The obesity resistant Lou/C rat as a model to study the metabolic impacts of white adipose tissue browning and of perinatal high-fat feeding

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Thesis

Reference

The obesity resistant Lou/C rat as a model to study the metabolic impacts of white adipose tissue browning and of perinatal high-fat

feeding

POHER, Anne-Laure

Abstract

Mon travail de thèse est basé sur l'étude de l'obésité et des comorbidités qui lui sont associées. Leur prévalence a augmenté dans le monde entier entraînant des conséquences socio-économiques majeures. La plupart des modèles animaux développent une obésité quand ils sont nourris en diète enrichie en lipides, faisant d'eux de bons modèles de développement de l'obésité. Cependant, nous étudions un modèle de rats résistant au développement de l'obésité : le rat Lou/C. Deux aspects distincts de son métabolisme ont été étudiés : le rôle des cellules beiges exprimant UCP1 d'une part, et l'impact à l'âge adulte d'une diète riche en lipides durant la période périnatale, d'autre part. Nous avons d'abord essayé de comprendre le rôle métabolique d'une surexpression d'UCP1 dans un tissu ectopique, plus précisément dans le tissu adipeux sous-cutané. Le deuxième projet était centré sur les conséquences d'une diète périnatale enrichie en lipides sur l'homéostasie métabolique à l'âge adulte chez les rats Wistar et Lou/C.

POHER, Anne-Laure. The obesity resistant Lou/C rat as a model to study the metabolic impacts of white adipose tissue browning and of perinatal high-fat feeding. Thèse de doctorat : Univ. Genève, 2016, no. Sc. 4951

URN : urn:nbn:ch:unige-869212

DOI : 10.13097/archive-ouverte/unige:86921

Available at:

http://archive-ouverte.unige.ch/unige:86921

Disclaimer: layout of this document may differ from the published version.

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1 UNIVERSITE DE GENEVE

Département de biologie cellulaire FACULTE DES SCIENCES

Professeur Jean-Claude Martinou

Département de médecine interne FACULTE DE MEDECINE Professeur Françoise Rohner-Jeanrenaud _____________________________________________________________________

The obesity resistant Lou/C rat as a model to study the metabolic impacts of white adipose tissue browning and of perinatal high-fat feeding

THESE

présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Anne-Laure POHER de

Paris (France)

Thèse n° 4951

Atelier d’impression Repromail Genève

2016

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2

Remerciements

Je souhaite remercier tout d’abord les membres du jury, le Professeur Jean-Claude Martinou, ainsi que le Professeur Abdul Dulloo pour avoir accepté de s’intéresser à mon travail de thèse, en le lisant et le corrigeant. Je remercie également le Professeur Jean-Paul Giacobino pour les discussions scientifiques et les nombreuses idées apportées tout au long de ma thèse.

Je remercie grandement le Professeur Françoise Rohner-Jeanrenaud qui m’a offert la possibilité de réaliser une thèse dans son laboratoire et transmis une partie de son savoir scientifique. Elle s’est toujours tenue disponible pour de très riches discussions, m’a fait confiance en laissant prendre les directions souhaitées et m’a ouvert les portes de la recherche scientifique.

Je tiens à remercier tous les collègues passés, et présents : Christelle, merci de m’avoir formée, à Jordi pour beaucoup trop de choses impossibles à résumer ici, à Aurélie pour ta gentillesse, ton amitié et ton accueil dès le premier jour, à Jacqueline, ma maman de thèse qui a été d’une patience infinie avec moi, à Nicolas, pour les pauses cafés et le reste. Un grand merci à Laurent avec qui le travail est très stimulant en plus d’être un plaisir. Un grand merci aussi à l’équipe de Pierre Fabre et particulièrement à Céline à qui je dois une partie de ma formation, mais également des mois très riches humainement à Castres.

Merci à mes parrain et marraine de thèse, Sophie et Michelange. Vous avez tous les deux été très présents, votre porte m’a toujours été ouverte. Merci pour le très grand soutien et la confiance que vous m’avez accordés.

Je souhaite remercier le Professeur Dominique Belin pour son aide et son soutien dans les moments plus difficiles, ainsi que pour les agréables discussions.

Merci à Tamara et Corine pour leur disponibilité au moindre problème administratif, ainsi qu’à Yannick, Hervé, et Christian pour leur aide technique. Merci à Marie et Danielle de la plateforme d’histologie qui ont toujours fait un travail remarquable et dans les meilleurs délais. Je tiens à remercier toute l’équipe de l’animalerie et particulièrement Anthony pour sa précieuse aide avec les animaux.

Merci aux amies, Sarah sans qui je n’arriverais toujours pas à préparer une solution correctement, Joanna mon amie toujours présente tout au long de ma thèse, Marion mon amie de course d’abord puis amie tout court, Anne-Sophie, toujours au-dessus de zéro grâce à toi, Inès uma nova e grande

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5 amizade, Lucie merci pour ta grande gentillesse et ton oreille toujours attentive, bref mes piliers de thèse. Merci à Yalin et Melis pour leurs discussions scientifiques toujours très constructives et leur amitié tout au long de cette thèse. Merci aussi à tous les amis pour les soirées et apéros pendant ces années pas toujours évidentes, Alex toujours présent et d’une patience infinie, Clotilde, Emilie, Daria, Leticia, Kévin, Carolyn, Lan, Matteo, Daniele, Byung Ho, Nicolas C, Dorothea, Ayman, Jade, Romain, Nicolas S, Claire, Salvatore, tout le groupe Foti, ainsi que tous ceux avec qui j’ai pu partager de bons moments. Un petit mot spécial pour Valentina, toujours un sourire à qui je pense et dont je n’oublierai pas la grande joie de vivre.

Merci aux amis dans le sud qui ont toujours été présents et d’un immense soutien, Emilie, Nico et Cora, Benjamin, Sébastien, Manon, Delphine, Maud, Lionel, Tiffany, François.

Enfin un immense merci à mes parents et à mes frères, ou plutôt bravo à eux qui ont également vécu une thèse et sans qui ça n’aurait pas été possible. Ils m’ont soutenue, écoutée et épaulée, et continuent de le faire.

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Abstract

The studies carried out for my thesis work concern obesity and its metabolic co- morbidities, whose prevalence steadily increases worldwide with major socio-economical consequences. Most animal models develop obesity when fed high caloric diets are thus obesity-prone. In contrast, we studied a rat model of resistance to the development of diet- induced obesity, the Lou/C rat. Two different aspects of metabolic homeostasis were studied comparing the Lou/C obesity-resistant to the Wistar obesity-prone rat. In the first study, the Lou/C rat was used to investigate the role of UCP1-positive beige cells on metabolism. In the second study, the impact of a HF diet during the perinatal period on body composition and glucose metabolism at adulthood was determined in both Lou/C and Wistar rats.

