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The gut microbiota participates in the control of energy homeostasis partly through fermentation of dietary fibers hence producing short-chain fatty acids (SCFAs), which in turn promote the secretion of the incretin Glucagon-Like Peptide-1 (GLP-1) by binding to the SCFA receptors FFAR2 and FFAR3 on enteroendocrine L-cells. We have previously shown that activation of the nuclear Farnesoid X Receptor (FXR) decreases the L-cell response to glucose. Here, we investigated whether FXR also regulates the SCFA-induced GLP-1 secretion. GLP-1 secretion in response to SCFAs was evaluated ex vivo in murine colonic biopsies and in colonoids of wild-type (WT) and FXR knock-out (KO) mice, in vitro in GLUTag and NCI-H716 L-cells activated with the synthetic FXR agonist GW4064 and in vivo in WT and FXR KO mice after prebiotic supplementation. SCFA-induced GLP-1 secretion was blunted in colonic biopsies from GW4064-treated mice and enhanced in FXR KO colonoids. In vitro FXR activation inhibited GLP-1 secretion in response to SCFAs and FFAR2 synthetic ligands, mainly by decreasing FFAR2 expression and downstream Gαq-signaling. FXR KO mice displayed elevated colonic FFAR2 mRNA levels and increased plasma GLP-1 levels upon local supply of SCFAs with prebiotic supplementation. Our results demonstrate that FXR activation decreases L-cell GLP-1 secretion in response to inulin-derived SCFA by reducing FFAR2 expression and signaling. Inactivation of intestinal FXR using bile acid sequestrants or synthetic antagonists in combination with prebiotic supplementation may be a promising therapeutic approach to boost the incretin axis in type 2 diabetes.

Background & Aims Peroxisome proliferator-activated receptors (PPARs) have been implicated in non-alcoholic steatohepatitis (NASH) pathogenesis, mainly based on animal data. Gene expression data in NASH patients are scarce. We studied liver PPARα, β/δ, and γ expression in a large cohort of obese patients assessed for presence of NAFLD at baseline and 1year follow-up. Methods Patients presented to the obesity clinic underwent a hepatic work-up. If NAFLD was suspected, liver biopsy was performed. Gene expression was studied by mRNA quantification. Patients were reassessed after 1year. Results 125 patients were consecutively included in the study, of which 85 patients had paired liver biopsy taken at 1year of follow-up. Liver PPARα expression negatively correlated with the presence of NASH ( p =0.001) and with severity of steatosis ( p =0.003), ballooning ( p =0.001), NASH activity score ( p =0.008) and fibrosis ( p =0.003). PPARα expression was positively correlated to adiponectin (R 2 =0.345, p =0.010) and inversely correlated to visceral fat (R 2 =−0.343, p 2 =−0.411, p 2 =−0.233, p =0.012). Liver PPARβ/δ and PPARγ expression did not correlate with any histological feature nor with glucose metabolism or serum lipids. At 1year, correlation of PPARα expression with liver histology was confirmed. In longitudinal analysis, an increase in expression of PPARα and its target genes was significantly associated with histological improvement ( p =0.008). Conclusion Human liver PPARα gene expression negatively correlates with NASH severity, visceral adiposity and insulin resistance and positively with adiponectin. Histological improvement is associated with an increase in expression of PPARα and its target genes. These data might suggest that PPARα is a potential therapeutic target in NASH.

Context: Bile acids (BAs) are signaling molecules controlling energy homeostasis that can be both toxic and protective for the liver. BA alterations have been reported in obesity, insulin resistance (IR), and nonalcoholic steatohepatitis (NASH). However, whether BA alterations contribute to NASH independently of the metabolic status is unclear. Objective: To assess BA alterations associated with NASH independently of body mass index and IR. Design and Setting: Patients visiting the obesity clinic of the Antwerp University Hospital (a tertiary referral facility) were recruited from 2006 to 2014. Patients: Obese patients with biopsy-proven NASH (n = 32) and healthy livers (n = 26) were matched on body mass index and homeostasis model assessment of IR. Main Outcome Measures: Transcriptomic analyses were performed on liver biopsies. Plasma concentrations of 21 BA species and 7 alpha-hydroxy-4-cholesten-3-one, a marker of BA synthesis, were determined by liquid chromatography-tandem mass spectrometry. Plasma fibroblast growth factor 19 was measured by enzyme-linked immunosorbent assay. Results: Plasma BA concentrations did not correlate with any hepatic lesions, whereas, as previously reported, primary BA strongly correlated with IR. Transcriptomic analyses showed unaltered hepatic BA metabolism in NASH patients. In line, plasma 7 alpha-hydroxy-4-cholesten-3-one was unchanged in NASH. Moreover, no sign of hepaticBAaccumulation or activation of BA receptors-farnesoid X, pregnane X, and vitamin Dreceptors-was found. Finally, plasma fibroblast growth factor 19, secondary-to-primary BA, and free-to-conjugated BA ratios were similar, suggesting unaltered intestinal BA metabolism and signaling. Conclusions: In obese patients, BA alterations are related to the metabolic phenotype associated with NASH, especially IR, but not liver necroinflammation.

Type 2 diabetes (T2D) is hallmarked by insulin resistance, impaired insulin secretion, and increased hepatic glucose production. The worldwide increasing prevalence of T2D calls for efforts to understand its pathogenesis in order to improve disease prevention and management. Recent genome-wide association studies have revealed strong associations between the CDKN2A/B locus and T2D risk. The CDKN2A/B locus contains genes encoding cell cycle inhibitors, including p16Ink4a, which have not yet been implicated in the control of hepatic glucose homeostasis. Here, we show that p16Ink4a deficiency enhances fasting-induced hepatic glucose production in vivo by increasing the expression of key gluconeogenic genes. p16Ink4a downregulation leads to an activation of PKA-CREB-PGC1α signaling through increased phosphorylation of PKA regulatory subunits. Taken together, these results provide evidence that p16Ink4a controls fasting glucose homeostasis and could as such be involved in T2D development.

Intestinal bile acid (BA) sequestration efficiently lowers plasma glucose concentrations in type 2 diabetes (T2D) patients. Because BAs act as signaling molecules via receptors, including the G protein-coupled receptor TGR5 and the nuclear receptor FXR (farnesoid X receptor), to regulate glucose homeostasis, BA sequestration, which interrupts the entero-hepatic circulation of BAs, constitutes a plausible action mechanism of BA sequestrants. An increase of intestinal L-cell glucagon-like peptide-1 (GLP-1) secretion upon TGR5 activation is the most commonly proposed mechanism, but recent studies also argue for a direct entero-hepatic action to enhance glucose utilization. We discuss here recent findings on the mechanisms of sequestrant-mediated glucose lowering via an increase of splanchnic glucose utilization through entero-hepatic FXR signaling.

