CREB-Regulated Transcriptional Coactivator 2 Proteome Landscape is Modulated by SREBF1.

cAMP response element-binding protein (CREB) regulated transcriptional coactivator 2 (CRTC2) is a critical transcription factor that maintains glucose homeostasis by activating CREB. Energy homeostasis is maintained through multiple pathways; therefore, CRTC2 may interact with other transcription factors, particularly under metabolic stress. CRTC2 liver-specific KO mice were created, and the global proteome, phosphoproteome, and acetylome from liver tissue under high-fat diet conditions were analyzed using liquid chromatography-tandem mass spectrometry and bioinformatics analysis. Differentially regulated proteins (DRPs) were enriched in metabolic pathways, which were subsequently corroborated through animal experiments. The consensus DRPs from these datasets were used as seed proteins to generate a protein-protein interaction network using STRING, and GeneMANIA identified fatty acid synthase as a mutually relevant protein. In an additional local-protein-protein interaction analysis of CRTC2 and fatty acid synthase with DRPs, sterol regulatory element binding transcription factor 1 (SREBF1) was the common mediator. CRTC2-CREB and SREBF1 are transcription factors, and DNA-binding motif analysis showed that multiple CRTC2-CREB-regulated genes possess SREBF1-binding motifs. This indicates the possible induction by the CRTC2-SREBF1 complex, which is validated through luciferase assay. Therefore, the CRTC2-SREBF1 complex potentially modulates the transcription of multiple proteins that fine-tune cellular metabolism under metabolic stress.

Tcf7l2 in hepatocytes regulates de novo lipogenesis in diet-induced non-alcoholic fatty liver disease in mice.

AIMS/HYPOTHESIS: Non-alcoholic fatty liver disease (NAFLD) associated with type 2 diabetes may more easily progress towards severe forms of non-alcoholic steatohepatitis (NASH) and cirrhosis. Although the Wnt effector transcription factor 7-like 2 (TCF7L2) is closely associated with type 2 diabetes risk, the role of TCF7L2 in NAFLD development remains unclear. Here, we investigated how changes in TCF7L2 expression in the liver affects hepatic lipid metabolism based on the major risk factors of NAFLD development. METHODS: Tcf7l2 was selectively ablated in the liver of C57BL/6N mice by inducing the albumin (Alb) promoter to recombine Tcf7l2 alleles floxed at exon 5 (liver-specific Tcf7l2-knockout [KO] mice: Alb-Cre;Tcf7l2f/f). Alb-Cre;Tcf7l2f/f and their wild-type (Tcf7l2f/f) littermates were fed a high-fat diet (HFD) or a high-carbohydrate diet (HCD) for 22 weeks to reproduce NAFLD/NASH. Mice were refed a standard chow diet or an HCD to stimulate de novo lipogenesis (DNL) or fed an HFD to provide exogenous fatty acids. We analysed glucose and insulin sensitivity, metabolic respiration, mRNA expression profiles, hepatic triglyceride (TG), hepatic DNL, selected hepatic metabolites, selected plasma metabolites and liver histology. RESULTS: Alb-Cre;Tcf7l2f/f essentially exhibited increased lipogenic genes, but there were no changes in hepatic lipid content in mice fed a normal chow diet. However, following 22 weeks of diet-induced NAFLD/NASH conditions, liver steatosis was exacerbated owing to preferential metabolism of carbohydrate over fat. Indeed, hepatic Tcf7l2 deficiency enhanced liver lipid content in a manner that was dependent on the duration and amount of exposure to carbohydrates, owing to cell-autonomous increases in hepatic DNL. Mechanistically, TCF7L2 regulated the transcriptional activity of Mlxipl (also known as ChREBP) by modulating O-GlcNAcylation and protein content of carbohydrate response element binding protein (ChREBP), and targeted Srebf1 (also called SREBP1) via miRNA (miR)-33-5p in hepatocytes. Eventually, restoring TCF7L2 expression at the physiological level in the liver of Alb-Cre;Tcf7l2f/f mice alleviated liver steatosis without altering body composition under both acute and chronic HCD conditions. CONCLUSIONS/INTERPRETATION: In mice, loss of hepatic Tcf7l2 contributes to liver steatosis by inducing preferential metabolism of carbohydrates via DNL activation. Therefore, TCF7L2 could be a promising regulator of the NAFLD associated with high-carbohydrate diets and diabetes since TCF7L2 deficiency may lead to development of NAFLD by promoting utilisation of excess glucose pools through activating DNL. DATA AVAILABILITY: RNA-sequencing data have been deposited into the NCBI GEO under the accession number GSE162449 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE162449 ).

