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Cyclic adenosine monophosphate (cAMP) signaling is critical for regulating metabolic homeostasis in mammals. In particular, transcriptional regulation by cAMP response element-binding protein (CREB) and its coactivator, CREB-regulated transcription coactivator (CRTC), is essential for controlling the expression of critical enzymes in the metabolic process, leading to more chronic changes in metabolic flux. Among the CRTC isoforms, CRTC2 is predominantly expressed in peripheral tissues and has been shown to be associated with various metabolic pathways in tissue-specific manners. While initial reports showed the physiological role of CRTC2 in regulating gluconeogenesis in the liver, recent studies have further delineated the role of this transcriptional coactivator in the regulation of glucose and lipid metabolism in various tissues, including the liver, pancreatic islets, endocrine tissues of the small intestines, and adipose tissues. In this review, we discuss recent studies that have utilized knockout mouse models to delineate the role of CRTC2 in the regulation of metabolic homeostasis.

Prominin-1 (PROM1), also known as CD133, is expressed in hepatic progenitor cells (HPCs) and cholangiocytes of the fibrotic liver. In this study, we show that PROM1 is upregulated in the plasma membrane of fibrotic hepatocytes. Hepatocellular expression of PROM1 was also demonstrated in mice (Prom1CreER; R26TdTom) in which cells expressed TdTom under control of the Prom1 promoter. To understand the role of hepatocellular PROM1 in liver fibrosis, global and liver-specific Prom1-deficient mice were analyzed after bile duct ligation (BDL). BDL-induced liver fibrosis was aggravated with increased phosphorylation of SMAD2/3 and decreased levels of SMAD7 by global or liver-specific Prom1 deficiency but not by cholangiocyte-specific Prom1 deficiency. Indeed, PROM1 prevented SMURF2-induced SMAD7 ubiquitination and degradation by interfering with the molecular association of SMAD7 with SMURF2. We also demonstrated that hepatocyte-specific overexpression of SMAD7 ameliorated BDL-induced liver fibrosis in liver-specific Prom1-deficient mice. Thus, we conclude that PROM1 is necessary for the negative regulation of TGFβ signaling during liver fibrosis.

Objective Diet-induced obesity is often associated with nonalcoholic fatty liver disease (NAFLD), which instigates severe metabolic disorders, including cirrhosis, hepatocellular carcinoma, and type 2 diabetes. We have shown that hepatic depletion of CREB regulated transcription co-activator (CRTC) 2 protects mice from the progression of diet-induced fatty liver phenotype, although the exact mechanism by which CRTC2 modulates this process is elusive to date. Here, we investigated the role of hepatic CRTC2 in the instigation of NAFLD in mammals. Methods Crtc2 liver-specific knockout (Crtc2 LKO) mice and Crtc2 flox/flox (Crtc2 f/f) mice were fed a high fat diet (HFD) for 7–8 weeks. Body weight, liver weight, hepatic lipid contents, and plasma triacylglycerol (TG) levels were determined. Western blot analysis was performed to determine Sirtuin (SIRT) 1, tuberous sclerosis complex (TSC) 2, and mammalian target of rapamycin complex (mTORC) 1 activity in the liver. Effects of Crtc2 depletion on lipogenesis was determined by measuring lipogenic gene expression (western blot analysis and qRT-PCR) in the liver as well as Oil red O staining in hepatocytes. Effects of miR-34a on mTORC1 activity and hepatic lipid accumulation was assessed by AAV-miR-34a virus in mice and Ad-miR-34a virus and Ad-anti-miR-34a virus in hepatocytes. Autophagic flux was assessed by western blot analysis after leupeptin injection in mice and bafilomycin treatment in hepatocytes. Lipophagy was assessed by transmission electron microscopy and confocal microscopy. Expression of CRTC2 and p-S6K1 in livers of human NAFLD patients was assessed by immunohistochemistry. Results We found that expression of CRTC2 in the liver is highly induced upon HFD-feeding in mice. Hepatic depletion of Crtc2 ameliorated HFD-induced fatty liver disease phenotypes, with a pronounced inhibition of the mTORC1 pathway in the liver. Mechanistically, we found that expression of TSC2, a potent mTORC1 inhibitor, was enhanced in Crtc2 LKO mice due to the decreased expression of miR-34a and the subsequent increase in SIRT1-mediated deacetylation processes. We showed that ectopic expression of miR-34a led to the induction of mTORC1 pathway, leading to the hepatic lipid accumulation in part by limiting lipophagy and enhanced lipogenesis. Finally, we found a strong association of CRTC2, miR-34a and mTORC1 activity in the NAFLD patients in humans, demonstrating a conservation of signaling pathways among species. Conclusions These data collectively suggest that diet-induced activation of CRTC2 instigates the progression of NAFLD by activating miR-34a-mediated lipid accumulation in the liver via the simultaneous induction of lipogenesis and inhibition of lipid catabolism. Therapeutic approach to specifically inhibit CRTC2 activity in the liver could be beneficial in combating NAFLD in the future., Graphical abstract Image 1, Highlights • CRTC2 expression is elevated in livers of HFD-fed mice. • Liver-specific depletion of Crtc2 ameliorated HFD-induced fatty liver phenotype in mice. • Crtc2 knockout dampened miR-34a-mTORC1 pathway, leading to the inhibition of lipogenesis and the activation of lipophagy. • Close association of CRTC2 with mTORC1 activity was also demonstrated in the human NAFLD patients. • Diet-induced activation of CRTC2 instigates the progression of NAFLD by activating the miR-34a-mTORC1 pathway.

AMP-activated protein kinase (AMPK) plays a key role in controlling energy metabolism in response to physiological and nutritional status. Although AMPK activation has been proposed as a promising molecular target for treating obesity and its related comorbidities, the use of pharmacological AMPK activators has been met with contradictory therapeutic challenges. Here we show a regulatory mechanism for AMPK through its ubiquitination and degradation by the E3 ubiquitin ligase makorin ring finger protein 1 (MKRN1). MKRN1 depletion promotes glucose consumption and suppresses lipid accumulation due to AMPK stabilisation and activation. Accordingly, MKRN1-null mice show chronic AMPK activation in both liver and adipose tissue, resulting in significant suppression of diet-induced metabolic syndrome. We demonstrate also its therapeutic effect by administering shRNA targeting MKRN1 into obese mice that reverses non-alcoholic fatty liver disease. We suggest that ubiquitin-dependent AMPK degradation represents a target therapeutic strategy for metabolic disorders., AMPK activation has been suggested as treatment for obesity and its complications. Here the authors show that the ubiquitin ligase MKRN1 binds to AMPK and mediates its ubiquitination and degradation. Loss of MKRN1 leads to AMPK activation, increased glucose consumption and decreased lipid accumulation.

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.

