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Find video protocols related to scientific articles indexed in Pubmed.
AIMP1 negatively regulates adipogenesis by inhibiting PPAR?.
J. Cell. Sci.
PUBLISHED: 08-21-2014
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Adipogenesis is known to be controlled by the concerted actions of transcription factors and co-regulators. However, little is known about the mechanism of regulation of the transcription factors that control adipogenesis. In addition, the adipogenic roles of translational factors remain unclear. Here, we show that aminoacyl tRNA synthetase-interacting multifunctional protein 1 (AIMP1, also known as p43), an auxiliary factor that is associated with a macromolecular tRNA synthetase complex, negatively regulates adipogenesis through a direct interaction with the DNA-binding domain of peroxisome proliferator-activated receptor ? (PPAR?). We found that AIMP1 expression increases during adipocyte differentiation. Adipogenesis is augmented in AIMP1-deficient cells, as compared with control cells. AIMP1 exhibits high affinity for active PPAR? and interacts with the DNA-binding domain of PPAR?, thereby inhibiting its transcriptional activity. Thus, AIMP1 appears to function as a novel inhibitor of PPAR? that regulates adipocyte differentiation by preventing the transcriptional activation of PPAR?.
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Transforming growth factor-?1 signaling represses testicular steroidogenesis through cross-talk with orphan nuclear receptor Nur77.
PLoS ONE
PUBLISHED: 08-20-2014
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Transforming growth factor- ?1 (TGF-?1) has been reported to inhibit luteinizing hormone (LH) mediated-steroidogenesis in testicular Leydig cells. However, the mechanism by which TGF-?1 controls the steroidogenesis in Leydig cells is not well understood. Here, we investigated the possibility that TGF-?1 represses steroidogenesis through cross-talk with the orphan nuclear receptor Nur77. Nur77, which is induced by LH/cAMP signaling, is one of major transcription factors that regulate the expression of steroidogenic genes in Leydig cells. TGF-?1 signaling inhibited cAMP-induced testosterone production and the expression of steroidogenic genes such as P450c17, StAR and 3?-HSD in mouse Leydig cells. Further, TGF-?1/ALK5 signaling repressed cAMP-induced and Nur77-activated promoter activity of steroidogenic genes. In addition, TGF-?1/ALK5-activated Smad3 repressed Nur77 transactivation of steroidogenic gene promoters by interfering with Nur77 binding to DNA. In primary Leydig cells isolated from Tgfbr2flox/flox Cyp17iCre mice, TGF-?1-mediated repression of cAMP-induced steroidogenic gene expression was significantly less than that in primary Leydig cells from Tgfbr2flox/flox mice. Taken together, these results suggest that TGF-?1/ALK5/Smad3 signaling represses the expression of steroidogenic genes via the suppression of Nur77 transactivation in testicular Leydig cells. These findings may provide a molecular mechanism involved in the TGF-?1-mediated repression of testicular steroidogenesis.
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Small heterodimer partner blocks cardiac hypertrophy by interfering with GATA6 signaling.
Circ. Res.
PUBLISHED: 07-11-2014
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Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA-binding domain. Through interactions with other transcription factors, SHP regulates diverse biological events, including glucose metabolism in liver. However, the role of SHP in adult heart diseases has not yet been demonstrated.
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PKB/Akt phosphorylation of ERR? contributes to insulin-mediated inhibition of hepatic gluconeogenesis.
Diabetologia
PUBLISHED: 06-05-2014
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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 ? (ERR?) is a major transcriptional regulator of hepatic gluconeogenesis. In this study, we investigated insulin-dependent post-translational modifications (PTMs) altering the transcriptional activity of ERR? for the regulation of hepatic gluconeogenesis.
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SMILE upregulated by metformin inhibits the function of androgen receptor in prostate cancer cells.
Cancer Lett.
PUBLISHED: 05-19-2014
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Metformin, a diabetes drug, has been reported to inhibit the growth of prostate cancer cells. In this study, we investigated the effect and action mechanism of metformin on the function of androgen receptor (AR), a key molecule in the proliferation of prostate cancer cells. Metformin was found to reduce androgen-dependent cell growth and the expression of AR target genes by inhibiting AR function in prostate cancer cells such as LNCaP and C4-2 cells. Interestingly, metformin upregulated the protein level of small heterodimer partner-interacting leucine zipper (SMILE), a coregulator of nuclear receptors, and knockdown of SMILE expression with shRNA abolished the inhibitory effect of metformin on AR function. Further studies revealed that SMILE protein itself suppressed the transactivation of AR, and its ectopic expression resulted in the repressed expression of endogenous AR target genes, PSA and NKX3.1, in LNCaP cells. In addition, SMILE protein physically interacted with AR and competed with the AR coactivator SRC-1 to modulate AR transactivation. As expected, SMILE repressed androgen-dependent growth of LNCaP and C4-2 cells. Taken together, these results suggest that SMILE, which is induced by metformin, functions as a novel AR corepressor and may mediate the inhibitory effect of metformin on androgen-dependent growth of prostate cancer cells.
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The adipokine Retnla modulates cholesterol homeostasis in hyperlipidemic mice.