Prominin-1-Radixin axis controls hepatic gluconeogenesis by regulating PKA activity.

Prominin-1 (Prom1) is a major cell surface marker of cancer stem cells, but its physiological functions in the liver have not been elucidated. We analyzed the levels of mRNA transcripts in serum-starved primary WT (Prom1+/+ ) and KO (Prom1-/- ) mouse hepatocytes using RNA sequencing (RNA-seq) data, and found that CREB target genes were downregulated. This initial observation led us to determine that Prom1 deficiency inhibited cAMP response element-binding protein (CREB) activation and gluconeogenesis, but not cyclic AMP (cAMP) accumulation, in glucagon-, epinephrine-, or forskolin-treated liver tissues and primary hepatocytes, and mitigated glucagon-induced hyperglycemia. Because Prom1 interacted with radixin, Prom1 deficiency prevented radixin from localizing to the plasma membrane. Moreover, systemic adenoviral knockdown of radixin inhibited CREB activation and gluconeogenesis in glucagon-treated liver tissues and primary hepatocytes, and mitigated glucagon-elicited hyperglycemia. Based on these results, we conclude that Prom1 regulates hepatic PKA signaling via radixin functioning as an A kinase-anchored protein (AKAP).

The SMILE transcriptional corepressor inhibits cAMP response element-binding protein (CREB)-mediated transactivation of gluconeogenic genes.

Under fasting conditions, activation of several hepatic genes sets the stage for gluconeogenesis in the liver. cAMP response element-binding protein (CREB), CREB-regulated transcription coactivator 2 (CRTC2), and peroxisome proliferator-activated receptor γ coactivator 1-alpha (PGC-1α) are essential for this transcriptional induction of gluconeogenic genes. PGC-1α induction is mediated by activation of a CREB/CRTC2 signaling complex, and recent findings have revealed that small heterodimer partner-interacting leucine zipper protein (SMILE), a member of the CREB/ATF family of basic region-leucine zipper (bZIP) transcription factors, is an insulin-inducible corepressor that decreases PGC-1α expression and abrogates its stimulatory effect on hepatic gluconeogenesis. However, the molecular mechanism whereby SMILE suppresses PGC-1α expression is unknown. Here, we investigated SMILE's effects on the CREB/CRTC2 signaling pathway and glucose metabolism. We found that SMILE significantly inhibits CREB/CRTC2-induced PGC-1α expression by interacting with and disrupting the CREB/CRTC2 complex. Consequently, SMILE decreased PGC-1α-induced hepatic gluconeogenic gene expression. Furthermore, SMILE inhibited CREB/CRTC2-induced phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) gene expression by directly repressing the expression of these genes and by indirectly inhibiting the expression of PGC-1α via CREB/CRTC2 repression. Indeed, enhanced gluconeogenesis and circulating blood glucose levels in mice injected with an adenovirus construct containing a constitutively active CRTC2 variant (CRTC2-S171A) were significantly reduced by WT SMILE, but not by leucine zipper-mutated SMILE. These results reveal that SMILE represses CREB/CRTC2-induced PGC-1α expression, an insight that may help inform potential therapeutic approaches targeting PGC-1α-mediated regulation of hepatic glucose metabolism.

CREB/CRTC2 controls GLP-1-dependent regulation of glucose homeostasis.

