearts of the same animals as above. H-K, dP/dtmin (H), dP/dtmax (I), systolic strain rate (J) & LV ejection fraction (K; n=8-11/condition) in the same animals as above. L, Representative microscopic images of myocardial p21, Pah, Gch1, 2-SC & H3K9me3 immunofluorescence in the same animals as above (n=3-4/condition). For all microscopic images, magnification: 200x, scale-bar: 50 µm. Data are presented either as original images (D & L) or as mean ± SEM analyzed with ANOVA with Bonferroni post-hoc test (B-C, E-K); ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5 .

 5 Figure 5. BH4 treatment rescues age-related cardiac dysfunction. A, Plasma Phe levels in 12.5 month-old WT mice treated with intraperitoneal BH4 or vehicle (VEH; n=8-10/group). B, Systolic and diastolic arterial pressures in the same animals (n=9-10/group). C, Myocardial NOS activity in hearts of the same animals (n=9-10/group). D, Immunofluorescence of p21, Pah, Gch1 and 2-SC as above (n=4/group). E, Representative images of myocardial sirius red and vimentin staining with quantification (F-G; n=4/group). H-K, Heart weight to tibial length (H; HW/TL), dP/dtmin (I), dP/dtmax (J) & systolic strain rate (K; n=9-10/group) in mice treated as above. For microscopic images, magnification: 200x, scale-bar: 50 µm. Data are presented as original images (D-E) or mean ± SEM and analyzed with two-tailed unpaired t-test (A-C, F-K); ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6 .

 6 Figure 6. BH4 rescues age-related hepatic Pah decline. A, Representative Pah and Gch1 immunofluorescent images of livers in 2 and 15 month-old WT and p21 -/-mice (n=3/group). B, Relative transcript levels of Phe transporters in livers 2-and 15-month-old WT and p21 -/-mice (n=4-8/group). C, Representative immunofluorescence for Slc7a8 in livers of 2 and 15 month-old WT and p21 -/-mice (n=3/group). D, Representative immunofluorescent co-labeling for Slc7a8 and Pah in livers of 6-monthold vehicle-or Phe-treated WT mice (n=3/group). E, Hepatic Phe content in WT mice with indicated treatment and ages (n=7-10/group). F, Immunoblots of senescence markers 2-and 15-month-old WT as indicated (n=3/age). G-H, Expression of p21 (G) and Pah (H) in AML-12 hepatocytes as a function of Phe concentration. I-J, Immunoblots (I) of vehicle (VEH)-or Nutlin3a (NU)-treated AML-12 hepatocytes transfected with p21-targeting or scrambled siRNA (n=3/condition) and tyrosine levels in media thereof (J; n=6/group). K, Representative immunofluorescence of liver Pah expression in vehicle-and BH4-treated 12.5-month-old WT mice (n=3/group). L-M, Liver Phe content (L), hepatic Tyr/Phe ratio (M) in the same groups of mice as K (n=7-10/group). N, Standardized expression of PAH

Figure 7 .

 7 Figure 7. Dietary Phe restriction rejuvenates old hearts. A, Plasma Phe levels in 24 month-old WT mice fed Phe-deficient or control diet (Phe-and Phe+, respectively; n=10/group). B, Immunofluorescence of p21, Pah, Gch1, 2-SC and H3K9me3 as above (n=4/group). C-E, Sirius red and vimentin staining (Representative images: C; quantification: D-E; n=5/group). F-I, Heart weight to tibial length (HW/TL: n=11/group; F), dP/dtmin (G), dP/dtmax (n=9/group; H) & systolic strain rate (n=10-11/group; I) in mice treated as above. J, Cartoon summarizing age-dependent hepatic Phe catabolic decline, rise in blood Phe levels and ectopic Phe catabolism in the heart with potential therapeutic targets. For microscopic images, magnification: 200x, scale-bar: 50 µm. Data are presented as original images (B-C) or mean ± SEM and analyzed with two-tailed unpaired t-test (A, D-I); ns: nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001.

