Le tissu adipeux brun (TABr) est devenu ces dix dernières années le centre d’intérêt de nombreux laboratoires en raison de sa capacité à dissiper l’énergie apportée par les substrats sous forme de chaleur. Chez les mammifères, le développement du TABr intervient à la fin de la gestation et devient fonctionnel à la naissance. Sa capacité thermogénique permet aux nouveau-nés de s’adapter face à l'environnement extra-utérin, puis son activité régresse avec l’âge. Ceci suggère que l’environnement intra-utérin joue un rôle important dans la programmation de la physiologie et du métabolisme du TABr. Dans un modèle bien décrit de retard de croissance intra-utérin, qu’est la carence protéique maternelle (CP), la jeune progéniture CP est normoglycémique malgré un défaut de sécrétion de l'insuline mais développe avec l'âge une résistance à l'insuline et une hyperglycémie. Lors de mon arrivée au laboratoire, des résultats suggéraient un rôle du TABr dans les changements dynamiques du profil métabolique de la progéniture CP en fonction de l'âge. En effet, le TABr des rats CP est hyperactif à 3 mois par rapport aux animaux contrôles alors qu’il revient au niveau des contrôles chez la progéniture CP âgée de 18 mois, ce qui corrèle avec l’apparition des troubles métaboliques caractéristiques du diabète de type 2. Durant ma thèse, mon premier objectif a été de démontrer le rôle causal du TABr dans le maintien de l’homéostasie glucidique chez les jeunes animaux CP. Pour ce faire, nous avons exposé au froid de jeunes rats CP pour solliciter leur TABr et nous avons procédé à l’ablation chirurgicale de ce tissu. Nos résultats montrent que la jeune progéniture CP est mieux protégée que les contrôles à une exposition au froid grâce à l’activité thermogénique accrue de leur TABr. / Brown adipose tissue (BAT) has grown over the last ten years into the center of interest for many laboratories due to its capacity to burn energy derived from metabolic substrates into heat. Indeed, in mammals, the development of BAT occurs at the end of gestation to become fully functional at birth. Its thermogenic capacity allows newborns to face extrauterine environment, and thereafter its activity declines with age. This suggests that the intrauterine environment plays an important role in the programming of BAT physiology and metabolism. In a well-known model of intrauterine growth retardation (IUGR), the maternal protein restriction model (called LP for low protein), the young LP progeny is normoglycemic despite an insulin secretion defect but develops insulin resistance and hyperglycemia with age. When I started my thesis work, available results in the laboratory suggested a role of BAT in the dynamic changes of the LP progeny metabolic profile according to the age. Indeed, BAT of young LP rats is hyperactive at 3 months compared to controls while this activity drops back to control levels in old 18-months LP progeny, consistent with the appearance of a type 2 diabetic phenotype. During my thesis, the first objective was to search for the causal role of BAT in the maintenance of glucose homeostasis in young LP progeny. Using a first strategy, we exposed young LP progeny to a cold challenge to activate their BAT. In a second approach, we performed surgical ablation of their BAT. Our results show that young LP progeny is more protected against a cold challenge than controls, due to the high thermogenic capacity of their BAT. However, BAT ablation induces hyperglycemia in young LP animals showing that this tissue is required to maintain their normoglycemia. This work, published in Diabetes in March 2017, suggests that a deleterious fetal environment could reprogram BAT metabolism. The second objective of my thesis was to identify the molecular mechanisms allowing the maintenance of active BAT in young LP progeny. To do so, we compared two models of BAT activation, ie our LP model and a well-known model of BAT activation with an agonist of β-3 adrenergic receptors. In both cases, when BAT is active, we observed a global increase in microRNA (miRNA) expression associated to augmented miRNA machinery expression, and in particular AGO2 expression. Interestingly, when BAT is inactive in old LP animals, miRNA expression and miRNA machinery expression return to control levels. While activation of mature brown adipocytes in vitro leads to an increase in AGO2 protein expression, partial deletion of this protein is sufficient to decrease the thermogenic activity of these cells. Collectively our data suggest that AGO2 and increased miRNA expression contribute to BAT activation. The manuscript concerning this research is in the review process at Molecular Metabolism. In the third part of my PhD research efforts, I have found that in the BAT of young LP progeny several miRNAs are robustly downregulated. We have focused on let-7cp and miR-22-3p, which have the most severe decrease in expression. Our key finding is that these two miRNAs act synergistically to hinder mature brown adipocyte thermogenic activity. This work is in the process of being finalized for publication. In conclusion, during my PhD training I have revealed several novel findings, which lead to a better understanding of BAT physiology and its dysregulation in situations eventuating in perturbed glucose homeostasis. While additional efforts are certainly needed, these contributions advance our vision to leverage BAT as a promising target for the prevention and/or treatment of metabolic perturbations associated to obesity and type 2 diabetes.

