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Increased sugar consumption is a risk factor for features of the metabolic syndrome including obesity, hypertriglyceridemia, insulin resistance, diabetes, and nonalcoholic fatty liver disease. The gut epithelium, which plays a central role in dietary sugar digestion, absorption and metabolism has emerged a key actor of metabolic disorders. While the transcription factor ChREBP (Carbohydrate response element binding protein) has been established as a key player of the adaptive reprograming of cellular metabolism in various tissues upon glucose or fructose challenge, its specific contribution to the regulation of blood glucose upon dietary sugar intake was not previously addressed.We demonstrate here that ChREBP is abundantly expressed in the proximal gut epithelium, where carbohydrate digestion and absorption primarily occur and in particular L cells, which produce the glucoincretin GLP-1. The inducible deletion of ChREBP specifically in the mouse gut epithelium (ChΔGUT mice) resulted in the reduction of early glycemic excursion upon oral glucose load. Surprisingly, despite being associated with reduced GLP-1 production, loss of gut ChREBP activity significantly dampened glucose transepithelial flux, and thereby delayed glucose distribution to peripheral tissues. Among the underlying mechanisms, we unveil that ChΔGUT mice show an impaired expression of key intestinal hexose (glucose, galactose, fructose) transporters and metabolic enzymes as well as brush border dissacharidases. In agreement, intestinal ChREBP deficiency was accompanied by a precocious intolerance to both high-lactose and high-sucrose diets concomitant with mild galactose and severe fructose malabsorption syndromes.Altogether, our study demonstrates that, by transcriptionally orchestrating local digestion and absorption of dietary sugars, ChREBP activity in the mouse gut epithelium controls glucose appearance rate into systemic circulation and prevents against intolerance to mono- and disaccharides.

Summary While the physiological benefits of the fibroblast growth factor 21 (FGF21) hepatokine are documented in response to fasting, little information is available on Fgf21 regulation in a glucose-overload context. We report that peroxisome-proliferator-activated receptor α (PPARα), a nuclear receptor of the fasting response, is required with the carbohydrate-sensitive transcription factor carbohydrate-responsive element-binding protein (ChREBP) to balance FGF21 glucose response. Microarray analysis indicated that only a few hepatic genes respond to fasting and glucose similarly to Fgf21. Glucose-challenged Chrebp−/− mice exhibit a marked reduction in FGF21 production, a decrease that was rescued by re-expression of an active ChREBP isoform in the liver of Chrebp−/− mice. Unexpectedly, carbohydrate challenge of hepatic Pparα knockout mice also demonstrated a PPARα-dependent glucose response for Fgf21 that was associated with an increased sucrose preference. This blunted response was due to decreased Fgf21 promoter accessibility and diminished ChREBP binding onto Fgf21 carbohydrate-responsive element (ChoRE) in hepatocytes lacking PPARα. Our study reports that PPARα is required for the ChREBP-induced glucose response of FGF21., Graphical Abstract, Highlights • Fgf21 is a unique hepatic gene inducible by both catabolic and anabolic signals • The ChREBP-mediated induction of Fgf21 in hepatocytes requires PPARα • Loss of PPARα impairs Fgf21 promoter accessibility at the ChoRE • PPARα is required for the control of sucrose preference in vivo, FGF21 is a hepatokine with beneficial metabolic effects, including control of sucrose preference. Iroz et al. demonstrate that Fgf21 is a unique hepatic gene inducible by both fasting and glucose signals and that the transcription factors PPARα and ChREBP both regulate the endocrine control of sugar intake by hepatic FGF21.

Background AMP-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular energy status that contributes to restoration of energy homeostasis by slowing down ATP-consuming pathways and activating ATP-producing pathways. Unexpectedly, in different systems, AMPK is also required for proper cell division. In the current study, we evaluated the potential effect of the AMPK catalytic subunit, AMPKα1, on hepatocyte proliferation. Methods Hepatocyte proliferation was determined in AMPKα1 knockout and wild-type mice in vivo after two thirds partial hepatectomy, and in vitro in primary hepatocyte cultures. The activities of metabolic and cell cycle-related signaling pathways were measured. Results After partial hepatectomy, hepatocytes proliferated rapidly, correlating with increased AMPK phosphorylation. Deletion of AMPKα1 delayed liver regeneration by impacting on G1/S transition phase. The proliferative defect of AMPKα1-deficient hepatocytes was cell autonomous, and independent of energy balance. The priming phase, lipid droplet accumulation, protein anabolic responses and growth factor activation after partial hepatectomy occurred normally in the absence of AMPKα1 activity. By contrast, mRNA and protein expression of cyclin A2, a key driver of S phase progression, were compromised in the absence of AMPK activity. Importantly, AMPKα1 controlled cyclin A2 transcription mainly through the ATF/CREB element. Conclusions Our study highlights a novel role for AMPKα1 as a positive regulator of hepatocyte division occurring independently of energy balance.

