Project description:SREBP1c is a key transcription factor that regulates de novo lipogenesis during anabolic periods. However, the molecular mechanisms involved in the suppression of SREBP1c under nutritional deprivation are largely unknown. In this study, we demonstrate that the small ubiquitin-related modifier (SUMO) E3 ligase, a protein inhibitor of activated STAT Y (PIASy), sumoylates SREBP1c at Lys98, leading to suppression of the hepatic lipogenic program upon fasting-induced signals. In primary hepatocytes, ablation of PIASy stimulated intracellular lipid accumulation through the induction of SREBP1c and its target genes. Given that protein kinase A (PKA) plays important roles in catabolic responses, activated PKA enhances the sumoylation of SREBP1c and potentiates the interaction between SREBP1c and PIASy. Notably, overexpression of PIASy in obese db/db mice ameliorated hepatic steatosis, while suppression of PIASy in lean (wild-type) mice stimulated hepatic lipogenesis with increased expression of SREBP1c target genes. Furthermore, PKA-mediated SREBP1c phosphorylation augmented SREBP1c sumoylation, subsequently leading to degradation of SREBP1c via ubiquitination. Together, these data suggest that PKA-induced SREBP1c sumoylation by PIASy is a key regulatory mechanism to turn off hepatic lipogenesis during nutritional deprivation.
Project description:The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, maintenance of higher energy expenditure during the fast and reduced binge-eating during the feeding period. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.
Project description:Hepatic gluconeogenesis is essential for glucose homeostasis and also a therapeutic target for type 2 diabetes, but its mechanism is incompletely understood. Here, we report that Sam68, an RNA-binding adaptor protein and Src kinase substrate, is a novel regulator of hepatic gluconeogenesis. Both global and hepatic deletions of Sam68 significantly reduce blood glucose levels and the glucagon-induced expression of gluconeogenic genes. Protein, but not mRNA, levels of CRTC2, a crucial transcriptional regulator of gluconeogenesis, are >50% lower in Sam68-deficient hepatocytes than in wild-type hepatocytes. Sam68 interacts with CRTC2 and reduces CRTC2 ubiquitination. However, truncated mutants of Sam68 that lack the C- (Sam68ΔC) or N-terminal (Sam68ΔN) domains fails to bind CRTC2 or to stabilize CRTC2 protein, respectively, and transgenic Sam68ΔN mice recapitulate the blood-glucose and gluconeogenesis profile of Sam68-deficient mice. Hepatic Sam68 expression is also upregulated in patients with diabetes and in two diabetic mouse models, while hepatocyte-specific Sam68 deficiencies alleviate diabetic hyperglycemia and improves insulin sensitivity in mice. Thus, our results identify a role for Sam68 in hepatic gluconeogenesis, and Sam68 may represent a therapeutic target for diabetes.
Project description:SREBP1c is a key lipogenic transcription factor activated by insulin in the postprandial state. Although SREBP1c appears to be involved in suppression of hepatic gluconeogenesis, the molecular mechanism is not thoroughly understood. Here we show that CRY1 is activated by insulin-induced SREBP1c and decreases hepatic gluconeogenesis through FOXO1 degradation, at least, at specific circadian time points. SREBP1c(-/-) and CRY1(-/-) mice show higher blood glucose than wild-type (WT) mice in pyruvate tolerance tests, accompanied with enhanced expression of PEPCK and G6Pase genes. CRY1 promotes degradation of nuclear FOXO1 by promoting its binding to the ubiquitin E3 ligase MDM2. Although SREBP1c fails to upregulate CRY1 expression in db/db mice, overexpression of CRY1 attenuates hyperglycaemia through reduction of hepatic FOXO1 protein and gluconeogenic gene expression. These data suggest that insulin-activated SREBP1c downregulates gluconeogenesis through CRY1-mediated FOXO1 degradation and that dysregulation of hepatic SREBP1c-CRY1 signalling may contribute to hyperglycaemia in diabetic animals.