Nat Commun
PUBLISHED: 04-26-2014
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Hyperlipidemia is a well-recognized risk factor for atherosclerosis and can be regulated by adipokines. Expression of the adipokine resistin-like molecule alpha (Retnla) is regulated by food intake; whether Retnla has a role in the pathogenesis of hyperlipidemia and atherosclerosis is unknown. Here we report that Retnla has a cholesterol-lowering effect and protects against atherosclerosis in low-density lipoprotein receptor-deficient mice. On a high-fat diet, Retnla deficiency promotes hypercholesterolaemia and atherosclerosis, whereas Retnla overexpression reverses these effects and improves the serum lipoprotein profile, with decreased cholesterol in the very low-density lipoprotein fraction concomitant with reduced serum apolipoprotein B levels. We show that Retnla upregulates cholesterol-7-?-hydroxylase, a key hepatic enzyme in the cholesterol catabolic pathway, through induction of its transcriptional activator liver receptor homologue-1, leading to increased excretion of cholesterol in the form of bile acids. These findings define Retnla as a novel therapeutic target for treating hypercholesterolaemia and atherosclerosis.
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B-cell translocation gene 2 regulates hepatic glucose homeostasis via induction of orphan nuclear receptor Nur77 in diabetic mouse model.
Diabetes
PUBLISHED: 03-19-2014
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B-cell translocation gene 2 (BTG2) is a member of an emerging gene family that is involved in cellular functions. In this study, we demonstrate that BTG2 regulates glucose homeostasis via upregulation of Nur77 in diabetic mice. Hepatic BTG2 gene expression was elevated by fasting and forskolin. Overexpression of Btg2 increased the expression of hepatic gluconeogenic genes and blood glucose output and subsequently impaired glucose and insulin tolerance. Upregulation of the transcriptional activity of Nur77, gluconeogenic genes, and glucose production by forskolin was observed by Btg2 transduction, but not in Btg2 knockdown. BTG2-stimulated glucose production and glucose-6-phosphatase promoter activity were attenuated by dominant-negative Nur77. Coimmunoprecipitation and chromatin immunoprecipitation assays showed that BTG2 induced Nur77 occupancy on the glucose-6-phosphatase promoter via a physical interaction. Btg2 gene expression was increased in streptozotocin-treated and db/db mice. Finally, impairment of glucose homeostasis, such as the increase of blood glucose, glucose intolerance, and insulin intolerance, was elevated in diabetic mice, whereas this phenomenon was abolished in knockdown of Btg2. Together, these data suggest that BTG2 participates in the regulation of hepatic glucose homeostasis, which means that BTG2 might serve as a potential therapeutic target for combating metabolic dysfunction.
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Inverse agonist of estrogen-related receptor ? controls Salmonella typhimurium infection by modulating host iron homeostasis.
Nat. Med.
PUBLISHED: 01-17-2014
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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 ? (ERR?, encoded by Esrrg) modulates the intramacrophage proliferation of S. typhimurium by altering host iron homeostasis, and we demonstrate an antimicrobial effect of an ERR? inverse agonist. Hepatic ERR? expression was induced by S. typhimurium-stimulated interleukin-6 signaling, resulting in an induction of hepcidin and eventual hypoferremia in mice. Conversely, ablation of ERR? 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 ERR? ameliorated S. typhimurium-mediated hypoferremia through reduction of ERR?-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 Err? induces C-reactive protein gene expression through induction of ER-bound Bzip transmembrane transcription factor CREBH.
PLoS ONE
PUBLISHED: 01-01-2014
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The orphan nuclear receptor estrogen-related receptor-? (ERR?) is a constitutively active transcription factor regulating genes involved in several important cellular processes, including hepatic glucose metabolism, alcohol metabolism, and the endoplasmic reticulum (ER) stress response. cAMP responsive element-binding protein H (CREBH) is an ER-bound bZIP family transcription factor that is activated upon ER stress and regulates genes encoding acute-phase proteins whose expression is increased in response to inflammation. Here, we report that ERR? directly regulates CREBH gene expression in response to ER stress. ERR? bound to the ERR? response element (ERRE) in the CREBH promoter. Overexpression of ERR? by adenovirus significantly increased expression of CREBH as well as C-reactive protein (CRP), whereas either knockdown of ERR? or inhibition of ERR? by ERR? specific inverse agonist, GSK5182, substantially inhibited ER stress-mediated induction of CREBH and CRP. The transcriptional coactivator PGC1? was required for ERR? mediated induction of the CREBH gene as demonstrated by the chromatin immunoprecipitation (ChIP) assay showing binding of both ERR? and PGC1? on the CREBH promoter. The ChIP assay also revealed that histone H3 and H4 acetylation occurred at the ERR? and PGC1? binding site. Moreover, chronic alcoholic hepatosteatosis, as well as the diabetic obese condition significantly increased CRP gene expression, and this increase was significantly attenuated by GSK5182 treatment. We suggest that orphan nuclear receptor ERR? directly regulates the ER-bound transcription factor CREBH in response to ER stress and other metabolic conditions.
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Ursodeoxycholic acid inhibits liver X receptor ?-mediated hepatic lipogenesis via induction of the nuclear corepressor SMILE.
J. Biol. Chem.
PUBLISHED: 11-21-2013
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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 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 (AMPK)-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 AMPK phosphorylation, which leads to repression of LXR?-mediated hepatic lipogenic enzyme gene expression.
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Estrogen-related receptor gamma induces cardiac hypertrophy by activating GATA4.