  involving animals were approved by the Institutional Animal Care and Use Committee of the French National Institute of Health and Medical Research (INSERM)-Unit 955, Créteil, France (ComEth 15-001). Global p21 -/-mice backcrossed to C57BL/6 background for at least 10 generations (Jackson Laboratory, USA) as well as wild-type (WT) littermates were kept in individually ventilated cages in a high-health facility with 12-hour light-dark cycle, controlled temperature (20-22°C) and humidity. Water and chow were provided ad libitum.

  levels induce cardiac senescence and dysfunction, a phenotype inducible with Phe treatment in WT young mice. Secondly, we show that age-related increase in p21 suppresses hepatic Pah function, the latter crucial in controlling systemic Phe levels. Thirdly, p21 deficiency delays structural, functional, and molecular alterations in the aging myocardium whilst Pah cofactor BH4 or dietary Phe restriction reverses them. Collectively, our findings may provide diagnostic clues for cardiac aeing and open new therapeutic avenues to rejuvenate aged myocardium, an important challenge in the face of high cardiovascular burden in the growing population of elderly individuals. 369

TABLE OF CONTENTS

 OF

Table 1 :

 1 Surface

marker expression in ADSCs and committed preadipocytes (Modified from

Sarantopoulos, C. N. & al., Stem Cell Rev Rep 2018)

64

1.1) Cell cycle arrest

	As we previously indicated, stable cell cycle arrest is a feature of senescence, and occurs
	mainly in response to DNA damage following telomere attrition or other stresses. Cell cycle
	arrest is mediated by the activation of either the p53/p21 CIP1 or p16 INK4A /phospho-Rb tumor
	suppressor pathways, 176,204,207 and many cancer types are associated with mutations in
	those pathways.
	Persistent DDR activates ATR and ATM kinases which in turn activates p53 through Chk1 and
	Chk2. Then p53 activates p21 CIP1 to initiate cell cycle arrest by inhibiting cyclin dependent
	kinase 2 (CDK2), hence preventing the progression of cell cycle from G1 to S phase. CDK
	inhibition, which can also result directly from p16 INK4A activation (inhibits CDK4 or CDK6),
	blocks phosphorylation of Retinoblastoma tumor suppressor protein (Rb), and that prevents

E2F-mediated transcription of genes that are required for DNA replication, 195 (figure

22

).

Table 2 :

 2 Bibliography summarizing the current literature about inflammation, fibrosis, and senescence parameters in AT of HFD-induced obesity mouse models.

	Authors
		< 5 weeks	< 10 weeks	12 to 15 weeks	20 weeks	30 weeks
	Feng et al., Diabetes 2011	x
	Xu et al, JCI 2003	x
	Williams et al., PloSOne 2014	x		x
	Strissel et al., Diabetes 2007		x		x
	Wang et al., Nat med 2013
	Paglialunga et al., Diabetologia 2015	x
	Kumar et al., International Journal of Obesity 2018		x x	x x x	x x x
	Shirawaka et al., Plos One 2017					x x x
	Bennett et al., Obesity 2014

x Observed parameters: Inflammation Fibrosis Senescence

	Mouse model: Duration of HFD

1st study: Adipose tissue senescence time course assessment

  animal experiments were approved by the Institutional Animal Care and Use Committee of

	the French National Institute of Health and Medical Research (INSERM)-Unit 955, Créteil,
	France (ComEth 15-001).
	Methods
	All mice were housed in a specific pathogen free platform at a constant temperature (22°c),
	kept in individually ventilated cages with a 12-hour light-dark cycle and unrestricted access to
	a chow diet (CD, A0310, Safe Diets, France) and water. Chow diet composition corresponded
	to 13.5% of fat, 61.3% of carbohydrate and 25.2% of protein
	C57/BL6JRj male mice (referred as wild-type: WT) purchased from Janvier were kept in
	individually ventilated cages with 12-hour light-dark cycle, and controlled temperature (20-
	22°C) and humidity. All mice were randomly assigned to chow diet (CD, A0310, Safe Diets,
	France) or high-fat diet (HFD, D12492i, Research Diets, USA) regimen. HFD composition
	corresponded to 60% of fat, 20% of carbohydrate (including sucrose 7% of total composition)
	and 20% of protein. Water and food were provided ad libitum. Body weight, status of glucose
	homeostasis (metabolic tests: ITT, GTT) and heart function (echocardiography) were
	monitored at baseline and before sacrifice.