Project description:The transcription factor SREBP1c (sterol-regulatory-element-binding protein 1c) is highly expressed in adipose tissue and plays a central role in several aspects of adipocyte development including the induction of PPARgamma (peroxisome-proliferator-activated receptor gamma), the generation of an endogenous PPARgamma ligand and the expression of several genes critical for lipid biosynthesis. Despite its significance, the regulation of SREBP1c expression during adipogenesis is not well characterized. We have noted that in several models of adipogenesis, SREBP1c expression closely mimics that of known C/EBPbeta (CCAAT/enhancer-binding protein beta) targets. Inhibition of C/EBP activity during adipogenesis by expressing either the dominant-negative C/EBPbeta LIP (liver-enriched inhibitory protein) isoform, the co-repressor ETO (eight-twenty one/MTG8) or using siRNAs (small interfering RNAs) targeting either C/EBPbeta or C/EBPdelta significantly impaired early SREBP1c induction. Furthermore, ChIP (chromatin immunoprecipitation) assays identified specific sequences in the SREBP1c promoter to which C/EBPbeta and C/EBPdelta bind in intact cells, demonstrating that these factors may directly regulate SREBP1c expression. Using cells in which C/EBPalpha expression is inhibited using shRNA (short hairpin RNA) and ChIP assays we show that C/EBPalpha replaces C/EBPbeta and C/EBPdelta as a regulator of SREBP1c expression in maturing adipocytes. These results provide novel insight into the induction of SREBP1c expression during adipogenesis. Moreover, the findings of the present study identify an important additional mechanism via which the C/EBP transcription factors may control a network of gene expression regulating adipogenesis, lipogenesis and insulin sensitivity.
Project description:MicroRNAs (miRNAs) are small, regulatory non-coding RNAs that have potent effects on gene expression. Several miRNA are deregulated in cellular processes involved in human liver diseases and regulation of cellular processes. Recent studies have identified the involvement of miR-29 in hepatic fibrosis and carcinogenesis. Although several targets of miR-29 have been identified, there is limited information regarding the cell-type specific roles of miR-29 in the liver, and we sought to evaluate the role of this miRNA in hepatic pathobiology. We report the generation of a tissue-specific knockout mouse to evaluate the role of miR-29 in hepatic fibrosis and carcinogenesis in response to injury. We hypothesized that miR-29 contributes to the hepatocyte driven response to chronic cellular injury that results in fibrosis. In support of this hypothesis, fibrosis and mortality were enhanced in miR29 knockout mice in response to carbon tetrachloride. Genome-wide gene expression analysis identified an over-representation of genes associated with fibrosis. The oncofetal RNA H19 was modulated in a miR-29 dependent manner following exposure to carbon tetrachloride in vivo. The impact of a hepatocyte specific miR-29 knockout on survival following chronic hepatic injury in vivo implicates this miRNA as a potential target for intervention. These results provide evidence of the involvement of miR-29 in chronic hepatic injury, and suggest a role for deregulated hepatocyte expression of miR-29 in the response to hepatic injury, fibrosis and carcinogenesis.
Project description:Glucose levels in mammals are tightly controlled through multiple mechanisms to meet systemic energy demands. Downregulation of hepatic glucokinase (GCK) during fasting facilitates the transition of the liver from a glucose-consuming to a gluconeogenic organ. Here, we report the transcriptional regulation of hepatic GCK by a long non-coding RNA (lncRNA) named liver GCK repressor (lncLGR). lncLGR is induced by fasting, and physiological overexpression of lncLGR to mimic fasting levels effectively suppresses GCK expression and reduces hepatic glycogen content in mice. Consistently, lncLGR knockdown enhances GCK expression and glycogen storage in fasted mice. Mechanistically, lncLGR specifically binds to heterogenous nuclear ribonucleoprotein L (hnRNPL), which is further confirmed to be a transcriptional repressor of GCK in vivo. Finally, we demonstrate that lncLGR facilitates the recruitment of hnRNPL to the GCK promoter and suppresses GCK transcription. Our data establish a lncRNA-mediated mechanism that regulates hepatic GCK expression and glycogen deposition in a physiological context.