J. Mol. Cell. Cardiol.
PUBLISHED: 08-07-2013
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Estrogen-related receptor gamma (ERR?) is an orphan nuclear receptor that has biological roles mainly in metabolism and that controls metabolic switching in perinatal heart. In adult heart diseases, however, the functional roles of ERR? have not yet been elucidated. In the present study, we aimed to characterize the role of ERR? in cardiac hypertrophy. The functional roles of ERR? in the development of cardiac hypertrophy were examined in primary cultured cardiomyocytes and in animal models. ERR? expression was increased in hearts from human hypertrophic cardiomyopathy patients and in both cellular and animal models of cardiac hypertrophy. Transgenic overexpression in mouse heart as well as forced expression of ERR? in cardiomyocytes induced hypertrophic phenotypes. Knock-down of ERR? blocked agonist-induced hypertrophic phenotypes. ERR? bound directly to the proximal ERR-responsive element in the GATA4 promoter in a sequence-specific manner and thereby induced transcription. ERR?-induced hypertrophy was blocked by inhibition of GATA4. GSK-5182, an inverse agonist of ERR?, completely blocked cardiac hypertrophy in cardiomyocytes. It also prevented aortic banding-induced cardiac hypertrophy and fibrosis in mouse heart. These findings demonstrate a novel ERR?/GATA4 signal cascade in the development of cardiac hypertrophy and suggest GSK-5182 as a possible therapeutic.
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Total synthesis of eryvarin H and its derivatives and their biological activity as ERR? inverse agonist.
Org. Biomol. Chem.
PUBLISHED: 08-02-2013
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Total synthesis of eryvarin H and a biological investigation of its analogues as a potential inverse agonist of ERR? are described here. Among the 13 analogues prepared by the modular synthetic route, eryvarin H and compound showed meaningful ERR? inverse agonistic activities along with moderate selectivity over ER? and other nuclear receptors in the cell-based reporter gene assay.
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Inverse agonist of nuclear receptor ERR? mediates antidiabetic effect through inhibition of hepatic gluconeogenesis.
Diabetes
PUBLISHED: 06-17-2013
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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 ? (ERR?) in T2DM remains unknown. In this study, we show that the nuclear receptor ERR? is a major contributor to hyperglycemia under diabetic conditions by controlling hepatic glucose production. Hepatic ERR? 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 ERR? 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 ERR?-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.
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Transcriptional cross talk between orphan nuclear receptor ERR? and transmembrane transcription factor ATF6? coordinates endoplasmic reticulum stress response.
Nucleic Acids Res.
PUBLISHED: 05-28-2013
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Orphan nuclear receptor ERR? 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 ERR? gene remains to be elucidated. Here, we report that activating transcription factor 6? (ATF6?), an endoplasmic reticulum (ER)-membrane-bound basic leucine zipper (bZip) transcription factor, directly regulates ERR? gene expression in response to ER stress. ATF6? binds to ATF6? responsive element in the ERR? promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-? (PGC-1?) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6? and PGC1? on the ERR? promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6? and PGC1? binding site. Of interest, ERR? along with PGC1? induce ATF6? gene transcription upon ER stress. ERR? binds to an ERR? responsive element in the ATF6? promoter. ChIP assay confirmed that both ERR? and PGC1? bind to a site in the ATF6? 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 ERR? and ATF6? gene transcription.
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Insulin directly regulates steroidogenesis via induction of the orphan nuclear receptor DAX-1 in testicular Leydig cells.
J. Biol. Chem.
PUBLISHED: 04-15-2013
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Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-? (phospho-IR-?), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states.
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The orphan nuclear receptor small heterodimer partner negatively regulates pancreatic beta cell survival and hyperglycemia in multiple low-dose streptozotocin-induced type 1 diabetic mice.
Int. J. Biochem. Cell Biol.
PUBLISHED: 03-27-2013
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The small heterodimer partner (SHP; NR0B2) regulates the transcription of a variety of target genes and controls a variety of physiological functions in various tissues. However, the role of SHP in beta cell has not been fully determined yet. We used SHP knockout (SHP KO) mice to investigate the role of SHP in multiple low-dose streptozotocin (MLDS)-induced diabetes. Blood glucose and insulin levels were measured until 20 days, and intraperitoneal glucose tolerance and glucose-stimulated insulin secretion tests were performed. The expression of apoptotic genes and beta cell markers were detected by quantitative realtime-polymerase chain reaction, immunostaining and western blot analysis. SHP KO mice showed significantly lower blood glucose, higher insulin levels, and enhanced glucose tolerance compared with wild type (WT) mice after MLDS treatment. Moreover, beta cell mass and pancreatic insulin content were remarkably increased in SHP KO mice. In the response to glucose stimulation, islets of SHP KO showed increased insulin secretion via up-regulation of beta cell enriched transcription factors compared to WT mice after streptozotocin (STZ) treatment. In quantification for beta cell apoptosis at day 1 post STZ treatment, the SHP KO mice showed significantly increased anti-apoptotic gene expression and decreased release of apoptotic markers cytochrome c, smac/diablo, and only a few apoptotic beta cells were found in SHP KO pancreas through inactivation of caspase-3, compared to those of WT. These data demonstrate that SHP deficiency ameliorates hyperglycemia and preserves islet function by inhibiting apoptosis of pancreatic beta cells and up-regulating of their enriched transcriptional factors.
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Disruption of the cereblon gene enhances hepatic AMPK activity and prevents high-fat diet-induced obesity and insulin resistance in mice.
Diabetes
PUBLISHED: 01-24-2013
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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 ?1-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.
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Emodin regulates glucose utilization by activating AMP-activated protein kinase.