3 rd study: Adipose tissue senescence is mediated by increased ATP content after a short- term high fat diet exposure

	senescence, and another group for 10 weeks which was in parallel submitted to exercise
	training
	). p16-luciferase mice (Strain Code
	01XBT--B6.Cg-Cdkn2a tm3.1Nesh Tyr c-2J/Nci) were obtained from the NCI mouse repository
	via Dr Norman Sharpless (University of North Carolina). 289 During follow-up, animals
	underwent a monthly body-weight (BW) evaluations.
	Five-month-old C57/BL6JRj mice (WT, Janvier Labs, France) were subjected to chow diet (CD)
	and HFD (60% calories from fat, D12492, irradiated; Research Diets Inc., New Brunswick, NJ)
	for 1, 2 and 5 weeks to assess the kinetics of epididymal white adipose tissue (WAT)

  Quantitative RT-PCR analysis of metabolism-related genes in VAT; (n=7/group). Data are presented as mean ± SEM analysed with ANOVA with Tukey's post-hoc test; ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 as indicated.

). E-F,

  Ternacle, J., Wan, F., Sawaki, D., Surenaud, M., Pini, M., Mercedes, R., Ernande, L., Audureau, E., Dubois-Rande, J.L., Adnot, S., et al. (2017). Short-term high-fat diet compromises myocardial function: a radial strain rate imaging study. Eur Heart J CardiovascImaging 18, 1283-1291. van Deursen, J.M. (2014). The role of senescent cells in ageing. Nature 509, 439-446. Vergoni, B., Cornejo, P.J., Gilleron, J., Djedaini, M., Ceppo, F., Jacquel, A., Bouget, G., Ginet, C., Gonzalez, T.,Maillet, J., et al. (2016). DNA Damage and the Activation of the p53 Pathway Mediate Alterations in Metabolic and Secretory Functions of Adipocytes. Diabetes 65, 3062-3074. Xu, M., Pirtskhalava, T., Farr, J.N., Weigand, B.M., Palmer, A.K., Weivoda, M.M., Inman, C.L.,Ogrodnik, M.B., Hachfeld, C.M., Fraser, D.G., et al. (2018). Senolytics improve physical function and increase lifespan in old age. Nature medicine 24, 1246-1256. Yousefzadeh, M.J., Schafer, M.J., Noren Hooten, N., Atkinson, E.J., Evans, M.K., Baker, D.J., Quarles, E.K., Robbins, P.D., Ladiges, W.C.,LeBrasseur, N.K., et al. (2018). Circulating levels of monocyte chemoattractant protein-1 as a potential measure of biological age in mice and frailty in humans. AgingCell 17.

  My PhD thesis involved four studies with senescence as a common denominator. The studies 1 to 3 investigated the adipose tissue premature senescence induced in in vivo and in vitro models of caloric overload (Studies1 and 3) and in the context of chronological aging (Study 2). Study 4 explored naturally occurring cardiac senescence in chronological aging. Specifically, Study 1 was tasked to evaluate time-dependent early changes in adipose tissue in HFD-fed mice, finding ATM to undergo senescence and associated dysfunction as the earliest detectable abnormality reflected in impaired systemic glucose homeostasis and cardiac dysfunction. Study 2 aimed to dissect how VAT senescence in natural aging emerges, identifying ATM senescence to compromise functional integrity of adjacent adipose tissue and systemic glucose homeostasis. The main focus of Study 3 was to explore how experimental caloric overload may induce adipose tissue and preadipocyte senescence, pointing to excessive ATP levels as the immediate inducer of premature senescence. Study 4 was based on the observation that there is an age-dependent increase in blood Phe levels and from here experimentation established Phe-related adverse cardiac effects in natural aging and impaired hepatic Pah activity as an underlying cause.

