👤 Marthe Moldes

The Constitutive Androstane Receptor (CAR, NR1I3) has been newly described as a regulator of energy metabolism. A relevant number of studies using animal models of obesity suggest that CAR activation could be beneficial on the metabolic balance. However, this remains controversial and the underlying mechanisms are still unknown. This work aimed to investigate the effect of CAR activation on hepatic energy metabolism during physiological conditions, i.e. in mouse models not subjected to metabolic/nutritional stress. Gene expression profiling in the liver of CAR knockout and control mice on chow diet and treated with a CAR agonist highlighted CAR-mediated up-regulations of lipogenic genes, concomitant with neutral lipid accumulation. A strong CAR-mediated up-regulation of the patatin-like phospholipase domain-containing protein 3 (Pnpla3) was demonstrated. Pnpla3 is a gene whose polymorphism is associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD) development. This observation was confirmed in human hepatocytes treated with the antiepileptic drug and CAR activator, phenobarbital and in immortalized human hepatocytes treated with CITCO. Studying the molecular mechanisms controlling Pnpla3 gene expression, we demonstrated that CAR does not act by a direct regulation of Pnpla3 transcription or via the Liver X Receptor but may rather involve the transcription factor Carbohydrate Responsive Element-binding protein. These data provide new insights into the regulation by CAR of glycolytic and lipogenic genes and on pathogenesis of steatosis. This also raises the question concerning the impact of drugs and environmental contaminants in lipid-associated metabolic diseases. Show less

Nonalcoholic fatty liver disease (NAFLD) is associated with all features of the metabolic syndrome. Although deposition of excess triglycerides within liver cells, a hallmark of NAFLD, is associated with a loss of insulin sensitivity, it is not clear which cellular abnormality arises first. We have explored this in mice overexpressing carbohydrate responsive element-binding protein (ChREBP). On a standard diet, mice overexpressing ChREBP remained insulin sensitive, despite increased expression of genes involved in lipogenesis/fatty acid esterification and resultant hepatic steatosis (simple fatty liver). Lipidomic analysis revealed that the steatosis was associated with increased accumulation of monounsaturated fatty acids (MUFAs). In primary cultures of mouse hepatocytes, ChREBP overexpression induced expression of stearoyl-CoA desaturase 1 (Scd1), the enzyme responsible for the conversion of saturated fatty acids (SFAs) into MUFAs. SFA impairment of insulin-responsive Akt phosphorylation was therefore rescued by the elevation of Scd1 levels upon ChREBP overexpression, whereas pharmacological or shRNA-mediated reduction of Scd1 activity decreased the beneficial effect of ChREBP on Akt phosphorylation. Importantly, ChREBP-overexpressing mice fed a high-fat diet showed normal insulin levels and improved insulin signaling and glucose tolerance compared with controls, despite having greater hepatic steatosis. Finally, ChREBP expression in liver biopsies from patients with nonalcoholic steatohepatitis was increased when steatosis was greater than 50% and decreased in the presence of severe insulin resistance. Together, these results demonstrate that increased ChREBP can dissociate hepatic steatosis from insulin resistance, with beneficial effects on both glucose and lipid metabolism. Show less

Carbohydrate-responsive element-binding protein (ChREBP) is a key transcription factor that mediates the effects of glucose on glycolytic and lipogenic genes in the liver. We have previously reported that liver-specific inhibition of ChREBP prevents hepatic steatosis in ob/ob mice by specifically decreasing lipogenic rates in vivo. To better understand the regulation of ChREBP activity in the liver, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAc or O-GlcNAcylation), an important glucose-dependent posttranslational modification playing multiple roles in transcription, protein stabilization, nuclear localization, and signal transduction. O-GlcNAcylation is highly dynamic through the action of two enzymes: the O-GlcNAc transferase (OGT), which transfers the monosaccharide to serine/threonine residues on a target protein, and the O-GlcNAcase (OGA), which hydrolyses the sugar. To modulate ChREBP(OG) in vitro and in vivo, the OGT and OGA enzymes were overexpressed or inhibited via adenoviral approaches in mouse hepatocytes and in the liver of C57BL/6J or obese db/db mice. Our study shows that ChREBP interacts with OGT and is subjected to O-GlcNAcylation in liver cells. O-GlcNAcylation stabilizes the ChREBP protein and increases its transcriptional activity toward its target glycolytic (L-PK) and lipogenic genes (ACC, FAS, and SCD1) when combined with an active glucose flux in vivo. Indeed, OGT overexpression significantly increased ChREBP(OG) in liver nuclear extracts from fed C57BL/6J mice, leading in turn to enhanced lipogenic gene expression and to excessive hepatic triglyceride deposition. In the livers of hyperglycemic obese db/db mice, ChREBP(OG) levels were elevated compared with controls. Interestingly, reducing ChREBP(OG) levels via OGA overexpression decreased lipogenic protein content (ACC, FAS), prevented hepatic steatosis, and improved the lipidic profile of OGA-treated db/db mice. Taken together, our results reveal that O-GlcNAcylation represents an important novel regulation of ChREBP activity in the liver under both physiological and pathophysiological conditions. Show less

The adiponutrin/PNPLA3 (patatin-like phospholipase domain-containing protein 3) variant I148M has recently emerged as an important marker of human fatty liver disease. In order to understand the role of the adiponutrin/PNPLA3 protein, we investigated the regulation of its expression in both human and mouse hepatocytes. Adiponutrin/PNPLA3 and lipogenic enzyme expression was determined by real-time PCR analysis in a wide panel of analysis in vivo in the mouse liver and in vitro in murine hepatocytes and human hepatocyte cell lines infected with ChREBP or SREBP1c-expressing adenoviruses. We show that in the mouse liver, adiponutrin/PNPLA3 gene expression is under the direct transcriptional control of ChREBP (carbohydrate-response element-binding protein) and SREBP1c (sterol regulatory element binding protein1c) in response to glucose and insulin, respectively. In silico analysis revealed the presence of a ChoRE (carbohydrate response element) and of a SRE (sterol response element) binding site on the mouse adiponutrin/PNPLA3 gene promoter. Point mutation analysis in reporter gene assays identified the functional response of these two binding sites in the mouse adiponutrin/PNPLA3 promoter. In contrast, in human immortalized hepatocytes and in HepG2 hepatoma cells, only SREBP1c was able to induce adiponutrin/PNPLA3 expression, whereas ChREBP was unable to modulate its expression. All together, our results suggest that adiponutrin/PNPLA3 is regulated by two key factors of the glycolytic and lipogenic pathways, raising the question of its implication in the metabolism of carbohydrates and lipids. Show less