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.

The Wnt/beta-catenin signalling pathway appears to operate to maintain the undifferentiated state of preadipocytes by inhibiting adipogenic gene expression. To define the mechanisms regulating suppression of Wnt/beta-catenin signalling, we analysed the beta-catenin expression in response to activation of transcription factors that regulate adipogenesis. The results show an extensive down-regulation of nuclear beta-catenin that occurs during the first few days of differentiation of 3T3-L1 preadipocytes and coincides with the induction of the adipogenic transcription factors, C/EBPbeta (CCAAT-enhancer-binding protein) and PPARgamma (peroxisome-proliferator-activated receptor). To assess the role of each of these factors in this process, we conditionally overexpressed C/EBPbeta in Swiss mouse fibroblasts using the TET-off system. Abundant expression of C/EBPbeta alone had minimal effect on beta-catenin expression, whereas expression of C/EBPbeta, in the presence of dexamethasone, induced PPARgamma expression and caused a measurable decrease in beta-catenin. In addition, exposure of cells expressing both C/EBPbeta and PPARgamma to a potent PPARgamma ligand resulted in an even greater decrease in beta-catenin by mechanisms that involve the proteasome. Our studies also suggest a reciprocal relationship between PPARgamma activity and beta-catenin expression, since ectopic production of Wnt-1 in preadipocytes blocked the induction of PPARgamma gene expression. Moreover, by suppressing beta-catenin expression, ectopic expression of PPARgamma in Wnt-1-expressing preadipocytes rescued the block in adipogenesis after their exposure to the PPARgamma ligand, troglitazone.

OBJECTIVECarbohydrate-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.RESEARCH DESIGN AND METHODSO-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 ChREBPOG 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.RESULTSOur 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 ChREBPOG 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, ChREBPOG levels were elevated compared with controls. Interestingly, reducing ChREBPOG levels via OGA overexpression decreased lipogenic protein content (ACC, FAS), prevented hepatic steatosis, and improved the lipidic profile of OGA-treated db/db mice.CONCLUSIONSTaken together, our results reveal that O-GlcNAcylation represents an important novel regulation of ChREBP activity in the liver under both physiological and pathophysiological conditions.

b-catenin signaling can be both a physiological and oncogenic pathway in the liver. It controls compartmentalized gene expression, allowing the liver to ensure its essential metabolic function. It is activated by mutations in 20%-40% of hepatocellular carcinomas (HCCs) with specific metabolic features. We decipher the molecular determinants of b-catenindependent zonal transcription using mice with b-catenin-activated or -inactivated hepatocytes, characterizing in vivo their chromatin occupancy by T-cell factor (Tcf )24 and b-catenin, transcriptome, and metabolome. We find that Tcf-4 DNA bindings depend on bcatenin. Tcf-4/b-catenin binds Wnt-responsive elements preferentially around b-catenininduced genes. In contrast, genes repressed by b-catenin bind Tcf-4 on hepatocyte nuclear factor 4 (Hnf-4)-responsive elements. b-Catenin, Tcf-4, and Hnf-4a interact, dictating bcatenin transcription, which is antagonistic to that elicited by Hnf-4a. Finally, we find the drug/bile metabolism pathway to be the one most heavily targeted by b-catenin, partly through xenobiotic nuclear receptors. Conclusions: b-catenin patterns the zonal liver together with Tcf-4, Hnf-4a, and xenobiotic nuclear receptors. This network represses lipid metabolism and exacerbates glutamine, drug, and bile metabolism, mirroring HCCs with b-catenin mutational activation. (HEPATOLOGY 2014;59:2344-2357 See Editorial on Page 2080 T he adult liver is a quiescent organ, fully compartmentalized to accomplish its crucial metabolic role. Its vasculature gives rise to two distinct hepatocyte populations: one located in the vicinity of the portal vein and the other around the central vein. 1 Pericentral (PC) hepatocyte metabolism is complementary to that of periportal (PP) hepatocytes in terms of energy, ammonia, and xenobiotic metabolism. This complementarity arises as a result of the production of distinct specialized proteins in the two zones. 1,2 It has been demonstrated that the Wnt/b-catenin pathway is the master transcriptional regulator of this zonal metabolism, and that control is rendered by a Wnt morphogenetic concentration gradient high in PC hepatocytes and decreasing toward PP hepatocytes. 3,4