Bile acid metabolism is intimately linked to the control of energy homeostasis and glucose and lipid metabolism. The nuclear receptor farnesoid X receptor (FXR) plays a major role in the enterohepatic cycling of bile acids, but the impact of nutrients on bile acid homeostasis is poorly characterized. Metabolically active hepatocytes cope with increases in intracellular glucose concentrations by directing glucose into storage (glycogen) or oxidation (glycolysis) pathways, as well as to the pentose phosphate shunt and the hexosamine biosynthetic pathway. Here we studied whether the glucose nonoxidative hexosamine biosynthetic pathway modulates FXR activity. Our results show that FXR interacts with and is O-GlcNAcylated by O-GlcNAc transferase in its N-terminal AF1 domain. Increased FXR O-GlcNAcylation enhances FXR gene expression and protein stability in a cell type-specific manner. High glucose concentrations increased FXR O-GlcNAcylation, hence its protein stability and transcriptional activity by inactivating corepressor complexes, which associate in a ligand-dependent manner with FXR, and increased FXR binding to chromatin. Finally, in vivo fasting-refeeding experiments show that FXR undergoes O-GlcNAcylation in fed conditions associated with increased direct FXR target gene expression and decreased liver bile acid content. Conclusion: FXR activity is regulated by glucose fluxes in hepatocytes through a direct posttranslational modification catalyzed by the glucose-sensing hexosamine biosynthetic pathway. (Hepatology 2014;59:2022–2033)

Gene polymorphisms of TCF7L2 are associated with increased risk of type 2 diabetes and transcription factor 7-like 2 (TCF7L2) plays a role in hepatic glucose metabolism. We therefore addressed the impact of TCF7L2 isoforms on hepatocyte nuclear factor 4α (HNF4α) and the regulation of gluconeogenesis genes.Liver TCF7L2 transcripts were analysed by quantitative PCR in 33 non-diabetic and 31 type 2 diabetic obese individuals genotyped for TCF7L2 rs7903146. To analyse transcriptional regulation by TCF7L2, small interfering RNA transfection, luciferase reporter and co-immunoprecipitation assays were performed in human hepatoma HepG2 cells.In livers of diabetic compared with normoglycaemic individuals, five C-terminal TCF7L2 transcripts showed increased expression. The type 2 diabetes risk allele of rs7903146 positively correlated with TCF7L2 expression in livers from normoglycaemic individuals only. In HepG2 cells, transcript and TCF7L2 protein levels were increased upon incubation in high glucose and insulin. Of the exon 13 transcripts, six were increased in a glucose dose-responsive manner. TCF7L2 transcriptionally regulated 29 genes related to glucose metabolism, including glucose-6-phosphatase. In cultured HepG2 cells, TCF7L2 did not regulate HNF4Α and FOXO1 transcription, but did affect HNF4α protein expression. The TCF7L2 isoforms T6 and T8 (without exon 13 and with exon 15/14, respectively) specifically interacted with HNF4α.The different levels of expression of alternative C-terminal TCF7L2 transcripts in HepG2 cells, in livers of normoglycaemic individuals carrying the rs7901346 type 2 diabetes risk allele and in livers of diabetic individuals suggest that these transcripts play a role in the pathophysiology of type 2 diabetes. We also report for the first time a protein interaction in HepG2 cells between HNF4α and the T6 and T8 isoforms of TCF7L2, which suggests a distinct role for these specific alternative transcripts.

Objective Mechanisms explaining the relationship in non-alcoholic fatty liver disease (NAFLD), obesity, and insulin resistance are poorly understood. A genetic basis has been suggested. We studied the association between the genes patatin-like phospholipase domain-containing protein 3 (PNPLA3) and apolipoprotein C3 (APOC3) and metabolic and histological parameters of NAFLD in obese patients. Design and Methods Overweight and obese patients underwent a metabolic and liver assessment. If NAFLD was suspected, liver biopsy was proposed. APOC3 variant rs2854117 and PNPLA3 variant rs738409 were genotyped. Results Four hundred seventy patients were included (61.1% had liver biopsy). The percentage of patients with non-alcoholic steatohepatitis (NASH) was significantly different according to the PNPLA3 variant. After adjustment for age and body mass index, the PNPLA3 variant was associated with alanine aminotransferase (P < 0.001) and aspartate aminotransferase (P < 0.001). The PNPLA3 variant was associated with more severe features of steatohepatitis: steatosis (P < 0.001), lobular inflammation (P < 0.001), and ballooning (P = 0.002), but not with liver fibrosis, anthropometry, or insulin resistance. No significant difference in liver histology, anthropometric, or metabolic parameters was found between carriers and non-carriers of the APOC3 variant. Conclusions PNPLA3 polymorphism rs738409 was associated with NASH and the severity of necroinflammatory changes independently of metabolic factors. No association between APOC3 gene variant rs2854117 and histological or metabolic parameters of NAFLD was found.

Adaptation to fasting involves both Glucocorticoid Receptor (GRα) and Peroxisome Proliferator-Activated Receptor α (PPARα) activation. Given both receptors can physically interact we investigated the possibility of a genome-wide cross-talk between activated GR and PPARα, using ChIP- and RNA-seq in primary hepatocytes. Our data reveal extensive chromatin co-localization of both factors with cooperative induction of genes controlling lipid/glucose metabolism. Key GR/PPAR co-controlled genes switched from transcriptional antagonism to cooperativity when moving from short to prolonged hepatocyte fasting, a phenomenon coinciding with gene promoter recruitment of phosphorylated AMP-activated protein kinase (AMPK) and blocked by its pharmacological inhibition. In vitro interaction studies support trimeric complex formation between GR, PPARα and phospho-AMPK. Long-term fasting in mice showed enhanced phosphorylation of liver AMPK and GRα Ser211. Phospho-AMPK chromatin recruitment at liver target genes, observed upon prolonged fasting in mice, is dampened by refeeding. Taken together, our results identify phospho-AMPK as a molecular switch able to cooperate with nuclear receptors at the chromatin level and reveal a novel adaptation mechanism to prolonged fasting.

The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.

International audience; Growth hormone (GH) is a major metabolic regulator that functions by stimulating lipolysis, preventing protein catabolism, and decreasing insulin-dependent glucose disposal. Modulation of hepatic sensitivity to GH and the downstream effects on the GH/IGF1 axis are important events in the regulation of metabolism in response to variations in food availability. For example, during periods of reduced nutrient availability, the liver becomes resistant to GH actions. However, the mechanisms controlling hepatic GH resistance are currently unknown. Here, we investigated the role of 2 tetraspanning membrane proteins, leptin receptor overlapping transcript (LEPROT; also known as OB-RGRP) and LEPROT-like 1 (LEPROTL1), in controlling GH sensitivity. Transgenic mice expressing either human LEPROT or human LEPROTL1 displayed growth retardation, reduced plasma IGF1 levels, and impaired hepatic sensitivity to GH, as measured by STAT5 phosphorylation and Socs2 mRNA expression. These phenotypes were accentuated in transgenic mice expressing both proteins. Moreover, gene silencing of either endogenous Leprot or Leprotl1 in H4IIE hepatocytes increased GH signaling and enhanced cell-surface GH receptor. Importantly, we found that both LEPROT and LEPROTL1 expression were regulated in the mouse liver by physiologic and pathologic changes in glucose homeostasis. Together, these data provide evidence that LEPROT and LEPROTL1 influence liver GH signaling and that regulation of the genes encoding these proteins may constitute a molecular link between nutritional signals and GH actions on body growth and metabolism.