	Discussion
	Corn Oil		50	50
	Coconut Oil		0	0
	Mineral Mix S10001	35	35
	Sodium Bicarbonate	7.5	7.5
	Vitamin	Mix	10	10
	V10001
	Choline Bitrartrate	2	2
	Red Dye, FD&C #40	0	0.05
	Blue Dye, FD&C #1	0.05	0
	Total		1000.05	1000.05
	Protein		172	162
	Carbohydrate		686	696
	Sucrose		46.40	46.40
	Fat		50	50
	Fiber		50	50
	g%
	Protein		17	16
	Carbohydrate		69	70
	Sucrose		4.64	4.64
	Fat		5	5
	Fiber		5	5
	Total L-Amino Acids kcal	172	162
	Protein		688	648
	Corn Starch Carbohydrate		502.1 2742	512.1 2782
	Maltodextrin 10 Fat		125 450	125 450
	Sucrose Total		46.4 3880	46.4 3880
	kcal%
	Cellulose Protein		50 18	50 17

Acknowledgments/ Remerciements

Acknowledgements We thank for technical assistance Xavier Decrouy, Christelle Gandolphe, Wilfried Verbecq-Morlot (imaging), Florian Beghi (animal experiments), Adeline Henry and Aurelie Guguin (Flow cytometry). We also thank Pr. Norman Sharpless, University of North Carolina for the donation of mice; Dr. Johanne Seguin, Paris Descarte University for help with in vivo luciferase imaging; Pr. Elizabeth Blackburn, University of California, San Francisco for helpful discussions and suggestions. This work was supported by RHU CARMMA grant (ANR-15-RHUS-0003)

Acknowledgments.

This work was supported by ANR Grant RHU CARMMA (ANR-15-RHUS-0003). No conflict of interest relevant to this article were reported.

We are grateful to Prof. E. Blackburn (UCSF, San Francisco, USA) for inspiring discussion and wise comments, to Prof N. E. Sharpless (Chapel Hill, NC, USA) for providing the p16 LUC mice and to Dr J. Rieusset (INSERM U870, Lyon, France) and Dr A; Pizard for precious support in revision.

We thank C. Gandolphe and X. Decrouy (IMRB Imagery platform), M. Surenaud (IMRB Luminex platform) and the NGS platform of IMRB. We thank M. Breau, Y. Zhang, D. Altintas, M. Halfaoui, H. Liang (U955, Créteil), C. Pertuiset and M. Porceddu (Mitologics SAS, Créteil) and J. Seguin (Optical imaging platform LIOPA, Paris Descartes University) for technical contribution.

Acknowledgements

We are grateful to Prof. Elizabeth Blackburn, University of California, San Francisco for helpful discussions and suggestions. We thank Dr. Fatima Clerc, University of Paris Est Créteil, for providing AML-12 cells. Xavier Decrouy, Christelle Gandolphe, Wilfried Verbecq-Morlot of the Histology Core Facility and members of EP3 platform at IMRB U955 facility are gratefully acknowledged for their excellent technical support. This work was supported by the RHU-CARMMA Grant (ANR-15-RHUS-0003).

Disclosures

None.

Data analysis

Radial strain rate was analyzed offline with the EchoPac software (GE Medical Systems) by a single operator blinded to the identity of study groups.

Supplemental table: Used Taqman assays and antibodies

Taqman assays

Target gene Taqman assay ID Target gene Taqman assay ID Cdkn2a Mm00494449_m1 Acaca Mm01304257_m1 Cdkn1a Mm04205640_g1 Pparg Mm00440940_m1 Tp53 Mm01731290_g1 Pnpla2 Mm00503040_m1 Mfge8 Mm00500549_m1 Adipoq Mm00456425_m1 Timd4 Mm00724709_m1 Il6 Mm00446190_m1 Mertk Mm00434920_m1 Tnf Mm00443258_m1 Mrc1 Mm01329362_m1 Mif Mm01611157-g1 Spp1 Mm00436767_m1 Serpine1 Mm00435858_m1 Thbs1 Mm01335418_m1 Vegfa Mm01283063_m1 Sparc Mm00486332_m1 Il12b Mm01288989 Lgals3 Mm00802901_m1 Cxcl2 Mm00436450_m1 Tnc Mm00495662_m1 Ccl2 Mm00441242_m1 Postn Mm00450111_m1 Cxcl1 Mm04207460_m1 Pdgfra Mm01205760_m1 Ihnbb Mm03023992_m1 Lep Mm00434759_m1 Tgfb1 Mm01178820_m1 Cd36 Mm00432403_m1 Il12a Mm00434169_m1 Slc2a1 Mm00441480 Cxcl5 Mm00436451_g1 Ucp1 Mm01244861_m1 Csf2 Mm01290062 Pgc1a Mm01208835_m1 Csf1 Mm00432686 Slc2a4 Mm00436615_m1 Il1b Mm00434228_m1 Plin1 Mm00558672_m1 Infg Mm01168134_m1 Fasn Mm00662319_m1 Actb Data availability. All data are stored in a centralized and secured Clinical and Translational Research data Repository (CARMMA CTRDR) available at IMRB. Data and resource sharing are available upon request and presentation of a protocol to RHU CARMMA consortium.