Glucagon-like peptide 1 (GLP-1) is a major incretin that controls glucose homeostasis. The secretion of mature GLP-1 is regulated via GPCRs, including bile acid receptor G protein-coupled bile acid receptor 1, which uses cAMP signaling to enhance the exocytosis of GLP-1-containing vesicles. However, the role of cAMP-mediated transcription has not been clearly demonstrated to date. In this study, we explored the role of cAMP response element-binding protein/CREB-regulated transcription coactivator 2 (CREB/CRTC2)-dependent transcription on GLP-1 secretion in the L cells. We found that the reduced CREB/CRTC2 activity impaired the cAMP-dependent increase in GLP-1 secretion, whereas expression of constitutively active CRTC2 increased GLP-1 exocytosis from the L cells. Close investigation revealed that expression of not only proglucagon but also PC1/3, an endopeptidase for GLP-1 maturation, is transcriptionally regulated by CREB/CRTC2. Furthermore, expression of peroxisome proliferator-activating receptor coactivator 1 α is also reduced upon depletion of CRTC2, leading to the decreased expression of oxidative phosphorylation (OxPhos) genes, reduced ATP levels, and calcium concentrations in the L cells. Finally, we observed that intestine-specific CRTC2 knockout mice displayed reduced GLP-1 expression, leading to the lower plasma GLP-1 levels, impaired glucose tolerance, and decreased insulin-containing ß cells in pancreatic islets. Our data show that the CREB/CRTC2-dependent transcriptional pathway is critical for regulating glucose homeostasis by controlling production of GLP-1 from the L cells at the level of transcription, maturation, and exocytosis.-Lee, J.-H., Wen, X., Cho, H., Koo, S.-H. CREB/CRTC2 controls GLP-1-dependent regulation of glucose homeostasis.

Prdm4 induction by the small molecule butein promotes white adipose tissue browning.

Increasing the thermogenic activity of adipocytes holds promise as an approach to combating human obesity and related metabolic diseases. We identified induction of mouse PR domain containing 4 (Prdm4) by the small molecule butein as a means to induce expression of uncoupling protein 1 (Ucp1), increase energy expenditure, and stimulate the generation of thermogenic adipocytes. This study highlights a Prdm4-dependent pathway, modulated by small molecules, that stimulates browning of white adipose tissue.

Fast food diet-induced non-alcoholic fatty liver disease exerts early protective effect against acetaminophen intoxication in mice.

BACKGROUND: Acetaminophen (APAP) is a readily available and safe painkiller. However, its overdose is the most common cause of acute liver injury (ALI). Many predisposing factors contribute to susceptibility to APAP-induced ALI. Non-alcoholic fatty liver disease (NAFLD), the major cause of chronic liver disease, is considered an important predictor of APAP-induced ALI, although the exact mechanism controversial. In this study, we aimed to elucidate the effects of NAFLD on APAP-induced ALI. METHODS: Two groups of mice, normal chow (NC) diet-fed and fast food (FF) diet-fed mice for 14 weeks, were further divided into two subgroups: intraperitoneally injected with either saline (NC-S and FF-S groups) or APAP (NC-A and FF-A groups). Biochemical tests, histological analysis, quantitative PCR, and western blotting were conducted. RESULTS: Alanine aminotransferase (ALT) level (199.0 ± 39.0 vs. 63.8 ± 7.4 IU/L, p < 0.05) and NAFLD activity score (0 vs. 4.5 ± 0.22) were significantly higher in mice in FF-S group than those in NC-S group. ALI features such as ALT level (8447.8 ± 1185.3 vs. 836.6 ± 185.1 IU/L, p < 0.001) and centrizonal necrosis were prominent and mRNA levels of Trib3 (RR, 1.81) was high in mice in the NC-A group. Levels of CYP2E1 and anti-inflammatory molecules such as PPAR-γ, p62, and NRF2 were high in mice in the FF-A group. CONCLUSIONS: Our results showed that while the FF diet clearly induced non-alcoholic steatohepatitis and metabolic syndrome, NAFLD also attenuates APAP-induced ALI by inducing anti-inflammatory molecules such as PPAR-γ.

Vibrio vulnificus Secretes an Insulin-degrading Enzyme That Promotes Bacterial Proliferation in Vivo.

We describe a novel insulin-degrading enzyme, SidC, that contributes to the proliferation of the human bacterial pathogen Vibrio vulnificus in a mouse model. SidC is phylogenetically distinct from other known insulin-degrading enzymes and is expressed and secreted specifically during host infection. Purified SidC causes a significant decrease in serum insulin levels and an increase in blood glucose levels in mice. A comparison of mice infected with wild type V. vulnificus or an isogenic sidC-deletion strain showed that wild type bacteria proliferated to higher levels. Additionally, hyperglycemia leads to increased proliferation of V. vulnificus in diabetic mice. Consistent with these observations, the sid operon was up-regulated in response to low glucose levels through binding of the cAMP-receptor protein (CRP) complex to a region upstream of the operon. We conclude that glucose levels are important for the survival of V. vulnificus in the host, and that this pathogen uses SidC to actively manipulate host endocrine signals, making the host environment more favorable for bacterial survival and growth.