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Obesity results from an excess of adipose tissue and is a major risk factor for type 2 diabetes, cardiovascular disease, and cancer. Adipose tissue exists in two main forms: white adipose tissue (WAT), which stores energy as triglycerides, and brown adipose tissue (BAT), which dissipates stored energy as heat. White adipose tissue is composed of several subcutaneous and visceral depots, each possessing distinct molecular and functional characteristics. Brown-like adipocytes can emerge in WAT depots in response to cold or beta-adrenergic stimulation. These cells have been called “beige” or “brite” (brown-in-white) cells. The reduction of obesity in mice treated with beta-adrenergic agonists is correlated with the emergence of beige cells. Beige cell development occurs most readily in subcutaneous depots, and to the least extent in visceral depots. Understanding the molecular mechanisms underlying beige cell development in different WAT depots may be important in discovering new therapies against obesity and related diseases. Our lab has previously discovered that Tyrosine Kinase 2 (Tyk2), an important mediator of cytokine signaling, promotes the development of classical brown adipose tissue. Due to the lack of functional BAT, Tyk2-knockout (Tyk2-/-) mice become grossly obese with age and develop several symptoms of the metabolic syndrome. In the present study, we have found a potential role of Tyk2 in the development of beige cells. Here, we show that mRNA expression of BAT-selective genes (UCP1, Cidea, Cox8b, and Elovl3) is significantly reduced in subcutaneous WAT of Tyk2-knockout (Tyk2-/-) mice compared to wild-type mice. Surprisingly, BAT-selective genes are induced in Tyk2-/- subcutaneous WAT by acute starvation. These findings suggest that Tyk2 is required for the development of beige cells under ambient conditions, and that the need for Tyk2 in beige cell development is bypassed during nutritional stress, a stimulus of the sympathetic response.

Adipose tissue plays a crucial role in nutrient and energy homeostasis. At the time of worldwide pandemy of obesity and consequent metabolic syndrome, a great effort is made to find new treatments with potential to preserve insulin sensitivity, or even counteract development of obesity and type 2 diabetes. There are three principal possibilities how the adipose tissue biology can contribute to this goal: 1) induction of UCP1-dependent energy dissipation in brown adipose tissue; 2) conversion of white adipose depots to brown-like tissue (i.e. "browning"); and 3) stimulation of UCP1-independent thermogenesis in white adipose tissue. This thesis is based on two published works and one article under preparation. Generaly, it is focused on three different approaches targeting the above mentioned processes in adipose tissue of laboratory mouse: 1) diet supplementation with bile acids; 2) combination treatment of ω-3 polyunsaturated fatty acids and calorie restriction; and 3) cold exposure. In the experiments with administration of bile (specifically chenodeoxycholic) acid to mice, we confirm specific induction of UCP1 in both brown and subcutaneous white adipose tissue, as well as reversion of obesity in the response to the treatment. Nevertheless, most of the acute beneficial effects are mediated by...

