High fat feeding increases hepatic fat accumulation and is associated with hepatic insulin resistance. AMP Activated Protein Kinase (AMPK) is thought to inhibit lipid synthesis by the acute inhibition of glycerol-3-phosphate acyltransferase (GPAT) activity and transcriptional regulation via SREBP-1c. The purpose of this study was to determine if chronic activation of AMPK prevented an increase in GPAT1 activity in rats fed a high fat diet. Rats were fed a control (C), or a high fat (HF) diet (60% fat) for 6 weeks and injected with saline or a daily AICAR dose of 0.5 mg/g body weight. Chronic AMPK activation by AICAR injections resulted in a significant reduction in hepatic triglyceride accumulation in both the C and HF fed animals (C, 5.5±0.7; C+AICAR, 2.7 ±0.3; HF, 21.8±3.3; and HF+AICAR, 8.0±1.8 mg/g liver). HF feeding caused an increase in total GPAT and GPAT1 activity which was not affected by chronic AMPK activation (GPAT1 activity vs. C, C+AICAR, 92±19%; HF, 186±43%; HF+AICAR, 234±62%). Markers of oxidative capacity, including citrate synthase activity and cytochrome c abundance, were not affected by chronic AICAR treatment. Interestingly, HF feeding caused a significant increase in LCAD (up 66% from C), a marker of fatty acid oxidation capacity. These results suggest that chronic AMPK activation limits hepatic triglyceride accumulation independent of a reduction in total GPAT1 activity.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / O estudo teve como objetivo avaliar os efeitos da rosuvastatina (ST) e darosiglitazona sobre a resistência à insulina (RI), morfologia do fígado e do tecido adiposo em camundongos alimentados com dieta hiperlipídica (HF). O tratamento com rosuvastatina resultou em uma acentuada melhoria na sensibilidade à insulina caracterizada pela melhor depuração da glicose durante o teste de tolerância à insulina e uma redução do índice HOMA-IR em 70% (P = 0,0008). O grupo tratado com rosuvastatina apresentou redução no ganho massa corporal (-8%, P <0,01) e menor depósito de gordura visceral (-60%, P <0,01) em comparação com o grupo HF não tratado. Em comparação com camundongos HF, animais do grupo HF+ST reduziram significativamente a massa hepática e a esteatose hepática (-6%; P <0,05% e -21; P <0,01, respectivamente). O grupo HF+ST, reduziu os níveis de triglicerídeos hepáticos em 58% comparado com o grupo HF (P <0,01). Além disso, a expressão de SREBP-1c (proteína 1c ligadora do elemento regulado por esteróis) foi reduzido em 50% no fígado dos animais HF + ST (P <0,01) em comparação com o grupo HF. Os níveis de resistina foram menores no grupo HF + ST comparado com o grupo HF (44% a menos, P <0,01). Em conclusão, demonstramos que camundongos alimentados com dieta HF tratados com rosuvastatina melhoram a sensibilidade à insulina, com redução da esteatose hepática. Além disso, ST reduziu o ganho de massa corporal, melhorou os níveis circulantes de colesterol e triglicerídeo plasmático, com menor conteúdo de hepático de triglicerídeo, que foi concomitante com menor resistina e aumento da adiponectina. / The study aimed to evaluate the effects of rosuvastatin (ST) and rosiglitazone on insulin resistance (IR) and liver and adipose tissue morphologies in mice fed a high-fat (HF) diet. Our data show that treatment with rosuvastatin resulted in a marked improvement in insulin sensitivity characterised by enhanced glucose clearance during insulin tolerance and a decrease in the HOMA-IR index level by 70% (P=0.0008). The group of mice treated with rosuvastatin exhibited reduced body mass gain (-8%; P<0.01) and visceral fat pad thickness (-60%; P<0.01)compared with the untreated HF group. In comparison with HF mice, HF+ST mice showed a significant reduction in hepatomegaly and liver steatosis (-6%; P<0.05 and -21%; P<0.01, respectively). In HF+ST mice, the hepatictriglyceride levels were reduced by 58% compared with the HF group (P <0.01). In addition, the expression of SREBP-1c (sterol regulatory element-binding protein) was decreased by 50% in the livers of HF+ST mice (P<0.01) compared with the HF mice. The levels of resistin were lower in the HF+ST group compared with the HF group (44% less, P< 0.01). In conclusion, we demonstrated that rosuvastatin-treated mice fed HF has been improving in insulin sensitivity, with decreased steatosis found in HF mice. Furthermore, ST reduced body mass gain, improved the circulating levels of plasma cholesterol and triglycerides and reduced hepatic triglycerides, which was concomitant with lower resistin and increased total adiponectin.