Tumor necrosis factor-␣ (TNF␣) is involved in the physiological and biological abnormalities found in two opposite metabolic situations: cachexia and obesity. In an attempt to identify novel genes and proteins that could mediate the effects of TNF␣ on adipocyte metabolism and development, we have used a differential display technique comparing 3T3-L1 cells exposed or not to the cytokine. We have isolated a novel adipose cDNA encoding a TNF␣-inducible 470-amino acid protein termed TIARP, with six putative transmembrane regions flanked by a large amino-terminal and a short carboxyl-terminal domain, a structure reminiscent of channel and transporter proteins. Commitment into the differentiation process is required for cytokine responsiveness. The differentiation process per se is accompanied by a sharp emergence of TIARP mRNA transcripts, in parallel with the expression of the protein at the plasma membrane. Transcripts are present at high levels in white and brown adipose tissues, and are also detectable in liver, kidney, heart, and skeletal muscle. Whereas the biological function of TIARP is presently unknown, its pattern of expression during adipose conversion and in response to TNF␣ exposure as a transmembrane protein mainly located at the cell surface suggest that TIARP might participate in adipocyte development and metabolism and mediate some TNF␣ effects on the fat cell as a channel or a transporter. Tumor necrosis factor-alpha (TNF␣)1 exerts a wide range of effects on cells and tissues. In addition to its immunological functions, TNF␣ also markedly alters adipose tissue development and metabolism. Surprisingly, TNF␣ seems to be involved in the pathophysiology of two opposite metabolic disorders (1). High plasma levels of TNF␣ likely play an important role in the onset of cachectic states observed during cancer or severe infectious diseases (2). By contrast, more recent studies have indicated that the cytokine is overexpressed in adipose tissue of obese rodents or humans, and that this locally produced TNF␣ may be involved in the obesity-linked insulin resistance (3). Thus, since abnormalities in its production or action are associated with alterations in body fat mass, TNF␣ is likely an important effector of adipose tissue development and metabolism in vivo.Many in vitro studies also support the view that TNF␣ has profound effects on lipid metabolism and adipocyte differentiation. TNF␣ was reported to inhibit lipid storage by reducing synthesis and activity of several proteins essential for lipogenesis, such as lipoprotein lipase (4) and acetyl-coenzyme A carboxylase (5), or by inhibition in the expression and/or function of the insulin-sensitive glucose transporter GLUT4 pathway (6). Otherwise TNF␣ is able to stimulate lipolysis in adipocytes by different mechanisms (7,8). In addition to the above effects on lipid storage or mobilization, TNF␣ potently inhibits adipose conversion and even causes a dramatic de-differentiation of adipocytes in culture (9). Prevention of adipose conversion by TNF␣ has been es...

Background & Aims: In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis, a regulated form of necrotic cell death mediated by the receptorinteracting protein kinase (RIPK) 1. We herein assessed the potential of RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL), as therapeutic targets and markers of activity in NAFLD. Methods: C57/BL6J-mice were fed a normal chow diet (NCD) or high fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in either a prophylactic or a curative treatment of HFD-fed mice, and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. Results: Both prophylactic and curative treatments of HFD-fed mice with RIPA-56, caused a down-regulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide (NSA), a specific inhibitor of human MLKL, and by knocking out (KO) MLKL in fat-loaded AML-12 mouse hepatocytes. MLKL KO in steatotic hepatocytes, caused an activation of the mitochondrial respiration, and an increase in b-oxidation. Along with MLKL decreased activation, RIPK3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with the activity. Conclusion: The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis by an MLKL-dependent mechanism that involves at least partly an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD.

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Adiponutrin is a newly identified nonsecreted adipocyte protein regulated by changes in energy balance in rodents. We documented the influence of energy balance modification on adiponutrin gene expression in humans. We investigated the mRNA expression in sc adipose tissue of nonobese women and in obese women during 2-d very low-calorie diet (VLCD) and subsequent refeeding as well as before and after a VLCD of 3 wk (21-d VLCD). The adiponutrin mRNA levels of the nonobese and obese women were not different (P > 0.05). Two-day VLCD reduced the average level of adiponutrin mRNA expression by 36% (P ‫؍‬ 0.0016), whereas refeeding elevated the mRNA level by 31% (P ‫؍‬ 0.004). The 3-wk VLCD caused a dramatic 58% fall of the adiponutrin mRNA expression level (P ‫؍‬ 0.001). The mRNA level was negatively correlated with fasting glucose (Rho ‫؍‬ ؊0.62; P < 0.0001), and subjects with high adiponutrin mRNA level had an increased insulin sensitivity. A DIPOSE TISSUE IS a highly active organ with numerous metabolic and endocrine functions (1, 2). Adipocyte-produced proteins include secreted (e.g. leptin, adiponectin, TNF-␣, IL-6, resistin, etc.) and nonsecreted (e.g. hormone-sensitive lipase, perilipin, glucose transporter GLUT4, etc.) molecules. It has been documented that adipocyte-produced proteins have a wide range of effects on energy homeostasis, carbohydrate and lipid metabolism, inflammation, and immunity as well as on insulin sensitivity. Dysregulated expression and/or dysfunction of genes encoding adipocyte-synthesized factors have been found in obesity and obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, and dyslipidemia (3-7). Clinical investigations examining the physiology and pathophysiology of adipocyte-produced proteins are therefore important for the understanding of complex human metabolic diseases such as obesity, diabetes mellitus, hypertension, and related cardiovascular complications (8, 9).A novel nonsecreted adipocyte protein, adiponutrin, was recently discovered in a preadipose cell line and found to be mainly expressed in adipose tissue (10). Identified as a transmembrane protein, adiponutrin is composed of 413 amino acid residues. The corresponding 3.2-kb mRNA appears early and is markedly increased during in vitro 3T3-L1 preadipocyte differentiation (10). In rodents, adiponutrin gene expression is regulated by changes in nutrition and energy balance. Its level decreases upon fasting and rapidly increases with refeeding or feeding with a high-carbohydrate diet (10, 11). Moreover, an increased expression of adiponutrin is observed in brown and white adipose tissues in (fa/fa) obese Zucker rat (10). Although the adiponutrin function is still unknown, these features suggest a possible contribution of adiponutrin to energy homeostasis and adipocyte function.In humans, there is no available information regarding adiponutrin gene expression in adipose tissue, and its regulation by the variation of energy balance is unknown. Using real time quantitative RT-PCR, we investigat...