Metabolic diseases such as obesity and type 2 diabetes are recognized as independent risk factors for hepatocellular carcinoma (HCC). Hyperinsulinemia, a hallmark of these pathologies, is suspected to be involved in HCC development. The molecular adapter growth factor receptor binding protein 14 (Grb14) is an inhibitor of insulin receptor catalytic activity, highly expressed in the liver. To study its involvement in hepatocyte proliferation, we specifically inhibited its liver expression using a short hairpin RNA strategy in mice. Enhanced insulin signaling upon Grb14 inhibition was accompanied by a transient induction of S-phase entrance by quiescent hepatocytes, indicating that Grb14 is a potent repressor of cell division. The proliferation of Grb14-deficient hepatocytes was cell-autonomous as it was also observed in primary cell cultures. Combined Grb14 down-regulation and insulin signaling blockade using pharmacological approaches as well as genetic mouse models demonstrated that Grb14 inhibition–mediated hepatocyte division involved insulin receptor activation and was mediated by the mechanistic target of rapamycin complex 1–S6K pathway and the transcription factor E2F1. In order to determine a potential dysregulation in GRB14 gene expression in human pathophysiology, a collection of 85 human HCCs was investigated. This revealed a highly significant and frequent decrease in GRB14 expression in hepatic tumors when compared to adjacent nontumoral parenchyma, with 60% of the tumors exhibiting a reduced Grb14 mRNA level. Conclusion: Our study establishes Grb14 as a physiological repressor of insulin mitogenic action in the liver and further supports that dysregulation of insulin signaling is associated with HCC. (Hepatology 2017;65:1352-1368).

OBJECTIVE Bariatric surgery causes durable weight loss. Gut hormones are implicated in obesity pathogenesis, dietary failure, and mediating gastrointestinal bypass (GIBP) surgery weight loss. In mice, we determined the effects of diet-induced obesity (DIO), subsequent dieting, and GIBP surgery on ghrelin, peptide YY (PYY), and glucagon-like peptide-1 (GLP-1). To evaluate PYY’s role in mediating weight loss post-GIBP, we undertook GIBP surgery in PyyKO mice. RESEARCH DESIGN AND METHODS Male C57BL/6 mice randomized to a high-fat diet or control diet were killed at 4-week intervals. DIO mice underwent switch to ad libitum low-fat diet (DIO-switch) or caloric restriction (CR) for 4 weeks before being killed. PyyKO mice and their DIO wild-type (WT) littermates underwent GIBP or sham surgery and were culled 10 days postoperatively. Fasting acyl-ghrelin, total PYY, active GLP-1 concentrations, stomach ghrelin expression, and colonic Pyy and glucagon expression were determined. Fasting and postprandial PYY and GLP-1 concentrations were assessed 30 days postsurgery in GIBP and sham pair-fed (sham.PF) groups. RESULTS DIO progressively reduced circulating fasting acyl-ghrelin, PYY, and GLP-1 levels. CR and DIO-switch caused weight loss but failed to restore circulating PYY to weight-appropriate levels. After GIBP, WT mice lost weight and exhibited increased circulating fasting PYY and colonic Pyy and glucagon expression. In contrast, the acute effects of GIBP on body weight were lost in PyyKO mice. Fasting PYY and postprandial PYY and GLP-1 levels were increased in GIBP mice compared with sham.PF mice. CONCLUSIONS PYY plays a key role in mediating the early weight loss observed post-GIBP, whereas relative PYY deficiency during dieting may compromise weight-loss attempts.

COUP-TFII has an important role in regulating metabolism in vivo. We showed this previously by deleting COUP-TFII from pancreatic beta cells in heterozygous mutant mice, which led to abnormal insulin secretion. Here, we report that COUP-TFII expression is reduced in the pancreas and liver of mice refed with a carbohydrate-rich diet and in the pancreas and liver of hyperinsulinemic and hyperglycemic mice. In pancreatic beta cells, COUP-TFII gene expression is repressed by secreted insulin in response to glucose through Foxo1 signaling. Ex vivo COUP-TFII reduces insulin production and secretion. Our results suggest that beta cell insulin secretion is under the control of an autocrine positive feedback loop by alleviating COUP-TFII repression. In hepatocytes, both insulin, through Foxo1, and high glucose concentrations repress COUP-TFII expression. We demonstrate that this negative glucose effect involves ChREBP expression. We propose that COUP-TFII acts in a coordinate fashion to control insulin secretion and glucose metabolism.