Cyclic AMP Response Element-binding Protein H (CREBH) Mediates the Inhibitory Actions of Tumor Necrosis Factor α in Osteoblast Differentiation by Stimulating Smad1 Degradation.

Endoplasmic reticulum (ER) stress transducers, such as old astrocyte specifically induced substance (OASIS) and activating transcription factor 6 (ATF6), which are induced by bone morphogenetic protein 2 (BMP2), regulate bone formation and osteoblast differentiation. Here, we examined the role of cAMP response element-binding protein H (CREBH), a member of the same family of ER membrane-bound basic leucine zipper (bZIP) transcription factors as OASIS and ATF6, in osteoblast differentiation and bone formation. Proinflammatory cytokine TNFα increased CREBH expression by up-regulating the nuclear factor-κB (NF-κB) signaling pathway in osteoblasts, increased the level of N-terminal fragment of CREBH in the nucleus, and inhibited BMP2 induction of osteoblast specific gene expression. Overexpression of CREBH suppressed BMP2-induced up-regulation of the osteogenic markers runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OC) in MC3T3-E1 cells and primary osteoblasts, as well as BMP2-induced ALP activity and OC protein production. In contrast, knockdown of CREBH attenuated the inhibitory effect of TNFα on BMP2-induced osteoblast differentiation. Mechanistic studies revealed that CREBH increased the expression of Smad ubiquitination regulatory factor 1 (Smurf1), leading to ubiquitin-dependent degradation of Smad1, whereas knockdown of CREBH inhibited TNFα-mediated degradation of Smad1 by Smurf1. Consistent with these in vitro findings, administration of Ad-CREBH inhibited BMP2-induced ectopic and orthotopic bone formation in vivo. Taken together, these results suggest that CREBH is a novel negative regulator of osteoblast differentiation and bone formation.

Retinoic acid-related orphan receptor alpha reprograms glucose metabolism in glutamine-deficient hepatoma cells.

UNLABELLED: The metabolism of glutamine and glucose is recognized as a promising therapeutic target for the treatment of cancer; however, targeted molecules that mediate glutamine and glucose metabolism in cancer cells have not been addressed. Here, we show that restricting the supply of glutamine in hepatoma cells, including HepG2 and Hep3B cells, markedly increased the expression of retinoic acid-related orphan receptor alpha (RORα). Up-regulation of RORα in glutamine-deficient hepatoma cells resulted from an increase in the level of cellular reactive oxygen species and in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate reduced (NADP+ /NADPH) ratio, which was consistent with a reduction in the glutathione/glutathione disulfide (GSH/GSSG) ratio. Adenovirus (Ad)-mediated overexpression of RORα (Ad-RORα) or treatment with the RORα activator, SR1078, reduced aerobic glycolysis and down-regulated biosynthetic pathways in hepatoma cells. Ad-RORα and SR1078 reduced the expression of pyruvate dehydrogenase kinase 2 (PDK2) and inhibited the phosphorylation of pyruvate dehydrogenase and subsequently shifted pyruvate to complete oxidation. The RORα-mediated decrease in PDK2 levels was caused by up-regulation of p21, rather than p53. Furthermore, RORα inhibited hepatoma growth both in vitro and in a xenograft model in vivo. We also found that suppression of PDK2 inhibited hepatoma growth in a xenograft model. These findings mimic the altered glucose utilization and hepatoma growth caused by glutamine deprivation. Finally, tumor tissue from 187 hepatocellular carcinoma patients expressed lower levels of RORα than adjacent nontumor tissue, supporting a potential beneficial effect of RORα activation in the treatment of liver cancer. CONCLUSION: RORα mediates reprogramming of glucose metabolism in hepatoma cells in response to glutamine deficiency. The relationships established here between glutamine metabolism, RORα expression and signaling, and aerobic glycolysis have implications for therapeutic targeting of liver cancer metabolism.