OBJECTIVE—The angiotensin type 1 receptor blocker (ARB) and peroxisome proliferator–activated receptor (PPAR) γ modulator telmisartan has been recently demonstrated to reduce plasma triglycerides in nondiabetic and diabetic hypertensive patients. The present study investigates the molecular mechanisms of telmisartans hypolipidemic actions, in particular its effect on the PPARα pathway. RESEARCH DESIGN AND METHODS—Regulation of PPARα target genes by telmisartan was studied by real-time PCR and Western immunoblotting in vitro and in vivo in liver/skeletal muscle of mice with diet-induced obesity. Activation of the PPARα ligand binding domain (LBD) was investigated using transactivation assays. RESULTS—Telmisartan significantly induced the PPARα target genes carnitine palmitoyl transferase 1A (CPT1A) in human HepG2 cells and acyl-CoA synthetase long-chain family member 1 (ACSL1) in murine AML12 cells in the micromolar range. Telmisartan-induced CPT1A stimulation was markedly reduced after small interfering RNA–mediated knockdown of PPARα. Telmisartan consistently activated the PPARα-LBD as a partial PPARα agonist. Despite high in vitro concentrations required for PPARα activation, telmisartan (3 mg · kg−1 · day−1) potently increased ACSL1 and CPT1A expression in liver from diet-induced obese mice associated with a marked decrease of hepatic and serum triglycerides. Muscular CPT1B expression was not affected. Tissue specificity of telmisartan-induced PPARα target gene induction may be the result of previously reported high hepatic concentrations of telmisartan. CONCLUSIONS—The present study identifies the ARB/PPARγ modulator telmisartan as a partial PPARα agonist. As a result of its particular pharmacokinetic profile, PPARα activation by telmisartan seems to be restricted to the liver. Hepatic PPARα activation may provide an explanation for telmisartan's antidyslipidemic actions observed in recent clinical trials.

Purpose of review Bile acids are amphiphilic molecules synthesized from cholesterol exclusively in the liver that are essential for effective absorption of dietary fat. In addition to this classical role', bile acids act as signalling molecules that control their own metabolism by activating the nuclear receptor, famesoid X receptor. Recent findings Recent work demonstrates that famesoid X receptor exerts metabolic control beyond bile acid homeostasis, notably effects on HDL, triglyceride and glucose metabolism. Farnesoid X receptor influences insulin sensitivity of tissues that are not part of the enterohepatic circulation, for example, adipose tissue. Certain metabolic effects in the liver appear to be mediated via farnesoid X receptor-stimulated release of an intestinal growth factor. In addition, novel signalling pathways independent of farnesoid X receptor have been identified that may contribute to bile acid-mediated metabolic regulation. Summary Farnesoid X receptor represents a potentially attractive target for treatment of various aspects of the metabolic syndrome and for prevention of atherosclerosis. Yet, in view of its pleiotropic effects and apparent species-specificity, it is evident that successful interference of the farnesoid X receptor signalling system will require the development of gene-specific and/or organ-specific famesoid X receptor modulators and extensive testing in human models of disease.

Bile acids are signalling molecules, which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex bile acids in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces glucagon-like peptide-1 (GLP-1) production by L cells which potentiates beta-cell glucose-induced insulin secretion. Whether FXR is expressed in L cells and controls GLP-1 production is unknown. Here, we show that FXR activation in L cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycaemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes.

Conditional gene knockout technology is a powerful tool to study the function of a gene in a specific tissue, organ or cell lineage. The most commonly used procedure applies the Cre-LoxP strategy, where the choice of the Cre driver promoter is critical to determine the efficiency and specificity of the system. However, a considered choice of an appropriate promoter does not always protect against the risk of unwanted recombination and the consequent deletion of the gene in other tissues than the desired one(s), due to phenomena of non-specific activation of the Cre transgene. Furthermore, the causes of these phenomena are not completely understood and this can potentially affect every strain of Cre-mice. In our study on the deletion of a same gene in two different tissues, we show that the incidence rate of non-specific recombination in unwanted tissues depends on the Cre driver strain, ranging from 100 %, rendering it useless (aP2-Cre strain), to ~5 %, which is still compatible with their use (RIP-Cre strain). The use of a simple PCR strategy conceived to detect this occurrence is indispensable when producing a tissue-specific knockout mouse. Therefore, when choosing the Cre-driver promoter, researchers not only have to be careful about its tissue-specificity and timing of activation, but should also include a systematical screening in order to exclude mice in which atypical recombination has occurred and to limit the unnecessary use of laboratory animals in uninterpretable experiments.

Signal transducer and activator of transcription 5 (STAT5) is activated by numerous cytokines that control blood cell development. STAT5 was also shown to actively participate in leukemogenesis. Among the target genes involved in cell growth, STAT5 had been shown to activate cyclin D1 gene expression. We now show that thrombopoietin-dependent activation of the cyclin D1 promoter depends on the integrity of a new bipartite proximal element that specifically binds STAT5A and -B transcription factors. We demonstrate that the stable recruitment of STAT5 to this element in vitro requires the integrity of an adjacent octamer element that constitutively binds the ubiquitous POU homeodomain protein Oct-1. We observe that cytokine-activated STAT5 and Oct-1 form a unique complex with the cyclin D1 promoter sequence. We find that STAT5 interacts with Oct-1 in vivo, following activation by different cytokines in various cellular contexts. This interaction involves a small motif in the carboxy-terminal region of STAT5 which, remarkably, is similar to an Oct-1 POU-interacting motif present in two well-known partners of Oct-1, namely, OBF-1/Bob and SNAP190. Our data offer new insights into the transcriptional regulation of the key cell cycle regulator cyclin D1 and emphasize the active roles of both STAT5 and Oct-1 in this process.