In fasting or diabetes, gluconeogenic genes are transcriptionally activated by glucagon stimulation of the cAMP-protein kinase A (PKA)–CREB signaling pathway. Previous work showed pyruvate dehydrogenase kinase (PDK) inhibition in skeletal muscle increases pyruvate oxidation, which limits the availability of gluconeogenic substrates in the liver. However, this study found upregulation of hepatic PDK4 promoted glucagon-mediated expression of gluconeogenic genes, whereas knockdown or inhibition of hepatic PDK4 caused the opposite effect on gluconeogenic gene expression and decreased hepatic glucose production. Mechanistically, PDK4 deficiency decreased ATP levels, thus increasing phosphorylated AMPK (p-AMPK), which increased p-AMPK–sensitive phosphorylation of cyclic nucleotide phosphodiesterase 4B (p-PDE4B). This reduced cAMP levels and consequently p-CREB. Metabolic flux analysis showed that the reduction in ATP was a consequence of a diminished rate of fatty acid oxidation (FAO). However, overexpression of PDK4 increased FAO and increased ATP levels, which decreased p-AMPK and p-PDE4B and allowed greater accumulation of cAMP and p-CREB. The latter were abrogated by the FAO inhibitor etomoxir, suggesting a critical role for PDK4 in FAO stimulation and the regulation of cAMP levels. This finding strengthens the possibility of PDK4 as a target against diabetes.

Olfactory receptors (ORs) are present in tissues outside the olfactory system; however, the function of these receptors remains relatively unknown. Here, we determined that olfactory receptor 544 (Olfr544) is highly expressed in the liver and adipose tissue of mice and regulates cellular energy metabolism and obesity. Azelaic acid (AzA), an Olfr544 ligand, specifically induced PKA-dependent lipolysis in adipocytes and promoted fatty acid oxidation (FAO) and ketogenesis in liver, thus shifting the fuel preference to fats. After 6 weeks of administration, mice fed a high-fat diet (HFD) exhibited a marked reduction in adiposity. AzA treatment induced expression of PPAR-α and genes required for FAO in the liver and induced the expression of PPAR-γ coactivator 1-α (Ppargc1a) and uncoupling protein-1 (Ucp1) genes in brown adipose tissue (BAT). Moreover, treatment with AzA increased insulin sensitivity and ketone body levels. This led to a reduction in the respiratory quotient and an increase in the FAO rate, as indicated by indirect calorimetry. AzA treatment had similar antiobesogenic effects in HFD-fed ob/ob mice. Importantly, AzA-associated metabolic changes were completely abrogated in HFD-fed Olfr544–/– mice. To our knowledge, this is the first report to show that Olfr544 orchestrates the metabolic interplay between the liver and adipose tissue, mobilizing stored fats from adipose tissue and shifting the fuel preference to fats in the liver and BAT.

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 role of a glucagon/cAMP-dependent protein kinase–inducible coactivator PGC-1α signaling pathway is well characterized in hepatic gluconeogenesis. However, an opposing protein kinase B (PKB)/Akt-inducible corepressor signaling pathway is unknown. A previous report has demonstrated that small heterodimer partner–interacting leucine zipper protein (SMILE) regulates the nuclear receptors and transcriptional factors that control hepatic gluconeogenesis. Here, we show that hepatic SMILE expression was induced by feeding in normal mice but not in db/db and high-fat diet (HFD)-fed mice. Interestingly, SMILE expression was induced by insulin in mouse primary hepatocyte and liver. Hepatic SMILE expression was not altered by refeeding in liver-specific insulin receptor knockout (LIRKO) or PKB β-deficient (PKBβ−/−) mice. At the molecular level, SMILE inhibited hepatocyte nuclear factor 4–mediated transcriptional activity via direct competition with PGC-1α. Moreover, ablation of SMILE augmented gluconeogenesis and increased blood glucose levels in mice. Conversely, overexpression of SMILE reduced hepatic gluconeogenic gene expression and ameliorated hyperglycemia and glucose intolerance in db/db and HFD-fed mice. Therefore, SMILE is an insulin-inducible corepressor that suppresses hepatic gluconeogenesis. Small molecules that enhance SMILE expression would have potential for treating hyperglycemia in diabetes.

Liver plays a crucial role in controlling energy homeostasis in mammals, although the exact mechanism by which it influences other peripheral tissues has yet to be addressed. Here we show that Creb regulates transcriptional co-activator (Crtc) 2 is a major regulator of whole-body energy metabolism. Crtc2 liver-specific knockout lowers blood glucose levels with improved glucose and insulin tolerance. Liver-specific knockout mice display increased energy expenditure with smaller lipid droplets in adipose depots. Both plasma and hepatic Fgf21 levels are increased in Crtc2 liver-specific knockout mice, as a result of the reduced miR-34a expression regulated by Creb/Crtc2 and the induction of Sirt1 and Pparα. Ectopic expression of miR-34a reverses the metabolic changes in knockout liver. We suggest that Creb/Crtc2 negatively regulates the Sirt1/Pparα/Fgf21 axis via the induction of miR-34a under diet-induced obesity and insulin-resistant conditions., CREB-regulated transcription coactivator 2, CRTC2, has been associated with regulation of glucose and lipid homeostasis. Here Han et al. show that Creb/Crtc2 modulates lipid and glucose metabolism by inhibiting the expression of mi-R34 that, in turn, represses the expression of Sirt1 and PPARα and consequently Fgf21 levels.

Objective Nuclear factor interleukin-3 regulated (NFIL3) has been known as an important transcriptional regulator of the development and the differentiation of immune cells. Although expression of NFIL3 is regulated by nutritional cues in the liver, the role of NFIL3 in the glucose metabolism has not been extensively studied. Thus, we wanted to explore the potential role of NFIL3 in the control of hepatic glucose metabolism. Materials/Methods Mouse primary hepatocytes were cultured to perform western blot analysis, Q-PCR and chromatin immunoprecipitation assay. 293 T cells were cultured to perform luciferase assay. Male C57BL/6 mice (fed a normal chow diet or high fat diet for 27 weeks) as well as ob / ob mice were used for experiments with adenoviral delivery. Results We observed that NFIL3 reduced glucose production in hepatocytes by reducing expression of gluconeogenic gene transcription. The repression by NFIL3 required its basic leucine zipper DNA binding domain, and it competed with CREB onto the binding of cAMP response element in the gluconeogenic promoters. The protein levels of hepatic NFIL3 were decreased in the mouse models of genetic- and diet-induced obesity and insulin resistance, and ectopic expression of NFIL3 in the livers of insulin resistant mice ameliorated hyperglycemia and glucose intolerance, with concomitant reduction in expression of hepatic gluconeogenic genes. Finally, we witnessed that knockdown of NFIL3 in the livers of normal chow-fed mice promoted elevations in the glucose levels and expression of hepatic gluconeogenic genes. Conclusions In this study, we showed that NFIL3 functions as an important regulator of glucose homeostasis in the liver by limiting CREB-mediated hepatic gluconeogenesis. Thus, enhancement of hepatic NFIL3 activity in insulin resistant state could be potentially beneficial in relieving glycemic symptoms in the metabolic diseases.