Study 3: Supplemental tables Used Taqman-MGB probes for qRT-PCR analysis Target gene Taqman assay ID Adipoq Mm00456425_m1 Adrb3 Mm02601819_g1 ATP5a1 Mm00431960_m1 Cdkn1a Mm04205640_g1 Cdkn2a Mm00494449_m1 Col1a Mm00801666_g1 Cox5b (Gm11273) Mm01229713_g1 Ctgf Mm01192933_g1 Cycs Mm01621048_s1 Fasn Mm00662319_m1 Fn1 Mm01256744_m1 Glb1 Mm00515342_m1 Hif1a Mm00468869_m1 Igf1 Mm00439560_m1 Il6 Mm00446190_m1 Lep Mm00434759_m1 Lipe/Hsl Mm00495359_m1 Lox Mm00495386_m1 Mcp1/Ccl2 Mm00441242_m1 Mfn2 Mm00500120_m1 Nrf1 Mm01135606_m1 Pai1/Serpine1 Mm00435858_m1 PdgfRa Mm00440701_m1 Plin1 Mm00558672_m1 Pnpla2 Mm00503040_m1 Ppara Mm00440939_m1 Ppargc1α Mm01208835_m1 Prdm16 Mm00712556_m1 P2rx7 Mm01199500_m1 Slc2a4 Mm00436615_m1 Tgfb1 Mm01178820_m1 Timp1 Mm01341361_m1 Tnf Mm00443258_m1 Trp53 Mm01731290_g1 Ucp1 Mm01244861_m1

List of abbreviations AdPLA2

Adipose-specific phospholipase A2 STUDY 1

STUDY 2

Conflict of interest statement

The authors have declared that no conflict of interest exists.

ABSTRACT

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin-resistance are not fully elucidated.

The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high fat diet (HFD, 1 to 10 weeks) in 5-months-old male C57BL/6J mice and the potential role of mitochondria.

We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence-associated ß-galactosidase activity and cyclin-dependent kinase inhibitor 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors and immune cells, together with adipocytes. WAT senescence was associated with a higher glycolysis and mitochondrial activity leading to enhanced ATP content in HFD-derived preadipocytes, as compared with chow diet-derived preadipocytes. One-month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while decreasing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss.

We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover the critical role of HFD-induced elevated ATP as a local danger signal initiating WAT senescence.

Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence and metabolic abnormalities.

Supportive information

 Supplementary methods  Supplementary tables: 1, 2  Supplementary figure legends STUDY 4

Supplementary material

Dysregulated phenylalanine catabolism is a defining characteristic of cardiac aging

Gabor Czibik, MD, PhD, 1,* § Zaineb Mezdari, MSc, 1* Dogus Murat Altintas, PhD, 1* Juliette Bréhat, MSc, 1 Maria Pini, PhD, 1 Elisabeth Riant, MD, 1,2 Costin Radu, MD, 1,3 Thomas d'Humières, MD, 1,2 Marielle Breau, PhD, 1 Hao Liang, MSc, 1 Cecile Martin, PhD, 1 Azania Abatan, BSc, 1 Rizwan Sarwar, MRCP, PhD, 4 Ophélie Marion, MSc, 1 Suzain Naushad, MD, 1 Yanyan Zhang, MD, PhD, 1 Maissa Halfaoui, MSc, 1 Nadine Suffee, PhD, 5,6 Didier Morin, PhD, 1 Serge Adnot, MD, PhD, 1,8 , Stéphane Hatem, MD, PhD, 5,6,9 Arash Yavari, MRCP, DPhil, 4,7 Daigo Sawaki, MD, PhD, 1 ‡ Geneviève Derumeaux, MD, PhD 1, 8 ‡ § and digital images were acquired using an imaging system (Azure Biosystems). Membranes were usually stripped of antibodies and reprobed with an antibody targeting a protein serving as loading control (α-actinin for hearts & β-actin for all others). For this procedure, membranes were placed in Guanidine-HCl-based stripping solution (6M Guanidine-HCl, 0.2% Nonidet P-40, 0.1M βmercaptoethanol, 20mM Tris-HCl, pH7.5 and 100 mM 2-mercaptoethanol) and incubated for 10 minutes at room temperature with gentle agitation, followed by extensive washing and re-blocking. 8