An independent role of nonalcoholic fatty liver disease (NAFLD) in the development of cardiovascular disease has been suggested, probably mediated through increased levels of prothrombotic factors. Therefore, we examined whether NAFLD is linked to a prothrombotic state, independently of metabolic risk factors in a large single-center cohort of overweight/obese patients. Patients presenting to the obesity clinic underwent a detailed metabolic and liver assessment, including an extensive panel of coagulation factors. If NAFLD was suspected, a liver biopsy was proposed. A series of 273 consecutive patients (65% female) with a liver biopsy were included (age, 44 +/- 0.76 years; body mass index: 39.6 +/- 0.40 kg/m(2)). Increase in fibrinogen, factor VIII, and von Willebrand factor and decrease in antithrombin III correlated with metabolic features, but not with liver histology. Levels of plasminogen activator inhibitor-1 (PAI-1) increased significantly with increasing severity of steatosis (P

See related articles, pages 1402–1409 The incidence of obesity-associated disorders, such as type 2 diabetes and the metabolic syndrome, is continuously increasing worldwide.1 These disorders are characterized by abnormalities in glucose and lipid metabolism, putting patients at increased risk for macro- and microvascular complications.2 Although statin treatment, which primarily targets elevated plasma low-density lipoprotein (LDL)-cholesterol levels, lowers cardiovascular morbidity and mortality in patients with type 2 diabetes,3 it is increasingly clear that a significant residual cardiovascular risk remains in these patients,3–5 which is partly attributable to the typical atherogenic lipoprotein profile (ALP) characterized by hypertriglyceridemia and low high-density lipoprotein (HDL)-cholesterol concentrations.6 Post hoc analysis of statin trials, such as PROVE-IT TIMI 22, have identified plasma triglycerides as a determinant of cardiovascular risk in patients achieving LDL-cholesterol goals.5 Plasma triglyceride concentrations are determined by the balance between clearance/uptake and production of triglyceride (TG)-rich lipoproteins (7). Dysregulation of this balance results in the development of hypertriglyceridemia.7 Triglycerides in very-low-density lipoproteins (VLDL) and chylomicrons are hydrolyzed by lipoprotein lipase, thus allowing their conversion to remnant and subsequently to LDL particles. This process is controlled by specific apolipoprotein (apo) constituents, such as apoCII and apoAV, which facilitate TG-rich lipoprotein clearance/lipolysis, whereas apoCIII delays it.8 ApoCIII is a 79-aa glycoprotein synthesized in the liver and the intestine and a major component of the apoB-containing TG-rich lipoproteins and HDL.8 Plasma apoCIII levels are positively correlated with plasma …

International audience; The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes.

Bile acid metabolism is intimately linked to the control of energy homeostasis and glucose and lipid metabolism. The nuclear receptor farnesoid X receptor (FXR) plays a major role in the enterohepatic cycling of bile acids, but the impact of nutrients on bile acid homeostasis is poorly characterized. Metabolically active hepatocytes cope with increases in intracellular glucose concentrations by directing glucose into storage (glycogen) or oxidation (glycolysis) pathways, as well as to the pentose phosphate shunt and the hexosamine biosynthetic pathway. Here we studied whether the glucose nonoxidative hexosamine biosynthetic pathway modulates FXR activity. Our results show that FXR interacts with and is O-GlcNAcylated by O-GlcNAc transferase in its N-terminal AF1 domain. Increased FXR O-GlcNAcylation enhances FXR gene expression and protein stability in a cell type-specific manner. High glucose concentrations increased FXR O-GlcNAcylation, hence its protein stability and transcriptional activity by inactivating corepressor complexes, which associate in a ligand-dependent manner with FXR, and increased FXR binding to chromatin. Finally, in vivo fasting-refeeding experiments show that FXR undergoes O-GlcNAcylation in fed conditions associated with increased direct FXR target gene expression and decreased liver bile acid content.FXR activity is regulated by glucose fluxes in hepatocytes through a direct posttranslational modification catalyzed by the glucose-sensing hexosamine biosynthetic pathway.

Introduction Nous avons recemment demontre que l'activation du recepteur nucleaire aux acides biliaires Farnesoid X Receptor (FXR) dans les cellules enteroendocrines de type L diminue la transcription du proglucagon et la secretion de GLP-1 en reponse au glucose. Il existe de nombreux autres secretagogues de GLP-1. Nous nous interessons en particulier aux acides gras a chaine courte (SCFA), metabolites produits par le microbiote intestinal par fermentation des polysaccharides et des fibres non digeres. Ils se lient a leur recepteur membranaire GPR43 et induisent la secretion de GLP-1 par les cellules L. L'objectif de notre etude est d'etudier le role de FXR dans la reponse des cellules L aux SCFA. Materiels et Methodes FXR a ete active par son agoniste synthetique GW4064 in vitro dans la lignee de cellules L murines GLUTag et in vivo chez la souris. Des tests de secretion de GLP-1 en reponse aux SCFA ont ete realises in vitro avec les cellules L GLUTag et ex vivo avec des explants de colon de ces souris. L'expression du gene codant GPR43 a ete evaluee par qPCR dans le colon des souris traitees par le GW4064, de souris invalidees pour FXR (FXR KO) et de souris traitees avec le colesevelam, sequestrant des acides biliaires. Resultats L'activation de FXR diminue la secretion de GLP-1 en reponse au butyrate (l'un des SCFA) a la fois in vitro par les cellules GLUTag et egalement ex vivo par les explants de colon de souris. En parallele, l'activation de FXR in vivo diminue l'ARNm de GPR43 dans le colon. A l'inverse, les souris FXR KO presentent une augmentation de l'ARNm de GPR43 dans le colon, de meme chez les souris traitees avec le colesevelam. Conclusions L'activation de FXR diminue l'expression de GPR43 et la secretion de GLP-1 en reponse au butyrate, un SCFA, par les cellules L enteroendocrines.

Metabolic diseases reach epidemic proportions. A better knowledge of the associated alterations in the metabolic pathways in the liver is necessary. These studies need in vitro human cell models. Several human hepatoma models are used, but the response of many metabolic pathways to physiological stimuli is often lost. Here, we characterize two human hepatocyte cell lines, IHH and HepaRG, by analysing the expression and regulation of genes involved in glucose and lipid metabolism. Our results show that the glycolysis pathway is activated by glucose and insulin in both lines. Gluconeogenesis gene expression is induced by forskolin in IHH cells and inhibited by insulin in both cell lines. The lipogenic pathway is regulated by insulin in IHH cells. Finally, both cell lines secrete apolipoprotein B-containing lipoproteins, an effect promoted by increasing glucose concentrations. These two human cell lines are thus interesting models to study the regulation of glucose and lipid metabolism.

Liver fat is increased in carriers of the minor G allele in rs738409 (I148M amino acid substitution) in patatin-like phospholipase domain-containing 3 (PNPLA3)/adiponutrin. We studied transcriptional regulation of PNPLA3 in immortalized human hepatocytes (IHH) and human hepatoma cells (HuH7) and the impact of PNPLA3 I148M mutant on hepatocyte triglyceride metabolism. Studies in IHH showed that silencing of the carbohydrate response element-binding protein (ChREBP) abolished induction of PNPLA3 mRNA by glucose. Glucose-dependent binding of ChREBP to a newly identified carbohydrate response element in the PNPLA3 promoter was demonstrated by chromatin immunoprecipitation. Adenoviral overexpression of mouse ChREBP in IHH failed to induce PNPLA3 mRNA. [3H]acetate or [3H]oleate incorporation with 1-h pulse labeling or 18-h [3H]oleate labeling in HuH7 cells showed no effect of PNPLA3 I148M on triglyceride (TG) synthesis in the absence of free fatty acid (FFA) loading. Increased [3H]oleate accumulation into triglycerides in I148M-expressing cells was observed after 18 h of labeling in the presence of 200 μM FFA-albumin complexes. This was accompanied by increased PNPLA3 protein levels. The rate of hydrolysis of [3H]TG during lipid depletion was decreased significantly by PNPLA3 I148M. Our results suggest that PNPLA3 is regulated in human hepatocytes by glucose via ChREBP. PNPLA3 I148M enhances cellular accumulation of [3H]TG in the presence of excess FFA, which is known to stabilize PNPLA3 protein. These data do not exclude an effect of PNPLA3 I148M on hepatocyte lipogenesis but show that the mutant increases the stability of triglycerides.