Uncoupling protein 1 (UCP1) is a protein responsible for thermogenesis by using protons formed via the respiratory chain in the mitochondria. UCP1 is normally expressed and activated in brown adipose tissue (BAT). This tissue is specific of mammals and is particularly involved in thermogenesis during hibernation. In rodents, this tissue is localised in the interscapular area. In human new-borns, BAT is also present in the interscapular region, but it disappears with age. As a matter of fact, it was long thought that adult humans have no BAT, nor UCP1 protein expression. A great interest about UCP1 has been renewed these last years thanks to a major discovery published in three different articles in the “New England Journal of Medicine”, revealing the presence of UCP1 expression and activity in adult humans in adipocytes, mainly localized in the neck, supraclavicular area and along the aorta. Although these adipocytes express UCP1, they are different from brown adipocytes in terms of their origin and appearance. They are scattered within white adipose tissue depots and their size is

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7 intermediary between that of white and brown adipocytes. In contrast to UCP1 in brown adipocytes, UCP1 in these cells need a stimulus to be activated. Moreover, they do not express Myf5 at the cell surface, a brown adipocyte marker. They were named beige adipocytes.

Several questions are linked to these beige cells and notably what is their role in energy expenditure, as well as in glucose homeostasis. As they are present in adult humans, understanding how to induce their differentiation and/or activation and what are the metabolic consequences of such inductions could be therapeutically relevant for the treatment of obesity and type 2 diabetes.

To address these questions we decided to work with Lou/C rats. In fact this strain is leaner and more insulin sensitive than its control, from which it originates, the Wistar rat. Our group previously showed that Lou/C rats express Ucp1 in the inguinal subcutaneous WAT depot (iWAT). This model thus represents an interesting tool to study the metabolic consequences of UCP1 induction in ectopic tissues.

The first aim of my thesis project was therefore to characterise and understand the metabolic role of UCP1 overexpression in ectopic tissues, more precisely in iWAT. To this end, we compared Wistar rats, which express UCP1 only in BAT, with Lou/C rats, which express UCP1 in BAT and WAT. We treated the animals with a specific β3 agonist, as UCP1 is mainly regulated by the sympathetic nervous system through the β3 adrenergic pathway. We observed a high increase in UCP1 expression in BAT of Wistar rats, whereas this induction occurred in both BAT and WAT in Lou/C rats. We further noticed that UCP1 overexpression in WAT of Lou/C rats improved their insulin sensitivity by acting on WAT glucose uptake exclusively. We then investigated the mechanisms involved in such an effect of the β3 agonist

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8 treatment in Lou/C rats. We observed that it involves an activation of fatty acid cycling, comprising a concomitant increase in lipolysis, de novo lipogenesis and glyceroneogenesis.

The second project was focused on the consequences of a perinatal HF diet on metabolic homeostasis at adulthood in Wistar and Lou/c rats. As obesity is developing more and more in young people, it is of major interest to understand what are the consequences of such an early obesity occurrence on metabolism later in life.

As in the first study, we used the Lou/C rat a model of resistance to the development of obesity and compared it to the obesity prone Wistar rat. We first observed that at 3 month of age, the perinatal high fat diet did not impair the metabolism of both Wistar and Lou/C rats if the animals were maintained on a chow diet. However, at 4 months of age, the obesisty- prone Wistar rats fed a HF diet at adulthood were more glucose intolerant if they already have received a perinatal HF diet. On the contrary, no impairment was observed in Lou/C rats.

These data suggest that the modifications occurring during the perinatal period in response to a HF diet impair the deleterious impact of a lipid enriched diet later in life.

All the studies of my thesis work were performed in Lou/C rat vs Wistar rats, because rats are metabolically closer to humans than mice, and because UCP1 promotor is very similar between humans and rats, whereas it is not the case for mice. Moreover, the obesity-resistant Lou/C rat model is of great interest, as it may help, it allows understanding the mechanisms responsible for obesity resistance.

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Résumé

Mon travail de thèse est basé sur l’étude de l’obésité et des comorbidités qui lui sont associées.

Leur prévalence a augmenté dans le monde entier entraînant des conséquences socio-économiques majeures. La plupart des modèles animaux développent une obésité quand ils sont nourris en diète enrichie en lipides, faisant d’eux de bons modèles de développement de l’obésité. Cependant, nous étudions un modèle de rats résistant au développement de l’obésité : le rat Lou/C. Deux aspects distincts de son métabolisme ont été étudiés : le rôle des cellules beiges exprimant UCP1 d’une part, et l’impact à l’âge adulte d’une diète riche en lipides durant la période périnatale, d’autre part.

La protéine découplante 1 (UCP1), est une protéine responsable de la thermogénèse en utilisant les protons formés par la chaîne respiratoire mitochondriale. UCP1 est normalement exprimée et activée dans le tissu adipeux brun. Cette protéine spécifique des mammifères, est particulièrement impliquée dans la thermogénèse nécessaire aux animaux entrant en hibernation. Chez le rongeur, ce tissu se situe dans la région interscapulaire. Chez l’homme, on trouve sa présence dans cette même région chez le nouveau-né mais il disparait avec l’âge. Il a longtemps été présumé que les hommes une fois adultes, ne possédaient plus de tissu adipeux brun. Il y a eu un regain d’intérêt pour ce tissu suite à une série de publications dans le journal “New England Journal of Medicine”, démontrant qu’UCP1 ainsi que le tissu adipeux brun étaient effectivement présents chez l’homme. Dans notre espèce, UCP1 est exprimé dans les adipocytes situés essentiellement dans le cou, la région supraclaviculaire, ainsi que le long de l’aorte. Bien que ces adipocytes expriment UCP1, ils ont une apparence et une origine différentes des adipocytes bruns présents dans le tissu adipeux brun. Ils sont dispersés au sein des adipocytes blancs, leur taille étant comprise entre celle des adipocytes blancs et bruns. Par ailleurs, ils n’expriment pas Myf5 à leur surface, un marqueur des adipocytes bruns. On les nomme pour ces raisons des adipocytes beiges.

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10 Plusieurs questions sont liées à ces adipocytes beiges et notamment leur rôle dans la dépense énergétique, ainsi que dans l’homéostasie du glucose. Dans la mesure où leur présence chez l’homme a été démontrée, comprendre comment induire leur différenciation et/ou activation, ainsi que ses conséquences métaboliques, pourrait être d’un grand intérêt thérapeutique dans le cadre du traitement de l’obésité, ainsi que du diabète de type 2.

Pour répondre à ces questions, nous avons décidé de travailler sur le rat Lou/C. En effet, cette souche est plus mince et plus sensible à l’insuline que son contrôle, le rat Wistar dont il est issu. Notre groupe a précédemment démontré que le rat Lou/C exprime UCP1 dans le tissu adipeux blanc sous-cutané inguinal. Ce modèle représente donc un outil très intéressant pour l’étude des conséquences métaboliques d’une induction d’UCP1 dans un tissu ectopique.