J. Biol. Chem.
PUBLISHED: 01-09-2013
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AMP-activated protein kinase has been described as a key signaling protein that can regulate energy homeostasis. Here, we aimed to characterize novel AMP-activated kinase (AMPK)-activating compounds that have a much lower effective concentration than metformin. As a result, emodin, a natural anthraquinone derivative, was shown to stimulate AMPK activity in skeletal muscle and liver cells. Emodin enhanced GLUT4 translocation and [(14)C]glucose uptake into the myotube in an AMPK-dependent manner. Also, emodin inhibited glucose production by suppressing the expression of key gluconeogenic genes, such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, in hepatocytes. Furthermore, we found that emodin can activate AMPK by inhibiting mitochondrial respiratory complex I activity, leading to increased reactive oxygen species and Ca(2+)/calmodulin-dependent protein kinase kinase activity. Finally, we confirmed that a single dose administration of emodin significantly decreased the fasting plasma glucose levels and improved glucose tolerance in C57Bl/6J mice. Increased insulin sensitivity was also confirmed after daily injection of emodin for 8 days using an insulin tolerance test and insulin-stimulated PI3K phosphorylation in wild type and high fat diet-induced diabetic mouse models. Our study suggests that emodin regulates glucose homeostasis in vivo by AMPK activation and that this may represent a novel therapeutic principle in the treatment of type 2 diabetic models.
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Hepatic cannabinoid receptor type 1 mediates alcohol-induced regulation of bile acid enzyme genes expression via CREBH.
PLoS ONE
PUBLISHED: 01-01-2013
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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.
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Small heterodimer partner-targeting therapy inhibits systemic inflammatory responses through mitochondrial uncoupling protein 2.
PLoS ONE
PUBLISHED: 01-01-2013
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The orphan nuclear receptor, small heterodimer partner (SHP), appears to play a negative regulatory role in innate immune signaling. Emerging evidence warrants further study on the therapeutic targeting of SHP to suppress excessive and deleterious inflammation. Here we show that fenofibrate, which targets SHP, is required for inhibiting systemic inflammation via mitochondrial uncoupling protein 2 (UCP2). In vivo administration of fenofibrate ameliorated systemic inflammatory responses and increased survival upon experimental sepsis through SHP. An abundance of SHP was observed in mice fed fenofibrate and in cultured macrophages through LKB1-dependent activation of the AMP-activated protein kinase pathway. Fenofibrate significantly blocked endotoxin-triggered inflammatory signaling responses via SHP, but not via peroxisome proliferator-activated receptor (PPAR)-?. In addition to the known mechanism by which SHP modulates innate signaling, we identify a new role of fenofibrate-induced SHP on UCP2 induction, which is required for the suppression of inflammatory responses through modulation of mitochondrial ROS production. These data strongly suggest that the SHP-inducing drug fenofibrate paves the way for novel therapies for systemic inflammation by targeting SHP.
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BMP2 protein regulates osteocalcin expression via Runx2-mediated Atf6 gene transcription.
J. Biol. Chem.
PUBLISHED: 11-18-2011
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Bone morphogenetic protein 2 (BMP2) activates unfolded protein response (UPR) transducers, such as PERK and OASIS, in osteoblast cells. ATF6, a bZIP transcription factor, is also a UPR transducer. However, the involvement of ATF6 in BMP2-induced osteoblast differentiation has not yet been elucidated. In the present study, BMP2 treatment was shown to markedly induce the expression and activation of ATF6 with an increase in alkaline phosphatase (ALP) and OC expression in MC3T3E1 cells. In contrast, ATF6 activation by BMP2 was not observed in the Runx2(-/-) primary calvarial osteoblasts, and Runx2 overexpression recovered BMP2 action. BMP2 stimulated ATF6 transcription by enhancing the direct binding of Runx2 to the osteoblast-specific cis-acting element 2 (OSE2, ACCACA, -205 to -200 bp) motif of the Atf6 promoter region. In addition, the overexpression of ATF6 increased the Oc promoter activity by enhancing the direct binding to a putative ATF6 binding motif (TGACGT, -1126 to -1121 bp). The inhibition of ATF6 function with the dominant negative form of ATF6 (DN-ATF6) blocked BMP2- or Runx2-induced OC expression. Interestingly, OASIS, which is structurally similar to ATF6, did not induce Oc expression. ALP and Alizarin red staining results confirmed that BMP2-induced matrix mineralization was also dependent on ATF6 in vitro. Overall, these results suggest that BMP2 induces osteoblast differentiation through Runx2-dependent ATF6 expression, which directly regulates Oc transcription.
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Curcumin differentially regulates endoplasmic reticulum stress through transcriptional corepressor SMILE (small heterodimer partner-interacting leucine zipper protein)-mediated inhibition of CREBH (cAMP responsive element-binding protein H).