The liver is a major target of injury in obese patients. Non-alcoholic fatty liver disease (NAFLD) is present in 60-90% of obese Americans and can range from simple steatosis to the more severe non-alcoholic steatohepatitis (NASH). The onset of a chronic inflammatory reaction marks the progression from simple steatosis to NASH and the expansion of adipose tissue is strongly associated with angiogenesis. Therefore, we determined the serum concentration of inflammatory [tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL6)] and angiogenic [vascular endothelial growth factor A (VEGF)] cytokines and soluble VEGF receptors 1 and 2 (sVEGFR1, sVEGFR2) in the serum of an obese population with simple steatosis and NASH compared to healthy controls. Moreover, we determined the TNF alpha, IL6, VEGF, VEGFR1 and VEGFR2 gene expression in the liver of these simple steatosis and NASH patients. The population consisted of 30 obese patients, which were diagnosed with simple steatosis and 32 patients with NASH and compared to 30 age-and-sex matched healthy controls. Mean serum TNF alpha levels were elevated in the serum of simple steatosis and NASH patients compared to healthy controls, reaching significance in NASH patients. IL6 was significantly increased in simple steatosis and NASH patients compared to the healthy controls. VEGF levels were significantly elevated in patients with simple steatosis and borderline significantly elevated in NASH patients compared to the serum levels of healthy control subjects. The concentration of sVEGFR1 was significantly increased in serum of simple steatosis and NASH patients compared to controls. sVEGFR2 concentration was not significantly different in the three groups. TNF alpha mRNA expression was higher in NASH patients compared to simple steatosis patients. Hepatic gene expression of VEGF, VEGFR1 and VEGFR2 were slightly decreased in NASH patients compared to simple steatosis patients. These data indicate the involvement of inflammatory (TNF alpha and IL6), angiogenic (VEGF) cytokines and sVEGFR1 in the pathophysiology of NAFLD. (C) 2012 Elsevier Ltd. All rights reserved.

Objective— Peroxisome proliferator–activated receptor-α (PPARα) is a ligand-activated transcription factor that controls lipid metabolism and inflammation. PPARα is activated by fibrates, hypolipidemic drugs used in the treatment of dyslipidemia. Previous studies assessing the influence of PPARα agonists on atherosclerosis in mice yielded conflicting results, and the implication of PPARα therein has not been assessed. The human apolipoprotein E2 knock-in (apoE2-KI) mouse is a model of mixed dyslipidemia, atherosclerosis, and nonalcoholic steatohepatitis (NASH). The aim of this study was to analyze, using homo- and heterozygous PPARα-deficient mice, the consequences of quantitative variations of PPARα gene levels and their response to the synthetic PPARα agonist fenofibrate on NASH and atherosclerosis in apoE2-KI mice. Methods and Results— Wild-type (+/+), heterozygous (+/−), and homozygous (−/−) PPARα-deficient mice in the apoE2-KI background were generated and subjected to a Western diet supplemented with fenofibrate or not supplemented. Western diet–fed PPARα−/− apoE2-KI mice displayed an aggravation of liver steatosis and inflammation compared with PPARα+/+ and PPARα+/− apoE2-KI mice, indicating a role of PPARα in liver protection. Moreover, PPARα expression was required for the fenofibrate-induced protection against NASH. Interestingly, fenofibrate treatment induced a similar response on hepatic lipid metabolism in PPARα+/+ and PPARα+/− apoE2-KI mice, whereas, for a maximal antiinflammatory response, both alleles of the PPARα gene were required. Surprisingly, atherosclerosis development was not significantly different among PPARα+/+, PPARα+/−, and PPARα−/− apoE2-KI mice. However, PPARα gene level determined both the antiatherosclerotic and vascular antiinflammatory responses to fenofibrate in a dose-dependent manner. Conclusion— These results demonstrate a necessary but quantitatively different role of PPARα in the modulation of liver metabolism, inflammation, and atherogenesis.

International audience; Type 2 diabetes (T2D) is a growing health problem worldwide, but the currently available strategies for therapy and prevention are insufficient. Recent observations indicate that bile acid homeostasis is altered in T2D. Bile acids are metabolic regulators that act as signaling molecules through receptor-dependent and -independent pathways. The most prominent signaling molecules mediating bile acid signaling are the nuclear receptor farnesoid X receptor (FXR) and the membrane receptor TGR5. Both are implicated in the regulation of lipid, glucose, and energy metabolism. Dysregulation of these pathways might contribute to the development of T2D and associated complications. Interestingly, data from studies with bile acids or bile acid sequestrants indicate that the manipulation of bile acid homeostasis might be an attractive approach for T2D therapy. In this review, we summarize the mechanisms of bile acid-mediated metabolic control that might be relevant in the pathogenesis of T2D.

OBJECTIVE Bile acids (BA) participate in the maintenance of metabolic homeostasis acting through different signaling pathways. The nuclear BA receptor farnesoid X receptor (FXR) regulates pathways in BA, lipid, glucose, and energy metabolism, which become dysregulated in obesity. However, the role of FXR in obesity and associated complications, such as dyslipidemia and insulin resistance, has not been directly assessed. RESEARCH DESIGN AND METHODS Here, we evaluate the consequences of FXR deficiency on body weight development, lipid metabolism, and insulin resistance in murine models of genetic and diet-induced obesity. RESULTS FXR deficiency attenuated body weight gain and reduced adipose tissue mass in both models. Surprisingly, glucose homeostasis improved as a result of an enhanced glucose clearance and adipose tissue insulin sensitivity. In contrast, hepatic insulin sensitivity did not change, and liver steatosis aggravated as a result of the repression of β-oxidation genes. In agreement, liver-specific FXR deficiency did not protect from diet-induced obesity and insulin resistance, indicating a role for nonhepatic FXR in the control of glucose homeostasis in obesity. Decreasing elevated plasma BA concentrations in obese FXR-deficient mice by administration of the BA sequestrant colesevelam improved glucose homeostasis in a FXR-dependent manner, indicating that the observed improvements by FXR deficiency are not a result of indirect effects of altered BA metabolism. CONCLUSIONS Overall, FXR deficiency in obesity beneficially affects body weight development and glucose homeostasis.