HBx induces the proliferation of hepatocellular carcinoma cells via AP1 over-expressed as a result of ER stress.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and chronic hepatitis B virus (HBV) infection is the most common risk factor for HCC. The HBV proteins can induce oncogenic or synergy effects with a hyperproliferative response on transformation into HCC. CREBH (cAMP-responsive, element-binding protein H), activated by stress in the endoplasmic reticulum (ER), is an ER-resident transmembrane bZIP (basic leucine zipper) transcription factor that is specifically expressed in the liver. In the present study, we address the role played by CREBH activated by ER stress in HBV-induced hepatic cell proliferation. We confirmed CREBH activation by ER stress and showed that it occurred as a result of/via hepatitis B virus X (HBx)-induced ER stress. CREBH activated by HBx increased the expression of AP-1 target genes through c-Jun induction. Under pathological conditions such as liver damage or liver regeneration, activated CREBH may have an important role to play in hepatic inflammation and cell proliferation, as an insulin receptor with dual functions under these conditions. We showed that CREBH activated by HBx interacted with HBx protein, leading to a synergistic effect on the expression of AP-1 target genes and the proliferation of HCC cells and mouse primary hepatocytes. In conclusion, in HBV-infected hepatic cells or patients with chronic HBV, CREBH may induce proliferation of hepatic cells in co-operation with HBx, resulting in HCC.

Ursodeoxycholic acid inhibits liver X receptor α-mediated hepatic lipogenesis via induction of the nuclear corepressor SMILE.

Small heterodimer partner interacting leucine zipper protein (SMILE) has been identified as a nuclear corepressor of the nuclear receptor (NRs) family. Here, we examined the role of SMILE in the regulation of nuclear receptor liver X receptor (LXR)-mediated sterol regulatory element binding protein-1c (SREBP-1c) gene expression. We found that SMILE inhibited T0901317 (T7)-induced transcriptional activity of LXR, which functions as a major regulator of lipid metabolism by inducing SREBP-1c, fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) gene expression. Moreover, we demonstrated that SMILE physically interacts with LXR and represses T7-induced LXR transcriptional activity by competing with coactivator SRC-1. Adenoviral overexpression of SMILE (Ad-SMILE) attenuated fat accumulation and lipogenic gene induction in the liver of T7 administered or of high fat diet (HFD)-fed mice. Furthermore, we investigated the mechanism by which ursodeoxycholic acid (UDCA) inhibits LXR-induced lipogenic gene expression. Interestingly, UDCA treatment significantly increased SMILE promoter activity and gene expression in an adenosine monophosphate-activated kinase-dependent manner. Furthermore, UDCA treatment repressed T7-induced SREBP-1c, FAS, and ACC protein levels, whereas knockdown of endogenous SMILE gene expression by adenovirus SMILE shRNA (Ad-shSMILE) significantly reversed UDCA-mediated repression of SREBP-1c, FAS, and ACC protein levels. Collectively, these results demonstrate that UDCA activates SMILE gene expression through adenosine monophosphate-activated kinase phosphorylation, which leads to repression of LXR-mediated hepatic lipogenic enzyme gene expression.

Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation.

UNLABELLED: Sterol regulatory element binding protein1c (SREBP1c) is a key transcription factor for de novo lipogenesis during the postprandial state. During nutritional deprivation, hepatic SREBP1c is rapidly suppressed by fasting signals to prevent lipogenic pathways. However, the molecular mechanisms that control SREBP1c turnover in response to fasting status are not thoroughly understood. To elucidate which factors are involved in the inactivation of SREBP1c, we attempted to identify SREBP1c-interacting proteins by mass spectrometry analysis. Since we observed that ring finger protein20 (RNF20) ubiquitin ligase was identified as one of SREBP1c-interacting proteins, we hypothesized that fasting signaling would promote SREBP1c degradation in an RNF20-dependent manner. In this work, we demonstrate that RNF20 physically interacts with SREBP1c, leading to degradation of SREBP1c via ubiquitination. In accordance with these findings, RNF20 represses the transcriptional activity of SREBP1c and turns off the expression of lipogenic genes that are targets of SREBP1c. In contrast, knockdown of RNF20 stimulates the expression of SREBP1c and lipogenic genes and induces lipogenic activity in primary hepatocytes. Furthermore, activation of protein kinase A (PKA) with glucagon or forskolin enhances the expression of RNF20 and potentiates the ubiquitination of SREBP1c via RNF20. In wild-type and db/db mice, adenoviral overexpression of RNF20 markedly suppresses FASN promoter activity and reduces the level of hepatic triglycerides, accompanied by a decrease in the hepatic lipogenic program. Here, we reveal that RNF20-induced SREBP1c ubiquitination down-regulates hepatic lipogenic activity upon PKA activation. CONCLUSION: RNF20 acts as a negative regulator of hepatic fatty acid metabolism through degradation of SREBP1c upon PKA activation. Knowledge regarding this process enhances our understanding of how SREBP1c is able to turn off hepatic lipid metabolism during nutritional deprivation.