Le premier aspect étudié dans mon projet de thèse était donc de caractériser et d’essayer de comprendre le rôle métabolique d’une surexpression d’UCP1 dans un tissu ectopique, plus précisément dans le tissu adipeux sous-cutané inguinal (WATi). Pour cela, nous avons comparé le rat Wistar, qui n’exprime UCP1 que dans le tissu adipeux brun, avec le rat Lou/C qui, lui, l’exprime à la fois dans les tissus adipeux bruns et blancs. Nous avons traité les animaux avec un agoniste β3 adrénergique spécifique, afin d’induire UCP1 qui est principalement régulée par le système nerveux sympathique via la voie de signalisation β3 adrénergique. Nous avons observé une augmentation de l’expression d’UCP1 dans le BAT des Wistar, alors que cette augmentation a été observée dans le BAT et le WAT des Lou/C. L’une des conséquences de ce traitement est une augmentation de la sensibilité à l’insuline, en partie grâce à une augmentation de la captation de glucose par le WATi chez le Lou/C. Afin de comprendre le mécanisme sous-jacent à cette amélioration de l’homéostasie glucidique, nous avons analysé le métabolisme lipidique et observé que cela impliquait une activation du cycle

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11 des acides gras comprenant simultanément une augmentation de la lipolyse, de la lipogenèse de novo et de la glycéronéogénèse.

Le deuxième projet était centré sur les conséquences d’une diète périnatale enrichie en lipides sur l’homéostasie métabolique à l’âge adulte chez les rats Wistar et Lou/C. Comme l’obésité se développe de plus en plus chez les jeunes, il est en effet du plus grand intérêt de comprendre quelles sont les conséquences métaboliques à long terme d’une malnutrition précoce.

Nous avons utilisé le modèle du rat Lou/C comparé à celui du Wistar afin de comprendre comment cette souche réagit à une exposition pénatale à une diète riche en lipides sur son métabolisme glucidique à l’âge adulte. Nous avons tout d’abord observé qu’à l’âge de 3 mois, la diète riche en lipides prise en périnatal n’altère pas le métabolisme des Wistar et des Lou/C s’ils sont nourris avec une alimentation normale dès le sevrage.

Cependant, à l’âge de 4 mois, les Wistar recevant une diète riche en lipides à l’âge audulte développent une intolérance au glucose plus forte s’ils ont été soumis à une diète grasse plus jeunes. En revanche, les rats Lou/C n’ont pas de modification de l’homéostasie glucidique sous le même régime. Cela semble indiquer que la diète périnatale peut influencer le métabolisme des adultes chez le Wistar, mais pas chez le rat Lou/C.

Ces études ont été réalisées sur les rats Lou/C et Wistar parce que les rats sont métaboliquement plus proches des humains que les souris. De plus, les rats Lou/C présentent un grand intérêt parce qu’ils sont résistants à l’obésité, qu’elle soit due à une diète riche en lipides ou à l’âge. Donc, comprendre les mécanismes qui mènent à ce phénotype pourrait permettre de trouver de nouvelles voies thérapeutiques dans la lutte contre le développement de l’obésité et pour une l’amélioration de la sensibilité à l’insuline.

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List of abbreviations

Aldhs: retinaldehyde dehydrogenases aP2: adipocyte protein2

ARC: arcuate nucleus

ATGL: Adipose triglyceride lipase ATP: adenosine triphosphate BAT: brown adipose tissue BMI: body mass index

BMP: bone morphogenic protein BW: body weight

C/EBP: CCAAT/Enhancer binding protein cAMP: cyclic adenosine mono phosphate CCK: Cholecystokinin

CD: cluster differentiation

Cd137: tumour necrosis factor receptor superfamily, member 9 CD36: cluster of differentiation 36, or FAT (fatty acid translocase) CGI-58: Comparative Gene Identification-58

ChREBP: Carbohydrate-responsive element-binding protein Cidea: Cell death activator A

CNS: central nervous system DAG: diacylglycerol

DIO: diet-induced obesity

DIO2: Type 2 iodothyronine deionidase DMH: dorso medial hypothalamus ECM: extracellular matrix

EE: energy expenditure

EHC: euglycemic hyperinsulinemic clamp

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13 eWAT: epidydimal white adipose tissue

FAs: fatty acids FFA: free fatty acids

FGF21: Fibroblast growth factor 21 FI: food intake

Fndc5: fibronectin type III domain containing 5 G3P: glycerol-3-phosphate

GLP1: glucagon-like peptide 1 GLUT: glucose transporter

gWAT: gonadal white adipose tissue HFD: high fat diet

HGP: hepatic glucose production HGP: hepatic glucose production HSL: Hormone sensitive lipase Ig: immunoglobulin

IGF-1: insulin growth factor-1 IL-6: interleukin 6

IR: insulin receptor

IRS: insulin receptor substrate LCFA: long chain fatty acids LH: lateral hypothalamus Lhx8: LIM homeobox protein 8 LPL: lipoprotein lipase

MAG: mono-acyl-glycerol

Mapk: Mitogen-activated protein kinase mWAT: mesenterix white adipose tissue NEFA: non esterified fatty acid

NTS: nucleus tractus solitarius

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14 ObRb: long leptin receptor isoform

PEPCK: Phosphoenolpyruvate carboxykinase

Pgc1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha PI3K: phosphoinositide 3-kinase

PIP2: Phosphatidylinositol-4,5-bisphosphate PIP3: phosphatidylinositol 3,4,5 trisphosphate PKA: protein kinase A

PKB: protein kinase B PKC: protein kinase C

Pparα: Peroxisome proliferator-activated receptor alpha PPARγ: Peroxisome proliferator-activated receptor gamma PRDM16: PR domain containing 16

Pref-1: Preadipocyte factor-1 PRLR: prolactin receptor

prWAT: perirenal white adipocyte tissue Pten: Phosphatase and TENsin homolog PVN: paraventricular nucleus

Rar: retinoic acid receptor

scWAT: sub cutaneous white adipose SNS: sympathetic nervous system Tbx1: T-Box 1

Tcf21 (transcription factor 21) and TG: triglyceride

TGF-β: Transforming growth factor beta:

TLE3 (transducin-like enhancer of split 3) Tmem26 (transmembrane protein 26) or TNF-α: tumour necrosis factor α

UCP1: uncoupling protein 1

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15 VLDL: very low density lipoproteins

VMH: ventromedial hypothalamic nucleus VTA: ventral tegmental area

vWAT: visceral white adipose tissue WAT: white adipose tissue

Zic1 (zinc finger protein of the cerebellum 1) are specific for brown adipocytes, β3R: beta 3 adreno receptor

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Introduction

Energy homeostasis is maintained thanks to an equilibrium between food intake and energy expenditure (EE). EE is the sum of all energy used by the body. It is composed of basal metabolic rate, physical activity and thermogenesis.

The basal metabolic rate is the minimal energy required to maintain homeostasis at rest and thermoneutrality. It corresponds to the energy required to maintain an organism’s vital functions, such as those of the heart, brain and lungs. It represents approximately two third of the global EE and varies according to the lean mass. Physical activity represents 15 to 20%

of total EE in sedentary adults, but this percentage can vary according to the type, degree and duration of physical activity. Finally, thermogenesis corresponds to the amount of energy required to digest food, store nutrients and maintain body temperature in mammals (non- shivering thermogenesis).