Project description:BackgroundPCSK9 has emerged as a key regulator of serum LDL-C metabolism by promoting the degradation of hepatic LDL receptor (LDLR). In this study, we investigated the effect of fasting on serum PCSK9, LDL-C, and hepatic LDLR expression in hamsters and further delineated the molecular pathways involved in fasting-induced repression of PCSK9 transcription.ResultsFasting had insignificant effects on serum total cholesterol and HDL-C levels, but reduced LDL-C, triglyceride and insulin levels. The decrease in serum LDL-C was accompanied by marked reductions of hepatic PCSK9 mRNA and serum PCSK9 protein levels with concomitant increases of hepatic LDLR protein amounts. Fasting produced a profound impact on SREBP1 expression and its transactivating activity, while having modest effects on mRNA expressions of SREBP2 target genes in hamster liver. Although PPARα mRNA levels in hamster liver were elevated by fasting, ligand-induced activation of PPARα with WY14643 compound in hamster primary hepatocytes did not affect PCSK9 mRNA or protein expressions. Further investigation on HNF1α, a critical transactivator of PCSK9, revealed that fasting did not alter its mRNA expression, however, the protein abundance of HNF1α in nuclear extracts of hamster liver was markedly reduced by prolonged fasting.ConclusionFasting lowered serum LDL-C in hamsters by increasing hepatic LDLR protein amounts via reductions of serum PCSK9 levels. Importantly, our results suggest that attenuation of SREBP1 transactivating activity owing to decreased insulin levels during fasting is primarily responsible for compromised PCSK9 gene transcription, which was further suppressed after prolonged fasting by a reduction of nuclear HNF1α protein abundance.
Project description:Lipin 1 regulates cellular lipid homeostasis through roles in glycerolipid synthesis (through phosphatidic acid phosphatase activity) and transcriptional coactivation. Lipin 1-deficient individuals exhibit episodic disease symptoms that are triggered by metabolic stress, such as stress caused by prolonged fasting. We sought to identify critical lipin 1 activities during fasting. We determined that lipin 1 deficiency induces widespread alternative mRNA splicing in liver during fasting, much of which is normalized by refeeding. The role of lipin 1 in mRNA splicing was largely independent of its enzymatic function. We identified interactions between lipin 1 and spliceosome proteins, as well as a requirement for lipin 1 to maintain homeostatic levels of spliceosome small nuclear RNAs and specific RNA splicing factors. In fasted Lpin1-/- liver, we identified a correspondence between alternative splicing of phospholipid biosynthetic enzymes and dysregulated phospholipid levels; splicing patterns and phospholipid levels were partly normalized by feeding. Thus, lipin 1 influences hepatic lipid metabolism through mRNA splicing, as well as through enzymatic and transcriptional activities, and fasting exacerbates the deleterious effects of lipin 1 deficiency on metabolic homeostasis.
Project description:Following their activation in response to inflammatory signals, innate immune cells secrete T-cell-polarizing cytokines that promote the differentiation of naive CD4 T cells into T helper (Th) cell subsets. Among these, Th17 cells play a prominent role in the development of a number of autoimmune diseases. Although regarded primarily as an immunosuppressant signal, cAMP has been found to mediate pro-inflammatory effects of macrophage-derived prostaglandin E2 (PGE2) on Th17 cells. Here we show that PGE2 enhances Th17 cell differentiation via the activation of the CREB co-activator CRTC2. Following its dephosphorylation, CRTC2 stimulates the expression of the cytokines IL-17A and IL-17F by binding to CREB over both promoters. CRTC2-mutant mice have decreased Th17 cell numbers, and they are protected from experimental autoimmune encephalitis, a model for multiple sclerosis. Our results suggest that small molecule inhibitors of CRTC2 may provide therapeutic benefit to individuals with autoimmune disease.