OBJECTIVE—Metformin is an antidiabetic drug commonly used to treat type 2 diabetes. The aim of the study was to determine whether metformin regulates hepatic gluconeogenesis through the orphan nuclear receptor small heterodimer partner (SHP; NR0B2). RESEARCH DESIGN AND METHODS—We assessed the regulation of hepatic SHP gene expression by Northern blot analysis with metformin and adenovirus containing a constitutive active form of AMP-activated protein kinase (AMPK) (Ad-AMPK) and evaluated SHP, PEPCK, and G6Pase promoter activities via transient transfection assays in hepatocytes. Knockdown of SHP using siRNA SHP was conducted to characterize the metformin-induced inhibition of hepatic gluconeogenic gene expression in hepatocytes, and metformin–and adenovirus SHP (Ad-SHP)–mediated hepatic glucose production was measured in B6-Lepob/ob mice. RESULTS—Hepatic SHP gene expression was induced by metformin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), and Ad-AMPK. Metformin-induced SHP gene expression was abolished by adenovirus containing the dominant negative form of AMPK (Ad-DN-AMPK), as well as by compound C. Metformin inhibited hepatocyte nuclear factor-4α–or FoxA2-mediated promoter activity of PEPCK and G6Pase, and the inhibition was blocked with siRNA SHP. Additionally, SHP knockdown by adenovirus containing siRNA SHP inhibited metformin-mediated repression of cAMP/dexamethasone-induced hepatic gluconeogenic gene expression. Furthermore, oral administration of metformin increased SHP mRNA levels in B6-Lepob/ob mice. Overexpression of SHP by Ad-SHP decreased blood glucose levels and hepatic gluconeogenic gene expression in B6-Lepob/ob mice. CONCLUSIONS—We have concluded that metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of SHP.

In response to microbial infection, expression of the defensin-like peptide hepcidin (encoded by Hamp) is induced in hepatocytes to decrease iron release from macrophages. To elucidate the mechanism by which Salmonella enterica var. Typhimurium (S. typhimurium), an intramacrophage bacterium, alters host iron metabolism for its own survival, we examined the role of nuclear receptor family members belonging to the NR3B subfamily in mouse hepatocytes. Here, we report that estrogen-related receptor γ (ERRγ, encoded by Esrrg) modulates the intramacrophage proliferation of S. typhimurium by altering host iron homeostasis, and we demonstrate an antimicrobial effect of an ERRγ inverse agonist. Hepatic ERRγ expression was induced by S. typhimurium-stimulated interleukin-6 signaling, resulting in an induction of hepcidin and eventual hypoferremia in mice. Conversely, ablation of ERRγ mRNA expression in liver attenuated the S. typhimurium-mediated induction of hepcidin and normalized the hypoferremia caused by S. typhimurium infection. An inverse agonist of ERRγ ameliorated S. typhimurium-mediated hypoferremia through reduction of ERRγ-mediated hepcidin mRNA expression and exerted a potent antimicrobial effect on the S. typhimurium infection, thereby improving host survival. Taken together, these findings suggest an alternative approach to control multidrug-resistant intracellular bacteria by modulating host iron homeostasis.

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Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of gluconeogenic enzyme gene expression. Here, we show that SHP inhibited protein kinase A-mediated transcriptional activity of cAMP-response element-binding protein (CREB), a major regulator of glucose metabolism, to modulate hepatic gluconeogenic gene expression. Deletion analysis of phosphoenolpyruvate carboxykinase (PEPCK) promoter demonstrated that SHP inhibited forskolin-mediated induction of PEPCK gene transcription via inhibition of CREB transcriptional activity. In vivo imaging demonstrated that SHP inhibited CREB-regulated transcription coactivator 2 (CRTC2)-mediated cAMP-response elementdriven promoter activity. Furthermore, overexpression of SHP using adenovirus SHP decreased CRTC2-dependent elevations in blood glucose levels and PEPCK or glucose-6-phosphatase (G6Pase) expression in mice. SHP and CREB physically interacted and were co-localized in vivo. Importantly, SHP inhibited both wild type CRTC2 and S171A (constitutively active form of CRTC2) coactivator activity and disrupted CRTC2 recruitment on the PEPCK gene promoter. In addition, metformin or overexpression of a constitutively active form of AMPK (Ad-CA-AMPK) inhibited S171A-mediated PEPCK and G6Pase gene expression, and hepatic glucose production and knockdown of SHP partially relieved the metformin-and Ad-CA-AMPK-mediated repression of hepatic gluconeogenic enzyme gene expression in primary rat hepatocytes. In conclusion, our results suggest that a delayed effect of metformin-mediated induction of SHP gene expression inhibits CREB-dependent hepatic gluconeogenesis.Glucose homeostasis is regulated by the opposing actions of insulin and glucagon (1-3), and glucose production in the liver is controlled primarily by gluconeogenesis (4). The regulation of hepatic gluconeogenesis involves the transcriptional regulation of key metabolic enzymes, including PEPCK 6 and G6Pase. The gluconeogenic program is largely regulated at the level of transcription and the process is coordinated by CREB via its direct binding to the cAMP-response element (CRE) site on the promoter of PEPCK, G6Pase, or PGC-1␣ (PPAR␥ coactivator-1␣) (5).Metformin has been shown to activate AMP-activated protein kinase (AMPK) via an LKB1-dependent mechanism (6). AMPK is a serine/threonine kinase that functions as an intracellular energy sensor and has been implicated in the modulation of glucose and fatty acid metabolism (7). AMPK is activated by physiological stimuli, including exercise, muscle contraction, and hormones, such as adiponectin and leptin, as well as by physiological stresses, glucose deprivation, hypoxia, oxidative stress, and osmotic shock conditions (8, 9). In the liver, activation of AMPK suppresses hepatic gluconeogenesis acutely by direct phosphorylation of its substrates, including CREB-binding protein (CBP) (10), CRTC2 (11), and GSK3␤ (glycogen synthase kinase 3␤) (12). Recent studies also suggest that AMPK induces SHP gene expression and inhibits hepatic g...