Cyclic AMP promotes chronic expression of target genes mainly by protein kinase A–dependent activation of CREB transcription factor machineries in the metabolic tissues. Here, we wanted to elaborate whether CREB-regulated transcription factor (CRTC)2 and its negative regulator salt-inducible kinase (SIK)2 are involved in the transcriptional control of the metabolic pathway in adipocytes. SIK2 knockout (SIK2 KO) mice exhibited higher blood glucose levels that were associated with impaired glucose and insulin tolerance. Hypertriglyceridemia was apparent in SIK2 KO mice, mainly due to the increased lipolysis from white adipocytes and the decreased fatty acid uptake in the peripheral tissues. Investigation of white adipocytes revealed the increases in fat cell size and macrophage infiltration, which could be linked to the metabolic anomaly that is associated in these mice. Interestingly, SIK2 KO promoted the enhancement in the CRTC2-CREB transcriptional pathway in white adipocytes. SIK2 KO mice displayed increased expression of activating transcription factor (ATF)3 and subsequent downregulation of GLUT4 expression and reduction in high–molecular weight adiponectin levels in the plasma, leading to the reduced glucose uptake in the muscle and white adipocytes. The effect of SIK2-dependent regulation of adipocyte metabolism was further confirmed by in vitro cell cultures of 3T3 L1 adipocytes and the differentiated preadipocytes from the SIK2 or CRTC2 KO mice. Collectively, these data suggest that SIK2 is critical in regulating whole-body glucose metabolism primarily by controlling the CRTC2-CREB function of the white adipocytes.

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 (LXRα)-mediated sterol regulatory element binding protein-1c (SREBP-1c) gene expression. We found that SMILE inhibited T0901317 (T7)-induced transcriptional activity of LXRα, 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 LXRα and represses T7-induced LXRα 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 LXRα-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 LXRα-mediated hepatic lipogenic enzyme gene expression.

Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder with diverse pathological manifestations and is often associated with abnormal regulation of hepatic glucose production. Many nuclear receptors known to control the hepatic gluconeogenic program are potential targets for the treatment of T2DM and its complications. Nevertheless, the therapeutic potential of the estrogen-related receptor γ (ERRγ) in T2DM remains unknown. In this study, we show that the nuclear receptor ERRγ is a major contributor to hyperglycemia under diabetic conditions by controlling hepatic glucose production. Hepatic ERRγ expression induced by fasting and diabetic conditions resulted in elevated levels of gluconeogenic gene expression and blood glucose in wild-type mice. Conversely, ablation of hepatic ERRγ gene expression reduced the expression of gluconeogenic genes and normalized blood glucose levels in mouse models of T2DM: db/db and diet-induced obesity (DIO) mice. In addition, a hyperinsulinemic-euglycemic clamp study and long-term studies of the antidiabetic effects of GSK5182, the ERRγ-specific inverse agonist, in db/db and DIO mice demonstrated that GSK5182 normalizes hyperglycemia mainly through inhibition of hepatic glucose production. Our findings suggest that the ability of GSK5182 to control hepatic glucose production can be used as a novel therapeutic approach for the treatment of T2DM.

Liver plays a major role in maintaining glucose homeostasis in mammals. Under fasting conditions, hepatic glucose production is critical as a source of fuel to maintain the basic functions in other tissues, including skeletal muscle, red blood cells, and the brain. Fasting hormones glucagon and cortisol play major roles during the process, in part by activating the transcription of key enzyme genes in the gluconeogenesis such as phosphoenol pyruvate carboxykinase (PEPCK) and glucose 6 phosphatase catalytic subunit (G6Pase). Conversely, gluconeogenic transcription is repressed by pancreatic insulin under feeding conditions, which effectively inhibits transcriptional activator complexes by either promoting post-translational modifications or activating transcriptional inhibitors in the liver, resulting in the reduction of hepatic glucose output. The transcriptional regulatory machineries have been highlighted as targets for type 2 diabetes drugs to control glycemia, so understanding of the complex regulatory mechanisms for transcription circuits for hepatic gluconeogenesis is critical in the potential development of therapeutic tools for the treatment of this disease. In this review, the current understanding regarding the roles of two key transcriptional activators, CREB and FoxO1, in the regulation of hepatic gluconeogenic program is discussed. [BMB Reports 2013; 46(12): 567-574]

Diet-induced obesity is a major contributing factor to the progression of hepatic insulin resistance. Increased free fatty acids in liver enhances endoplasmic reticulum (ER) stress and production of reactive oxygen species (ROS), both are directly responsible for dysregulation of hepatic insulin signaling. BI-1, a recently studied ER stress regulator, was examined to investigate its association with ER stress and ROS in insulin resistance models. To induce obesity and insulin resistance, BI-1 wild type and BI-1 knock-out mice were fed a high-fat diet for 8 weeks. The BI-1 knock-out mice had hyperglycemia, was associated with impaired glucose and insulin tolerance under high-fat diet conditions. Increased activity of NADPH-dependent CYP reductase-associated cytochrome p450 2E1 (CYP2E1) and exacerbation of ER stress in the livers of BI-1 knock-out mice was also observed. Conversely, stable expression of BI-1 in HepG2 hepatocytes was shown to reduce palmitate-induced ER stress and CYP2E1-dependent ROS production, resulting in the preservation of intact insulin signaling. Stable expression of CYP2E1 led to increased ROS production and dysregulation of insulin signaling in hepatic cells, mimicking palmitate-mediated hepatic insulin resistance. We propose that BI-1 protects against obesity-induced hepatic insulin resistance by regulating CYP2E1 activity and ROS production.

In this study, we explored the effects of Bax Inhibitor-1 (BI-1) on ApoB aggregation in high-fat diet (HFD)-induced hepatic lipid accumulation. After 1 week on a HFD, triglycerides and cholesterol accumulated more in the liver and were not effectively secreted into the plasma, whereas after 8 weeks, lipids were highly accumulated in both the liver and plasma, with a greater effect in BI-1 KO mice compared with BI-1 WT mice. ApoB, a lipid transfer protein, was accumulated to a greater extent in the livers of HFD-BI-1 KO mice compared with HFD-BI-1 WT mice. Excessive post-translational oxidation of protein disulfide isomerase (PDI), intra-ER ROS accumulation and folding capacitance alteration were also observed in HFD-BI-1 KO mice. Higher levels of endoplasmic reticulum (ER) stress were consistently observed in KO mice compared with the WT mice. Adenovirus-mediated hepatic expression of BI-1 in the BI-1 KO mice rescued the above phenotypes. Our results suggest that BI-1-mediated enhancement of ApoB secretion regulates hepatic lipid accumulation, likely through regulation of ER stress and ROS accumulation.

Glucose homeostasis is tightly controlled by hormonal regulation of hepatic glucose production. Dysregulation of this system is often associated with insulin resistance and diabetes, resulting in hyperglycemia in mammals. Here, we show that the orphan nuclear receptor estrogen-related receptor γ (ERRγ) is a novel downstream mediator of glucagon action in hepatic gluconeogenesis and demonstrate a beneficial impact of the inverse agonist GSK5182. Hepatic ERRγ expression was increased by fasting-dependent activation of the cAMP-response element-binding protein-CRTC2 pathway. Overexpression of ERRγ induced Pck1 and G6PC gene expression and glucose production in primary hepatocytes, whereas abolition of ERRγ gene expression attenuated forskolin-mediated induction of gluconeogenic gene expression. Deletion and mutation analyses of the Pck1 promoter showed that ERRγ directly regulates the Pck1 gene transcription via ERR response elements of the Pck1 promoter as confirmed by ChIP assay and in vivo imaging analysis. We also demonstrate that GSK5182, an inverse agonist of ERRγ, specifically inhibits the transcriptional activity of ERRγ in a PGC-1α dependent manner. Finally, the ERRγ inverse agonist ameliorated hyperglycemia through inhibition of hepatic gluconeogenesis in db/db mice. Control of hepatic glucose production by an ERRγ-specific inverse agonist is a new potential therapeutic approach for the treatment of type 2 diabetes.