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / O estudo teve como objetivo avaliar os efeitos da rosuvastatina (ST) e darosiglitazona sobre a resistência à insulina (RI), morfologia do fígado e do tecido adiposo em camundongos alimentados com dieta hiperlipídica (HF). O tratamento com rosuvastatina resultou em uma acentuada melhoria na sensibilidade à insulina caracterizada pela melhor depuração da glicose durante o teste de tolerância à insulina e uma redução do índice HOMA-IR em 70% (P = 0,0008). O grupo tratado com rosuvastatina apresentou redução no ganho massa corporal (-8%, P <0,01) e menor depósito de gordura visceral (-60%, P <0,01) em comparação com o grupo HF não tratado. Em comparação com camundongos HF, animais do grupo HF+ST reduziram significativamente a massa hepática e a esteatose hepática (-6%; P <0,05% e -21; P <0,01, respectivamente). O grupo HF+ST, reduziu os níveis de triglicerídeos hepáticos em 58% comparado com o grupo HF (P <0,01). Além disso, a expressão de SREBP-1c (proteína 1c ligadora do elemento regulado por esteróis) foi reduzido em 50% no fígado dos animais HF + ST (P <0,01) em comparação com o grupo HF. Os níveis de resistina foram menores no grupo HF + ST comparado com o grupo HF (44% a menos, P <0,01). Em conclusão, demonstramos que camundongos alimentados com dieta HF tratados com rosuvastatina melhoram a sensibilidade à insulina, com redução da esteatose hepática. Além disso, ST reduziu o ganho de massa corporal, melhorou os níveis circulantes de colesterol e triglicerídeo plasmático, com menor conteúdo de hepático de triglicerídeo, que foi concomitante com menor resistina e aumento da adiponectina. / The study aimed to evaluate the effects of rosuvastatin (ST) and rosiglitazone on insulin resistance (IR) and liver and adipose tissue morphologies in mice fed a high-fat (HF) diet. Our data show that treatment with rosuvastatin resulted in a marked improvement in insulin sensitivity characterised by enhanced glucose clearance during insulin tolerance and a decrease in the HOMA-IR index level by 70% (P=0.0008). The group of mice treated with rosuvastatin exhibited reduced body mass gain (-8%; P<0.01) and visceral fat pad thickness (-60%; P<0.01)compared with the untreated HF group. In comparison with HF mice, HF+ST mice showed a significant reduction in hepatomegaly and liver steatosis (-6%; P<0.05 and -21%; P<0.01, respectively). In HF+ST mice, the hepatictriglyceride levels were reduced by 58% compared with the HF group (P <0.01). In addition, the expression of SREBP-1c (sterol regulatory element-binding protein) was decreased by 50% in the livers of HF+ST mice (P<0.01) compared with the HF mice. The levels of resistin were lower in the HF+ST group compared with the HF group (44% less, P< 0.01). In conclusion, we demonstrated that rosuvastatin-treated mice fed HF has been improving in insulin sensitivity, with decreased steatosis found in HF mice. Furthermore, ST reduced body mass gain, improved the circulating levels of plasma cholesterol and triglycerides and reduced hepatic triglycerides, which was concomitant with lower resistin and increased total adiponectin.

Plusieurs études ont démontré que les agonistes du PPARgamma, dont la pioglitazone (PGZ), améliorent les paramètres métaboliques et histologiques de la stéatohépatite non-alcoolique (NASH) chez l'homme et la souris, et qu’ils ont des effets bénéfiques sur la fibrose hépatique chez le rat. Les mécanismes d’action sont mal connus. La NASH, caractérisée par de la stéatose, des lésions hépatocytaires, de l’inflammation et une fibrose variable, est considérée comme une complication hépatique du syndrome métabolique. L'obésité, un des facteurs de risque pour le développement de la NASH, est caractérisée par de faibles taux d'adiponectine sérique. Cette adipocytokine, dont l'expression génique est régulée par le PPARgamma, possède des propriétés anti-stéatosique et anti-fibrotique chez la souris. L'activité intracellulaire de l'adiponectine est médiée via ses récepteurs spécifiques qui activent la protéine kinase AMPK