Transcriptional cross talk between orphan nuclear receptor ERRγ and transmembrane transcription factor ATF6α coordinates endoplasmic reticulum stress response.

Orphan nuclear receptor ERRγ is a member of nuclear receptor superfamily that regulates several important cellular processes including hepatic glucose and alcohol metabolism. However, mechanistic understanding of transcriptional regulation of the ERRγ gene remains to be elucidated. Here, we report that activating transcription factor 6α (ATF6α), an endoplasmic reticulum (ER)-membrane-bound basic leucine zipper (bZip) transcription factor, directly regulates ERRγ gene expression in response to ER stress. ATF6α binds to ATF6α responsive element in the ERRγ promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6α and PGC1α on the ERRγ promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6α and PGC1α binding site. Of interest, ERRγ along with PGC1α induce ATF6α gene transcription upon ER stress. ERRγ binds to an ERRγ responsive element in the ATF6α promoter. ChIP assay confirmed that both ERRγ and PGC1α bind to a site in the ATF6α promoter that exhibits histone H3 and H4 acetylation. Overall, for the first time our data show a novel pathway of cross talk between nuclear receptors and ER-membrane-bound transcription factors and suggest a positive feed-forward loop regulates ERRγ and ATF6α gene transcription.

TCF7L2 modulates glucose homeostasis by regulating CREB- and FoxO1-dependent transcriptional pathway in the liver.

Peripheral insulin resistance contributes to the development of type 2 diabetes. TCF7L2 has been tightly associated with this disease, although the exact mechanism was largely elusive. Here we propose a novel role of TCF7L2 in hepatic glucose metabolism in mammals. Expression of medium and short isoforms of TCF7L2 was greatly diminished in livers of diet-induced and genetic mouse models of insulin resistance, prompting us to delineate the functional role of these isoforms in hepatic glucose metabolism. Knockdown of hepatic TCF7L2 promoted increased blood glucose levels and glucose intolerance with increased gluconeogenic gene expression in wild-type mice, in accordance with the PCR array data showing that only the gluconeogenic pathway is specifically up-regulated upon depletion of hepatic TCF7L2. Conversely, overexpression of a nuclear isoform of TCF7L2 in high-fat diet-fed mice ameliorated hyperglycemia with improved glucose tolerance, suggesting a role of this factor in hepatic glucose metabolism. Indeed, we observed a binding of TCF7L2 to promoters of gluconeogenic genes; and expression of TCF7L2 inhibited adjacent promoter occupancies of CREB, CRTC2, and FoxO1, critical transcriptional modules in hepatic gluconeogenesis, to disrupt target gene transcription. Finally, haploinsufficiency of TCF7L2 in mice displayed higher glucose levels and impaired glucose tolerance, which were rescued by hepatic expression of a nuclear isoform of TCF7L2 at the physiological level. Collectively, these data suggest a crucial role of TCF7L2 in hepatic glucose metabolism; reduced hepatic expression of nuclear isoforms of this factor might be a critical instigator of hyperglycemia in type 2 diabetes.

PKB/Akt phosphorylation of ERRγ contributes to insulin-mediated inhibition of hepatic gluconeogenesis.

AIMS/HYPOTHESIS: Insulin resistance, a major contributor to the pathogenesis of type 2 diabetes, leads to increased hepatic glucose production (HGP) owing to an impaired ability of insulin to suppress hepatic gluconeogenesis. Nuclear receptor oestrogen-related receptor γ (ERRγ) is a major transcriptional regulator of hepatic gluconeogenesis. In this study, we investigated insulin-dependent post-translational modifications (PTMs) altering the transcriptional activity of ERRγ for the regulation of hepatic gluconeogenesis. METHODS: We examined insulin-dependent phosphorylation and subcellular localisation of ERRγ in cultured cells and in the liver of C57/BL6, leptin receptor-deficient (db/db), liver-specific insulin receptor knockout (LIRKO) and protein kinase B (PKB) ß-deficient (Pkbß (-/-)) mice. To demonstrate the role of ERRγ in the inhibitory action of insulin on hepatic gluconeogenesis, we carried out an insulin tolerance test in C57/BL6 mice expressing wild-type or phosphorylation-deficient mutant ERRγ. RESULTS: We demonstrated that insulin suppressed the transcriptional activity of ERRγ by promoting PKB/Akt-mediated phosphorylation of ERRγ at S179 and by eliciting translocation of ERRγ from the nucleus to the cytoplasm through interaction with 14-3-3, impairing its ability to promote hepatic gluconeogenesis. In addition, db/db, LIRKO and Pkbß (-/-) mice displayed enhanced ERRγ transcriptional activity due to a block in PKBß-mediated ERRγ phosphorylation during refeeding. Finally, the phosphorylation-deficient mutant ERRγ S179A was resistant to the inhibitory action of insulin on HGP. CONCLUSIONS/INTERPRETATION: These results suggest that ERRγ is a major contributor to insulin action in maintaining hepatic glucose homeostasis.

Estrogen-related receptor γ controls hepatic CB1 receptor-mediated CYP2E1 expression and oxidative liver injury by alcohol.

BACKGROUND: The hepatic endocannabinoid system and cytochrome P450 2E1 (CYP2E1), a key enzyme causing alcohol-induced reactive oxygen species (ROS) generation, are major contributors to the pathogenesis of alcoholic liver disease. The nuclear hormone receptor oestrogen-related receptor γ (ERRγ) is a constitutively active transcriptional activator regulating gene expression. OBJECTIVE: To investigate the role of ERRγ in the alcohol-mediated regulation of CYP2E1 and to examine the possibility to control alcohol-mediated oxidative stress and liver injury through an ERRγ inverse agonist. DESIGN: For chronic alcoholic hepatosteatosis study, C57BL/6J wild-type and CB1(-/-) mice were administered alcohol for 4 weeks. GSK5182 and chlormethiazole (CMZ) were given by oral gavage for the last 2 weeks of alcohol feeding. Gene expression profiles and biochemical assays were performed using the liver or blood of mice. RESULTS: Hepatic ERRγ gene expression induced by alcohol-mediated activation of CB1 receptor results in induction of CYP2E1, while liver-specific ablation of ERRγ gene expression blocks alcohol-induced expression of CYP2E1 in mouse liver. An ERRγ inverse agonist significantly ameliorates chronic alcohol-induced liver injury in mice through inhibition of CYP2E1-mediated generation of ROS, while inhibition of CYP2E1 by CMZ abrogates the beneficial effects of the inverse agonist. Finally, chronic alcohol-mediated ERRγ and CYP2E1 gene expression, ROS generation and liver injury in normal mice were nearly abolished in CB1(-/-) mice. CONCLUSIONS: ERRγ, as a previously unrecognised transcriptional regulator of hepatic CB1 receptor, controls alcohol-induced oxidative stress and liver injury through CYP2E1 induction, and its inverse agonist could ameliorate oxidative liver injury due to chronic alcohol exposure.

The role of BCAA metabolism in metabolic health and disease.

It has long been postulated that dietary restriction is beneficial for ensuring longevity and extending the health span of mammals, including humans. In particular, a reduction in protein consumption has been shown to be specifically linked to the beneficial effect of dietary restriction on metabolic disorders, presumably by reducing the activity of the mechanistic target of rapamycin complex (mTORC) 1 and the reciprocal activation of AMP-activated protein kinase (AMPK) and sirtuin pathways. Although it is widely used as a dietary supplement to delay the aging process in humans, recent evidence suggests that branched-chain amino acids (BCAAs) might be a major cause of the deteriorating effect of a protein diet on aging and related disorders. In this review, we delineate the regulation of metabolic pathways for BCAAs at the tissue-specific level and summarize recent findings regarding the role of BCAAs in the control of metabolic health and disease in mammals.

Regulation of hepatic lipogenesis by asymmetric arginine methylation.