Activated cannabinoid 1 receptor (CB1R) signaling has been implicated in the development of phenotypes associated with fatty liver, insulin resistance, and impaired suppression of hepatic glucose output. Endoplasmic reticulum stress-associated liver-specific transcription factor CREBH is emerging as a critical player in various hepatic metabolic pathways and regulates hepatic gluconeogenesis in diet-induced obese settings. In this study, we elucidated the critical role of CREBH in mediating CB1R signaling to regulate glucose homeostasis in primary rat and human hepatocytes. mRNA and protein levels and glucose production were analyzed in primary rat and human hepatocytes. ChIP assays were performed together with various transcriptional analyses using standard techniques. CB1R activation by 2-arachidonoylglycerol (2-AG) specifically induced CREBH gene expression via phosphorylation of the JNK signaling pathway and c-Jun binding to the AP-1 binding site in the CREBH gene promoter. 2-AG treatment significantly induced hepatic gluconeogenic gene expression and glucose production in primary hepatocytes, and we demonstrated that the CREBH binding site mutant significantly attenuated 2-AG-mediated activation of the gluconeogenic gene promoter. Endogenous knockdown of CREBH led to ablation of 2-AG-induced gluconeogenic gene expression and glucose production, and the CB1R antagonist AM251 or insulin exhibited repression of CREBH gene induction and subsequently inhibited gluconeogenesis in both rat and human primary hepatocytes. These results demonstrate a novel mechanism of action of activated CB1R signaling to induce hepatic gluconeogenesis via direct activation of CREBH, thereby contributing to a better understanding of the endocannabinoid signaling mechanism involved in regulating the hepatic glucose metabolism.

Dysregulation of lipid metabolism is a key feature of metabolic disorder related to side effects of antipsychotic drugs. Here, we investigated the molecular mechanism by which second-generation atypical antipsychotic drugs (AAPDs) affect hepatic lipid metabolism in liver. AAPDs augmented hepatic lipid accumulation by activating expression of sterol regulatory element-binding protein (SREBP) transcription factors, with subsequent induction of downstream target genes involved in lipid and cholesterol synthesis in hepatocytes. We confirmed the direct involvement of SREBPs on AAPD-induced expression of lipogenic and cholesterogenic genes by utilization of adenovirus for dominant negative SREBP (Ad-SREBP-DN). Interestingly, AAPDs significantly decreased phosphorylation of AMPKα and expression of fatty acid oxidation genes. Treatment of constitutive active AMPK restored AAPD-mediated dysregulation of genes involved in both lipid synthesis and fatty acid oxidation. Moreover, AAPDs decreased transcriptional activity of PPARα, a critical transcriptional regulator for controlling hepatic fatty acid oxidation, via an AMPK-dependent manner. Close investigations revealed that mutations at the known p38 MAPK phosphorylation sites (S6/12/21A), but not mutations at the putative AMPKα phosphorylation sites (S167/373/453A), block AAPD-dependent reduction of PPARα transcriptional activity, suggesting that p38 MAPK might be also involved in the regulatory pathway as a downstream effector of AAPDs/AMPK. Taken together, these data suggest that AAPD-stimulated hepatic dysregulation of lipid metabolism could result from the inhibition of AMPK activity, and pharmaceutical means to potentiate AMPK activity would contribute to restore hepatic lipid homeostasis that occurs during AAPD treatment.

OBJECTIVE Diet-induced obesity (DIO) is linked to peripheral insulin resistance—a major predicament in type 2 diabetes. This study aims to identify the molecular mechanism by which DIO-triggered endoplasmic reticulum (ER) stress promotes hepatic insulin resistance in mouse models. RESEARCH DESIGN AND METHODS C57BL/6 mice and primary hepatocytes were used to evaluate the role of LIPIN2 in ER stress-induced hepatic insulin resistance. Tunicamycin, thapsigargin, and lipopolysaccharide were used to invoke acute ER stress conditions. To promote chronic ER stress, mice were fed with a high-fat diet for 8–12 weeks. To verify the role of LIPIN2 in hepatic insulin signaling, adenoviruses expressing wild-type or mutant LIPIN2, and shRNA for LIPIN2 were used in animal studies. Plasma glucose, insulin levels as well as hepatic free fatty acids, diacylglycerol (DAG), and triacylglycerol were assessed. Additionally, glucose tolerance, insulin tolerance, and pyruvate tolerance tests were performed to evaluate the metabolic phenotype of these mice. RESULTS LIPIN2 expression was enhanced in mouse livers by acute ER stress–inducers or by high-fat feeding. Transcriptional activation of LIPIN2 by ER stress is mediated by activating transcription factor 4, as demonstrated by LIPIN2 promoter assays, Western blot analyses, and chromatin immunoprecipitation assays. Knockdown of hepatic LIPIN2 in DIO mice reduced fasting hyperglycemia and improved hepatic insulin signaling. Conversely, overexpression of LIPIN2 impaired hepatic insulin signaling in a phosphatidic acid phosphatase activity–dependent manner. CONCLUSIONS These results demonstrate that ER stress–induced LIPIN2 would contribute to the perturbation of hepatic insulin signaling via a DAG-protein kinase C ε–dependent manner in DIO mice.

Liver plays a major role in regulating energy homeostasis in mammals. During feeding conditions, excessive glucose is converted into a preferred storage form of energy sources as triacylglycerol in liver via a collective metabolic pathway termed lipogenesis. Sterol regulatory element-binding protein 1c is a master regulator for this process by activating number of enzyme genes, such as Fasn or Acaca, that are involved in this pathway at the transcriptional level. Here we show that the salt-inducible kinase (SIK) family of proteins regulates the hepatic lipogenesis by modulating SREBP-1c activity. Overexpression of SIK1 inhibits hepatic expression of lipogenic genes, such as Fasn, whereas knockdown of SIK1 in liver greatly enhances their expression. Regulation of the Fasn gene by SIK kinases is mediated at the level of transcription via phosphorylation and inactivation of nuclear SREBP-1c. Among candidate sites for SIK-dependent regulation of SREBP-1c, the serine 329 residue is shown to be a critical regulatory site for SIK-mediated repression of SREBP-1c activity by in vitro kinase assay and reverse transcription-PCR analysis in primary hepatocytes. Finally, reduced hepatic triacylglycerol levels and lipogenic gene expression by adenoviral SIK1 transgenic expression are restored to normal levels by co-infection of mutant SREBP-1c, suggesting that SIK-dependent regulation of hepatic lipogenesis is indeed mediated through the phosphorylation of SREBP-1c in vivo. The process for the development of nonalcoholic fatty liver involves de novo lipogenesis via the activation of SREBP-1c. Modulation of SREBP-1c activity by SIK proteins would provide an attractive means for the regulation of such diseases.

