Adipocyte Glucocorticoid Receptor Activation With High Glucocorticoid Doses Impairs Healthy Adipose Tissue Expansion by Repressing Angiogenesis.

In humans, glucocorticoids (GCs) are commonly prescribed because of their anti-inflammatory and immunosuppressive properties. However, high doses of GCs often lead to side effects, including diabetes and lipodystrophy. We recently reported that adipocyte glucocorticoid receptor (GR)-deficient (AdipoGR-KO) mice under corticosterone (CORT) treatment exhibited a massive adipose tissue (AT) expansion associated with a paradoxical improvement of metabolic health compared with control mice. However, whether GR may control adipose development remains unclear. Here, we show a specific induction of hypoxia-inducible factor 1α (HIF-1α) and proangiogenic vascular endothelial growth factor A (VEGFA) expression in GR-deficient adipocytes of AdipoGR-KO mice compared with control mice, together with an increased adipose vascular network, as assessed by three-dimensional imaging. GR activation reduced HIF-1α recruitment to the Vegfa promoter resulting from Hif-1α downregulation at the transcriptional and posttranslational levels. Importantly, in CORT-treated AdipoGR-KO mice, the blockade of VEGFA by a soluble decoy receptor prevented AT expansion and the healthy metabolic phenotype. Finally, in subcutaneous AT from patients with Cushing syndrome, higher VEGFA expression was associated with a better metabolic profile. Collectively, these results highlight that adipocyte GR negatively controls AT expansion and metabolic health through the downregulation of the major angiogenic effector VEGFA and inhibition of vascular network development.

The Goto-Kakizaki rat is a spontaneous prototypical rodent model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is characterized by an oligo-anovulation, hyperandrogenism and polycystic ovarian morphology combined with major metabolic disturbances. However, despite the high prevalence and the human and economic consequences of this syndrome, its etiology remains unknown. In this study, we show that female Goto-Kakizaki (GK) rats, a type 2 diabetes mellitus model, encapsulate naturally all the reproductive and metabolic hallmarks of lean women with PCOS at puberty and in adulthood. The analysis of their gestation and of their fetuses demonstrates that this PCOS-like phenotype is developmentally programmed. GK rats also develop features of ovarian hyperstimulation syndrome. Lastly, a comparison between GK rats and a cohort of women with PCOS reveals a similar reproductive signature. Thus, this spontaneous rodent model of PCOS represents an original tool for the identification of the mechanisms involved in its pathogenesis and for the development of novel strategies for its treatment.

Insulin activates hepatic Wnt/ß-catenin signaling through stearoyl-CoA desaturase 1 and Porcupine.

The Wnt/ß-catenin pathway plays a pivotal role in liver structural and metabolic homeostasis. Wnt activity is tightly regulated by the acyltransferase Porcupine through the addition of palmitoleate. Interestingly palmitoleate can be endogenously produced by the stearoyl-CoA desaturase 1 (SCD1), a lipogenic enzyme transcriptionally regulated by insulin. This study aimed to determine whether nutritional conditions, and insulin, regulate Wnt pathway activity in liver. An adenoviral TRE-Luciferase reporter was used as a readout of Wnt/ß-catenin pathway activity, in vivo in mouse liver and in vitro in primary hepatocytes. Refeeding enhanced TRE-Luciferase activity and expression of Wnt target genes in mice liver, revealing a nutritional regulation of the Wnt/ß-catenin pathway. This effect was inhibited in liver specific insulin receptor KO (iLIRKO) mice and upon wortmannin or rapamycin treatment. Overexpression or inhibition of SCD1 expression regulated Wnt/ß-catenin activity in primary hepatocytes. Similarly, palmitoleate added exogenously or produced by SCD1-mediated desaturation of palmitate, induced Wnt signaling activity. Interestingly, this effect was abolished in the absence of Porcupine, suggesting that both SCD1 and Porcupine are key mediators of insulin-induced Wnt/ß-catenin activity in hepatocytes. Altogether, our findings suggest that insulin and lipogenesis act as potential novel physiological inducers of hepatic Wnt/ß-catenin pathway.

Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease.

BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. METHODS: C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. RESULTS: When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation 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, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in ß-oxidation in steatotic hepatocytes. Along with decreased MLKL 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 activity. CONCLUSION: The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. LAY SUMMARY: There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity.

WISP1 (Wnt1-inducible signaling pathway protein-1, also known as CCN4) is a member of the CCN family able to mediate cell growth, transformation and survival in a tissue-specific manner. Here, we report that WISP1 expression was highly increased in preadipocytes and decreased during adipocyte differentiation. Moreover, we observed an increase in WISP1 gene expression in adipose tissue from both diet-induced and leptin-deficient ob/ob obese mice, suggesting that WISP1 could be involved in the pathophysiological onset of obesity. Interestingly, overexpression of WISP1 in 3T3-F442A cells prevented adipocyte differentiation via downregulation of peroxisome proliferator-activated receptor (PPARγ) transcriptional activity thereby attenuating the expression of adipogenic markers. Conversely, silencing of WISP1 enhanced adipocyte differentiation. We further show that the inactivation of PPARγ transcriptional activity was mediated, at least in part, by a direct physical association between WISP1 and PPARγ, followed by proteasome-dependent degradation of PPARγ. These results suggest for the first time that WISP1 interacts with PPARγ and that this interaction results in the inhibition of PPARγ activity. Taken together our results suggest that WISP1 functions as a negative regulator of adipogenesis.

NOV/CCN3: A New Adipocytokine Involved in Obesity-Associated Insulin Resistance.

Identification of new adipokines that potentially link obesity to insulin resistance represents a major challenge. We recently showed that NOV/CCN3, a multifunctional matricellular protein, is synthesized and secreted by adipose tissue, with plasma levels highly correlated with BMI. NOV involvement in tissue repair, fibrotic and inflammatory diseases, and cancer has been previously reported. However, its role in energy homeostasis remains unknown. We investigated the metabolic phenotype of NOV(-/-) mice fed a standard or high-fat diet (HFD). Strikingly, the weight of NOV(-/-) mice was markedly lower than that of wild-type mice but only on an HFD. This was related to a significant decrease in fat mass associated with an increased proportion of smaller adipocytes and to a higher expression of genes involved in energy expenditure. NOV(-/-) mice fed an HFD displayed improved glucose tolerance and insulin sensitivity. Interestingly, the absence of NOV was associated with a change in macrophages profile (M1-like to M2-like), in a marked decrease in adipose tissue expression of several proinflammatory cytokines and chemokines, and in enhanced insulin signaling. Conversely, NOV treatment of adipocytes increased chemokine expression. Altogether, these results show that NOV is a new adipocytokine that could be involved in obesity-associated insulin-resistance.

Activation of the Constitutive Androstane Receptor induces hepatic lipogenesis and regulates Pnpla3 gene expression in a LXR-independent way.

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.

Oleuropein activated AMPK and induced insulin sensitivity in C2C12 muscle cells.

AIMS: Oleuropein has been recognized as an important medicinal compound because of its various biological properties, including anti-cancer, antidiabetic and anti-atherosclerotic activities. Here, we evaluate the antioxidant activity as well as the mechanism of the hypoglycemic effects of oleuropein in C2C12 cells and we establish the mechanism underlying these effects. MAIN METHODS: To perform this study, C2C12 cells viability was analyzed via MTT assay and the antioxidant activity was investigated by ROS and TBARS assays. Also, the effect of oleuropein on AMPK and PI3 kinase signaling pathways was evaluated. KEY FINDINGS: Treatment with oleuropein was able to protect cells against H2O2 induced stress in cells. On the other hand, the molecular bases of its actions have been scarcely understood. Oleuropein significantly enhanced glucose consumption and the phosphorylation of AMPK (AMP-activated protein kinase/ACC (acetyl-CoA carboxylase)) and MAPKs (mitogen-activated protein kinases), but not PI3 kinase (Phosphatidylinositol 3-kinase)/Akt. However, the co-treatment of oleuropein and insulin improved the insulin sensitivity via insulin-dependent (PI3 kinase/Akt) and insulin independent (AMPK/ACC) pathways. These results could be confirmed from the findings of GLUT4 translocation which was strongly enhanced in the case of oleuropein. SIGNIFICANCE: Our results provide important insights for the possible mechanism of action of oleuropein as a therapeutic agent in diabetic patients.