En dehors de son rôle dans la régulation de la balance hydrosodée, le récepteur minéralocorticoïde (MR) est un facteur de transcription hormono-dépendant qui exerce des effets pro-adipogéniques et anti-thermogéniques in vitro, mais son rôle dans la régulation du métabolisme énergétique in vivo n’a jamais été précisément étudié. Dans ce travail, nous avons montré que les souris surexprimant le MR humain (Tg) ont une résistance à l’obésité induite par le régime hyperlipidique. Ceci s’accompagne d’un défaut de développement de la masse adipeuse comme en témoignent des surfaces adipocytaires plus petites en histomoprhométrie et une diminution de l’expression de gènes impliqués dans l’adipogenèse tels que PPARγ2. Ce défaut d’adipogenèse n’est pas dû à une altération de la capacité intrinsèque des préadipocytes surexprimant le MR, isolés de la fraction stroma vasculaire, mais probablement à une modification de la polarisation macrophagique analysée par la technique du FACS. Ces résultats soulignent un impact immuno-métabolique de la surexpression du MR in vivo. Par ailleurs, dans notre modèle adipocytaire brun, nous démontrons que les corégulateurs du MR ont un profil d’expression différentiel pouvant rendre compte d’une coopération moléculaire au cours de la différenciation adipocytaire des cellules T37i. De plus, nous confirmons in vitro l’effet inhibiteur de l’aldostérone sur l’expression de UCP1 (Uncoupling protein 1). Enfin nous démontrons in vivo que la surexpression du MR dans le tissu adipeux brun des souris Tg induit une diminution de l’induction l’expression de UCP1 par une exposition au froid. L’ensemble de ces résultats apporte une meilleure compréhension du rôle du MR dans la régulation du métabolisme énergétique et devrait ouvrir des nouvelles perspectives thérapeutiques innovantes tels que l’utilisation de modulateurs sélectifs du MR dans le traitement des troubles métaboliques / Besides its role in the regulation of sodium homeostasis, the mineralocorticoid receptor (MR) is a hormone-dependent transcription factor that exerts pro-adipogenic and anti- thermogenic effects in vitro, but its role in vivo in the regulation of energy balance has never been precisely studied. In this study, we show that human MR overexpressing mice (Tg) were resistant to high fat diet-induced obesity. This was associated with a defect of fat mass as evidenced by smaller adipocyte size analyzed by histomorphometric study and a decreased expression of genes involved in adipogenesis such as PPARγ2. This alteration in adipogenesis was not related to a defect of the intrinsic capacity of MR overexpressing preadipocytes to differentiate into adipocytes, but probably to a change in macrophage polarization studied by FACS analysis. These results indicate an immuno-metabolic impact of MR overexpression in vivo. Moreover, in our brown adipocyte model, we demonstrate that MR coregulators have a differential expression profile, consistent with a coordinated and physiologically relevant cooperation occuring during brown adipogenesis. In addition, we confirm in vitro the inhibitory effect of aldosterone on UCP1 expression (Uncoupling protein 1). Finally, we demonstrate in vivo that MR overexpression in brown adipose tissue of Tg mice induced a decrease in the cold-induced UCP1 expression. Taken together, these results provide a better understanding of MR involvement in the regulation of energy metabolism and should open new therapeutic oportunities such as the use of selective MR modulators in the management of metabolic disorders.