International audience; Unlike the adjustable gastric banding procedure (AGB), Roux-en-Y gastric bypass surgery (RYGBP) in humans has an intriguing effect: a rapid and substantial control of type 2 diabetes mellitus (T2DM). We performed gastric lap-band (GLB) and entero-gastro anastomosis (EGA) procedures in C57Bl6 mice that were fed a high-fat diet. The EGA procedure specifically reduced food intake and increased insulin sensitivity as measured by endogenous glucose production. Intestinal gluconeogenesis increased after the EGA procedure, but not after gastric banding. All EGA effects were abolished in GLUT-2 knockout mice and in mice with portal vein denervation. We thus provide mechanistic evidence that the beneficial effects of the EGA procedure on food intake and glucose homeostasis involve intestinal gluconeogenesis and its detection via a GLUT-2 and hepatoportal sensor pathway.

Pancreatic islet beta cell differentiation and function are dependent upon a group of transcription factors that maintain the expression of key genes and suppress others. Knockout mice with the heterozygous deletion of the gene for chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) or the complete disruption of the gene for hepatocyte nuclear factor 4alpha (HNF4alpha) in pancreatic beta cells have similar insulin secretion defects, leading us to hypothesize that there is transcriptional cross talk between these two nuclear receptors. Here, we demonstrate specific HNF4alpha activation of a reporter plasmid containing the COUP-TFII gene promoter region in transfected pancreatic beta cells. The stable association of the endogenous HNF4alpha with a region of the COUP-TFII gene promoter that contains a direct repeat 1 (DR-1) binding site was revealed by chromatin immunoprecipitation. Mutation experiments showed that this DR-1 site is essential for HNF4alpha transactivation of COUP-TFII. The dominant negative suppression of HNF4alpha function decreased endogenous COUP-TFII expression, and the specific inactivation of COUP-TFII by small interfering RNA caused HNF4alpha mRNA levels in 832/13 INS-1 cells to decrease. This positive regulation of HNF4alpha by COUP-TFII was confirmed by the adenovirus-mediated overexpression of human COUP-TFII (hCOUP-TFII), which increased HNF4alpha mRNA levels in 832/13 INS-1 cells and in mouse pancreatic islets. Finally, hCOUP-TFII overexpression showed that there is direct COUP-TFII autorepression, as COUP-TFII occupies the proximal DR-1 binding site of its own gene in vivo. Therefore, COUP-TFII may contribute to the control of insulin secretion through the complex HNF4alpha/maturity-onset diabetes of the young 1 (MODY1) transcription factor network operating in beta cells.

A long-standing paradox in the pathophysiology of metabolic diseases is the selective insulin resistance of the liver. It is characterized by a blunted action of insulin to reduce glucose production, contributing to hyperglycemia, while de novo lipogenesis remains insulin sensitive, participating in turn to hepatic steatosis onset. The underlying molecular bases of this conundrum are not yet fully understood. Here, we established a model of selective insulin resistance in mice by silencing an inhibitor of insulin receptor catalytic activity, the growth factor receptor binding protein 14 (Grb14) in liver. Indeed, Grb14 knockdown enhanced hepatic insulin signaling but also dramatically inhibited de novo fatty acid synthesis. In the liver of obese and insulin-resistant mice, downregulation of Grb14 markedly decreased blood glucose and improved liver steatosis. Mechanistic analyses showed that upon Grb14 knockdown, the release of p62/sqstm1, a partner of Grb14, activated the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2), which in turn repressed the lipogenic nuclear liver X receptor (LXR). Our study reveals that Grb14 acts as a new signaling node that regulates lipogenesis and modulates insulin sensitivity in the liver by acting at a cross-road between the insulin receptor and the p62-Nrf2-LXR signaling pathways.

Hepatic glucokinase (GK) catalyzes the phosphorylation of glucose to glucose 6-phosphate (G6P), a step which is essential for glucose metabolism in liver as well as for the induction of glycolytic and lipogenic genes. The sterol regulatory element-binding protein-1c (SREBP-1c) has emerged as a major mediator of insulin action on hepatic gene expression, but the extent to which its transcriptional effect is caused by an increased glucose metabolism remains unclear. Through the use of hepatic GK knockout mice (hGK-KO) we have shown that the acute stimulation by glucose of l-pyruvate kinase (l-PK), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and Spot 14 genes requires GK expression. To determine whether the effect of SREBP-1c requires GK expression and subsequent glucose metabolism, a transcriptionally active form of SREBP-1c was overexpressed both in vivo and in primary cultures of control and hGK-KO hepatocytes. Our results demonstrate that the synergistic action of SREBP-1c and glucose metabolism via GK is necessary for the maximal induction of l-PK, ACC, FAS, and Spot 14 gene expression. Indeed, in hGK-KO hepatocytes overexpressing SREBP-1c, the effect of glucose on glycolytic and lipogenic genes is lost because of the impaired ability of these hepatocytes to efficiently metabolize glucose, despite a marked increase in low K(m) hexokinase activity. Our studies also reveal that the loss of glucose effect observed in hGK-KO hepatocytes is associated with a decreased in the carbohydrate responsive element-binding protein (ChREBP) gene expression, a transcription factor suggested to mediate glucose signaling in liver. Decreased ChREBP gene expression, achieved using small interfering RNA, results in a loss of glucose effect on endogenous glycolytic (l-PK) and lipogenic (FAS, ACC) gene expression, thereby demonstrating the direct implication of ChREBP in glucose action. Together these results support a model whereby both SREBP-1c and glucose metabolism, acting via ChREBP, are necessary for the dietary induction of glycolytic and lipogenic gene expression in liver.

Background & Aims Despite major public health concern, therapy for non-alcoholic fatty liver, the liver manifestation of the metabolic syndrome often associated with insulin resistance (IR), remains elusive. Strategies aiming to decrease liver lipogenesis effectively corrected hepatic steatosis and IR in obese animals. However, they also indirectly increased mitochondrial long-chain fatty acid oxidation (mFAO) by decreasing malonyl-CoA, a lipogenic intermediate, which is the allosteric inhibitor of carnitine palmitoyltransferase 1 (CPT1A), the key enzyme of mFAO. We thus addressed whether enhancing hepatic mFAO capacity, through a direct modulation of liver CPT1A/malonyl-CoA partnership, can reverse an already established hepatic steatosis and IR in obese mice. Methods Adenovirus-mediated liver expression of a malonyl-CoA-insensitive CPT1A (CPT1mt) in high-fat/high-sucrose (HF/HS) diet-induced or genetically ( ob / ob ) obese mice was followed by metabolic and physiological investigations. Results In association with increased hepatic mFAO capacity, liver CPT1mt expression improved glucose tolerance and insulin response to a glucose load in HF/HS and ob / ob mice, showing increased insulin sensitivity, and corrected IR in ob / ob mice. Surprisingly, hepatic steatosis was not affected in CPT1mt-expressing obese mice, indicating a clear dissociation between hepatic steatosis and IR. Moreover, liver CPT1mt expression rescued HF/HS-induced impaired hepatic insulin signaling at the level of IRS-1, IRS-2, Akt, and GSK-3β, most likely through the observed decrease in the HF/HS-induced accumulation of lipotoxic lipids, oxidative stress, and JNK activation. Conclusions Enhancing hepatic mFAO capacity is sufficient to reverse a state of IR and glucose intolerance in obese mice independently of hepatic steatosis.

Obesity is a metabolic disorder often associated with type 2 diabetes, insulin resistance, and hepatic steatosis. Leptin-deficient (ob/ob) mice are a well-characterized mouse model of obesity in which increased hepatic lipogenesis is thought to be responsible for the phenotype of insulin resistance. We have recently demonstrated that carbohydrate responsive element–binding protein (ChREBP) plays a key role in the control of lipogenesis through the transcriptional regulation of lipogenic genes, including acetyl-CoA carboxylase and fatty acid synthase. The present study reveals that ChREBP gene expression and ChREBP nuclear protein content are significantly increased in liver of ob/ob mice. To explore the involvement of ChREBP in the physiopathology of hepatic steatosis and insulin resistance, we have developed an adenovirus-mediated RNA interference technique in which short hairpin RNAs (shRNAs) were used to inhibit ChREBP expression in vivo. Liver-specific inhibition of ChREBP in ob/ob mice markedly improved hepatic steatosis by specifically decreasing lipogenic rates. Correction of hepatic steatosis also led to decreased levels of plasma triglycerides and nonesterified fatty acids. As a consequence, insulin signaling was improved in liver, skeletal muscles, and white adipose tissue, and overall glucose tolerance and insulin sensitivity were restored in ob/ob mice after a 7-day treatment with the recombinant adenovirus expressing shRNA against ChREBP. Taken together, our results demonstrate that ChREBP is central for the regulation of lipogenesis in vivo and plays a determinant role in the development of the hepatic steatosis and of insulin resistance in ob/ob mice.

Introduction L’accumulation ectopique des lipides contribue a l’instauration d’une intolerance au glucose et d’une insulinoresistance. Le transport des acides gras a chaine longue (AGCL), catalyse par la carnitine palmitoyltransferase 1 hepatique (CPT1A), represente l’etape majeur du controle de l’oxydation mitochondriale des AGCL suite a l’inhibition de la CPT1A par le malonyl-CoA, un intermediaire de la lipogenese. Le but de l’etude est de determiner si une augmentation de l’oxydation hepatique des AGCL peut contrecarrer la steatose hepatique et l’insulinoresistance. Materiels et methodes Nous avons exprime par approche adenovirale une CPT1 constitutivement active (CPT1mt), i.e. insensible au malonyl-CoA, dans le foie de souris genetiquement obeses (ob/ob) ou rendues obeses via un regime riche en lipides et en saccharose (HFD). Apres 16 jours, les consequences physiologiques et metaboliques ont ete etudiees. Resultats Chez les souris mises sur regime standard (ST), l’expression hepatique de la CPT1mt entraine une augmentation respective de 1,4 et 3 du flux d’oxydation hepatique des AGCL et de la production hepatique de s-hydroxybutyrate. Une augmentation similaire est observee chez les souris HFD-CPT1mt. L’expression de la CPT1mt n’affecte pas la steatose hepatique des souris HFD et ob/ob mais diminue leur intolerance au glucose et leur insulino-resistance. Pour comprendre ces effets, nous avons etudie l’expression genique de plusieurs acteurs impliques dans le metabolisme glucido-lipidique du foie. Nos resultats indiquent que l’expression de la CPT1mt induit des enzymes cles impliquees dans la glycolyse, la lipogenese, l’esterification et l’export des lipoproteines a tres faible densite chez les souris ST et HFD. D’autres etudes sont en cours afin de determiner l’impact de l’expression hepatique de la CPT1mt sur la sensibilite du foie a l’insuline. Conclusion Nos resultats suggerent : 1/l’existence d’une dissociation entre steatose hepatique et insulinoresistance, et 2/que la CPT1A representerait une cible pour ameliorer la tolerance au glucose et la sensibilite a l’insuline chez des souris obeses.

Introduction La steatose hepatique et la resistance a l’insuline sont en augmentation constante dans nos societes industrialisees. Cependant, bien que souvent correlees la relation de causalite entre l’apparition de la steatose hepatique et le developpement de la resistance a l’insuline n’est pas toujours etablie. Le facteur de transcription ChREBP est central dans le controle transcriptionnnel de la voie de synthese de novo des acides gras par la lipogenese, voie metabolique qui contribuerait pour 30 % au stockage des triglycerides dans la steatose. L’objectif de notre etude est de determiner si une elevation de la lipogenese via la surexpression de ChREBP dans le foie de souris entraine une steatose hepatique associee a une resistance a l’insuline. Materiels et methodes Par une approche adenovirale nous avons surexprime une forme constitutivement active de ChREBP dans le foie souris C57Bl/6J. Apres 30 jours, les especes lipidiques hepatiques ont ete dosees par spectrometrie de masse et des tests de sensibilite a l’insuline ont ete realises. Resultats L’induction de la lipogenese via ChREBP entraine une steatose hepatique mais n’est pas, de maniere surprenante, associee au developpement d’une resistance a l’insuline. La composition en acides gras totaux dans le foie des souris surexprimant ChREBP revele une augmentation significative des acides gras mono-insatures, tels que le palmitoleate (C16 : 1, n-7) et l’oleate (C18 : 1, n-9). En culture primaire d’hepatocytes, la surexpression de ChREBP previent les effets deleteres des acides gras satures (palmitate, C16 : 0) sur la signalisation insulinique via une augmentation des concentrations des acides gras mono-insatures insulino-sensibilisateurs neo-synthetises par la voie de la lipogenese. Conclusion La voie de la lipogenese serait une voie de sensibilite a l’insuline en permettant la synthese d’especes lipidiques insulino-protectrices.

Introduction La carnitine palmitoyltransferase 1 (CPT1) represente le site majeur de controle de l’oxydation des acides gras a chaine longue (AGCL) du fait de sa capacite a etre inhibee par le malonyl-CoA, premier intermediaire de la lipogenese. Dans le foie d’animaux obeses ou diabetiques, l’existence d’une lipogenese accrue conduit a une augmentation du malonyl-CoA qui entraine une diminution de l’oxydation mitochondriale des AGCL. Materiels et methodes Nous avons developpe un nouveau modele de souris transgeniques (Tg-CPT1mt) exprimant specifiquement dans le foie (promoteur de la transthyretine) une CPT1 insensible au malonyl-CoA (CPT1mt). Resultats L’expression homozygote de la CPT1mt entraine une letalite embryonnaire. Chez les souris heterozygotes a l’etat nourri, l’expression de la CPT1mt entraine une diminution de la sensibilite de la CPT1 hepatique vis-a-vis du malonyl-CoA similaire a celle observee apres un jeune chez des souris temoins (WT). A l’âge de 3 mois, les souris Tg-CPT1mt presentent a l’etat nourri des concentrations circulantes de triglycerides et d’acides gras libres significativement plus faibles que chez les souris WT. Chez les souris Tg-CPT1mt âgees de 10 mois, seule la triglyceridemie est diminuee et ceci s’accompagne d’une diminution du poids corporel. Quel que soit leur âge, les souris Tg-CPT1mt ont a l’etat nourri une diminution du poids des tissus adipeux (epididymaire, visceral et sous cutane) et du contenu hepatique en glycogene. De plus, la glycemie des souris Tg-CPT1mt est plus faible a l’etat nourri comme a l’etat a jeun. Seules les souris Tg-CPT1mt âgees de 10 mois ont une tolerance au glucose et une sensibilite a l’insuline ameliorees. Chez ces souris, des experiences de clamp euglycemique-hyperinsulinemique suggerent que la diminution de la glycemie observee ne resulte pas d’une diminution de la production hepatique de glucose, mais d’une amelioration de l’utilisation basale de glucose. Conclusion Ces resultats suggerent que les souris Tg-CPT1mt presentent un phenotype evolutif et constituent un modele pertinent pour l’etude des consequences d’une augmentation de l’oxydation des AGCL dans le foie sur l’obesite, l’insulinoresistance et la steatose hepatique.

Introduction L’accumulation de lipides dans des tissus autres que le tissu adipeux contribue a l’instauration d’une intolerance au glucose et d’une insulinoresistance. Du fait de son inhibition par le malonyl-CoA (un intermediaire de la lipogenese), la carnitine palmitoyltransferase 1 (CPT1) mitochondriale represente le site majeur du controle de l’oxydation mitochondriale des acides gras a chaine longue (AGCL). Nous avons precedemment montre que l’expression d’une CPT1 mutee insensible au malonyl-CoA, par une approche adenovirale (Ad-CPT1mt), dans des cultures primaires d’hepatocytes de rat permettait d’abolir les effets du glucose et de l’insuline en reorientant le metabolisme des AGCL vers l’oxydation aux depens de l’esterification sans alterer l’utilisation du glucose. Materiels et methodes Afin de determiner les consequences metaboliques de cette augmentation de la capacite du foie a oxyder les AGCL in vivo, nous avons infecte des souris C57Bl6/J avec 109 particules infectieuses d’Ad-CPT1mt/souris. Resultats Chez des souris sous regime standard, l’injection d’Ad-CPT1mt permet l’expression dans le foie d’une CPT1mt active et insensible au malonyl-CoA au moins pendant 3 semaines. L’expression de cette CPT1mt entraine une augmentation de 43 % du poids du foie, une diminution de 25 % du contenu hepatique en glycogene ainsi qu’une amelioration de la tolerance au glucose des 8 jours post-infection, sans modifier la sensibilite a l’insuline. Des souris placees sur regime hypercalorique riche en lipides et en saccharose pendant 5 mois developpent une obesite, une steatose hepatique et une intolerance au glucose mais pas d’insulinoresistance. L’expression de la CPT1mt chez ces souris n’a pas d’effet sur l’obesite apres 3 semaines mais entraine une augmentation de 56 % du poids du foie et ameliore la tolerance au glucose 15 jours apres l’infection adenovirale. Conclusion Ces resultats indiquent que l’expression de la CPT1mt in vivo dans le foie de souris permet d’ameliorer la tolerance au glucose. Ainsi, la diminution de la sensibilite de la CPT1 au malonyl-CoA pourrait constituer une cible pharmacologique pour ameliorer la tolerance au glucose.

Introduction L’adaptateur moleculaire Grb14 est un inhibiteur endogene de la signalisation de l’insuline qui se lie sur le recepteur active par l’insuline et en bloque l’activite catalytique. L’objectif de notre etude a ete d’inhiber a court terme l’expression de Grb14 dans le foie afin d’en etudier les consequences sur la regulation du metabolisme glucido-lipidique. Materiels et Methodes Un adenovirus exprimant un ARN interferant controle ou dirige contre Grb14 (shGrb14) a ete injecte a des souris C57Bl6 ou ob/ob. Des tests de tolerance au glucose et de sensibilite a l’insuline ont ete realises 4 jours apres l’injection. Les animaux ont ete sacrifies dans differents etats nutritionnels apres 7 jours. Les parametres metaboliques et hepatiques ont ete etudies par RT-PCRq, Western blot et dosages biochimiques. Resultats La signalisation de l’insuline est augmentee dans le foie des souris injectees avec le shGrb14 (pAkt, pERK). Ces souris presentent egalement une amelioration de la tolerance au glucose et une diminution de la glycemie quel que soit leur etat nutritionnel. Ce phenotype est associe a une diminution de l’expression des genes cles de la neoglucogenese (PEPCK, G6Pase) dans le foie des souris a jeun. Parallelement, et de facon inattendue, on observe une diminution du stockage de glycogene ainsi qu’une diminution de l’expression des genes de la lipogenese (SREBP-1c, ChREBP, ACC, FAS, SCD1) dans le foie des souris a l’etat renourri, ce qui entraine une diminution du contenu en triglycerides hepatiques. De plus, l’inhibition de Grb14 dans le foie de souris ob/ob entraine une diminution de 70 % de la steatose hepatique. Discussion Le paradoxe des effets du shGrb14 sur la neoglucogenese d’une part et sur l’inhibition de la synthese de glycogene et la lipogenese d’autre part suggere que Grb14 pourrait controler la signalisation de l’insuline en agissant egalement a une etape distale en aval du recepteur. Conclusion L’inhibition de Grb14 dans le foie entraine une amelioration de l’effet de l’insuline sur la production hepatique de glucose et une inhibition de la lipogenese. Cest effets combines de Grb14 pourraient etre benefiques pour la prevention de l’obesite et du diabete de type 2, pathologies caracterisees par une intolerance au glucose et une steatose hepatique.

Introduction La steatose hepatique non-alcoolique est caracterisee par une accumulation excessive de triglycerides (TG). La persistance de cet etat peut conduire sous l’effet d’un stress oxydant a une steatohepatite non alcoolique (SHNA). Chez l’homme, la SHNA est associee a une lipogenese accrue generatrice de malonyl-CoA (M-CoA), l’inhibiteur de l’enzyme cle de la β-oxydation mitochondriale des acides gras (OAGmit) : la carnitine palmitoyltransferase 1A (CPT1-A). La presente etude a pour but de determiner si une augmentation de l’OAGmit via l’expression d’une CPT1-A, constitutivement active, i.e. Insensible au M-CoA (CPT1mt), dans le foie de souris permet de contrecarrer la SHNA induite par un regime deficient en methionine et choline (DMC). Materiels et Methodes Des souris transgeniques exprimant constitutivement et specifiquement dans le foie une CPT1mt sont mises sur un regime temoin ou DMC pendant 30 jours. Apres sacrifice, l’analyse histologique du foie, le dosage de certains parametres seriques et la mesure de l’expression hepatique de certains genes sont realises. Resultats : Le regime DMC provoque une perte de poids (-39 %) et une fonte du tissu adipeux independamment du genotype des souris. Parallelement, le regime DMC induit chez les souris temoins une steatose hepatique (accumulation de TG), une augmentation du taux circulant d’alanine aminotransferase (ALAT), et une augmentation de l’expression genique des cytochromes P450 CYP4a10 et CYP2e1, de la cytokine TNF-α et de la chimiokine MCP-1 marqueurs de l’inflammation. De maniere interessante, bien qu’il n’y ait aucun effet sur la steatose hepatique, les souris transgeniques CPT1mt mises sur regime DMC montrent une moindre augmentation d’ALAT (-31 %) et de l’expression de TNF-α (-94 %), MCP-1 (-80 %), CYP4a10 (-54 %) et CYP2e1 (- 39 %). Conclusion L’augmentation de l’OAGmit hepatique via l’expression d’une CPT1mt permet, sans diminuer la steatose, d’attenuer la SHNA induite par un regime DCM en contrecarrant la voie microsomale d’oxydation des acides gras catalysee par les CYPs et generatrice de stress oxydant. Ces resultats suggerent que l’augmentation de l’OAGmit hepatique constitue une cible de choix pour attenuer et/ou prevenir le developpement de la SHNA.

Introduction La steatose hepatique non alcoolique, frequemment associee a l’obesite, la resistance a l’insuline et le diabete de type 2, se caracterise par une accumulation excessive de triglycerides (TG) dans le foie. Les acides gras utilises pour la synthese des TG proviennent du pool plasmatique ou des acides gras neosynthetises a partir du glucose par la voie de la lipogenese. Recemment, le facteur de transcription ChREBP a ete identifie comme un determinant central du controle transcriptionnnel de la lipogenese. Ainsi, son inhibition ciblee dans le foie corrige la steatose hepatique des souris ob/ob. L’objectif de notre etude a ete de determiner si la surexpression de ChREBP dans le foie suffit a entrainer une steatose hepatique associee a une resistance a l’insuline. Materiels et methodes Un adenovirus exprimant l’ADN complementaire d’une forme constitutivement active de ChREBP a ete injecte a des souris C57Bl/6J. Apres 7 et 9 jours, des tests de sensibilite a l’insuline et de tolerance au glucose ont ete realises. Le dosage d’especes lipidiques par spectrometrie de masse et la coloration de coupes de foies a l’huile rouge ont permis d’etudier la mise en place d’une eventuelle steatose hepatique. Resultats La surexpression de ChREBP dans le foie induit de facon significative l’expression des genes de la glycolyse (L-PK) et de la lipogenese (ACC, FAS, SCD1) et ceci sans augmentation de l’expression de SREBP-1c, le mediateur des effets transcriptionnels de l’insuline sur ces genes. La surexpression de ChREBP s’accompagne d’une accumulation importante des lipides hepatiques visualises par coloration a l’huile rouge. De facon interessante, les souris surexprimant ChREBP ne sont pas resistantes a l’action de l’insuline et presentent au contraire une tolerance au glucose et une sensibilite a l’insuline ameliorees. Conclusion L’ensemble de nos resultats indique que l’induction de la lipogenese via ChREBP est suffisante pour induire une steatose hepatique mais suggere que l’accumulation intra-hepatique de lipides n’est pas toujours associee a un etat de resistance a l’insuline.

Introduction ChREBP, le mediateur des effets transcriptionnels du glucose, a ete recemment implique dans le developpement de la resistance a l’insuline et de la steatose hepatique chez les souris ob/ob . L’activite de ChREBP est stimulee par le metabolisme du glucose qui controle sa localisation nucleaire et son activite transcriptionnelle en modulant sa phosphorylation. Cependant, des donnees recentes suggerent que d’autres modifications post-traductionnelles seraient necessaires. Notre hypothese est que la O -GlcNAc, glycosylation dont les niveaux dependent du glucose et qui module l’activite de nombreux facteurs de transcription, serait impliquee dans la regulation de ChREBP. Materiels et methodes Pour cela, trois groupes ont ete etudies : 1. des souris a jeun 24 h ; 2. des souris realimentees par un regime hyperglucidique apres la periode de jeune ; 3. des souris a jeun gavees par de la glucosamine, permettant d’augmenter les niveaux de O -GlcNAc. Resultats L’expression de ChREBP est faible a jeun et augmente lorsque les souris sont realimentees par le regime hyperglucidique. L’augmentation de l’expression de ChREBP coincide avec sa dephosphorylation, sa translocation nucleaire et l’induction de ses genes cibles. Le gavage par la glucosamine permet d’augmenter les concentrations proteiques de ChREBP dans le foie de souris a jeun a des niveaux comparables a ceux des souris nourries. Cette induction est concomitante a une augmentation de la glycosylation globale et propre de ChREBP. Cependant, le traitement par la glucosamine ne permet pas l’activation de ChREBP qui reste cytosolique et phosphoryle. Des experiences d’inhibition du proteasome realisees en cultures d’hepatocytes suggerent que ChREBP est degrade par ce biais et que la O -GlcNAc le protege de cette degradation. Discussion En conclusion, la faible glycosylation de ChREBP a jeun entrainerait sa degradation. L’apport en glucides en induisant a la fois la glycosylation et la dephosphorylation de ChREBP contribuerait a la stimulation de son activite. Conclusion Nos resultats, en demontrant le controle de ChREBP par la O -GlcNAc, apportent une meilleure comprehension des mecanismes de regulation de ce facteur cle du metabolisme glucidique et lipidique.