Objective— Hypertriglyceridemia and fatty liver are common in patients with type 2 diabetes, but the factors connecting alterations in glucose metabolism with plasma and liver lipid metabolism remain unclear. Apolipoprotein CIII (apoCIII), a regulator of hepatic and plasma triglyceride metabolism, is elevated in type 2 diabetes. In this study, we analyzed whether apoCIII is affected by altered glucose metabolism. Methods and Results— Liver-specific insulin receptor–deficient mice display lower hepatic apoCIII mRNA levels than controls, suggesting that factors other than insulin regulate apoCIII in vivo. Glucose induces apoCIII transcription in primary rat hepatocytes and immortalized human hepatocytes via a mechanism involving the transcription factors carbohydrate response element–binding protein and hepatocyte nuclear factor-4α. ApoCIII induction by glucose is blunted by treatment with agonists of farnesoid X receptor and peroxisome proliferator-activated receptor-α but not liver X receptor, ie, nuclear receptors controlling triglyceride metabolism. Moreover, in obese humans, plasma apoCIII protein correlates more closely with plasma fasting glucose and glucose excursion after oral glucose load than with insulin. Conclusion— Glucose induces apoCIII transcription, which may represent a mechanism linking hyperglycemia, hypertriglyceridemia, and cardiovascular disease in type 2 diabetes.

International audience; Farnesoid X receptor (FXR) is highly expressed in liver and intestine where it controls bile acid (BA), lipid and glucose homeostasis. Here we show that FXR is expressed and functional, as assessed by target gene expression analysis, in human islets and beta-cell lines. FXR is predominantly cytosolic-localized in the islets of lean mice, but nuclear in obese mice. Compared to FXR+/+ mice, FXR-/- mice display a normal architecture and beta-cell mass but the expression of certain islet-specific genes is altered. Moreover, glucose-stimulated insulin secretion (GSIS) is impaired in the islets of FXR-/- mice. Finally, FXR activation protects human islets from lipotoxicity and ameliorates their secretory index.

The bile acid receptor farnesoid X receptor (FXR) is expressed in adipose tissue, but its function remains poorly defined. Peroxisome proliferator-activated receptor-gamma (PPAR gamma) is a master regulator of adipocyte differentiation and function. The aim of this study was to analyze the role of FXR in adipocyte function and to assess whether it modulates PPAR gamma action. Therefore, we tested the responsiveness of FXR-deficient mice (FXR-/-) and cells to the PPAR gamma activator rosiglitazone. Our results show that genetically obese FXR-/-/ob/ob mice displayed a resistance to rosiglitazone treatment. In vitro, rosiglitazone treatment did not induce normal adipocyte differentiation and lipid droplet formation in FXR-/- mouse embryonic fibroblasts (MEFs) and preadipocytes. Moreover, FXR-/- MEFs displayed both an increased lipolysis and a decreased de novo lipogenesis, resulting in reduced intracellular triglyceride content, even upon PPAR gamma activation. Retroviral-mediated FXR re-expression in FXR-/- MEFs restored the induction of adipogenic marker genes during rosiglitazone-forced adipocyte differentiation. The expression of Wnt/beta-catenin pathway and target genes was increased in FXR-/- adipose tissue and MEFs. Moreover, the expression of several endogenous inhibitors of this pathway was decreased early during the adipocyte differentiation of FXR-/- MEFs. These findings demonstrate that FXR regulates adipocyte differentiation and function by regulating two counteracting pathways of adipocyte differentiation, the PPAR gamma and Wnt/beta-catenin pathways.

Glucocorticoid receptor alpha (GRalpha) and peroxisome proliferator-activated receptor alpha (PPARalpha) are transcription factors with clinically important immune-modulating properties. Either receptor can inhibit cytokine gene expression, mainly through interference with nuclear factor kappaB (NF-kappaB)-driven gene expression. The present work aimed to investigate a functional cross-talk between PPARalpha- and GRalpha-mediated signaling pathways. Simultaneous activation of PPARalpha and GRalpha dose-dependently enhances transrepression of NF-kappaB-driven gene expression and additively represses cytokine production. In sharp contrast and quite unexpectedly, PPARalpha agonists inhibit the expression of classical glucocorticoid response element (GRE)-driven genes in a PPARalpha-dependent manner, as demonstrated by experiments using PPARalpha wild-type and knockout mice. The underlying mechanism for this transcriptional antagonism relies on a PPARalpha-mediated interference with the recruitment of GRalpha, and concomitantly of RNA polymerase II, to GRE-driven gene promoters. Finally, the biological relevance of this phenomenon is underscored by the observation that treatment with the PPARalpha agonist fenofibrate prevents glucocorticoid-induced hyperinsulinemia of mice fed a high-fat diet. Taken together, PPARalpha negatively interferes with GRE-mediated GRalpha activity while potentiating its antiinflammatory effects, thus providing a rationale for combination therapy in chronic inflammatory disorders.

FXR (farnesoid X receptor), a nuclear receptor activated by BAs (bile acids), is a key factor in the regulation of BA, lipid and carbohydrate metabolism. The recent development of synthetic FXR agonists and knockout mouse models has accelerated the discovery of FXR target genes. In the present study, we identify human fetuin-B as a novel FXR target gene. Treatment with FXR agonists increased fetuin-B expression in human primary hepatocytes and in the human hepatoma HepG2 cell line. In contrast, fetuin-B expression was not responsive to FXR agonist treatment in murine primary hepatocytes. Fetuin-B induction by FXR agonist was abolished upon FXR knockdown by siRNA (small interfering RNA). In addition to the previously described P1 promoter, we show that the human fetuin-B gene is also transcribed from an alternative promoter, termed P2. Transcription via the P2 promoter was induced by FXR agonist treatment, whereas P1 promoter activity was not sensitive to FXR agonist treatment. Two putative FXR-response elements [IR-1 (inverted repeat-1)] were identified in the region –1.6 kb upstream of the predicted P2 transcriptional start site. Both motifs bound FXR–RXR (retinoid X receptor) complexes in vitro and were activated by FXR in transient transfection reporter assays. Mutations in the IR-1 sites abolished FXR–RXR binding and activation. Taken together, these results identify human fetuin-B as a new FXR target gene in human hepatocytes.

Introduction L'invalidation conditionnelle in vivo par la strategie Cre-Lox est un outil puissant pour identifier la fonction d'un gene/d'une proteine specifiquement dans un tissu ou un type cellulaire donne. Le choix du promoteur guidant l'expression de la recombinase Cre est fondamental pour determiner la specificite tissulaire de l'invalidation genique. Cependant, le choix d'un promoteur approprie ne permet pas d'empecher les recombinaisons non-specifiques dues au phenomene d'activation non-specifique du gene codant la Cre dans les tissus non cibles. Materiels et methodes Un meme gene d'interet a ete invalide dans 2 types cellulaires differents (cellule beta pancreatique et adipocyte) en croisant une souche de souris floxee pour le gene d'interet avec respectivement une souche transgenique RIP (Rat Insulin Promoter) -Cre (Tg (Ins2-Cre) 23Herr) et une souche transgenique aP2 (adipocyte protein 2) -Cre (aP2-CreSI). Pour detecter une potentielle recombinaison non-specifique, une recherche systematique de l'allele « nul » (allele floxe et recombine par la l'enzyme Cre-recombinase) en plus de l'allele floxe et de l'allele sauvage a ete realisee par PCR sur l'ADN extrait de biopsie caudale et des principaux tissus metaboliques dans les souris issues des deux elevages. Resultats 1) L'utilisation systematique d'une simple PCR permet de detecter les recombinaisons non-specifiques dans le genotypage sur biopsie caudale, et ainsi d'eliminer les souris problematiques de l'elevage. 2) Le taux de recombi-naison non-specifique est dependant de la souche Cre, allant de 5 % (RIP-Cre, la souche est alors utilisable pour generer les souris invalidees pour le gene d'interet) a 100 % (aP2-Cre, rendant la souche inutilisable). 3) L'analyse de l'ADN de differents tissus metabolique (foie, pancreas, tissus adipeux) par la meme procedure de PCR montre que lorsque la recombinaison non-specifique est presente dans l'ADN caudal, elle est retrouvee aussi dans les autres organes analyses. Conclusion Une recherche systematique de recombinaison non-specifique devrait etre mise en œuvre par simple PCR au cours du genotypage dans toutes les souches murines obtenues par ce systeme. Declaration d’interet Les auteurs declarent ne pas avoir d'interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Recently, several studies have suggested a potential role of FXR in the control of hepatic carbohydrate metabolism, but its contribution to the maintenance of peripheral glucose homeostasis remains to be established. FXR-deficient mice display decreased adipose tissue mass, lower serum leptin concentrations, and elevated plasma free fatty acid levels. Glucose and insulin tolerance tests revealed that FXR deficiency is associated with impaired glucose tolerance and insulin resistance. Moreover, whole-body glucose disposal during a hyperinsulinemic euglycemic clamp is decreased in FXR-deficient mice. In parallel, FXR deficiency alters distal insulin signaling, as reflected by decreased insulin-dependent Akt phosphorylation in both white adipose tissue and skeletal muscle. Whereas FXR is not expressed in skeletal muscle, it was detected at a low level in white adipose tissue in vivo and induced during adipocyte differentiation in vitro. Moreover, mouse embryonic fibroblasts derived from FXR-deficient mice displayed impaired adipocyte differentiation, identifying a direct role for FXR in adipocyte function. Treatment of differentiated 3T3-L1 adipocytes with the FXR-specific synthetic agonist GW4064 enhanced insulin signaling and insulin-stimulated glucose uptake. Finally, treatment with GW4064 improved insulin resistance in genetically obese ob/ob mice in vivo. Although the underlying molecular mechanisms remain to be unraveled, these results clearly identify a novel role of FXR in the regulation of peripheral insulin sensitivity and adipocyte function. This unexpected function of FXR opens new perspectives for the treatment of type 2 diabetes.

mRNA translation is mainly regulated at the level of initiation, a process that involves the synergistic action of the 5′ cap structure and the 3′ poly(A) tail at the ends of eukaryotic mRNA. The eukaryote initiation factor 4G(eIF4G) is a pivotal scaffold protein that forms a critical link between mRNA cap structure, poly(A) tail, and the small ribosomal subunit. There are two functional homologs of eIF4G in mammals, the original eIF4G, renamed eIF4GI, and eIF4GII that functionally complements eIF4GI. To date, biochemical and functional analysis have not identified differential activities for eIF4GI and eIF4GII. In this report, we demonstrate that eIF4GII, but not eIF4GI, is selectively recruited to capped mRNA at the onset of cell differentiation. This recruitment is coincident with a strong and long-lasting phosphorylation of eIF4E and the release of 4E-BP1, a suppressor of eIF4E function, from the cap structure, without a concomitant change in 4E-BP1's phosphorylation. Our data further indicate that cytokines such as thrombopoietin can differentially regulate eIF4GI/II activities. These results provide the first evidence that eIF4GI/II does fulfill selective roles in mammalian cells.

Introduction Des variants genetiques du facteur de transcription TCF7L2 sont fortement associes a la susceptibilite au diabete de type 2 (DT2) et suscitent beaucoup d’interet. Recemment, TCF7L2 a ete implique dans le metabolisme hepatique du glucose. Notre objectif a ete de determiner si les transcrits alternatifs de TCF7L2 inhibent differemment la transcription de l’enzyme glucose-6- phosphatase (G6PC). Materiels et methodes Nous avons evalue l’expression hepatique de TCF7L2 par PCR-quantitative exon-specifique dans des echantillons humains (64 patients obeses normoglycemiques ou DT2 de la cohorte ABOS) et dans les cellules HepG2. L’impact de l’inhibition de TCF7L2 par ARNi sur l’expression de G6PC a ete evalue. Par la technique du gene rapporteur luciferase, nous avons analyse les interactions possibles entre la regulation transcriptionnelle de G6PC par TCF7L2, HNF4alpha et FOXO1. En realisant des co-immunoprecipitations, nous avons analyse les interactions proteiniques de TCF7L2 avec HNF4alpha dans les cellules HepG2. Resultats Dans les cellules HepG2, tous les transcrits de TCF7L2 contenant l’exon 13 sont induits par le glucose de maniere concentration dependante. L’expression de ces transcrits est aussi augmentee dans le foie d’individus diabetiques, sauf ceux ayant un C-terminal-court (T3 et T5). La transfection d’ARNi dans les cellules HepG2, a montre que l’expression de G6PC est reprimee par TCF7L2 independamment de la presence d’insuline et de l’activite de FOXO1. Nous avons pu montrer que l’inhibition de l’expression de HNF4alpha par TCF7L2 n’est pas indispensable pour la repression transcriptionelle de G6PC. Par contre, les transcrits T6 et T8, avec un C-terminal long mais sans exon 13, interagissent avec la proteine HNF4alpha dans les cellules HepG2. Conclusion Les transcrits T3 et T5 de TCF7L2 pourraient jouer un role different dans les anomalies du metabolisme hepatique lie au DT2 en comparaison aux transcrits T6 et T8 qui interagissent specifiquement avec la proteine HNF4alpha dans les cellules HepG2.