Il existe une relation étroite entre le cancer du sein et le poids. Si l’excès de poids est un facteur de risque de survenue du cancer du sein, l’obésité au diagnostic et les variations de poids (± 5 % du poids initial) au cours des traitements sont associées à un mauvais pronostic. La perte et le gain de poids résultent d’un déséquilibre de la balance énergétique (apports et dépenses). Les caractéristiques et les causes de ces variations ne sont pas encore totalement décrites. La prise de masse grasse semble être reconnue comme un facteur de mauvais pronostic d’où l’importance de bien comprendre l’évolution de la composition corporelle au cours des traitements du cancer du sein.Dans ce contexte, les objectifs de cette thèse ont été de caractériser la variation de poids et de composition corporelle tout au long des traitements du cancer du sein et d’étudier les facteurs impliqués dans le déséquilibre de la balance énergétique dont l’activité physique et certaines composantes de la dépense énergétique de repos.Les travaux menés dans le cadre de cette thèse se sont articulés autour de trois essais cliniques. En premier lieu, l’essai MétaCa2 qui vise à décrire l’évolution à long terme du poids et de la composition corporelle de patientes ménopausées atteintes d’un cancer du sein non métastatique (en moyenne 3 ans post-chimiothérapie). Les résultats cette étude ont montré qu’une perte de poids pendant la chimiothérapie est corrélée à une prise de poids dans la suite des traitements adjuvants. Cette étude a également permis de mettre évidence une prise de poids et de masse grasse au cours de l’hormonothérapie dans la population globale et plus particulièrement chez les patientes en excès de masse grasse au diagnostic. L’augmentation du temps assis entre la fin de la chimiothérapie et le début de l’hormonothérapie a été identifiée comme un facteur lié à la prise de masse grasse à long terme.Nous avons ensuite exploré une hypothèse avancée par notre équipe qui suggère que la chimiothérapie pourrait avoir un impact sur le tissu adipeux brun. Celui-ci est impliqué dans la thermogénèse adaptative, donc dans les dépenses énergétiques, et pourrait ainsi contribuer à la prise de poids. Pour cela, nous avons réalisé une étude ancillaire au protocole AVATAXHER sur 109 patientes atteintes d’un cancer du sein HER2+ et traitées par chimiothérapie néoadjuvante et thérapie ciblée anti-HER2. Les résultats ont mis en évidence une diminution significative de l’activité métabolique du tissu adipeux brun après une cure de chimiothérapie dans la population générale et plus particulièrement dans le sous-groupe de patientes qui prennent du poids au cours de la chimiothérapie.L’activité physique constitue une possible stratégie de prévention individuelle sur les variations de poids et de composition corporelle. Dans la sous-population de patientes HER2+, elle pourrait également permettre de limiter la cardiotoxicité des traitements standards utilisés. Nous avons mis en place un essai interventionnel prospectif afin d’étudier la faisabilité d’une intervention d’activité physique à domicile pour des patientes en cours de chimiothérapie néoadjuvante + thérapie ciblée pour un cancer du sein HER2 positif. L’intervention consiste à atteindre ou maintenir un niveau d’activité physique correspondant aux recommandations internationales soit 150 minutes par semaine à une intensité modérée grâce à un programme personnalisé combinant activité physique de type aérobie et renforcement musculaire. Ces résultats originaux ont ainsi conduit à apporter de nouvelles connaissances quant aux variations de poids et de composition corporelle au cours des traitements du cancer du sein à un stade précoce et sur les causes possibles du déséquilibre énergétique. / There is a close relationship between breast cancer and weight. If weight excess is a risk factor for the onset of breast cancer, obesity at diagnosis and weight variations (± 5 % of initial weight) during treatement are associated with poor prognosis. Weight loss and weight gain result in an energy imbalance (energy intake and energy expenditure). The caracteristics and causes of these variations are not well described. Fat mass gain seems to be recognized as a factor of poor prognosis hence the importance to understand body composition evolution during breast cancer treatment.In this context, the aims of this thesis were to characterized weight and body compostion variation throughout treatment and to study the factors involved in the energy imbalance of which physical activity and certain components of resting energy expenditure.This thesis works have been centered around three clinical trials. Firstly, MétaCa2 trial that attempted to describe the long term evolution of weight and body composition among no-metastatic postmenopausal breast cancer patients (on average 3 years post-chemotherapy). The results of this study have shown that weight loss during chemotherapy is associated with a later weight gain during adjuvant period of treatment. This study has also demonstrated a weight and fat mass gain during endocrine therapy in the overall population and more especially for the patients with an initial fat mass excess. The increase in time spent sitting between the end of chemotherapy and the initiation of endocrine therapy has been identified has a factor associated with long term fat mass gain.Then, we have explored a hypothesis, supported by our team, suggesting that chemotherapy could have an impact on brown adipose tissue. This one is implied in the adaptative thermogenesis and so in energy expenditure and could contribute to weight gain. For this end, we realised an ancillary study to AVATAXHER trial among 109 HER2+ breast cancer patients treated with neoadjuvant chemotherapy and HER2 targeted therapy. The results have highlighted a significant decrease of BAT metabolic activity after one course of chemotherapy in the overall population and specifically in the subgroup of patients who gained weight during chemotherapy. Physical activity constitutes a possible strategy for individual prevention against weight and body composition variation. In the HER2+ subpopulation, it could also limit the cardiotoxicity of standard treatements. We have set up a prospective interventional trial in order to study the feasibility of a home-based physical activity intervention among HER2+ breast cancer patients currently treated with neoadjuvant chemotherapy + targeted therapy. The objective of the intervention is to achieve or maintain a physical activity level corresponding to the international recommendations, i.e. at least 150 minutes of moderate-intensity physical activity per week thanks to a personalised program combining aerobic physical activity and muscular strengthening.These original results have contributed to provide new knowledge about weight and body composition variation during early breast cancer treatments and also about some potential causes imply in energy imbalance.