BACKGROUND AND AIMS: Hepatic lipogenesis is elevated in nutrient abundant conditions to convert the excess carbohydrate into triacylglycerol (TAG). Fatty acyl moiety of TAG is eventually transported into adipose tissues by very low density lipoprotein, leading to the accumulation of TAG as a preferred storage form of excess energy. Disruption of the balance between TAG clearance and synthesis leads to the accumulation of lipids in the liver, leading to the progression of non-alcoholic fatty liver disease (NAFLD) including non-alcoholic steatohepatitis. Protein arginine methyltransferase (PRMT) 6 has been linked to the various metabolic processes including hepatic gluconeogenesis, muscle atrophy and lipodystrophy in mouse models. However, the role of PRMT6 in the control of hepatic lipogenesis has not been elucidated to date. METHODS: We assessed the interaction between PRMT6 and LXR alpha by using co-immunoprecipitation assay. The specific arginine residue of LXR alpha that is methylated by PRMT6 was assessed by LC-MS/MS assay and the functional consequences of LXR alpha methylation was explored by mSREBP-1c luciferase assay. The effect of PRMT6 on hepatic lipogenesis was assessed by adenovirus-mediated ectopic expression of PRMT6 or knockdown of PRMT6 via shRNA in hepatocytes. Finally, the role of PRMT6 in hepatic lipid metabolism in vivo was explored by either ectopic expression of LXR alpha mutant that is defective in PRMT6-mediated arginine methylation or knockdown of PRMT6 in liver. RESULTS: We found that promoter activity of sterol regulatory element binding protein (SREBP) 1c is robustly activated by PRMT6. Interestingly, we demonstrated that PRMT6 binds to LXR alpha, a transcription factor for SREBP-1c, via its LXXLL motif, leading to the asymmetric dimethylation of an arginine residue and activation of this protein. Indeed, ectopic expression of PRMT6 in hepatocytes led to the enhanced expression of LXR alpha target genes in the lipogenic pathway. Conversely, genetic or pharmacological inhibition of PRMT6 diminished expression of lipogenic genes and the lipid accumulation in primary hepatocytes. Mechanistically, we found that asymmetric dimethylation of LXR alpha led to the dissociation of small heterodimer partner (SHP), a transcriptional co-inhibitor of this factor, resulting in the activation of LXR alpha-mediated transcriptional process. Finally, we showed that disruption of asymmetric dimethylation of LXR alpha in the liver led to the diminished expression of genes in the lipogenesis, resulting in the reduced hepatic lipid accumulation in high fat diet-fed mice in vivo. CONCLUSIONS: We showed that PRMT6 modulates LXR alpha activity by conferring asymmetric dimethylation of arginine 253, thus blocking SHP-mediated inhibition and promoting hepatic lipid accumulation. These results suggest that PRMT6 is critical in the control of lipid homeostasis by regulation of LXR alpha-mediated lipogenesis in the liver.

Activation of cannabinoid receptor type 1 (Cb1r) disrupts hepatic insulin receptor signaling via cyclic AMP-response element-binding protein H (Crebh)-mediated induction of Lipin1 gene.

Activation of hepatic cannabinoid 1 receptor (Cb1r) signaling has been implicated in the development of phenotypes associated with fatty liver, hypertriglyceridemia, and insulin resistance. In the current study, we have elucidated the critical role of endoplasmic reticulum-bound transcription factor cyclic AMP-response element-binding protein H (Crebh) in mediating activated Cb1r signaling in inducing phosphatidic acid phosphatase Lipin1 gene expression and subsequently deregulating hepatic insulin receptor signaling. Cb1r agonist (2-arachidonoylglycerol (2-AG)) treatment induced Lipin1 gene expression in a Crebh-dependent manner via recruiting CREBH to the endogenous Lipin1 gene promoter. Adenoviral overexpression of Crebh or 2-AG treatment in mice induced Lipin1 gene expression to increase the hepatic diacylglycerol (DAG) level and phosphorylation of protein kinase Cε (PKCε). This in turn inhibited hepatic insulin receptor signaling. Knockdown of Crebh or Cb1r antagonism attenuated 2-AG-mediated induction of Lipin1 gene expression and decreased DAG production in mouse liver and subsequently restored insulin receptor signaling. Similarly, knockdown of Lipin1 attenuated the 2-AG-induced increase in the DAG level and PKCε phosphorylation. Finally, shRNA-mediated knockdown of Crebh partially but significantly blunted Lipin1 expression and the DAG level in db/db mice. These results demonstrate a novel mechanism by which Cb1r signaling induces Lipin1 gene expression and increases DAG production by activating Crebh, thereby deregulating insulin receptor signaling pathway and lipid homeostasis.