et/ou le PPARalpha. Une fois activée, l’AMPK induit les voies cataboliques de production d’énergie (telles que l'oxydation des acide gras) et inhibe les voies consommant de l’ATP (telles que la lipogenèse). L'activation du PPARalpha augmente l'oxydation des acides gras et inhibe la réponse inflammatoire. Le but de notre travail est d’évaluer l'implication de la voie PGZ–adiponectine–AMPK et/ou PPARalpha dans la prévention de la NASH et de la fibrose hépatique. Nous avons tout d’abord évalué l'effet de la PGZ sur la fibrose hépatique chez la souris. Nos observations montrent que, contrairement aux résultats observés chez le rat, la PGZ n’inhibe pas le développement de la fibrose hépatique chez la souris in vivo. Ces résultats ont été confirmés par des études sur les cellules stellaires hépatiques (HSCs), les cellules effectrices de la fibrose, in vitro. Dans une seconde étude, nous avons évalué l'impact de l’AMPK sur la fibrose hépatique in vivo et sur l’activation des HSCs in vitro. Nous avons constaté que l’AMPK jouait un rôle dans le contrôle de la trans-différentiation des HSCs in vitro mais pas dans le développement de la fibrose hépatique chez la souris in vivo. Finalement, nous avons évalué l'hypothèse que l'effet bénéfique de la PGZ sur la NASH résulte de la stimulation de l'AMPK et/ou du PPARalpha par l’adiponectine. Nos résultats ont montrés que cet effet de la PGZ était strictement dépendant de l’adiponectine mais ne semblait pas impliquer l'AMPK ni le PPARalpha. Nous avons également identifié SREBP-1c, régulant la lipogenèse de novo, comme cible thérapeutique potentielle pour le développement de la NASH. Les résultats obtenus dans le cadre de ce travail de thèse fournissent une meilleure compréhension de l’axe PPARgamma–adiponectine–AMPK dans le contrôle du développement de la NASH et de la fibrose hépatique chez la souris. / Several studies have demonstrated that peroxisome proliferator-activated receptor gamma (PPARg) agonists, such as pioglitazone (PGZ), improve metabolic parameters and histology of nonalcoholic steatohepatitis (NASH) development in humans and mice, and have beneficial effects on liver fibrosis in rats. NASH, characterized by steatosis, hepatocellular damage, inflammation and variable fibrosis, is recognised as the hepatic complication of the metabolic syndrome. Obesity, one of the risk factors for NASH development, is characterized by low serum adiponectin levels. This adipocytokine, of which gene expression is regulated by PPARg, demonstrates anti-steatotic and anti-fibrotic properties in mice. Intracellular activity of adiponectin is mediated through its specific receptors which activate AMP-activated protein kinase (AMPK) and PPARalpha. Once activated, AMPK switches on catabolic pathways (such as fatty acid oxidation and glycolysis) and switches off ATP-consuming pathways (such as lipogenesis). Activation of PPARalpha increases fatty acid oxidation and reduces inflammatory reaction. The aim of the present work is to analyse the activation of the axis PGZ-adiponectin-AMPK and/or PPARalpha as a way to control NASH and hepatic fibrosis development. We first evaluated the effect of PGZ on hepatic fibrosis in mice. We observed that, by contrast with results in rats, PGZ did not prevent hepatic fibrosis development in vivo in mice. These results were confirmed by in vitro studies on the key effector cells of fibrogenesis, the hepatic stellate cells (HSCs). We then assessed the impact of AMPK on hepatic fibrosis in vivo and on HSC trans-differentiation/activation phenomenon in vitro. We found that AMPK played a role in the control of HSC trans-differentiation in vitro but was not implicated in the wound-healing fibrosis in vivo in mice. Finally, we tested the hypothesis that the beneficial effect of PGZ on steatohepatitis results from the adiponectin-dependent stimulation of AMPK and/or PPARalpha. We found that this preventive effect was clearly dependent of adiponectin but did not involve AMPK or PPARalpha activation. We have also identified SREBP-1c, implicated in the regulation of de novo lipogenesis, as a potential therapeutic target for the control of the development of NASH. The present thesis provides a better understanding of the axis PPARg–adiponectin–AMPK in the control of NASH and hepatic fibrosis development in mouse.