<p>The domestication of the pig began about 9 000 years ago and many of the existing domestic breeds have been selected for phenotypic traits like lean meat and fast growth. Domestic pigs are phenotypically very different from the ancestral wild boar that has adapted to survive in their natural environment. Because of their divergence, crosses between domestic pigs and wild boars are suitable for constructing genetic maps and Quantitative trait locus (QTL) analyses. A cross between the Large White and the European wild boar was thus initiated in the late 1980s. A major QTL for fat deposition and growth, denoted <i>FAT1</i>, was found on chromosome 4. The aim of this thesis was to further characterise the <i>FAT1</i> locus and to identify the causative gene(s) and mutation(s). We have identified new markers and constructed a high-resolution linkage and RH map of the <i>FAT1</i> QTL interval. We also performed comparative mapping to the human genome and showed that the pig chromosome 4 is homologous to human chromosomes 1 and 8. The gene order is very well conserved between the two species. In parallel we have narrowed down the <i>FAT1</i> QTL interval by repeated backcrossing to the domestic Large White breed for six generations. The QTL could be confirmed for fatness but not for growth. Furthermore, the data strongly suggested that there might be more than one gene underlying the <i>FAT1</i> QTL. Depending on which hypothesis to consider, the one- or two-loci model, the <i>FAT1</i> interval can be reduced to 3,3 or 20 centiMorgan (cM), respectively, based on the backcross experiments. In the last study we confirm the two-loci model with one locus primarily effecting abdominal fat and another locus primarily effecting subcutaneous fat. We have identified a missense mutation in the <i>RXRG</i> gene which is in strong association with the abdominal fat QTL and the mutation is a potential candidate for that locus.</p><p>Brown adipose tissue (BAT) is a specific type of fat essential for non-shivering thermogenesis in mammals. Piglets appear to lack BAT and rely on shivering as the main mechanism for thermoregulation. Uncoupling protein 1 (<i>UCP1</i>) gene is exclusively expressed in BAT and its physiological role is to generate heat by uncoupling oxidative phosphorylation. We show that the <i>UCP1</i> gene has been disrupted in the pig lineage about 20 years ago. The inactivation of <i>UCP1</i> provides a genetic explanation for the poor thermoregulation in piglets. </p>

Einleitung<br /> Säugetiere haben zwei verschiedene Arten von Fettgewebe: das weiße Fettgewebe, welches vorwiegend zur Lipidspeicherung dient, und das braune Fettgewebe, welches sich durch seine Fähigkeit zur zitterfreien Thermogenese auszeichnet. Weiße und braune Adipozyten sind beide mesodermalen Ursprungs. Die Mechanismen, die zur Entwicklung von Vorläuferzellen in den weißen oder braunen Fettzellphenotyp führen, sind jedoch unbekannt. Durch verschiedene experimentelle Ansätze konnte gezeigt werden, daß diese Adipocyten vermutlich durch die Differenzierung zweier Typen unterschiedlicher Vorläuferzellen entstehen: weiße und braune Preadipozyten. Von dieser Hypothese ausgehend, war das Ziel dieser Studie, die Genexpression weißer und brauner Preadipozyten auf Unterschiede systematisch zu analysieren.<br /> <br /> Methoden<br /> Die zu vergleichenden Zellen wurden aus primären Zellkulturen weißer und brauner Preadipozyten des dsungarischen Zwerghamsters gewonnen. „Representational Difference Analysis“ wurde angewandt, um potentiell unterschiedlich exprimierte Gene zu isolieren. Die daraus resultierenden cDNA Fragmente von Kandidatengenen wurden mit Hilfe der Microarraytechnik untersucht. Die Expression dieser Gene wurde in braunen und weißen Fettzellen in verschiedenen Differenzierungsstadien und in braunem und weißem Fettgewebe verglichen.