The Peutz-Jegher syndrome tumor-suppressor gene encodes a protein-threonine kinase, LKB1, which phosphorylates and activates AMPK [adenosine monophosphate (AMP)–activated protein kinase]. The deletion of LKB1 in the liver of adult mice resulted in a nearly complete loss of AMPK activity. Loss of LKB1 function resulted in hyperglycemia with increased gluconeogenic and lipogenic gene expression. In LKB1-deficient livers, TORC2, a transcriptional coactivator of CREB (cAMP response element–binding protein), was dephosphorylated and entered the nucleus, driving the expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which in turn drives gluconeogenesis. Adenoviral small hairpin RNA (shRNA) for TORC2 reduced PGC-1α expression and normalized blood glucose levels in mice with deleted liver LKB1, indicating that TORC2 is a critical target of LKB1/AMPK signals in the regulation of gluconeogenesis. Finally, we show that metformin, one of the most widely prescribed type 2 diabetes therapeutics, requires LKB1 in the liver to lower blood glucose levels.

Insulin resistance is a major hallmark in the development of type 2 diabetes, which is characterized by an impaired ability of insulin to inhibit glucose output from the liver and to promote glucose uptake in muscle. The nuclear hormone receptor coactivator PGC-1 (peroxisome proliferator-activated (PPAR)-gamma coactivator-1) has been implicated in the onset of type 2 diabetes. Hepatic PGC-1 expression is elevated in mouse models of this disease, where it promotes constitutive activation of gluconeogenesis and fatty acid oxidation through its association with the nuclear hormone receptors HNF-4 and PPAR-alpha, respectively. Here we show that PGC-1-deficient mice, generated by adenoviral delivery of PGC-1 RNA interference (RNAi) to the liver, experience fasting hypoglycemia. Hepatic insulin sensitivity was enhanced in PGC-1-deficient mice, reflecting in part the reduced expression of the mammalian tribbles homolog TRB-3, a fasting-inducible inhibitor of the serine-threonine kinase Akt/PKB (ref. 6). We show here that, in the liver, TRB-3 is a target for PPAR-alpha. Knockdown of hepatic TRB-3 expression improved glucose tolerance, whereas hepatic overexpression of TRB-3 reversed the insulin-sensitive phenotype of PGC-1-deficient mice. These results indicate a link between nuclear hormone receptor and insulin signaling pathways, and suggest a potential role for TRB-3 inhibitors in the treatment of type 2 diabetes.

Fasting glucose homeostasis is maintained in part through cAMP (adenosine 3′,5′-monophosphate)– dependent transcriptional control of hepatic gluconeogenesis by the transcription factor CREB (cAMP response element–binding protein) and its coactivator CRTC2 (CREB-regulated transcriptional coactivator 2). We showed that PRMT6 (protein arginine methyltransferase 6) promotes fasting-induced transcriptional activation of the gluconeogenic program involving CRTC2. Mass spectrometric analysis indicated that PRMT6 associated with CRTC2. In cells, PRMT6 mediated asymmetric dimethylation of multiple arginine residues of CRTC2, which enhanced the association of CRTC2 with CREB on the promoters of gluconeogenic enzyme–encoding genes. In mice, ectopic expression of PRMT6 promoted higher blood glucose concentrations, which were associated with increased expression of genes encoding gluconeogenic factors, whereas knockdown of hepatic PRMT6 decreased fasting glycemia and improved pyruvate tolerance. The abundance of hepatic PRMT6 was increased in mouse models of obesity and insulin resistance, and adenovirus-mediated depletion of PRMT6 restored euglycemia in these mice. We propose that PRMT6 is involved in the regulation of hepatic glucose metabolism in a CRTC2-dependent manner.

Transcription of a number of genes involved in lipogenesis is stimulated by dietary carbohydrate in the mammalian liver. Both insulin and increased glucose metabolism have been proposed to be initiating signals for this process, but the pathways by which these effectors act to alter transcription have not been resolved. We have previously defined by electrophoretic mobility shift assay a factor in nuclear extracts from rat liver, designated the carbohydrate-responsive factor (Cho- RF), that binds to liver-type pyruvate kinase and S(14) promoters at sites critical for regulation by carbohydrate. The sterol regulatory element binding protein-1c (SREBP-1c) has also emerged as a major transcription factor involved in this nutritional response. In this study, we examined the relationship between SREBP-1c and ChoRF in lipogenic gene induction. The two factors were found to possess distinct DNA binding specificities both in vitro and in hepatocytes. Reporter constructs containing binding sites for ChoRF were responsive to glucose but not directly to insulin. On the other hand, reporter constructs with an SREBP-1c site responded directly to insulin. The S(14) gene possesses binding sites for both ChoRF and SREBP, and both sites were found to be functionally important for the response of this promoter to glucose and insulin in hepatocytes. Consequently, we propose that SREBP-1c and ChoRF are independent transcription factors that mediate signals generated by insulin and glucose, respectively. For many lipogenic enzyme genes, these two factors may provide an integrated signaling system to support the overall nutritional response to dietary carbohydrate.

Sterol regulatory element binding proteins (SREBPs) play key roles in lipid homeostasis from yeast to humans.1,2 In mammals, three different SREBP isoforms, including SREBP1a, SREBP1c (also known as ADD1), and SREBP2, are encoded by two genes: SREBF1 and SREBF2. SREBP1 regulates fatty acid metabolism, whereas SREBP2 controls cholesterol metabolism.3 When the cellular sterol level is low, SREBP cleavage-activating protein (SCAP) escorts the SREBP precursors from the endoplasmic reticulum (ER) to the Golgi, where SREBPs are cleaved by Site-1 and Site-2 proteases.4 Subsequently, the mature forms of SREBPs are translocated into the nucleus and stimulate the expression of target genes.5,6 SREBP1a and SREBP1c are generated through transcription from alternative promoters and splicing from a single SREBF1 gene. In metabolic tissues such as adipose tissue and liver, SREBP1c is the predominant isoform of SREBP1.1,7 SREBP1c governs de novo lipogenesis by stimulating its target genes, including fatty acid synthase (FASN), acetyl-CoA carboxylase1 (ACC1), steroyl-CoA desaturase1 (SCD1), and long-chain fatty acid elongase (ELOVL6).8–10 Furthermore, SREBP1c is sensitively regulated by nutritional and hormonal changes to achieve energy balance. For example, SREBP1c is suppressed by fasting, whereas SREBP1c is activated by feeding in adipose tissue and liver.11,12 In parallel, the expression of most lipogenic genes, including FASN, ACC1, and SCD1, is also modulated in a fashion analogous to that of nutritionally regulated SREBP1c.12–14 Accordingly, it has been reported that various hormones, such as insulin, glucagon, and adrenaline, participate in the regulation of SREBP1c and its target lipogenic genes.15,16 Insulin, a key postprandial hormone, stimulates the expression and activity of SREBP1c to accommodate anabolic processes, such as fatty acid synthesis, upon feeding.12,17 In contrast, glucagon, a key catabolic hormone, suppresses the activity of SREBP1c in fasting states, leading to a decrease in lipogenesis.18 In the nucleus, mature SREBPs are very unstable and are rapidly degraded by the proteasome.19,20 Previous reports have shown that SREBP1 is phosphorylated by glycogen synthase kinase-3 beta (GSK-3β), which leads to F-box and WD repeat domain-containing7 (FBXW7)-dependent ubiquitination of SREBP1.21,22 However, a recent in vivo study revealed that inhibition of FBXW7 does not alter the expression of SREBP1c or lipogenic genes in the liver.23 Although SREBP1c-mediated lipogenic program in liver is rapidly repressed by nutritional deprivations, the factors that are involved in the suppression of SREBP1c activity during fasting have not been thoroughly characterized. The finding that ring finger protein20 (RNF20) ubiquitin ligase was identified as one of the novel SREBP1c-interating proteins led us to test whether fasting signaling would promote SREBP1c degradation in an RNF20-dependent manner. In this study, we demonstrate that RNF20 promotes polyubiquitination and degradation of SREBP1c. Overexpression of RNF20 represses SREBP1c activity, leading to a decrease in the expression of lipogenic genes. In obese db/db mice, RNF20 overexpression alleviates hepatic steatosis by reducing the lipogenic program by way of SREBP1c down-regulation. Furthermore, activated PKA, a major signaling cascade that mediates the fasting state, induces degradation of SREBP1c by increasing RNF20 expression. Taken together, these data suggest that RNF20 plays a critical role in the regulation of hepatic lipid metabolism by modulating the protein stability and transcriptional activity of SREBP1c during hormonal changes.