Body weight (BW) depends on the balance between caloric intake and total EE. A positive energy balance is usually characterized by a large excess of food consumption coupled with a decrease in physical activity, inducing a reduction in overall EE. This imbalance leads to lipid storage as triglycerides (TG) in white adipose tissue (WAT). With time, this phenomenon induces the development of an obesity syndrome.

1) Energy imbalance

1.1 Obesity and WAT

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17 Obesity is characterized by an excess in lipid storage and is defined by the body mass index (BMI). It corresponds to the weight (in kg) divided by the square height (m²). The WHO defines overweight with a BMI equal or up to 25 and obesity if it is equal or superior to 30 [1].

BMI is the most useful way for measuring overweight or obesity because, in a population, it is independent of sex or age. However, this value remains approximate because it does not provide any information with regard to body composition. In fact, it does not always correlate with the metabolic risk in the sense that many obese persons are not diabetic, whereas some overweight individuals are [2]. Obesity does not induce death by itself, but it induces co- morbidities that will threaten life. The main complication of obesity is type 2 diabetes.

Overweight and obesity are increasing in the world. Children obesity is one of the main challenges for public health in the 21^st century. According to the WHO, it is a worldwide problem affecting many countries, especially where socio-economical conditions are poor.

The prevalence of obesity in adults, as well as in children increases very quickly. Thus, in 2010, it was estimated that more than 42 millions of children were overweight in the world, 35 millions of them living in developing countries. Overweight or obese children have a high risk of staying obese when adult and are more susceptible to develop diseases linked with obesity, such as T2D and CVD at an early age. Although the underlying mechanisms are unravelled as yet, the main cause of obesity is a modification of food behaviour, with an increase in the consumption of carbohydrates and lipids, as well as deficiencies in vitamins and minerals, linked with a lower physical activity [1].

Predisposition toward the development of obesity could occur before or during the perinatal period (weeks after birth), or even before, during pregnancy of the mother. Thus,

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18 mother behaviour could influence energy metabolism and insulin sensitivity of the new-born [3].

To summarize, the etiology of obesity is due to both genetic and environmental changes, including those that occur during the foetal or the perinatal period [4, 5].

1.2 Central control of food intake

In normal individuals, there is a good equilibrium between energy intake and energy expenditure over time, in order to maintain a stable body weight. This is controlled by neurons localised in the mediobasal hypothalamus mainly, but also in other areas interacting with factors and hormones produced in peripheral tissues [6]. The main humoral effector is leptin, secreted by adipose tissue [7]. The plasma leptin levels are proportional to the percent fat mass. Leptin accesses to the brain by a specific transport mechanism [8]. A dysregulation of the central leptin signalling induces hyperphagia, leading to an increase in body weight gain [9]. Insulin also reduces food intake by acting centrally [10]. Moreover, a reduction in central insulin signalling leads to the development of a mild-obesity syndrome [11, 12]. Leptin action in the brain is mainly focused on the nucleus tractus solitarius (NTS) neurons in the hindbrain, and in the hypothalamic arcuate nucleus (ARC) in the forebrain [13, 14]. In the ARC, there are various neuronal subpopulations. One of them co-expresses neuropeptide Y (NPY) and agouti- related protein (AgRP). These two neuropeptides stimulate food intake and are inhibited by leptin, as demonstrated in ob/ob mice completely lacking leptin [15]. In contrast, another neuron subpopulation with the ARC expresses pro-opiomelanocortin (POMC) and is activated by leptin [16]. When cleaved by pro-hormone convertases, POMC gives different peptides, such as α-melanocyte stimulating hormone (α-MSH), responsible for satiety by activation of

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19 the melanocortin pathway [12]. POMC neurons are a very heterogeneous neuronal population. In fact, all of them do not respond to leptin, and those which do not, are responsive to insulin signalling. Surprisingly, these neurons react in contrast to the leptin- sensitive POMC neurons [17]. The opioid peptide, β-endorphin is another peptide produced by POMC cleavage. It was recently demonstrated that POMC activation through the secretion of this peptide is essential for the activity of the Cannabinoid receptor 1 (CB1R) [18]. This apparent paradox (CBR are known to activate pathways involved in food intake induction) demonstrates the complexity of the systems regulating food intake.

If leptin remains the main hormone involved in the regulation of food intake, many others are involved. Indeed, central regulation of food intake is also mediated by various hormones secreted by the gastrointestinal (GI) tract. In contrast to hormones secreted by WAT (like leptin) that play a role in the long-term FI regulation, the GI-produced circulating factors are mainly involved in the short-term regulation of food intake. For example, ghrelin is secreted by the stomach during fasting and it increases FI by exerting opposite effects on hypothalamic neuropeptides compared with leptin. Cholecystokinin (CCK) or glucagon-like peptide-1 (GLP- 1) are secreted by the intestinal tract and induce satiety signals [12].

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20 Figure 1: Central regulation of food intake. Taken from Neurobiology of food intake in health and disease, Morton et al, Nat Rev Neurosci. 2014 Jun;15(6):367-78. The arcuate nucleus of the hypothalamus (ARC) is the main nucleus in the regulation of FI, but the paraventricular nucleus (PVN), or the nucleus of the solitary tract (NTS) are also involved in this process.

The brain structures involved in food intake regulation continue their development after birth, during the perinatal period. This period starts approximately after 15 days of intrauterine life and continues during the first weeks after neonatal life [19, 20]. Rodents are fed by suckling until 15 days postnatally, after which they start to feed themselves independently [21]. Hypothalamic circuits responsible for food intake and energy homeostasis later in life are formed during this period [22]. In rodents, leptin levels are very high during the two first weeks of the perinatal period, in comparison with levels at adulthood [23]. But leptin has no anorexigenic effect before four weeks of postnatal life, suggesting other important roles during this period [24]. In fact, leptin acquires its anorexigenic capacity when ARC

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21 neurons (POMC, AgRP and NPY) projections are fully developed [23, 25]. In contrast, recent evidence suggests that 13-15 days after birth, leptin activates orexigenic pathways (AgRP and NPY neurons) [26]. Orexigenic neurons send their projections mainly to the lateral hypothalamic area (LH), the ventromedial hypothalamic nucleus (VMH), and the dorsomedial hypothalamic nucleus (DMH) [27]. Moreover, the presence of the long leptin receptor isoform, ObRb, which is responsible for the effects of leptin has been demonstrated in LH neurons [28].

In rats, LH neurons send their projections to the ventral tegmental area (VTA), an area involved in reward function. This appears to occur at the end of the first week of life. Thus, it seems that in contrast to what was described in the ARC, leptin action could regulate the onset of independent feeding [29]. Prominent literature indicates that the regulation of food intake at adulthood is modulated during the perinatal period, notably by the mother feeding habits. For example, a HF diet can predispose to the development of obesity or T2D later in life [30-32].

Many studies in humans demonstrated a probable link between brain development, neuronal plasticity and circulating leptin levels [33].

1.3 Glucose metabolism

1.3.1 Insulin pathway

The main source of energy is glucose. Even if FAs are also a source of energy, the brain and skeletal muscles use glucose as a major source of energy. Skeletal muscle and adipose tissue take up glucose through glucose transporter 4 (GLUT4), present at the surface of the membrane after its translocation from endosomes in the intracellular space. The GLUT4 translocation process is activated by insulin [34].

Insulin is a hormone secreted by β cells in the endocrine pancreas, in response to an increase in glycaemia during postprandial conditions [35]. β cells are endocrine cells grouped in cluster

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22 named islets of Langerhans. Another population, α cells are also present in islets and are responsible for glucagon production, a hyperglycaemic hormone. Glucagon activates hepatic glucose production, whereas insulin induces an increase in glucose uptake, mainly by skeletal muscles, liver and WAT. Glucose entry in β cells results in an increase in intracellular adenosine triphosphate (ATP) levels. ATP promotes the closure of potassium channels, resulting in a membrane depolarisation that allows Ca²⁺ entrance, the signal for insulin exocytosis [36].

In skeletal muscles or adipose tissue, cell glucose uptake is activated by insulin binding to its receptor. The insulin receptor (IR) is composed of two extracellular α subunits and two intracellular β subunits. Its activation by insulin induces an autophosphorylation of various tyrosine residues on β subunits, as well activation of different insulin receptor substrates (IRS1, 2, 3, 4) [37]. IRS1 activates phosphatidyl inositol 3-kinase (PI3K), an enzyme that phosphorylates phosphatidyl inositol 2 phosphate (PIP2) into phosphatidyl inositol 3 phosphate (PIP3). PIP3 will then activate protein kinase B (PKB), also known as Akt, and PKC subunits λ and ζ [38]. PKB and PKC finally induce GLUT4 translocation to the membrane.

Figure 1: Insulin signalling pathway. In skeletal muscles and adipocytes, insulin phosphorylates the insulin receptor (IR), inducing phosphorylation of a cascade of different proteins, leading to the translocation of GLUT4, a glucose transporter which is needed for cell glucose uptake in insulin-dependent tissues.

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23 1.3.2 Glucose homeostasis

Glucose homeostasis is the regulation of glycaemia (closely adjusted between 0.8 and 1.2 g/L) in the body by all different cellular and molecular physiological mechanisms. In fact, glycaemia is the consequence of energy fluxes, in which mechanisms involved in glucose utilisation are in equilibrium with those responsible for glucose production processes. This dynamic equilibrium allows for very precise energetic homeostasis. For example, during a meal, the organism is able to first detect nutrient entrance, then to activate mechanisms involved in storage and inhibit processes involved in glucose production. On the contrary, between meals or during a fasting period, pathways involved in glucose storage are inhibited, while glucose production by organs such as the liver, kidneys, or the intestine is activated to provide glucose to gluco-dependent tissues (mainly the brain). Glucose fluxes are closely linked with lipid fluxes and their co-dysregulations lead to the development of T2D.

The liver is the main organ able to produce glucose, as well as to store it. During a starvation period or physical activity, glucose is produced from glycogen catabolism (glycogenolysis) and from metabolites such as glycerol, lactate or amino acids (neoglucogenesis). During a meal, glucose is stored as glycogen (glyconeogenesis) to be released when needed. Glucose can be stored as TG as well (de novo lipogenesis).

During a meal, 75% of glucose is used by skeletal muscles for glycogen synthesis [39].

This occurs through Akt2 activation, leading to an increase in GLUT4 translocation and glycogen synthesis [40, 41]. Mice with a specific deletion of IR in skeletal muscles have a lower glucose uptake induced by insulin, resulting in the development of insulin resistance.

Interestingly, glucose uptake induced by physical exercise is normal in these mice [42, 43].

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24 In WAT, glucose uptake is mostly dependent on insulin signalling and has a regulation similar to skeletal muscles [44]. Glucose uptake in WAT represents around 5% to 10% of the whole body glucose utilisation, being thus considered a minor tissue responsible for glucose metabolism during the postprandial period [45, 46]. However, as will be discussed below, this can change according to the situations.

1.4 Type 2 diabetes

Diabetes corresponds to a chronic increase in glucose concentrations in the blood, also named hyperglycaemia. In type 2 diabetes (T2D), hyperglycaemia is due to alterations in glucose metabolism and insulin secretion that occur slowly and insidiously. Although T2D is usually detected after the age of 40, it also concerns young persons, including teenagers and even children.

The disease starts with a decrease in insulin sensitivity (insulin-resistance), mainly due to the presence of excess fat deposition, hence obesity and also of a sedentary life style. To counteract insulin-resistance, β cells produce more insulin, inducing an increase in β cells mass, leading to an overall hyperinsulinemia. At this stage, the disease is reversible by the consumption of a healthier food and the adoption of a better life style. At a certain point of insulin resistance, β cells cannot increase their insulin production any more. Finally, β cells drastically reduce insulin production and secretion, and insulin becomes inadequate, leading to a chronical hyperglycaemia.

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25 Glucose homeostasis in insulin-dependent tissues is impaired in T2D diabetes. This is particularly relevant in the liver, skeletal muscles and adipose tissue. The presence of obesity is well known to favour insulin resistance that impairs both glucose and lipid metabolism in the liver [47]. An ectopic accumulation of lipids in the liver is associated with insulin resistance in this tissue [48]. Hepatic glucose uptake and glycogen deposition are impaired, gluconeogenesis and de novo lipogenesis are activated, and fatty acid delivery, as well as triglyceride esterification and secretion are accelerated [49]. All these disruptions lead to a global impairment of glucose and lipid metabolism in the liver [50]. In fact, a DAG accumulation leads to an increase in PKCε, the main PKC isoform in the liver [51, 52]. In humans, a variation of both DAG and PKCε are very good indicators to predict the evolution of insulin sensitivity in lean or obese subjects [53, 54]. Of note, hepatic steatosis can be present independently from insulin resistance, as shown in a knock-down of "gene identification 58" (CGI-58) that activates adipose triglyceride lipase (ATGL) and induces the development of a hepatic steatosis, as well as an accumulation of DAG without any hepatic insulin resistance [55, 56].

In skeletal muscles, an increase in intramyocellular lipids (IMCL), frequently observed in obesity, is correlated with muscle insulin resistance [44]. But, as no correlation between TG content and muscle insulin resistance was found, it seems that other types of lipids are involved in insulin resistance [57]. Thus, it was demonstrated that IML metabolites, such as DAG lead to insulin resistance due to a defect in insulin-dependent glucose transport in skeletal muscles [58]. HF diet in rodents and humans increases the DAG content in skeletal muscles, leading to an increase in PI3K through an increase in IRS-1 phosphorylation [57-59].

This leads to the activation of PKCθ and δ notably, impairing the insulin signalling pathway [60, 61].

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26 Even if WAT has a minor contribution effect in the total glucose uptake, its role in insulin sensitivity is essential. In fact, insulin action in WAT is multiple: it activates glucose transport, promotes lipid uptake and lipogenesis, while inhibiting lipolysis [44, 62].

Perturbation of these different mechanisms can drastically impair glucose metabolism. For example, KO mice for GLUT4 specifically in WAT exhibit impairment of insulin sensitivity in skeletal muscles and in the liver [63]. Carbohydrate response element binding protein (ChREBP) is a transcription factor regulating both glycolysis and lipogenesis [64]. Mice overexpressing GLUT4 in adipose tissue (AG4OX) are obese but more insulin sensitive. A deletion of ChREBP in this model reverses the phenotype to the same insulin sensitivity as in wild-type mice, thereby demonstrating that ChREBP drives GLUT4 action in WAT [65]. A new class of lipids, recently discovered, the fatty acid esters of hydroxyl-fatty acids (FAHFAs), correlates with insulin sensitivity, improving insulin secretion and glucose tolerance in mice.

Interestingly, ChREBP is involved in the synthesis of the FAHFAs, as demonstrated in the AG4OX mice model [66]. In humans, WAT glucose uptake is also directly related to insulin sensitivity and could play a significant role in lipid metabolism [67].

Inflammation in WAT, frequently encountered in obesity syndromes appears to be related to WAT insulin resistance [68]. This is likely mediated by the secretion of cytokines, such as tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6) released by macrophages present in the tissue. A global inflammation induces insulin resistance, notably by decreasing the insulin-induced inhibition of lipolysis. An increase in TNF-α was thus shown to alter lipolysis through perturbation of HSL, perilipin and ATGL activity [69].

The main factor responsible for insulin resistance is probably the higher release of non- esterified fatty acids (NEFA). In fact, an acute injection of NEFA increases insulin resistance, whereas this parameter can be improved by using antilipolytic agents [70, 71]. Moreover,

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27 obese patients suffering from T2D have higher circulating NEFA levels than “healthy” obese patients [72, 73]. Fat mass repartition is also an important factor responsible for the development of T2D development, as will be detailed below.

A genetic predisposition for T2D exists and systematic analysis of large patient cohorts permitted to identify genes associated with an increased risk to develop the disease. They are very heterogeneous and none of them can predict the occurrence of the disease. For example, an APOC3 gene mutation predisposes to non-alcoholic fatty liver disease (NAFLD) and insulin resistance [74]. But, genetic factors by themselves cannot explain the huge increase in the development of T2D observed during these last years [75]. This increase is most correlated with age, and with the interaction between the genome and life style. Different factors induce T2D, including hypercholesterolemia, overweight, obesity and hypertension, sometimes for unknown reasons. Even if obesity is considered as the main risk factor for the development of type 2 diabetes, some lean individuals develop T2D and many obese patients never develop it. This paradox highlight the fact that general obesity alone cannot explain incidence of T2D development. In fact, fat mass repartition (subcutaneously or intraperitoneally) or presence of an inflammation state in white adipose tissue (WAT) correlates much better with cardiovascular or T2D development than the BMI [76].

2) WAT

WAT is the main energy storage tissue in the body, representing more than 75% of the energy stored, thus between 10 and 15 kg in a young 60 kg woman [77]. It is a highly plastic tissue that can represent between 5% until more than 60% of the total mass in adult humans

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28 [78]. In rodents, it is well described that metabolic adaptation during conditions such as fasting or temperature changes can modify both the anatomy and the physiology of this tissue [79].

2.1 Composition and anatomy

WAT is a heterogeneous tissue comprising several cell types. Thus, after a simple centrifugation, the tissue can be separated into two fractions depending on their different density: mature adipocytes and the stroma vascular fraction (SVF). In fact adipocytes, full of lipids, float at the surface of the samples, whereas other cell types are precipitated at the bottom of the tubes [80].

2.1.1 Stroma Vascular Fraction

An important number of different cell types are described as being part of the SVF, which thus consists in a very heterogeneous cell population. Pre-adipocytes are a very important cell type in the SVF, as it is an immature cell whose structure is close to that of a fibroblast, having the ability to differentiate into mature adipocytes. These cells are particularly important in obesity, known to be characterized by hypertrophy of adipocytes followed by hyperplasia [81]. As mature adipocytes cannot proliferate, the increase in cell number is due to the differentiation of pre-adipocytes [82]. Apart from pre-adipocytes, the SVF fraction also contains endothelial cells, nervous cells and macrophages [83]. Macrophages contained in the SVF represent 15% of all cells in the visceral WAT of a normal subject, and it can reach values of 45 to 60 % of this fat depot during the development of obesity [84].

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29 2.1.2 Adipocyte

Mature white adipocytes represent between one-third to two-thirds of the total WAT cells [85]. They are very big cells (150 μm), mainly filled by a unique lipid droplet. These cells contain a small and flattened nucleus and a small cytoplasm.

Figure 2: White adipocyte. Histology of white adipose tissue stained with hematoxylin eosin (HE). As schematized on the right panel, the cell has a unique and big lipid droplet. Cytoplasm, containing a flattened nucleus and few organelles lie flat on the periphery.

2.2 Repartition

WAT metabolic properties are different depending on their localisation in the body. The pathogenesis of obesity is affected by the different localisation of fat depots [79, 81]. Thus, in humans, accumulation of deep abdominal subcutaneous WAT, as well as of visceral WAT is associated with the development of T2D, as well as with cardiovascular diseases [86, 87].

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30 2.2.1 Subcutaneous WAT (scWAT)

Subcutaneous WAT is the fat depot which is most remodelled in case of body weight changes. In humans, there are two distinct scWAT layers: the superficial and the deep layer.

Thickness of scWAT depends on body localisation and sex. In fact, it develops preferentially in the abdominal region in men, whereas it is mainly located at the level of the hips and thighs in women. The superficial part of scWAT is positively correlated with plasma leptin concentrations and poorly related to insulin resistance, whereas the deep scWAT, located in the abdominal region is associated with insulin resistance, as is the case for vWAT [88].

Rodents have an anterior and a posterior scWAT. The anterior part includes mainly the interscapular and subscapular areas, while the posterior part is made of the inguinal, dorso- lumbar and the gluteal regions [79]. The anterior part of scWAT is more innervated and has a higher vascularization than the posterior part of the depot [89].

2.2.2 Visceral WAT (vWAT)

It is well recognized that development of this fat depot is responsible for a detrimental effect on metabolism and therefore represents a risk factor for the development of cardiovascular diseases, as well as dyslipidemia, insulin resistance and hypertension [90]. The metabolic syndrome is characterised by several criteria, one of them being the measurement of the waist circumference [91], which assesses the amount of vWAT, thereby demonstrating the importance of this tissue in metabolic diseases.

Visceral WAT is made of the retro- and the intraperitoneal fat depots, the latter consisting in the omental, mesenteric and, in rodents, in the epididymal WAT (eWAT) fat pad. The amount of vWAT positively correlates with glucose intolerance, plasma LDL cholesterol, triglyceride

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31 (TG) and cholesterol levels, as well as with hypertension and dyslipidaemia [92, 93]. Moreover, analysis of the insulin signalling pathway in both human scWAT and vWAT shows that vWAT expresses higher levels of various proteins involved in this pathway, as well as a better insulin sensitivity [94]. Visceral WAT is also more metabolically active, having a higher lipolytic activity and producing more of some adipokines [95].