Sirt1 (Sirtuine 1) est une protéine histone déacétylase dépendante de NAD+ qui stimule la néoglucogénèse et inhibe la glycolyse dans le foie, et qui augmente l’oxydation des acides gras dans le muscle strié squelettique. Le but de ce travail de thèse a été de définir les fonctions métaboliques de Sirt1 dans le muscle strié squelettique. Nous avons tout d’abord montré, à l’aide d’un modèle de souris déficientes pour le gène Sirt1, que Sirt1 régulait l’expression de l’hexokinase II et de SREBP-1c, protéine régulatrice de l’expression de l’hexokinase. De plus, un modèle d’électrotransfert de gènes permettait de mettre en évidence que Sirt1 régulait l’expression de SREBP-1c de façon LXR dépendante. Enfin, l’inhibition de Sirt1 par l’EX527 aboutissait à une diminution de la consommation de glucose chez des myotubes C2C12. Prises ensemble, ces données suggèrent un rôle important de Sirt1 dans la régulation du métabolisme du glucose dans le muscle strié squelettique. Dans un second temps, nous avons déterminé le rôle potentiel de Sirt1 lors d’un jeûne chez des myotubes C2C12. Un jeûne entraînait une augmentation de l’activité cathepsine B + L et une déphosphorylation des protéines AktS473, GSK3S21/S9, p70S6KT412 et S6 S235/S236 qui précédait une amyotrophie des myotubes. La renutrition aboutissait à une rephosphorylation de ces protéines et à un retour à la normale de la taille des myotubes. L’activité cathepsine B + L restait cependant élevée. Enfin, le niveau en ARNm de Sirt1 était augmenté de façon transitoire lors de la renutrition. D’autres mesures de marqueurs des voies protéolytiques et de l’activité de Sirt1 sont à envisager. Nos données ainsi que celles de la littérature suggèrent que Sirt1 pourrait avoir un rôle dans la régulation de l’autophagie lors du jeûne. Pour conclure, ce travail de thèse met en évidence un rôle pour Sirt1 dans la régulation du métabolisme du glucose dans le muscle strié squelettique et apporte de nouvelles perspectives dans l’étude de la régulation de ce métabolisme en conditions pathologiques / Sirt1 (Sirtuin 1) is a NAD+-dependent histone deacetylase, which stimulates gluconeogenesis and inhibits glycolysis in the liver, and which increases fatty acid oxidation in skeletal muscle. The aim of this thesis was to define the metabolic functions of Sirt1 in skeletal muscle. We first showed, using a mouse model lacking the Sirt1 gene, that Sirt1 regulated expression of hexokinase II and SREBP-1c, a protein that regulates hexokinase expression. In addition, a model of gene electrotransfer allowed us to show that Sirt1 regulated expression of SREBP-1c in a LXR-dependent manner. Finally, inhibition of Sirt1 by EX527 resulted in a decrease of glucose consumption in C2C12 myotubes. Taken together, these data suggest an important role of Sirt1 in the regulation of glucose metabolism in skeletal muscle. Secondly, we determined the potential role of Sirt1 during fasting in C2C12 myotubes. Fasting resulted in an increase in cathepsin B + L activity and a dephosphorylation of AktS473, GSK3S21/S9, p70S6KT412 and S6 S235/S236 preceding a myotubes atrophy. Refeeding led to a rephosphorylation of these proteins and a return to normal size of myotubes. However, cathepsin B + L activity remained elevated. Finally, the level of Sirt1 mRNA was transiently increased during refeeding. Other measures of proteolytic pathways and Sirt1 activity markers will be determined. Our data and those of the literature suggest that Sirt1 could play a role in autophagyregulation during fasting. To conclude, this thesis highlights a role for Sirt1 in the regulation of glucose metabolism in skeletal muscle and provides new perspectives in the study of regulation of this metabolism in pathological conditions

Sirt1 (Sirtuine 1) est une protéine histone déacétylase dépendante de NAD+ qui stimule la néoglucogénèse et inhibe la glycolyse dans le foie, et qui augmente l'oxydation des acides gras dans le muscle strié squelettique. Le but de ce travail de thèse a été de définir les fonctions métaboliques de Sirt1 dans le muscle strié squelettique. Nous avons tout d'abord montré, à l'aide d'un modèle de souris déficientes pour le gène Sirt1, que Sirt1 régulait l'expression de l'hexokinase II et de SREBP-1c, protéine régulatrice de l'expression de l'hexokinase. De plus, un modèle d'électrotransfert de gènes permettait de mettre en évidence que Sirt1 régulait l'expression de SREBP-1c de façon LXR dépendante. Enfin, l'inhibition de Sirt1 par l'EX527 aboutissait à une diminution de la consommation de glucose chez des myotubes C2C12. Prises ensemble, ces données suggèrent un rôle important de Sirt1 dans la régulation du métabolisme du glucose dans le muscle strié squelettique. Dans un second temps, nous avons déterminé le rôle potentiel de Sirt1 lors d'un jeûne chez des myotubes C2C12. Un jeûne entraînait une augmentation de l'activité cathepsine B + L et une déphosphorylation des protéines AktS473, GSK3S21/S9, p70S6KT412 et S6 S235/S236 qui précédait une amyotrophie des myotubes. La renutrition aboutissait à une rephosphorylation de ces protéines et à un retour à la normale de la taille des myotubes. L'activité cathepsine B + L restait cependant élevée. Enfin, le niveau en ARNm de Sirt1 était augmenté de façon transitoire lors de la renutrition. D'autres mesures de marqueurs des voies protéolytiques et de l'activité de Sirt1 sont à envisager. Nos données ainsi que celles de la littérature suggèrent que Sirt1 pourrait avoir un rôle dans la régulation de l'autophagie lors du jeûne. Pour conclure, ce travail de thèse met en évidence un rôle pour Sirt1 dans la régulation du métabolisme du glucose dans le muscle strié squelettique et apporte de nouvelles perspectives dans l'étude de la régulation de ce métabolisme en conditions pathologiques

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels.

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