T-cell factor 4 and ß-catenin chromatin occupancies pattern zonal liver metabolism in mice.

UNLABELLED: ß-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 ß-catenin-dependent zonal transcription using mice with ß-catenin-activated or -inactivated hepatocytes, characterizing in vivo their chromatin occupancy by T-cell factor (Tcf)-4 and ß-catenin, transcriptome, and metabolome. We find that Tcf-4 DNA bindings depend on ß-catenin. Tcf-4/ß-catenin binds Wnt-responsive elements preferentially around ß-catenin-induced genes. In contrast, genes repressed by ß-catenin bind Tcf-4 on hepatocyte nuclear factor 4 (Hnf-4)-responsive elements. ß-Catenin, Tcf-4, and Hnf-4α interact, dictating ß-catenin transcription, which is antagonistic to that elicited by Hnf-4α. Finally, we find the drug/bile metabolism pathway to be the one most heavily targeted by ß-catenin, partly through xenobiotic nuclear receptors. CONCLUSIONS: ß-catenin patterns the zonal liver together with Tcf-4, Hnf-4α, and xenobiotic nuclear receptors. This network represses lipid metabolism and exacerbates glutamine, drug, and bile metabolism, mirroring HCCs with ß-catenin mutational activation.

The lipogenic transcription factor ChREBP dissociates hepatic steatosis from insulin resistance in mice and humans.

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.

O-GlcNAcylation increases ChREBP protein content and transcriptional activity in the liver.

OBJECTIVE: 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 ß-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 METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Distinct regulation of adiponutrin/PNPLA3 gene expression by the transcription factors ChREBP and SREBP1c in mouse and human hepatocytes.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Peroxisome-proliferator-activated receptor gamma suppresses Wnt/beta-catenin signalling during adipogenesis.

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.

Regulation of semicarbazide-sensitive amine oxidase expression by tumor necrosis factor-alpha in adipocytes: functional consequences on glucose transport.

Membrane-associated semicarbazide-sensitive amine oxidase (SSAO) is mainly present in the media of aorta and in adipose tissue. Recent works have reported that SSAO activation can stimulate glucose transport of fat cells and promote adipose conversion. In this study, the murine 3T3-L1 preadipose cell line was used to investigate SSAO regulation by tumor necrosis factor-alpha (TNF-alpha), a cytokine that is synthesized in fat cells and known to be involved in obesity-linked insulin resistance. SSAO mRNA and protein levels, and enzyme activity were decreased by TNF-alpha in a dose- and time-dependent manner, without any change of SSAO affinity for substrates or inhibitors. SSAO inhibition caused by TNF-alpha was spontaneously reversed along the time after TNF-alpha removal. The decrease in SSAO expression also occurred in white adipose tissue of C57BL/6 mice treated with mTNF-alpha. Overall, we demonstrated that reduction in SSAO expression induced by the cytokine had marked repercussions on amine-stimulated glucose transport, in a dose- and time-dependent manner. This effect was more pronounced than the inhibiting effect of TNF-alpha on insulin-stimulated glucose transport. Moreover, the peroxisome proliferator-activated receptor gamma agonists thiazolidinediones did not reverse either TNF-alpha effect on amine-sensitive glucose transport or the inhibition of SSAO activity, whereas they antagonized TNF-alpha effects on insulin-sensitive glucose transport. These results demonstrate that TNF-alpha can strongly down-regulate SSAO expression and activity, and through this mechanism can dramatically reduce amine-stimulated glucose transport. This suggests a potential role of this regulatory process in the pathogenesis of glucose homeostasis dysregulations observed during diseases accompanied by TNF-alpha overproduction, such as cachexia or obesity.