J. Biol. Chem.
PUBLISHED: 10-12-2011
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Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), is a natural polyphenolic compound. Herein the effect of curcumin on endoplasmic reticulum (ER) stress responsive gene expression was investigated. We report that curcumin induces transcriptional corepressor small heterodimer partner-interacting leucine zipper protein (SMILE) gene expression through liver kinase B1 (LKB1)/adenosine monophosphate-activated kinase (AMPK) signaling pathway and represses ER stress-responsive gene transcription in an ER-bound transcription factor specific manner. cAMP responsive element-binding protein H (CREBH) and activating transcription factor 6 (ATF6) are both ER-bound bZIP family transcription factors that are activated upon ER stress. Of interest, we observed that both curcumin treatment and SMILE overexpression only represses CREBH-mediated transactivation of the target gene but not ATF6-mediated transactivation. Knockdown of endogenous SMILE significantly releases the inhibitory effect of curcumin on CREBH transactivation. Intrinsic repressive activity of SMILE is observed in the Gal4 fusion system, and the intrinsic repressive domain is mapped to the C terminus of SMILE spanning amino acid residues 203-269, corresponding to the basic region leucine zipper (bZIP) domain. In vivo interaction assay revealed that through its bZIP domain, SMILE interacts with CREBH and inhibits its transcriptional activity. Interestingly, we observed that SMILE does not interact with ATF6. Furthermore, competition between SMILE and the coactivator peroxisome proliferator-activated receptor ? (PGC-1?) on CREBH transactivation has been demonstrated in vitro and in vivo. Finally, chromatin immunoprecipitation assays revealed that curcumin decreases the binding of PGC-1? and CREBH on target gene promoter in a SMILE-dependent manner. Overall, for the first time we suggest a novel phenomenon that the curcumin/LKB1/AMPK/SMILE/PGC1? pathway differentially regulates ER stress-mediated gene transcription.
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Estrogen-related receptor ? (ERR?) is a novel transcriptional regulator of phosphatidic acid phosphatase, LIPIN1, and inhibits hepatic insulin signaling.
J. Biol. Chem.
PUBLISHED: 09-12-2011
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LIPINs have been reported to perform important roles in the regulation of intracellular lipid levels. Their mutations induce lipodystrophy, myoglobinuria, and inflammatory disorders. Recently, the phosphatidic acid phosphatase function of LIPINs has been associated with the perturbation of hepatic insulin receptor signaling via the diacylglycerol-mediated stimulation of PKC? activity. Here, we report that nuclear estrogen-related receptor (ERR) ? is a novel transcriptional regulator of LIPIN1. Overexpression of ERR? significantly increased LIPIN1 expression in primary hepatocytes, whereas the abolition of ERR? gene expression attenuated the expression of LIPIN1. Deletion and mutation analyses of the LIPIN1 promoter showed that ERR? exerts its effect on the transcriptional regulation of LIPIN1 via ERRE1 of the LIPIN1 promoter, as confirmed by ChIP assay. We also determined that the gene transcription of LIPIN1 by ERR? is controlled by the competition between PGC-1? and small heterodimer partner. Additionally, ERR? leads to the induction of hepatic LIPIN1 expression and diacylglycerol production in vivo. Finally, an inverse agonist of ERR?, GSK5182, restores the impaired insulin signaling induced by LIPIN1-mediated PKC? activation. Our findings indicate that the selective control of ERR? transcriptional activity by its specific inverse agonist could provide a novel therapeutic approach to the amelioration of impaired hepatic insulin signaling induced by LIPIN1-mediated PKC? activation.
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Cannabinoid receptor type 1 (CB1R) signaling regulates hepatic gluconeogenesis via induction of endoplasmic reticulum-bound transcription factor cAMP-responsive element-binding protein H (CREBH) in primary hepatocytes.
J. Biol. Chem.
PUBLISHED: 06-21-2011
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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.
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Nrf2 inhibits LXR?-dependent hepatic lipogenesis by competing with FXR for acetylase binding.
Antioxid. Redox Signal.
PUBLISHED: 06-13-2011
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The nuclear receptor liver X receptor-? (LXR?) stimulates lipogenesis, leading to steatosis. Nuclear factor erythroid-2-related factor-2 (Nrf2) contributes to cellular defense mechanism by upregulating antioxidant genes, and may protect the liver from injury inflicted by fat accumulation. However, whether Nrf2 affects LXR? activity is unknown. This study investigated the inhibitory role of Nrf2 in hepatic LXR? activity and the molecular basis.
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The orphan nuclear receptor SHP acts as a negative regulator in inflammatory signaling triggered by Toll-like receptors.
Nat. Immunol.
PUBLISHED: 04-21-2011
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The orphan nuclear receptor SHP (small heterodimer partner) is a transcriptional corepressor that regulates hepatic metabolic pathways. Here we identified a role for SHP as an intrinsic negative regulator of Toll-like receptor (TLR)-triggered inflammatory responses. SHP-deficient mice were more susceptible to endotoxin-induced sepsis. SHP had dual regulatory functions in a canonical transcription factor NF-?B signaling pathway, acting as both a repressor of transactivation of the NF-?B subunit p65 and an inhibitor of polyubiquitination of the adaptor TRAF6. SHP-mediated inhibition of signaling via the TLR was mimicked by macrophage-stimulating protein (MSP), a strong inducer of SHP expression, via an AMP-activated protein kinase-dependent signaling pathway. Our data identify a previously unrecognized role for SHP in the regulation of TLR signaling.
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Effect of 2,2,4,4-tetrahydroxybenzophenone (BP2) on steroidogenesis in testicular Leydig cells.