Co-immunoprecipitation

Ten million human AC-16 cardiomyocytes (see later) per condition were used for coimmunoprecipitation experiments. Cells were scraped in ice-cold 1x PBS, collected and pelleted for 5 minutes at 1000g. Pellets were resuspended in DA150 buffer (20mM HEPES pH 7.9, 150mM NaCl, 20% glycerol, 0,2mM EDTA and one Roche protease/phosphatase inhibitor tablet per 10 ml) and lysed for 30 minutes at 4°C. Lysates were cleared by centrifugation at 15000g for 15 minutes. 1% v/v of the supernatant was put aside for input fraction, whilst an IP-grade rabbit monoclonal antibody raised against p21 (Abcam: ab188224) was added to the rest of the supernatants at the concentration recommended by the manufacturer (1:30; or IgG as control). Subsequently, 20 µl of magnetic beads were added. Samples were incubated overnight at 4°C under constant agitation. Next day beads were washed 3 times in DA300 buffer (20 mM HEPES pH 7.9, 300 mM NaCl, 20% glycerol, 0,2 mM EDTA, 1 mM DTT and one Roche protease/phosphatase inhibitor tablet per 10 ml). Protein complexes were eluted at denaturing condition by heating the beads at 95°C for 10 minutes. Eluates were loaded onto a 12% Nupage gel for reprobing with an anti-ubiquitin antibody (Abcam, ab7254).

Histology

Formalin-fixed organs were embedded in paraffin. Cross-sections were cut into 7 µm thickness using a rotary microtome (Leica). To assess cardiac fibrosis, Sirius red staining was performed, followed by dehydration and mounting with Eukitt quick-hardening mounting medium (Sigma). To visualize cardiomyocyte hypertrophy, sections were incubated in 2 µg/mL Texas red-conjugated wheat germ agglutinin (WGA; Invitrogen) in 1x PBS at room temperature for 45 minutes. Slides were then mounted with fluorescent mounting media (Abcam). Images were acquired using a Zeiss fluorescent microscope.

Immunohistochemistry/immunofluorescence

Whenever a dispersed pattern was expected, immunohistochemistry was performed on paraffinembedded sections. Briefly, after rehydration, citrate buffer-assisted, heat-mediated antigen retrieval Mice were randomly assigned to experimental groups and data were acquired and analyzed blind to genotype, age or treatment. Statistical analyses were performed using GraphPad Prism Software (version 6). In all cases n numbers were raised to obtain Gaussian distribution and parametric tests were used. Accordingly, unpaired, two-tailed t-test was used to compare two groups, whilst more than two groups were compared using one-way analysis of variance (ANOVA), with Bonferroni post-hoc test for multiple comparisons. Two-way ANOVA was used to compare groups with time-dependent evolution of readouts, with Bonferroni post-hoc test for more than two groups. Data are presented as mean ± standard error of the mean or individual values. Annotations used: *p < 0.05, **p < 0.01, ***p < 0.001 compared to groups indicated. A p value of < 0.05 was considered significant.

➢ VAT senescence in natural aging

In 2018, our team identified that age-related cardiac decline was fuelled by a VAT-derived profibrotic secretome. In search of clues, we found that induction of matricellular proteins (i.e., OPN and galactin3)

and senescence markers was more discriminative than proinflammatory genes (which have been proposed to be a source of harmful effects under stress such as caloric overload resulting in a chronic, low-grade proinflammatory state) 322 to drive VAT aging. These results associated with a marked downregulation of efferocytosis-related genes. Whilst a role for various matricellular proteins including OPN has been shown in VAT remodeling in obesity, [323][324][325][326] we previously established VAT as the major source of OPN secretion in natural aging. 320 Moreover, we found that OPN increasingly and overwhelmingly colocalised with markers of The first focus was to explore early cell type-specific changes induced by HFD in young mice

(2 months old) and test if experimental interference will reverse relevant HFD-induced abnormalities. Accordingly, major findings of this study can be summarised as follows:

1. Macrophage senescence is the first pathology preceding inflammation and major tissue remodeling.

In vivo pharmacological clearance of macrophages restored normal cardiometabolic

status in mice fed a HFD for two weeks.