<br /> <br /> Ergebnisse<br /> 12 Gene, die in braunen und weißen Preadipozyten unterschiedlich exprimiert werden, konnten identifiziert werden. Drei Komplement Faktoren und eine Fettsäuren Desaturase werden in weißen Preadipozyten höher exprimiert; drei Struktur Gene (Fibronectin, Metargidin und a Actinin 4), drei Gene verbunden mit transkriptioneller Regulation (Necdin, Vigilin und das „small nuclear ribonucleoprotein polypeptide A“) sowie zwei Gene unbekannter Funktion werden in braunen Preadipozyten höher exprimiert. Mittels Clusteranalyse (oder Gruppenanalyse) wurden die gesamten Genexpressionsdaten charakterisiert. Dabei konnten die Gene in 4 typischen Expressionsmuster aufgeteilt werden: in weißen Preadipozyten höher exprimierte Gene, in braunen Preadipozyten höher exprimierte Gene, während der Differenzierung herunter regulierte Gene und während der Differenzierung hoch regulierte Gene.<br /> <br /> Schlußfolgerungen<br /> In dieser Studie konnte gezeigt werden, daß weiße und braune Preadipozyten aufgrund der Expression verschiedener Gene unterschieden werden können. Es wurden mehrere Kandidatengene zur Bestimmung weißer und brauner Preadipozyten identifiziert. Außerdem geht aus den Genexpressionsdaten hervor, daß funktionell unterschiedliche Gruppen von Genen eine wichtige Rolle bei der Differenzierung von weißen und braunen Preadipozyten spielen könnten, wie z.B. Gene des Komplementsystems und der extrazellulären Matrix. / Introduction<br /> Mammals have two types of adipose tissue: the lipid storing white adipose tissue and the brown adipose tissue characterised by its capacity for non-shivering thermogenesis. White and brown adipocytes have the same origin in mesodermal stem cells. Yet nothing is known so far about the commitment of precursor cells to the white and brown adipose lineage. Several experimental approaches indicate that they originate from the differentiation of two distinct types of precursor cells, white and brown preadipocytes. Based on this hypothesis, the aim of this study was to analyse the gene expression of white and brown preadipocytes in a systematic approach. <br /> <br /> Experimental approach<br /> The white and brown preadipocytes to compare were obtained from primary cell cultures of preadipocytes from the Djungarian dwarf hamster. Representational difference analysis was used to isolate genes potentially differentially expressed between the two cell types. The thus obtained cDNA libraries were spotted on microarrays for a large scale gene expression analysis in cultured preadipocytes and adipocytes and in tissue samples.<br /> <br /> Results<br /> 4 genes with higher expression in white preadipocytes (3 members of the complement system and a fatty acid desaturase) and 8 with higher expression in brown preadipocytes were identified. From the latter 3 coded for structural proteins (fibronectin, metargidin and a actinin 4), 3 for proteins involved in transcriptional regulation (necdin, vigilin and the small nuclear ribonucleoprotein polypeptide A) and 2 are of unknown function. Cluster analysis was applied to the gene expression data in order to characterise them and led to the identification of four major typical expression profiles: genes up-regulated during differentiation, genes down-regulated during differentiation, genes higher expressed in white preadipocytes and genes higher expressed in brown preadipocytes.<br /> <br /> Conclusion<br /> This study shows that white and brown preadipocytes can be distinguished by different expression levels of several genes. These results draw attention to interesting candidate genes for the determination of white and brown preadipocytes (necdin, vigilin and others) and furthermore indicate that potential importance of several functional groups in the differentiation of white and brown preadipocytes, mainly the complement system and extracellular matrix.