Toxicology
PUBLISHED: 03-21-2011
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Endocrine disruptors (EDs) affect the function of animal reproductive systems. Recently, 2,2,4,4-tetrahydroxybenzophenone (BP2), which is a component of UV protection products, was found to be an ED that interferes with the thyroid hormone (TH) axis. However, BP2 activity in the testis has not been well addressed. In this study, we have examined the effects of BP2 on steroidogenesis in testicular Leydig cells in connection with thyroid hormone signaling, which is known to play an important role in testicular development and function. Our study showed that BP2 affected the expression of steroidogenic enzyme genes in testicular Leydig cells, which is differentially regulated by thyroid hormone/thyroid hormone receptor (TR) signaling. In MA-10 Leydig cell line, TR/T3 signaling increased the expression of P450c17 and P450scc, while it decreased the expression of StAR and 3?-HSD. Interestingly, BP2 affected the expression of steroidogenic enzyme genes in a manner opposite to that of T3 signaling. BP2 downregulated the TR?/T3-activation of P450c17 and P450scc expression while enhancing the TR?/T3-repression of StAR and 3?-HSD expression. Transient transfection analyses with promoter-reporter constructs revealed that BP2 altered the expression of steroidogenic enzyme genes by affecting the cAMP and Nur77-activated promoter activity of P450c17, StAR, and 3?-HSD. Animal experiments with mice revealed that BP2 decreased the production of testosterone in the testis by affecting the expression of some steroidogenic enzyme genes in vivo. Together, these findings elucidate a molecular mechanism of BP2 action underlying testicular steroidogenesis and also suggest that BP2 acts, in part, as a thyroid antagonist that affects steroidogenesis in the testis.
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?-Lipoic acid prevents neointimal hyperplasia via induction of p38 mitogen-activated protein kinase/Nur77-mediated apoptosis of vascular smooth muscle cells and accelerates postinjury reendothelialization.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 09-09-2010
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To explore whether ?-lipoic acid (ALA), a naturally occurring antioxidant, inhibits neointimal hyperplasia by inducing apoptosis of vascular smooth muscle cells and to examine its potential effects on reendothelialization and platelet aggregation.
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AMPK-dependent repression of hepatic gluconeogenesis via disruption of CREB.CRTC2 complex by orphan nuclear receptor small heterodimer partner.
J. Biol. Chem.
PUBLISHED: 08-05-2010
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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 element-driven 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.
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Metformin induces osteoblast differentiation via orphan nuclear receptor SHP-mediated transactivation of Runx2.
Bone
PUBLISHED: 08-02-2010
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Metformin is an oral anti-diabetic drug of the biguanide class that is commonly used to treat type 2 diabetes mellitus. This study examined the molecular mechanism for the action of metformin on osteoblast differentiation. Metformin-induced mRNA expression of the osteogenic genes and small heterodimer partner (SHP) in MC3T3E1 cells were determined by RT-PCR and real-time PCR. Metformin increased significantly the expression of the key osteogenic genes, such as alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP) as well as SHP. Transient transfection assays were performed in MC3T3E1 cells to confirm the effects of metformin on SHP, OC and Runx2 promoter activities. Metformin increased the transcription of the SHP and OC genes, and the metformin effect was inhibited by dominant negative form of AMPK (DN-AMPK) or compound C (an inhibitor of AMPK). The adenoviral overexpression of SHP increased significantly the level of ALP staining and OC production. However, metformin did not have any significant effect on osteogenic gene expression, ALP staining and activity, and OC production in SHP null (SHP-/-) primary calvarial cells. Moreover, upstream stimulatory factor-1 (USF-1) specifically mediated metformin-induced SHP gene expression. In addition, metformin-induced AMPK activation increased the level of Runx2 mRNA and protein. However, USF-1 and SHP were not involved in metformin-induced Runx2 expression. Transient transfection and chromatin immunoprecipitation assays confirmed that metformin-induced SHP interacts physically and forms a complex with Runx2 on the osteocalcin gene promoter in MC3T3E1 cells. These results suggest that metformin may stimulate osteoblast differentiation through the transactivation of Runx2 via AMPK/USF-1/SHP regulatory cascade in mouse calvaria-derived cells.
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Systems-level analysis of gene expression data revealed NR0B2/SHP as potential tumor suppressor in human liver cancer.
Mol. Cells
PUBLISHED: 05-14-2010
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Nuclear receptors (NRs) play pivotal roles in cell growth, proliferation, differentiation and homeostasis. Recent progress demonstrates that NR is tightly linked to human disease such as cancer, diabetes and obesity. Here we explore NR expression profiles in human tissue using systematic approaches. NR gene profiles reveal that individual NR has its own gene expression signature depending on tissue type. Of many organs, NRs expression is enriched in liver. Expression of many NRs was significantly changed in liver cancer. Notably, NR0B2/SHP expression level was significantly decreased in human liver cancer but not in normal liver. In addition, expression of SHP is well associated with good prognosis. SHP gene network analysis based on microarray data in liver cancer shows that SHP regulates cell proliferation and metabolism related gene sets. Our systematic approaches suggest that loss of SHP expression in liver might be key genetic events during hepatocarcinogenesis.
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Transcriptional corepressor SHP recruits SIRT1 histone deacetylase to inhibit LRH-1 transactivation.
Nucleic Acids Res.
PUBLISHED: 04-07-2010
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Orphan nuclear receptor Small Heterodimer Partner (SHP; NR0B2) is a transcriptional corepressor of a wide variety of nuclear receptors (NRs). Here, we report that SHP recruits SIRT1, a class III histone deacetylase, in an NR-specific manner to inhibit transcriptional activity. SHP interacts and co-localizes specifically with SIRT1 in vivo and inhibition of SIRT1 activity leads to a recovery from the intrinsic repressive activity of SHP but not of DAX1. Furthermore, we observed that SIRT1 does not deacetylate SHP or LRH1. However, inhibition of either SIRT1 or SHP significantly diminished the repressive effect of SHP on LRH1 transactivity. LRH1-mediated activation of CYP7A1 and SHP gene transcription was significantly repressed by both SHP and SIRT1 whereas inhibition of SIRT1 activity by inhibitors or dominant negative SIRT1 or knockdown of SHP led to a significant release of this inhibitory effect. ChIP assays revealed that SHP recruits SIRT1 on LRH1 target gene promoters and SIRT1 deacetylated template-dependent histone H3 and H4 to inhibit transcription of LRH1 target genes. Finally, we demonstrated that inhibition of SIRT1 activity significantly reversed SHP-mediated inhibition of bile-acid synthesis by LRH1 overexpression, thereby suggesting a novel mechanism of SHP-mediated inhibition of LRH1-dependent bile-acid homeostasis via recruitment of SIRT1 histone deacetylase protein.