Visceral adiposity is a profound risk for T2DM and heart disease. 318,342,343 With caloric overload, experimentally realised by HFD, the majority of high-energy nutrients find their way into adipose tissue for storage. This happens at an expense of gradual remodeling and altered function. Intriguingly, early processes within the VAT that may later escalate are unknown. In the course of the present study, we explored this initial phase and found that VAT alterations confined to adipocyte hypertrophy and macrophage senescence, without signs of inflammation, change in macrophage number or fibrotic remodeling. Early HFD-induced WAT senescence is consistent with findings identified in Study 3 where the mice were slightly older

(5 months old vs 2 months old).

Intriguingly, an increase in CLS count characteristic of caloric overload was absent before 10 weeks of HFD. Moreover, whether the unaltered macrophage number and position reflect the resistance of resident macrophages to HFD-inflicted injury or their balanced but accelerated replacement from local pools, 344 peritoneal cavity 345 or bone marrow 288,346 remains to be explored. At any rate, despite subtle microscopic findings in VAT, at the organism level even 2 weeks of HFD was sufficient to impair glucose homeostasis and cardiac function.

Long-term HFD induces macrophage M1 polarisation, recruitment from bone marrow and AT fibrosis, all attributed an important role in the ensuing dysmetabolic state. [347][348][349] In contrast with these widely accepted abnormalities associated with long-term HFD regimen, we found no evidence for them up to 5-10 weeks of HFD. This led us to hypothesize that a senescenceassociated dysfunction of adipose tissue macrophages is a critical priming event. To directly

Conclusion -Perspectives

My PhD thesis involved studies investigating the adipose tissue senescence induced in in vivo and in vitro models of caloric overload or chronological aging together with exploring the remote effects on cardiometabolic parameters.

Specifically, Study 1 was tasked to evaluate time-dependent early changes in adipose tissue in HFD-fed mice, finding ATM to undergo senescence and associated dysfunction as the earliest detectable abnormality, occurring well before local or systemic inflammation as well as major tissue remodeling take place. Its key pathogenic role was underscored by restored glucose homeostasis and cardiac function after in vivo pharmacological clearance of macrophages.

Hence, it seems important to characterize these senescent macrophages deeper to understand the intricate mechanisms that shifted them into a senescent state and to learn how they induce metabolic and cardiac alterations. These exploratory studies will shed further light on early macrophage senescence and dysfunction in VAT and possibly other organs that may take us a step closer to improved therapeutic targets in T2DM.

In 2018, our team identified that age-related cardiac decline was fuelled by a VAT-derived profibrotic secretome. During my PhD thesis I participated in experiments aiming to extend our previous findings in natural aging by identifying age-related VAT remodeling and dysfunction as a consequence of adipose tissue macrophages (ATM) senescence (Study 2).

Senescence-associated impairment of ATM function emerged as matricellular protein OPNdependent macrophage dysfunction, such as efferocytosis. This study identified ATM senescence to compromise not only functional integrity of adjacent adipose tissue but also systemic glucose homeostasis. These observations suggest that macrophage-specific and senescence-targeted therapeutics (such as macrophage-directed pharmacological OPN inhibition) could be a viable strategy to control AT senescence with altered secretome and systemic metabolic decline in aging.

After the identification of the early and key role of macrophages in adipose tissue senescence, we aimed to explore other mechanisms potentially underlying the accumulation of senescent cells in AT. Thus, the main focus of Study 3 was to explore how experimental caloric overload may induce adipose tissue and preadipocyte senescence, pointing to excessive ATP levels as the immediate inducer of premature senescence. In this context, exercise, by controling local

SUPPLEMENTAL TABLE 1

Used antibodies for immunohistochemistry (IF) analysis