In rodents, vWAT is located in the thorax and in the abdomen cavities. It is intraperitoneal or retroperitonal (rpWAT). The rpWAT is mainly composed of mesenteric (mWAT), gonadal (gWAT) and perirenal (prWAT) WAT. Gonadal WAT is well developed in rodents, whereas mWAT is limited [79].

Figure 3: Fat mass repartition in mice. Taken from The adipose organ, Cinti, Prostaglandins Leukot Essent Fatty Acids. 2005 Jul;73(1):9-15 Repartition of adipose depots in mice. A: deep cervical, B: anterior subcutaneous, C: visceral mediastinic, D: visceral mesenteric, E: visceral retroperitoneal, F: visceral perirenal, periovarian, parametrial and perivesical, G: posterior subcutaneous.

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32 2.3 Development

In many species, WAT formation starts before birth. WAT expansion occurs immediately after birth and is due to an increase in both the size and number of adipocytes, known as hypertrophy and hyperplasia, respectively. Moreover, many studies suggest that WAT expansion in adult does not only result in an increase in adipocyte size, but also by an increase in cell number [96, 97]. Adipocyte hyperplasia was indeed observed in many rodent models [98, 99]. In humans, even if hyperplasia stays controversial, adult human preadipocytes were still shown to be able to differentiate into mature adipocytes in vitro [100], indicating the capacity to generate new mature adipocytes even at adulthood.

Moreover, the SVF fraction can differentiate in vitro into many different cell types, such as adipocytes, myocytes and osteoblasts [101], confirming the plasticity of WAT. It was recently shown that vWAT in humans arises from WT1 expressing cells, which is not the case for scWAT and BAT. The authors also demonstrated that vWAT has heterogeneous progenitors in the mesoderm [102].

This suggests the presence of multipotent precursors in human WAT, in addition to preadipocytes. However, molecular mechanisms involved in cell determination in adipocyte lineage remain poorly unravelled. The mechanisms involved in the differentiation of a fibroblast into a mature adipocyte is, however, well described and is called adipogenesis.

2.4 Differentiation

During the adipocyte differentiation process, acquisition of an adipocyte phenotype is characterised by chronologic changes in the expression of various genes. This is reflected by the appearance of early, intermediate and late markers and by the accumulation of TG. These

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33 changes are mainly seen at the translational level. Changes in gene expression during the early and late phases were mainly characterised in an adipocyte mouse cell line, the 3T3-L1 cells [103].

The first step at the beginning of differentiation consists in stopping the proliferation process. In fact, after an exponential phase of growth (regulated by mitogenic factors), adipoblasts, that look like fibroblasts, reach a confluence step that will stop the proliferation.

When cells are stopped at the G0/G1 phase of mitosis, they express early pre-adipocyte markers, like lipoprotein lipase (LPL) and α2 chain collagen type IV. Expression of preadipocyte factor 1 (pref-1) stops at this moment, as it has an inhibitory effect on adipocyte differentiation [104]. Preadipocytes can continue differentiation only if they pass through a clonal expansion phase, which includes several cycles of postconfluent mitosis [105].

However, primary adipocytes from human WAT do not need cell division to enter into the differentiation process [106].

At least two families of transcriptional factors are induced during the early phase of adipocyte differentiation: C/EBP and PPAR. PPARγ is expressed only in adipocytes, its expression is low but detectable in preadipocytes and increases during the differentiation process to maintain a high level in mature adipocytes. A transitory induction of C/EBP-β and C/EBP-δ precedes the induction of PPARγ; It decreases before the late phase, and is accompanied by an increase in C/EBP-α expression just before induction of specific adipocyte differentiation genes [107, 108]. During differentiation, morphology of the cell is completely remodelled: they pass from a fibroblastic morphology to a spherical form. Cytoskeleton extracellular matrix elements are drastically changed with a decrease of 90 to 95% of actin and tubulin synthesis during the early phase of differentiation in order to allow for a correct differentiation [109]. In 3T3-L1 cells, a

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34 decrease in procollagen type I and III and an increase in type IV collagen and chondroitin was measured [110, 111]. Different specific markers are expressed at the cell surface during these phases, as summarized by Figure 4.

Figure 4: Main markers identified during white adipocyte development. Taken from Weighing in on adipocyte precursors, Berry R, Cell Metab. 2014 Jan 7;19(1):8-20. Identification of different markers during white adipocyte differentiation.

During the last phase of differentiation, the adipocytes start to be able to synthesise FAs from glucose (de novo lipogenesis), and acquire their insulin sensitivity. Expression and activity of various proteins involved in TG metabolism, such as ATP citrate lyase, malic enzyme, acetyl Co-A carboxylase, stearoyl-CoA desaturase, glycerol-3- phosphate acyltransferase, glycerol-3-phosphate dehydrogenase, fatty acid synthase, and GLUT4 are increased between 10 and 100 times. The expression of genes encoding for other proteins indirectly involved in

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35 lipid metabolism also increases. This is the case for proteins such as aP2, CD36, perilipin, adipsin, leptin, PEPCK [103, 112, 113].

2.5 WAT as an endocrine organ

WAT has long been thought to be a mere storage tissue, only involved in FA release depending on the metabolic needs. But WAT is also a real endocrine organ able to receive hormonal signalling coming from the whole body and to react by secreting hormones named adipokines. These hormones have an impact on many target tissues, such as the liver or skeletal muscles and participate in the general control of energy balance. Some of these adipokines, like leptin or adiponectin activate neural circuits in the hypothalamus, mainly aimed at regulating EE and lipid catabolism [114]. Moreover, under stress, WAT secretes pro- or anti-inflammatory cytokines regulated by the WAT mass and the physiological state of the organism [115].

Table 1: Adipokines secreted by WAT and their impact on the tissue

name expression Main functions Leptin adipocytes Reflects fat mass

Satiety hormone, direct action on the hypothalamus

Stimulates lipolysis, inhibits lipogenesis, increases FA oxidation Adiponectin adipocytes Increases insulin sensitivity

Increases FA oxidation Anti-inflammatory action Il-6 adipocytes Pro-inflammatory cytokine

Inhibits insulin and leptin pathways TNFα macrophages Pro-inflammatory cytokine

Induces insulin resistance Increases lipolysis in adipocytes Expression increased in obesity Pref-1 preadipocytes Inhibits adipogenesis

Its overexpression in WAT impairs insulin sensitivity [116]

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36 TGFβ adipocytes/

macrophages

Growth factor

Anti-inflammatory cytokine

Induces proliferation, differentiation and apoptosis [117]

Expression increased in obesity MCP1 adipocytes/

macrophages

Anti-inflammatory chemokine

Recruits macrophages on inflammatory sites Increases lipolysis and leptin secretion 2.6 Lipid homeostasis

One of the main functions of WAT is to store excess energy as TG, which can be degraded into FAs to be used by other tissues in response to metabolic needs, such as during food restriction periods. Synthesis of TG from FAs is named lipogenesis. Adipocytes are able to store large amounts of TG in their lipid droplet surrounded by a protein named perilipin, without causing any lipotoxicity to the cell [118].