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Targeting orphan nuclear receptor SHP in the treatment of metabolic diseases.
Expert Opin. Ther. Targets
PUBLISHED: 03-17-2010
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The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) is an atypical nuclear receptor that contains a ligand-binding domain, but lacks the conserved DNA-binding domain. Since its discovery, SHP has been identified as a key transcriptional regulatory factor of genes involved in diverse metabolic pathways.
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Regulation of hepatic gluconeogenesis by an ER-bound transcription factor, CREBH.
Cell Metab.
PUBLISHED: 02-26-2010
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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-1alpha-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.
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Alpha-lipoic acid inhibits hepatic PAI-1 expression and fibrosis by inhibiting the TGF-beta signaling pathway.
Biochem. Biophys. Res. Commun.
PUBLISHED: 02-01-2010
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Accumulating evidence suggests that plasminogen activator inhibitor (PAI)-1 plays an important role in the development of hepatic fibrosis via its involvement in extracellular matrix remodeling. We previously reported that alpha-lipoic acid (ALA), a naturally occurring thiol antioxidant, prevents hepatic steatosis by inhibiting the expression of sterol regulatory element binding protein-1c. The aim of the present study was to determine whether ALA prevents hepatic PAI-1 expression and fibrosis through the inhibition of multiple TGF-beta-mediated molecular mediators. We investigated whether ALA inhibited the development of hepatic fibrosis in mice following bile duct ligation (BDL), an established animal model of liver fibrosis. We found that ALA markedly inhibited BDL-induced hepatic fibrosis and PAI-1 expression. We also found that ALA attenuated TGF-beta-stimulated PAI-1 mRNA expression, and inhibited PAI-1 promoter activity in liver cells; this effect was mediated by Smads and the JNK and ERK pathways. The results of the present study indicate that ALA inhibits hepatic PAI-1 expression through inhibition of TGF-beta-mediated molecular mediators, including Smad3, AP1, and Sp1, and prevents the development of BDL-induced hepatic fibrosis. These findings suggest that ALA may have a clinical application in preventing the development and progression of hepatic fibrosis.
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Orphan nuclear receptor DAX-1 acts as a novel corepressor of liver X receptor alpha and inhibits hepatic lipogenesis.
J. Biol. Chem.
PUBLISHED: 01-15-2010
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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.
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Involvement of induction and mitochondrial targeting of orphan nuclear receptor Nur77 in 6-OHDA-induced SH-SY5Y cell death.
Neurochem. Int.
PUBLISHED: 01-13-2010
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The orphan nuclear receptor Nur77 is a member of the nuclear receptor superfamily. Nur77 is known to regulate survival and death in response to extracellular stimuli, but it is unclear whether Nur77 is regulated by oxidative stress and contributes to the cytotoxicity in neurodegenerative diseases. Here we showed that (1) Nur77 was up-regulated, phosphorylated, and translocated from the nucleus into the cytosol and mitochondria by H(2)O(2) treatment in HEK293 cells, as well as in 6-hydroxy dopamine (6-OHDA)-treated dopaminergic SH-SY5Y cells, (2) oxidative stress-mediated cell death was exacerbated in Nur77-overexpressed cells and abolished by dominant-negative-Nur77 transfection, and (3) blockade of nuclear export attenuated 6-OHDA-induced SH-SY5Y cell death. Together, our results show that the nuclear export and targeting to mitochondria of Nur77 and resultant activation of apoptotic death may participate in the pathogenesis of Parkinsons disease.
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Foxa2 and Nurr1 synergistically yield A9 nigral dopamine neurons exhibiting improved differentiation, function, and cell survival.
Stem Cells
PUBLISHED: 01-06-2010
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Effective dopamine (DA) neuron differentiation from neural precursor cells (NPCs) is prerequisite for precursor/stem cell-based therapy of Parkinsons disease (PD). Nurr1, an orphan nuclear receptor, has been reported as a transcription factor that can drive DA neuron differentiation from non-dopaminergic NPCs in vitro. However, Nurr1 alone neither induces full neuronal maturation nor expression of proteins found specifically in midbrain DA neurons. In addition, Nurr1 expression is inefficient in inducing DA phenotype expression in NPCs derived from certain species such as mouse and human. We show here that Foxa2, a forkhead transcription factor whose role in midbrain DA neuron development was recently revealed, synergistically cooperates with Nurr1 to induce DA phenotype acquisition, midbrain-specific gene expression, and neuronal maturation. Thus, the combinatorial expression of Nurr1 and Foxa2 in NPCs efficiently yielded fully differentiated nigral (A9)-type midbrain neurons with clearly detectable DA neuronal activities. The effects of Foxa2 in DA neuron generation were observed regardless of the brain regions or species from which NPCs were derived. Furthermore, DA neurons generated by ectopic Foxa2 expression were more resistant to toxins. Importantly, Foxa2 expression resulted in a rapid cell cycle exit and reduced cell proliferation. Consistently, transplantation of NPCs transduced with Nurr1 and Foxa2 generated grafts enriched with midbrain-type DA neurons but reduced number of proliferating cells, and significantly reversed motor deficits in a rat PD model. Our findings can be applied to ongoing attempts to develop an efficient and safe precursor/stem cell-based therapy for PD.