Transforming growth factor (TGF)-beta, a pivotal cytokine involved in a variety of cellular functions, transmits signals through Smad-dependent canonical and Smad-independent noncanonical pathways. In contrast to the canonical TGF-beta pathway, it is unknown how noncanonical TGF-beta pathways are negatively regulated. Here we demonstrate that the inhibitory Smad Smad6, but not Smad7, negatively regulates TGF-beta 1-induced activation of the TRAF6-TAK1-p38 MAPK/JNK pathway, a noncanonical TGF-beta pathway. TGF-beta 1-induced Smad6 abolishes K63-linked polyubiquitination of TRAF6 by recruiting the A20 deubiquitinating enzyme in AML-12 mouse liver cells and primary hepatocytes. In addition, the knockdown of Smad6 or A20 in an animal model or cell culture system maintains TAK1 and p38 MAPK/JNK phosphorylation and increases apoptosis, emphasizing the crucial role of the Smad6-A20 axis in negative regulation of the TGF-beta 1-TRAF6-TAK1-p38 MAPK/JNK pathway. Therefore, our findings provide insight into the molecular mechanisms underlying negative regulation of noncanonical TGF-beta pathways.

Bile acids concentration in liver is tightly regulated to prevent cell damage. Previous studies have demonstrated that deregulation of bile acid homeostasis can lead to cholestatic liver disease. Recently, we have shown that ER-bound transcription factor Crebh is a downstream effector of hepatic Cb1r signaling pathway. In this study, we have investigated the effect of alcohol exposure on hepatic bile acid homeostasis and elucidated the mediatory roles of Cb1r and Crebh in this process. We found that alcohol exposure or Cb1r-agonist 2-AG treatment increases hepatic bile acid synthesis and serum ALT, AST levels in vivo alongwith significant increase in Crebh gene expression and activation. Alcohol exposure activated Cb1r, Crebh, and perturbed bile acid homeostasis. Overexpression of Crebh increased the expression of key bile acid synthesis enzyme genes via direct binding of Crebh to their promoters, whereas Cb1r knockout and Crebh-knockdown mice were protected against alcohol-induced perturbation of bile acid homeostasis. Interestingly, insulin treatment protected against Cb1r-mediated Crebh-induced disruption of bile acid homeostasis. Furthermore, Crebh expression and activation was found to be markedly increased in insulin resistance conditions and Crebh knockdown in diabetic mice model (db/db) significantly reversed alcohol-induced disruption of bile acid homeostasis. Overall, our study demonstrates a novel regulatory mechanism of hepatic bile acid metabolism by alcohol via Cb1r-mediated activation of Crebh, and suggests that targeting Crebh can be of therapeutic potential in ameliorating alcohol-induced perturbation of bile acid homeostasis.

Glucose homeostasis is tightly regulated to meet the energy requirements of the vital organs and maintain an individual's health. The liver has a major role in the control of glucose homeostasis by controlling various pathways of glucose metabolism, including glycogenesis, glycogenolysis, glycolysis and gluconeogenesis. Both the acute and chronic regulation of the enzymes involved in the pathways are required for the proper functioning of these complex interwoven systems. Allosteric control by various metabolic intermediates, as well as post-translational modifications of these metabolic enzymes constitute the acute control of these pathways, and the controlled expression of the genes encoding these enzymes is critical in mediating the longer-term regulation of these metabolic pathways. Notably, several key transcription factors are shown to be involved in the control of glucose metabolism including glycolysis and gluconeogenesis in the liver. In this review, we would like to illustrate the current understanding of glucose metabolism, with an emphasis on the transcription factors and their regulators that are involved in the chronic control of glucose homeostasis.

Glucose is a primary fuel for generating energy in basic daily activities. Thus, glucose homeostasis is tightly regulated by counter-regulatory hormones such as glucagon, cortisol, and insulin, which affect key organs including liver, skeletal muscle, pancreas, and adipocytes. Among metabolic tissues, liver plays a critical role in controlling glucose production under various hormonal and metabolic cues. Under fasting, acute activation of both glycogenolysis and gluconeogenesis is achieved by post-translational modification or allosteric activation of key rate-limiting enzymes, thereby enabling enhanced glucose production from the liver to maintain glucose homeostasis. More prolonged fasting or starvation leads to the chronic activation of gluconeogenesis that requires increased expression of key enzymes in the pathway, which is turned off under feeding conditions by the molecular events that are initiated by insulin. This process is normally achieved by the regulation of gene expression at the level of transcription. Recently, the transcriptional regulators of hepatic gluconeogenesis are considered as potential therapeutic targets for the treatment of type 2 diabetes. In this review, we would like to discuss the current knowledge regarding the key transcriptional activators and inhibitors of hepatic gluconeogenic program to provide the better insight into the control of glycemia in the disease status.

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.

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., Author Summary Previous genome-wide association studies revealed that TCF7L2 is a strong candidate for a type 2 diabetes gene. However, the direct involvement of TCF7L2 on hepatic glucose metabolism has been elusive to date. Here we show that TCF7L2 is critical in mediating transcriptional control of hepatic glucose production. We found that hepatic expression of nuclear isoforms of TCF7L2 was reduced in mouse models of insulin resistance. Acute depletion of TCF7L2 in the liver promoted glucose intolerance and up-regulation of gluconeogenic genes, while ectopic expression of TCF7L2 in DIO mice improved glucose tolerance. TCF7L2 was shown to bind to the gluconeogenic promoters, thereby interfering with the promoter occupancies of both CREB/CRTC2 and FoxO1 on their cognate sites. Furthermore, TCF7L2 haploinsufficiency promoted higher glucose levels with impaired glucose tolerance and increased hepatic glucose production in mice, and adenovirus-mediated TCF7L2 expression in the liver reversed the phenotype. We propose that TCF7L2 is a critical player in regulating glucose homeostasis in mammals by modulating hepatic glucose production.