Introduction Le diabete de type 2 (T2D) est un trouble metabolique de l'homeostasie du glucose. Il est caracterise par une hyperglycemie chronique qui resulte en partie d'une production excessive de glucose par le foie consequence au developpement d'une resistance a l'insuline. Le T2D est une pathologie multifactorielle a la fois genetique et environnementale. Recemment des etudes d'associations de genes (GWAS) dans differentes cohortes ont mis en evidence une forte correlation entre le locus CDKN2A et le risque de developpement du T2D en se basant sur certains parametres metaboliques tel que la glycemie a jeun. Le locus CDKN2A code pour des proteines regulatrices du cycle cellulaire dont la proteine p16INK4a. p16INK4a est largement decrite dans la litterature pour son role suppresseur de tumeurs et comme marqueur de senescence, cependant son role dans le controle de l'homeostasie hepatique du glucose n'a jamais ete rapporte Materiels et methodes Afin de determiner le role de p16INK4a dans le metabolisme hepatique du glucose, nous avons utilise in vivo des souris sauvages (p16+/+) et deficientes pour p16INK4a (p16-/-) et in vitro des hepatocytes primaires ainsi que la lignee AML12. Resultats Nous avons montre qu'apres un jeune, les souris p16-/- presentent une hypoglycemie moins prononcee qui se traduit par une expression hepatique plus elevee de genes de la neoglucogenese tels que PEPCK, G6Pase et PGC1a. De plus, les hepatocytes primaires de souris p16-/- presentent une meilleur reponse au glucagon que ceux des p16+/+. Enfin, nous avons montre que la diminution d'expression de p16INK4a par siRNA dans les AML12 suffit a induire l'expression des genes de la neoglucogenese et potentialise la reponse de ces cellules a differents stimuli gluconeogenique. L'effet observe depend de l'activation de la voie PKA-CREB-PGC1A. Conclusion L'ensemble de ces donnees montrent pour la premiere fois que p16INK4a pourrait jouer un role un cours du developpement du T2D.

Introduction Des variants genetiques de TCF7L2 ont ete fortement associes a la susceptibilite au diabete de type 2 (DT2). Nous avons montre que plusieurs transcrits alternatifs de TCF7L2 avaient des effets distincts sur la proliferation et la secretion d’insuline des cellules beta pancreatiques d’ilots humains isoles. Recemment, TCF7L2 a ete implique dans le metabolisme du glucose des cellules hepatiques et nous avons egalement observe une expression differentielle des transcrits TCF7L2 dans le foie humain diabetique. Etant donne que l’expression des enzymes cles de la neoglucogenese est fortement regulee par HNF4alpha et FOXO1, notre objectif a ete d’analyser l’interaction des transcrits de TCF7L2 avec ces facteurs de transcription dans les cellules hepatiques HepG2. Materiels et methodes Nous avons evalue l’expression de TCF7L2 par PCR quantitative specifique-au-exon dans les cellules HepG2 cultivees en presence de differentes concentrations de glucose et d’insuline. L’impact de l’inhibition de l’expression de TCF7L2 total ou de ses transcrits par ARNi sur l’expression des genes de la neoglucogenese a ete evalue. Par la technique du gene rapporteur luciferase, nous avons analyse le role de TCF7L2 dans la regulation de la transcription de la glucose-6-phosphatase (G6PC) et les interactions possibles entre TCF7L2, HNF4alpha et FOXO1. Resultats Notre analyse montre que l’expression des transcrits de TCF7L2, dont ceux qui sont induite dans le foie d’individus diabetiques, sont aussi induits dans les cellules HepG2 par glucose, de maniere dependante de la concentration. Grâce a la transfection d’ARNi dans les cellules HepG2, nous avons observe que la transcription de 29 genes de la neoglucogenese, dont G6PC, est reprimee par TCF7L2. La repression de la transcription de G6PC par TCF7L2 est independante de la presence de FOXO1 et de la regulation d’expression de HNF4alpha par TCF7L2. Par contre, certaines TCF7L2 transcrits interagir avec HNF4alpha dans les cellules HepG2. Conclusion Nos resultats suggerent que les transcrits alternatifs de TCF7L2 pourraient jouer un role important dans les anomalies du metabolisme hepatique du glucose dans le DT2.

Introduction L'incidence de l'obesite et des maladies metaboliques en general ne cesse d'augmenter a travers le monde. Ces desordres metaboliques sont associes a des perturbations de l'expression des genes impliques dans le metabolisme lipidique et glucidique et l'inflammation. L'expression d'un certain nombre de ces genes est regulee par les membres de la superfamille des recepteurs nucleaires. Le recepteur nucleaire Farnesoid X Receptor (FXR) est exprime dans le tissu adipeux, cependant son role reste encore inconnu. L'objectif de ce projet est d'elucider le role de FXR dans la fonction adipocytaire et de determiner les mecanismes moleculaires impliques dans ce processus. Resultats Nous montrons ainsi que les cellules MEFs (mouse embryonic fibroblasts) deficientes en FXR (FXR-/-) presentent une augmentation de la lipolyse et une diminution de la neolipogenese par rapport aux cellules MEFs controles (FXR +/+ ) ce qui a pour consequence une diminution des triglycerides stockees. Ce phenotype est associe a une diminution de l'expression des genes marqueurs de l'adipogenese et les genes codant pour les proteines des gouttelettes lipidiques a la fois dans les MEFs FXR -/- et les pre-adipocytes isolees a partir du tissu adipeux des souris FXR -/- . L'introduction de FXR, par la voie d'un retrovirus sur-exprimant FXR, restaure une differentiation adipocytaire normale. L'absence de FXR induit une augmentation de l'expression des genes de la voie Wnt/β-catenine et une augmentation de l'expression de la proteine β-catenine. D'ailleurs, l'expression des genes codant pour les inhibiteurs de cette voie de signalisation sont inhibes dans les MEFs FXR -/- . Un traitement par l'agoniste synthetique de PPARγ, la rosiglitazone, ne restaure pas une differenciation adipocytaire normale et des gouttelettes lipidiques comparables aux MEFs FXR +/+ . De meme, les souris FXR -/- sont resistantes au traitement rosiglitazone en comparaisons aux controles. Conclusion Cette etude montre clairement que FXR controle la differenciation adipocytaire en interferant avec la voie Wnt/β-catenine et la voie d'activation de PPARγ

The role of the nuclear receptor FXR in adaptive thermogenesis was investigated using FXR-deficient mice. Despite elevated serum bile acid concentrations and increased mRNA expression profiles of thermogenic genes in brown adipose tissue, FXR-deficiency did not alter energy expenditure under basal conditions. However, FXR-deficiency accelerated the fasting-induced entry into torpor in a leptin-dependent manner. FXR-deficient mice were also extremely cold-intolerant. These altered responses may be linked to a more rapid decrease in plasma concentrations of metabolic fuels (glucose, triglycerides) thus impairing uncoupling protein 1-driven thermogenesis. These results identify FXR as a modulator of energy homeostasis. (C) 2007 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

The purpose of this study was to determine whether bile acids (BAs) modulate hepatic pro-protein convertase subtilisin/kexin 9 (PCSK9) gene expression. Immortalized human hepatocytes were treated with various BAs. Chenodeoxycholic acid (CDCA) treatment specifically decreased both PCSK9 mRNA and protein contents. Moreover, activation of the BA-activated farnesoid X receptor (FXR) by its synthetic specific agonist GW4064 also decreased PCSK9 expression. Of functional relevance, coadministration of CDCA counteracted the statin-induced PCSK9 expression, leading to a potentiation of LDL receptor activity. This study suggests that a transcriptional repression of PCSK9 by CDCA or FXR agonists may potentiate the hypolipidemic effect of statins.