The highly developed endoplasmic reticulum (ER) structure of pancreatic beta-cells is a key factor in beta-cell function. Here we examined whether ER stress-induced activation of activating transcription factor (ATF)-6 impairs insulin gene expression via up-regulation of the orphan nuclear receptor small heterodimer partner (SHP; NR0B2), which has been shown to play a role in beta-cell dysfunction. We examined whether ER stress decreases insulin gene expression, and this process is mediated by ATF6. A small interfering RNA that targeted SHP was used to determine whether the effect of ATF6 on insulin gene expression is mediated by SHP. We also measured the expression level of ATF6 in pancreatic islets in Otsuka Long Evans Tokushima Fatty rats, a rodent model of type 2 diabetes. High glucose concentration (30 mmol/liter glucose) increased ER stress in INS-1 cells. ER stress induced by tunicamycin, thapsigargin, or dithiotreitol decreased insulin gene transcription. ATF6 inhibited insulin promoter activity, whereas X-box binding protein-1 and ATF4 did not. Adenovirus-mediated overexpression of active form of ATF6 in INS-1 cells impaired insulin gene expression and secretion. ATF6 also down-regulated pancreatic duodenal homeobox factor-1 and RIPE3b1/MafA gene expression and repressed the cooperative action of pancreatic duodenal homeobox factor-1, RIPE3b1/MafA, and beta-cell E box transactivator 2 in stimulating insulin transcription. The ATF6-induced suppression of insulin gene expression was associated with up-regulation of SHP gene expression. Finally, we found that expression of ATF6 was increased in the pancreatic islets of diabetic Otsuka Long Evans Tokushima Fatty rats, compared with their lean, nondiabetic counterparts, Long-Evans Tokushima Otsuka rats. Collectively, this study shows that ER stress-induced activation of ATF6 plays an important role in the development of beta-cell dysfunction.

BACKGROUND AND PURPOSEEndoplasmic reticulum (ER) stress has been implicated in the pathogeneses of insulin resistance and type 2 diabetes, and extracellular signal-regulated kinase (ERK) antagonist is an insulin sensitizer that can restore muscle insulin responsiveness in both tunicamycin-treated muscle cells and type 2 diabetic mice. The present study was undertaken to determine whether the chemical or genetic inhibition ER stress pathway targeting by ERK results in metabolic benefits in muscle cells. EXPERIMENTAL APPROACHER stress was induced in L6 myotubes using tunicamycin (5 mg·mL -1 ) or thapsigargin (300 nM) and cells were transfected with siRNA ERK or AMPKa2. Changes in ER stress and in the ERK and AMPK signalling pathways were explored by Western blotting. The phosphorylation levels of insulin receptor substrate 1 were analysed by immunoprecipitation and using glucose uptake assay. KEY RESULTSER stress dampened insulin-stimulated signals and glucose uptake, whereas treatment with the specific ERK inhibitor U0126 (25 mM) rescued impaired insulin signalling via AMPK activation. In db/db mice, U0126 administration decreased markers of insulin resistance and increased the phosphorylations of Akt and AMPK in muscle tissues. CONCLUSIONS AND IMPLICATIONSInhibition of ERK signalling pathways by a chemical inhibitor and knockdown of ERK improved AMPK and Akt signallings and reversed ER stress-induced insulin resistance in L6 myotubes. These findings suggest that ERK signalling plays an important role in the regulation of insulin signals in muscle cells under ER stress. AbbreviationsACC, acetyl-CoA carboxylase; Ad-DN-AMPK, AMPK adenovirus dominant negative; AMPK, AMP-activated protein kinase; ER, endoplasmic reticulum; PERK, RNA-activated protein kinase-like ER resident kinase; IRE-1, inositol-requiring kinase-1; siRNA, small interfering RNA; UPR, unfolded protein response; IRS-1, insulin receptor substrate-1; WT, wild type BJP British Journal of Pharmacology

A nonsense mutation in cereblon (CRBN) causes a mild type of mental retardation in humans. An earlier study showed that CRBN negatively regulates the functional activity of AMP-activated protein kinase (AMPK) in vitro by binding directly to the α1-subunit of the AMPK complex. However, the in vivo role of CRBN was not studied. For elucidation of the physiological functions of Crbn, a mouse strain was generated in which the Crbn gene was deleted throughout the whole body. In Crbn-deficient mice fed a normal diet, AMPK in the liver showed hyperphosphorylation, which indicated the constitutive activation of AMPK. Since Crbn-deficient mice showed significantly less weight gain when fed a high-fat diet and their insulin sensitivity was considerably improved, the functions of Crbn in the liver were primarily investigated. These results provide the first in vivo evidence that Crbn is a negative modulator of AMPK, which suggests that Crbn may be a potential target for metabolic disorders of the liver.

Background:The PAP function of LIPINs is involved in the regulation of intracellular lipid levels and hepatic insulin receptor signaling. Results: ERR␥-mediated induction of LIPIN1 results in the perturbation of hepatic insulin signaling through DAG-mediated activation of PKC⑀. Conclusion: ERR␥ is a novel transcriptional regulator of LIPIN1. Significance: An ERR␥ inverse agonist could ameliorate LIPIN1-mediated perturbation of hepatic insulin signaling.