Fasting promotes hepatic gluconeogenesis to maintain glucose homeostasis. The cAMP-response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is responsible for transcriptional activation of gluconeogenic genes and is critical for conveying the opposing hormonal signals of glucagon and insulin in the liver. Here, we show that suppressor of MEK null 1 (SMEK1) and SMEK2 [protein phosphatase 4 (PP4) regulatory subunits 3a and 3b, respectively] are directly involved in the regulation of hepatic glucose metabolism in mice. Expression of hepatic SMEK1/2 is up-regulated during fasting or in mouse models of insulin-resistant conditions in a Peroxisome Proliferator-Activated Receptor-gamma Coactivator 1α (PGC-1α)-dependent manner. Overexpression of SMEK promotes elevations in plasma glucose with increased hepatic gluconeogenic gene expression, whereas depletion of the SMEK proteins reduces hyperglycemia and enhances CRTC2 phosphorylation; the effect is blunted by S171A CRTC2, which is refractory to salt-inducible kinase (SIK)-dependent inhibition. Taken together, we would propose that mammalian SMEK/PP4C proteins are involved in the regulation of hepatic glucose metabolism through dephosphorylation of CRTC2.

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is a member of the nuclear receptor superfamily that can repress diverse nuclear receptors and has a key role in adreno-gonadal development. Our previous report has demonstrated that DAX-1 can inhibit hepatocyte nuclear factor 4alpha transactivity and negatively regulate gluconeogenic gene expression (Nedumaran, B., Hong, S., Xie, Y. B., Kim, Y. H., Seo, W. Y., Lee, M. W., Lee, C. H., Koo, S. H., and Choi, H. S. (2009) J. Biol. Chem. 284, 27511-27523). Here, we further expand the role of DAX-1 in hepatic energy metabolism. Transfection assays have demonstrated that DAX-1 can inhibit the transcriptional activity of nuclear receptor liver X receptor alpha (LXRalpha). Physical interaction between DAX-1 and LXRalpha was confirmed Immunofluorescent staining in mouse liver shows that LXRalpha and DAX-1 are colocalized in the nucleus. Domain mapping analysis shows that the entire region of DAX-1 is involved in the interaction with the ligand binding domain region of LXRalpha. Competition analyses demonstrate that DAX-1 competes with the coactivator SRC-1 for repressing LXRalpha transactivity. Chromatin immunoprecipitation assay showed that endogenous DAX-1 recruitment on the SREBP-1c gene promoter was decreased in the presence of LXRalpha agonist. Overexpression of DAX-1 inhibits T7-induced LXRalpha target gene expression, whereas knockdown of endogenous DAX-1 significantly increases T7-induced LXRalpha target gene expression in HepG2 cells. Finally, overexpression of DAX-1 in mouse liver decreases T7-induced LXRalpha target gene expression, liver triglyceride level, and lipid accumulation. Overall, this study suggests that DAX-1, a novel corepressor of LXRalpha, functions as a negative regulator of lipogenic enzyme gene expression in liver.

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is an atypical member of the nuclear receptor family and acts as a corepressor of a number of nuclear receptors. HNF4alpha (hepatocyte nuclear factor 4alpha) is a liver-enriched transcription factor that controls the expression of a variety of genes involved in cholesterol, fatty acid, and glucose metabolism. Here we show that DAX-1 inhibits transcriptional activity of HNF4alpha and modulates hepatic gluconeogenic gene expression. Hepatic DAX-1 expression is increased by insulin and SIK1 (salt-inducible kinase 1), whereas it is decreased in high fat diet-fed and diabetic mice. Coimmunoprecipitation assay from mouse liver samples depicts that endogenous DAX-1 interacts with HNF4alpha in vivo. In vivo chromatin immunoprecipitation assay affirms that the recruitment of DAX-1 on the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is inversely correlated with the recruitment of PGC-1alpha and HNF4alpha under fasting and refeeding, showing that DAX-1 could compete with the coactivator PGC-1alpha for binding to HNF4alpha. Adenovirus-mediated expression of DAX-1 decreased both HNF4alpha- and forskolin-mediated gluconeogenic gene expressions. In addition, knockdown of DAX-1 partially reverses the insulin-mediated inhibition of gluconeogenic gene expression in primary hepatocytes. Finally, DAX-1 inhibits PEPCK and glucose-6-phosphatase gene expression and significantly lowers fasting blood glucose level in high fat diet-fed mice, suggesting that DAX-1 can modulate hepatic gluconeogenesis in vivo. Overall, this study demonstrates that DAX-1 acts as a corepressor of HNF4alpha to negatively regulate hepatic gluconeogenic gene expression in liver.

Summary Endoplasmic reticulum (ER)-bound transcription factor families are shown to be involved in the control of various metabolic pathways. Here, we report a critical function of ER-bound transcription factor, CREBH, in the regulation of hepatic gluconeogenesis. Expression of CREBH is markedly induced by fasting or in the insulin-resistant state in rodents in a dexamethasone- and PGC-1α-dependent manner, which results in the accumulation of active nuclear form of CREBH (CREBH-N). Overexpression of constitutively active CREBH activates transcription of PEPCK-C or G6Pase by binding to its enhancer site that is distinct from the well-characterized CREB/CRTC2 regulatory sequences in vivo. Of interest, knockdown of CREBH in liver significantly reduces blood glucose levels without altering expression of genes involved in the ER stress signaling cascades in mice. These data suggest a crucial role for CREBH in the regulation of hepatic glucose metabolism in mammals.

FoxO transcription factors are important targets of insulin action. To better understand the role of FoxO proteins in the liver, we created transgenic mice expressing constitutively active FoxO1 in the liver using the alpha1-antitrypsin promoter. Fasting glucose levels are increased, and glucose tolerance is impaired in transgenic (TGN) versus wild type (WT) mice. Interestingly, fasting triglyceride and cholesterol levels are reduced despite hyperinsulinemia, and post-prandial changes in triglyceride levels are markedly suppressed in TGN versus WT mice. Activation of pro-lipogenic signaling pathways (atypical protein kinase C and protein kinase B) and the ability to suppress beta-hydroxybutyrate levels are not impaired in TGN. In contrast, de novo lipogenesis measured with (3)H(2)O is suppressed by approximately 70% in the liver of TGN versus WT mice after refeeding. Gene-array studies reveal that the expression of genes involved in gluconeogenesis, glycerol transport, and amino acid catabolism is increased, whereas genes involved in glucose utilization by glycolysis, the pentose phosphate shunt, lipogenesis, and sterol synthesis pathways are suppressed in TGN versus WT. Studies with adenoviral vectors in isolated hepatocytes confirm that FoxO1 stimulates expression of gluconeogenic genes and suppresses expression of genes involved in glycolysis, the shunt pathway, and lipogenesis, including glucokinase and SREBP-1c. Together, these results indicate that FoxO proteins promote hepatic glucose production through multiple mechanisms and contribute to the regulation of other metabolic pathways important in the adaptation to fasting and feeding in the liver, including glycolysis, the pentose phosphate shunt, and lipogenic and sterol synthetic pathways.