2.6.1 Lipogenesis

TG stored in adipocytes are synthetized from fatty acids (FA) and glycerol, after being transformed, respectively into acyl-CoA and glycerol-3-phosphate (G3P). Most FAs come from circulating plasma lipids, whereas G3P has two main origins: glycolysis and glyceroneogenesis.

Plasma circulating FA levels are mainly non esterified FA (NEFA) linked to albumin, or TG incorporated within lipoproteins, mainly very low density lipoproteins (VLDL) or chylomicrons.

TG are first hydrolysed by lipoprotein lipase (LPL), an enzyme linked to capillaries of WAT and skeletal muscles, in order to release FAs [119]. Its expression and activity is increased in WAT during the postprandial period, mainly under diet enriched in carbohydrates, due to a

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37 stimulatory effect of insulin. In contrast, LPL expression and activity are decreased during a starvation period or in response to a high fat diet (HFD) [120].

Long chain fatty acids (LCFA) need specific transporters to cross the plasma membrane [121]. White adipocytes express different FA transporters, such as "cluster of differentiation 36" (CD36), "fatty acid transport protein" (FATP) and "fatty acid binding protein" (FABP). CD36 is responsible for the majority of FA uptake [122]. Insulin improves this transport by stimulating the expression of these transporters, as well as their presence at the plasma membrane level [123].

FA are soluble in the cytosol only. To avoid cytotoxic effects, they are linked to a cytosolic protein, the FABP, which transports FAs to the action site of acyl-CoA synthase.

Human white adipocytes express two type of FABP: "adipocyte protein2" (AP2) (product of the FABP4 gene) and the "keratinocyte lipid-binding protein" (KLBP). AP2 is exclusively expressed in adipocytes and is the main molecule involved in FA transport, whereas KLPB is also expressed in macrophages [124]. The first step in FA metabolism after being linked to FABP is the activation of LCFA to LCFA-CoA through the acyl-CoA synthase. LCFA-CoA are then oxidized or orientated to the synthesis of more complex lipids, such as TG. Oxidation of LCFA- CoA occurs in the mitochondria after their transport inside this organelle by carnitine- palmitoyl transferase I (CPT1).

Another source of FA is through de novo lipogenesis, which is the synthesis of new FA molecules from non-lipid substrates, mainly carbohydrates in mammals. Expression and activity of enzymes involved in lipogenesis and glycolysis are closely related in the liver and WAT, which are lipogenic tissues. De novo lipogenesis is less active in humans than in rodents and it contributes to a small amount of TG production in adipocytes [125, 126].

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38 Synthesis of G3P is needed for the esterification of FA into TG. G3P is produced from glucose (via glycolysis) or from glyceroneogenesis [127]. The limiting step of this latter process is the cytosolic form of phosphoenol pyruvate carboxykinase (PEPCK). Relative contribution of glycolysis and glyceroneogenesis to produce G3P depends on nutritional and pharmacological factors. Global availability of G3P controls FA esterification coming from de novo lipogenesis or circulating lipids, as well as partial re-esterification of FA released by hydrolysis of TG.

Figure 6: Lipogenesis pathways in white adipocytes. These pathways enable energy storage as lipids. FA are transported as lipoproteins (chylomicrons and VLDL), FA and glucose are transformed into acylCoA and G3P, respectively, which are both needed for triacylglycerol synthesis (TAG on the Figure). DAG: diacylglycerol, MAG: monoacyglycerol, LPA:

lysophosphatidic acid, PA: phosphatidic acid.

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39 2.6.2 Lipolysis

In WAT, TG are hydrolysed when energy needs are not completely covered by food intake. TG in adipocytes are successively hydrolysed into diacylglycerols (DAG) and monoacylglycerols (MAG) to ultimately produce three molecules of FA and one molecule of glycerol. Glycerol produced by lipolysis (TG degradation into FAs and glycerol) is released into the bloodstream to be transformed to G3P by glycerol kinase in the liver or to be used by other tissues. This glycerol release depends in part on aquaporine, a canal protein in the plasma membrane. FAs produced by lipolysis are also released in the circulation or re-esterified directly as TG, inside the adipocytes. The intracellular FA cycling depends on G3P availability and on the re-esterification of FAs thanks to the triglyceride synthase. In the basal state, this cycling is negligible, but it increases during starvation periods, stress [128] or in pathological situations, such as hyperthyroidism [129]. The extent of FA re-esterification plays a role in the regulation of plasma FA levels.

Hormone sensitive lipase (HSL) plays a key role in hydrolysis of TG. Lipolysis is activated by hormones or mediators activating the adenylate cyclase system (glucagon, adrenaline, noradrenaline), inducing an intracellular increase in cyclic adenosine mono phosphate (cAMP) that phosphorylates protein kinase A, which will then phosphorylate HSL and perilipin.

Inhibition of HSL expression in mice induces an accumulation of DAG instead of TG. HSL hydrolyses DAG and then a MAG lipase releases the last FA and one glycerol. MAG lipase is the limiting enzyme in the release of glycerol and free fatty acids (FAs).

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40 Adipose triglyceride lipase (ATGL) is another lipase present in WAT. Inhibition of both ATGL and HSL induces an inhibition of 90% of TG hydrolysis [130]. In fact, TG hydrolysis depends mainly on ATGL activity and less on HSL activity.

Figure 7: Lipolysis pathways in white adipocytes. TG (TAG on the Figure) are degraded between meals when body needs energy. The FAs produced will be then be used by the mitochondria (β oxidation) to produce ATP or they will be exported to the tissues which are able to use FAs (mainly skeletal muscles, heart, liver, adipose tissue).

3) BAT

BAT is characteristic of mammals, more precisely of hibernating animals because this tissue is responsible for heat production by consuming lipids [131]. The association between BAT and hibernation was described for the first time by Gessner in 1551 in marmots.

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41 In contrast to white adipose tissue, brown adipose tissue (BAT), which is darker due to the presence of cytochrome c in the mitochondria and a higher degree of vascularization, is specialised in adaptive thermogenesis [132]. Even if the role of BAT has been well studied in rodents and human new-borns, its persistence and importance in adult humans are currently actively studied and its functions remain to be precisely determined [133-135].

3.1 Brown adipocyte

BAT is composed of brown adipocytes mainly. In contrast to white adipocytes that contain a unique and big lipid droplet, brown adipocytes are multilocular, have a central nucleus and a large cytoplasm that contains a lot of mitochondria. This tissue is highly vascularized and innervated.

Figure 8: Brown adipocyte. Histology of brown adipose tissue stained with hematoxylin eosin (HE). As schematized on the right panel, the cell has a central nucleus, a large cytoplasm full of active mitochondria and lot a small lipid droplets.

Brown adipocytes have a common origin with skeletal muscles, which is not the case for white adipocytes [136]. Thus, it was demonstrated by cell tracing that both brown adipocytes and myocytes express Myf5, strongly suggesting that they share a common precursor [137].

The obesity resistant Lou/C rat as a model to study the metabolic impacts of white adipose tissue browning and of perinatal high-fat feeding

Thesis

Reference