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Endoplasmic reticulum stress-induced activation of activating transcription factor 6 decreases cAMP-stimulated hepatic gluconeogenesis via inhibition of CREB.
Endocrinology
PUBLISHED: 12-18-2009
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The expression of genes encoding key hepatic gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), is regulated at the transcriptional level by a network of transcription factors and cofactors, including cAMP response element-binding protein (CREB). It has been suggested that increased endoplasmic reticulum (ER) stress in the liver impairs hepatic glucose metabolism. However, the direct effect of ER stress on hepatic gluconeogenesis is still not clear. Here, we investigated whether ER stress influences hepatic gluconeogenesis and whether this process is mediated by activating transcription factor 6 (ATF6) through the inhibition of cAMP-mediated activation of CREB. A cAMP stimulant, forskolin, and 8-bromoadenosine-cAMP increased PEPCK and G6Pase mRNA expression in H4IIE rat hepatoma cells, and ER stress induced by tunicamycin or thapsigargin decreased the expression of these genes in forskolin or 8-bromoadenosine-cAMP-treated cells. In a transient transfection study, ATF6 inhibited the PEPCK and G6Pase promoters. Also, adenovirus-mediated overexpression of ATF6 in H4IIE cells decreased forskolin-stimulated PEPCK and G6Pase gene expression. Moreover, the inhibition of endogenous ATF6 expression by small interfering RNAs restored the ER stress-induced suppression of PEPCK and G6Pase gene expression. Transient transfection of ATF6 inhibited transactivation by CREB on the PEPCK and G6Pase promoters, and a gel shift assay showed that Ad-ATF6 inhibits forskolin-stimulated CREB DNA-binding activity. Finally, we found that expression of ATF6 decreased fasting-induced PEPCK, G6Pase mRNA expression, and blood glucose levels in mice. Taken together, these data extend our understanding of ER stress and the regulation of liver gluconeogenesis by ATF6.
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Glucose stimulates cholesterol 7alpha-hydroxylase gene transcription in human hepatocytes.
J. Lipid Res.
PUBLISHED: 10-28-2009
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Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4alpha (HNF4alpha) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4alpha to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.
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Efficient discovery of selective small molecule agonists of estrogen-related receptor gamma using combinatorial approach.
J Comb Chem
PUBLISHED: 09-15-2009
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With the goal of discovering a selective agonist of estrogen-related receptor gamma (ERRgamma) with enhanced potency, we designed a series of small-molecule ligands derived from a known ERRgamma agonist, GSK4716, that can substantially potentiate the transcriptional activity of ERRgamma. Individual compounds among a 30-member library of acyl hydrazones were pre-evaluated through in silico docking studies on the receptor cavities of ERRgamma LBDs using X-ray crystal structures cocrystallized with GSK4716 and 4-OHT. This rational approach to achieve the enhanced potency in ERRgamma transcriptional activity with selectivity over ERRalpha/beta enables us to complete the construction of a focused library by carrying out microwave-assisted parallel synthesis with excellent yields and purities. Finally, we identified a more potent ERRgamma agonist, E6, with excellent selectivity over ERRalpha/beta.
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Hepatocyte growth factor family negatively regulates hepatic gluconeogenesis via induction of orphan nuclear receptor small heterodimer partner in primary hepatocytes.
J. Biol. Chem.
PUBLISHED: 08-31-2009
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Hepatic gluconeogenesis is tightly balanced by opposing stimulatory (glucagon) and inhibitory (insulin) signaling pathways. Hepatocyte growth factor (HGF) is a pleiotropic growth factor that mediates diverse biological processes. In this study, we investigated the effect of HGF and its family member, macrophage-stimulating factor (MSP), on hepatic gluconeogenesis in primary hepatocytes. HGF and MSP significantly repressed expression of the key hepatic gluconeogenic enzyme genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (Glc-6-Pase) and reduced glucose production. HGF and MSP activated small heterodimer partner (SHP) gene promoter and induced SHP mRNA and protein levels, and the effect of HGF and MSP on SHP gene expression was demonstrated to be mediated via activation of the AMP-activated protein kinase (AMPK) signaling pathway. We demonstrated that upstream stimulatory factor-1 (USF-1) specifically mediated HGF effect on SHP gene expression, and inhibition of USF-1 by dominant negative USF-1 significantly abrogated HGF-mediated activation of the SHP promoter. Elucidation of the mechanism showed that USF-1 bound to E-box-1 in the SHP promoter, and HGF increased USF-1 DNA binding on the SHP promoter via AMPK and DNA-dependent protein kinase-mediated pathways. Adenoviral overexpression of USF-1 significantly repressed PEPCK and Glc-6-Pase gene expression and reduced glucose production. Knockdown of endogenous SHP expression significantly reversed this effect. Finally, knockdown of SHP or inhibition of AMPK signaling reversed the ability of HGF to suppress hepatocyte nuclear factor 4alpha-mediated up-regulation of PEPCK and Glc-6-Pase gene expression along with the HGF- and MSP-mediated suppression of gluconeogenesis. Overall, our results suggest a novel signaling pathway through HGF/AMPK/USF-1/SHP to inhibit hepatic gluconeogenesis.
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Transcriptional corepressor SMILE recruits SIRT1 to inhibit nuclear receptor estrogen receptor-related receptor gamma transactivation.
J. