L’hépatokine FGF21 (Fibroblast Growth factor 21) joue un rôle primordial dans le contrôle de l’homéostasie énergétique. Des études chez l’Homme et l’animal mettent en évidence ses effets bénéfiques dans la lutte contre l’hyperglycémie, la dyslipidémie et l’obésité. Connue pour être induite en réponse au jeûne par le récepteur nucléaire PPARα (Proliferator Activated Receptor α), des études récentes suggèrent l’implication du facteur de transcription ChREBP (Carbohydrate Responsive Element Binding Protein) dans la réponse nutritionnelle de FGF21. Dans ce contexte, les objectifs de thèse ont été : 1) d’obtenir une meilleure compréhension de la régulation de FGF21 dans le foie par le jeûne et le glucose via les acteurs moléculaires ChREBP et PPARα ; 2) de déterminer la relevance physiologique de l’axe ChREBP-PPARα-FGF21 en réponse au glucose. Nos résultats mettent en évidence que l’expression hépatique de ChREBP est nécessaire à l’induction de FGF21 en réponse au glucose in vitro et in vivo. De manière inattendue, lorsque l’expression de PPARα est spécifiquement invalidée dans le foie, la réponse au glucose de FGF21 est diminuée de manière significative car ChREBP ne peut se lier à son élément de réponse de type ChoRE, présent sur le promoteur de fgf21. La réponse synergique de ChREBP et de PPARα sur FGF21 a été également mise en évidence dans des cultures primaires d’hépatocytes humains. Chez les souris invalidées pour PPARα dans le foie, l’absence de FGF21 circulant entraine une augmentation de la préférence au sucrose. Notre étude révèle l’existence d’un dialogue fonctionnel unique entre ChREBP et PPARα pour la régulation de FGF21 en réponse au glucose / The hepatokine FGF21 (Fibroblast Growth factor 21) plays an important role in the control of energy homeostasis. Studies in humans and animals have established FGF21 as an important therapeutic target for its beneficial effects on hyperglycemia, dyslipidemia and obesity. Induced in response to fasting by the PPARα nuclear receptor (Proliferator Activated Receptor α), recent studies suggest the involvement of ChREBP (Carbohydrate Responsive Element Binding) in the nutritional response of FGF21. In this context, the thesis objectives were: 1) to obtain a better understanding of the regulation of FGF21 in the liver by fasting and glucose via the molecular actors ChREBP and PPARα; 2) to determine the physiological relevance of the ChREBP-PPARα-FGF21 axis in response to glucose. Our results demonstrate that hepatic expression of ChREBP is necessary for the induction of FGF21 in response to glucose in vitro and in vivo. Unexpectedly, when PPARα expression is specifically invalidated in the liver, the glucose response of FGF21 is significantly decreased as ChREBP cannot bind to its ChoRE response element present on the fgf21 promoter. The synergistic response of ChREBP and PPARα to FGF21 was also demonstrated in primary cultures of human hepatocytes. In mice deficient for PPARα in the liver, the absence of circulating FGF21 leads to an increase in their preference to sucrose. Our study reveals the existence of a unique functional dialogue between ChREBP and PPARα for the regulation of FGF21 in response to glucose

Fibroblast Growth Factor 21 (FGF21) is an endocrine hormone derived from the liver that exerts pleiotropic effects on the body to maintain overall metabolic homeostasis. During the past decade, there has been an enormous effort to understand the physiological roles of FGF21 in regulating metabolism and to identify the mechanism for its potent pharmacological effects to reverse diabetes and obesity. Through both human and rodent studies, it is now evident that FGF21 is dynamically regulated by nutrient sensing and consequently functions as a critical regulator of nutrient homeostasis. In addition, recent studies with new genetic and molecular tools have provided critical insight into the actions of this exciting endocrine factor. Dissection of these FGF21-regulated pathways has tremendous potential for new targeted therapies to treat metabolic disease. The goals of this thesis are 1) to identify FGF21’s physiological role as a carbohydrate-regulated signal of macronutrient-specific satiety and 2) to determine the mechanism and tissues responsible for mediating the pharmacological effects of FGF21. To address the first goal, we used different FGF21 genetic knockout mouse models to determine if loss of FGF21 would affect macronutrient preference. We found that loss of FGF21 led to an increase in simple sugar intake whereas this had no effect on other macronutrients such as lipid or protein. To further characterize the relationship between carbohydrates and FGF21, in vitro and in vivo techniques revealed that FGF21 transcription in the liver increased in response to carbohydrate intake and this was dependent on the presence of a transcription factor activated by carbohydrates, ChREBP. We next addressed whether or not increased FGF21 levels would affect preference for simple sugars. We found that in response to increased circulating levels of FGF21, either through genetic overexpression or pharmacological administration, FGF21 would lead to a significant decrease in caloric and non-caloric sweeteners. Finally, we were able to determine that FGF21 was signaling to the hypothalamus to mediate this suppression of simple sugar intake through region specific knockout of the co-receptor beta-klotho. To address the pharmacological actions of FGF21, we generated an adipose-specific KLB KO mouse using mice that express Cre-recombinase under the adiponectin promoter. These mice lack the co-receptor for FGF21 in adipose tissue and are a more reliable adipose knockout model than previous studies that have used aP2-Cre mice. We were able to determine that the acute glucose lowering effects of FGF21 are mediated through direct signaling to adipose tissue and that FGF21 enhances insulin sensitivity by increasing glucose uptake in brown adipose tissue. However, FGF21 mediates its chronic effects, including lowering body weight and triglycerides, by signaling to some other non-adipose tissue. Overall our work has shown that FGF21 can significantly regulate glucose metabolism through multiple mechanisms.

Fibroblast Growth Factor 21 (FGF21) is a hormone that is produced from the liver which has pleiotropic effects. Physiologically, FGF21 increases energy expenditure, increases glucose uptake, enhances glucose tolerance, and increases peripheral insulin sensitivity. Pharmacologically, FGF21 reverses obesity and diabetes in animal models and significantly improves metabolic profiles in humans through unknown mechanisms. We hypothesized that the physiological actions of FGF21 may provide insights to explain FGF21’s beneficial pharmacological effects. The overall theme of this work was to identify the elusive mechanism by which FGF21 regulates energy homeostasis. In chapter 1, I review some adipokines and hepatokines that regulate energy homeostasis. In chapter 2, I provide background on fibroblast growth factors (FGFs), metabolic FGFs, and the tissue-specific effects of FGF21. In chapter 3, I will review the role of growth factors in thermoregulation. In chapter 4, we use tissue-specific loss of function models to investigate the trajectory of FGF21’s thermogenic effects during prolonged cold. In chapter 5, we specifically address the necessity and sufficiency of FGF21 signaling directly to adipose tissue, and the contribution of the adipokine adiponectin in mediating FGF21’s metabolic effects. In chapter 6, I summarize our results, reflect upon the ramifications of these results, and briefly address potential future experiments given our results on the physiological and pharmacological actions of FGF21 in adipose tissues.

Metformin has been used clinically since 1957 for its efficacy and safety as therapy for type 2 diabetes. Besides ameliorating hyperglycemia without risk of hypoglycemia, metformin also lowers plasma triglyceride levels. Furthermore, a wealth of data shows that metformin facilitates weight loss in mice as well as humans. Due to its numerous metabolic benefits, researchers and clinicians are interested in the possibility of using metformin as treatment to combat obesity and other metabolic disorders such as non-alcoholic fatty liver disease (NAFLD). Despite being the most commonly prescribed anti-diabetic, metformin’s complete mechanism(s) for weight loss or for lowering glucose and lipids remains an enigma. Our studies show that metformin-treated mice exhibited decreased caloric intake, providing a viable mechanism for metformin to bring about weight loss. Intriguingly, we found that metformin induces PRDM16 to promote browning of iWAT and increase expression of thermogenic genes such as UCP1 and DIO2. However, metformin did not appear to increase energy expenditure. It’s possible that metformin’s effect on energy expenditure was masked since energy expenditure measurements were taken when metformin-treated mice were still losing weight and were in a state of negative energy balance. Recently, there has been much attention given to AMPK activators as exercise mimetics. Metformin is known to activate AMPK and similarly brings about many beneficial effects as exercise such as alleviation of obesity-induced NAFLD. SIRT1 stimulation by resveratrol and delivery of exogenous FGF21 mimics metformin’s ability to combat obesity and improve NAFLD. Collectively, these results implicate metformin, resveratrol, and exogenous administration of FGF21 as beneficial therapies for weight loss and amelioration of NAFLD.

Background & aims Cytochrome P450 2A5 (CYP2A5) is induced by ethanol, and the ethanol induction of CYP2A5 is regulated by nuclear factor-erythroid 2-related factor 2 (NRF2). Cyp2a5 knockout (Cyp2a5−/−) mice develop more severe alcoholic fatty liver than Cyp2a5+/+ mice. Fibroblast growth factor 21 (FGF21), a PPARα-regulated liver hormone, is involved in hepatic lipid metabolism. Alcoholic and non-alcoholic fatty liver are enhanced in Pparα knockout (Pparα−/−) mice. This study investigates the relationship between the PPARα-FGF21 axis and the enhanced alcoholic fatty liver in Cyp2a5−/− mice. Methods Mice were fed the Lieber-Decarli ethanol diet to induce alcoholic fatty liver. Results More severe alcoholic fatty liver disease was developed in Cyp2a5−/− mice than in Cyp2a5+/+ mice. Basal FGF21 levels were higher in Cyp2a5−/− mice than in Cyp2a5+/+ mice, but ethanol did not further increase the elevated FGF21 levels in Cyp2a5−/− mice while FGF21 was induced by ethanol in Cyp2a5+/+ mice. Basal levels of serum FGF21 were lower in Pparα−/− mice than in Pparα+/+ mice; ethanol induced FGF21 in Pparα+/+ mice but not in Pparα−/− mice, whereas ethanol induced hypertriglyceridemia in Pparα−/− mice but not in Pparα+/+ mice. Administration of recombinant FGF21 normalized serum FGF21 and triglyceride in Pparα−/− mice. Alcoholic fatty liver was enhanced in liver-specific Fgf21 knockout mice. Pparα and Cyp2a5 double knockout (Pparα−/−/Cyp2a5−/−) mice developed more severe alcoholic fatty liver than Pparα+/+/Cyp2a5−/− mice. Conclusions These results suggest that CYP2A5 protects against the development of alcoholic fatty liver disease, and the PPARα-FGF21 axis contributes to the protective effects of CYP2A5 on alcoholic fatty liver disease.

Plusieurs études ont montré que certaines kinases situées dans le foie, « mammalian target of rapamycin » (mTOR), « adenosine monophosphate-activated protein kinase » (AMPK) et « general control non-depressible kinase 2 » (GCN2) répondent à la disponibilité en acides aminés.L’objectif de nos études a été de préciser le rôle de deux de ces voies, l’AMPK et GCN2, dans les adaptations du métabolisme énergétique et de la synthèse protéique en réponse aux variations en protéines du régime. Pour cela, des souris de type sauvage et des souris KO n’exprimant plus la voie AMPK ou GCN2 dans le foie ont été nourries pendant trois semaines avec un régime faible, normal ou fort en protéines. Les analyses ont montré que les souris KO-AMPK foie spécifique et nourries sous régime normoprotéique adaptent leur métabolisme hépatique notamment en sécrétant le facteur fibroblastique FGF21 ce qui leur permet de compenser l’absence d’AMPK et de présenter des profils d’oxydation normaux.Au contraire, les souris KO-AMPK foie spécifique nourries avec des régimes faibles ou forts en protéines présentent des altérations des profils d’oxydation des lipides et des glucides liées à une absence de modification du métabolisme hépatique.La délétion de GCN2 dans le foie, quant à elle, n’a d’effet que sous régime faible en protéines : les souris KO-GCN2 foie spécifique présentent une plus faible oxydation lipidique et une plus forte oxydation glucidique que les souris sauvages en période postprandiale dû à l’absence d’induction de la sécrétion de FGF21.Concernant le métabolisme des protéines, les kinases GCN2 et AMPK ne semblent pas impliquées dans l’intensité du flux de synthèse protéique dans le foie et en périphérie dans le muscle en période postprandiale.En conclusion, ces travaux montrent que les délétions de l’AMPK et de GCN2 hépatiques affectent le métabolisme énergétique mais pas le métabolisme protéique et que les conséquences dépendent de la composition du régime. / Several studies have reported that some kinases located in the liver respond to the availability of amino acids. These kinases are mammalian target of rapamycin '(mTOR), "adenosine monophosphate-activated protein kinase" (AMPK) and "general control non-depressible kinase 2" (GCN2).The aim of our study was to clarify the role of two of these signaling pathways, AMPK and GCN2 in the adaptations of energy and protein metabolism in response to the modulation of dietary protein content. Wild-type and liver AMPK-deficient or liver GCN2-deficient mice were fed either a low, a normal or high protein diet during three weeks. Analyzes showed that liver AMPK-deficient mice fed under a normo-protein diet exhibit an adapatation of liver metabolism and secret FGF21 which enables them to have normal postprandial oxidation profiles.In contrast, liver AMPK-deficient mice fed a low or a high protein diet exhibit an alteration in postprandial oxidation profiles. The deletion of GCN2 in the liver only has an effect under low protein diet as liver GCN2 deficient mice have a lower lipid oxidation and a higher carbohydrate oxidation linked to the absence of FGF21 secretion. Concerning protein metabolism, AMPK and GCN2 do not seem to be involved in protein synthesis rate in the posrprandial period in the liver and periphery in the postprandial muscle. In conclusion, these studies show that hepatic AMPK and GCN2 deletions affect energy metabolism, but not protein metabolism and that the consequences depend on diet composition.

The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and function of islet β-cells, the mechanisms are currently unknown. This thesis examines mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2) protein expression in mouse islets. Autofluorescence microscopy showed difference in NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets showed significant decreased mitochondrial energetics when acutely stimulated with high concentrations of glucose and palmitate. This decrease in energetics correlated with increased generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state. These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high fat loading and reduces insulin during nutrient stress.

The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and function of islet β-cells, the mechanisms are currently unknown. This thesis examines mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2) protein expression in mouse islets. Autofluorescence microscopy showed difference in NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets showed significant decreased mitochondrial energetics when acutely stimulated with high concentrations of glucose and palmitate. This decrease in energetics correlated with increased generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state. These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high fat loading and reduces insulin during nutrient stress.

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