SummaryInsulin resistance is a primary defect in type 2 diabetes characterized by impaired peripheral glucose uptake and insufficient suppression of hepatic glucose output. Insulin signaling inhibits liver glucose production by inducing nuclear exclusion of the gluconeogenic transcription factor FOXO1 in an Akt-dependent manner. Through the concomitant application of genome-scale functional screening and quantitative image analysis, we have identified PTP-MEG2 as a modulator of insulin-dependent FOXO1 subcellular localization. Ectopic expression of PTP-MEG2 in cells inhibited insulin-induced phosphorylation of the insulin receptor, while RNAi-mediated reduction of PTP-MEG2 transcript levels enhanced insulin action. Additionally, adenoviral-mediated depletion of PTP-MEG2 in livers of diabetic (db/db) mice resulted in insulin sensitization and normalization of hyperglycemia. These data implicate PTP-MEG2 as a mediator of blood glucose homeostasis through antagonism of insulin signaling, and suggest that modulation of PTP-MEG2 activity may be an effective strategy in the treatment of type 2 diabetes.

SummaryUnder fasting conditions, the cAMP-responsive CREB coactivator TORC2 promotes glucose homeostasis by stimulating the gluconeogenic program in liver. Following its nuclear translocation in response to elevations in circulating glucagon, TORC2 regulates hepatic gene expression via an association with CREB on relevant promoters. Here, we show that, in parallel with their effects on glucose output, CREB and TORC2 also enhance insulin signaling in liver by stimulating expression of the insulin receptor substrate 2 (IRS2) gene. The induction of hepatic IRS2 during fasting appears critical for glucose homeostasis; knockdown of hepatic IRS2 expression leads to glucose intolerance, whereas hepatic IRS2 overexpression attenuates the gluconeogenic program and reduces fasting glucose levels. By stimulating the expression of IRS2 in conjunction with gluconeogenic genes, the CREB:TORC2 pathway thus triggers a feedback response that limits glucose output from the liver during fasting.

Fasting triggers a series of hormonal cues that promote energy balance by inducing glucose output and lipid breakdown in the liver. In response to pancreatic glucagon and adrenal cortisol, the cAMP-responsive transcription factor CREB activates gluconeogenic and fatty acid oxidation programmes by stimulating expression of the nuclear hormone receptor coactivator PGC-1 (refs 2-5). In parallel, fasting also suppresses lipid storage and synthesis (lipogenic) pathways, but the underlying mechanism is unknown. Here we show that mice deficient in CREB activity have a fatty liver phenotype and display elevated expression of the nuclear hormone receptor PPAR-gamma, a key regulator of lipogenic genes. CREB inhibits hepatic PPAR-gamma expression in the fasted state by stimulating the expression of the Hairy Enhancer of Split (HES-1) gene, a transcriptional repressor that is shown here to be a mediator of fasting lipid metabolism in vivo. The coordinate induction of PGC-1 and repression of PPAR-gamma by CREB during fasting provides a molecular rationale for the antagonism between insulin and counter-regulatory hormones, and indicates a potential role for CREB antagonists as therapeutic agents in enhancing insulin sensitivity in the liver.

The rat acetyl-CoA carboxylase (ACC) α gene is transcribed from two promoters, denoted PI and PII, that direct regulated expression in a tissue-specific manner. Induction of ACC, the rate-controlling enzyme of fatty acid biosynthesis, occurs in the liver in response to feeding of a high carbohydrate, low fat diet, conditions that favor enhanced lipogenesis. This induction is mainly due to increases in PI promoter activity. We have used primary cultured hepatocytes from the rat to investigate glucose regulation of ACC expression. Glucose and insulin synergistically activated expression of ACC mRNAs transcribed from the PI promoter with little or no effect on PII mRNAs. Glucose treatment stimulated PI promoter activity in transfection assays and a glucose-regulated element was identified (−126/−102), homologous to those previously described in other responsive genes, including l-type pyruvate kinase, S14 and fatty acid synthase. Mutation of this element eliminated the response to glucose. This region of the ACC PI promoter was able to bind a liver nuclear factor designated ChoRF that interacts with other conserved glucose-regulated elements. This ACC PI element is also capable of conferring a strong response to glucose when linked to a heterologous promoter. We conclude that induction of ACC gene expression under lipogenic conditions in hepatocytes is mediated in part by the activation of a glucose-regulated transcription factor, ChoRF, which stimulates transcription from the PI promoter. Similar mechanisms operate on related genes permitting the coordinate induction of the lipogenic pathway.

Refeeding carbohydrate to fasted rats induces the transcription of genes encoding enzymes of fatty acid biosynthesis, e.g. fatty-acid synthase (FAS). Part of this transcriptional induction is mediated by insulin. An insulin response element has been described for the fatty-acid synthase gene region of -600 to +65, but the 2-3-fold increase in fatty-acid synthase promoter activity attributable to this region is small compared with the 20-30-fold induction in fatty-acid synthase gene transcription observed in fasted rats refed carbohydrate. We have previously reported that the fatty-acid synthase gene region between -7382 and -6970 was essential for achieving high in vivo rates of gene transcription. The studies of the current report demonstrate that the region of -7382 to -6970 of the fatty-acid synthase gene contains a carbohydrate response element (CHO-RE(FAS)) with a palindrome sequence (CATGTGn(5)GGCGTG) that is nearly identical to the CHO-RE of the l-type pyruvate kinase and S(14) genes. The glucose responsiveness imparted by CHO-RE(FAS) was independent of insulin. Moreover, CHO-RE(FAS) conferred glucose responsiveness to a heterologous promoter (i.e. l-type pyruvate kinase). Electrophoretic mobility shift assays demonstrated that CHO-RE(FAS) readily bound a unique hepatic ChoRF and that CHO-RE(FAS) competed with the CHO-RE of the l-type pyruvate kinase and S(14) genes for ChoRF binding. In vivo footprinting revealed that fasting reduced and refeeding increased ChoRF binding to CHO-RE(FAS). Thus, carbohydrate responsiveness of rat liver fatty-acid synthase appears to require both insulin and glucose signaling pathways. More importantly, a unique hepatic ChoRF has now been shown to recognize glucose responsive sequences that are common to three different genes: fatty-acid synthase, l-type pyruvate kinase, and S(14).

VLDL levels are elevated in type II diabetes, where they contribute to the risk of coronary heart disease. A study by Wolfrum and Stoffel (2006) shows that the forkhead protein Foxa2 stimulates hepatic VLDL production in concert with the coactivator PGC-1beta and that insulin inhibits this process by inactivating Foxa2.

During fasting periods, hepatic glucose production is enhanced by glucagon to provide fuels for other organs. This process is mediated via cAMP-dependent induction of the CREB regulated transcriptional coactivator (CRTC) 2, a critical transcriptional activator for hepatic gluconeogenesis. We have previously shown that CRTC2 activity is regulated by AMP activated protein kinase (AMPK) family members. Here we show that adiponectin and thiazolidinedione directly regulate AMPK to modulate CRTC2 activity in hepatocytes. Adiponectin or thiazolidinedione lowered glucose production from primary hepatocytes. Treatment of both reagents reduced gluconeogenic gene expression as well as cAMP-mediated induction of CRE reporter, suggesting that these reagents directly affect CREB/CRTC2- dependent transcription. Furthermore, adiponectin or thiazolidinedione mediated repression of CRE activity is largely blunted by co-expression of phosphorylation defective mutant CRTC2, underscoring the importance of serine 171 residue of this factor. Taken together, we propose that adiponectin and thiazolidinedione promote the modulation of AMPK-dependent CRTC2 activity to influence hepatic gluconeogenesis.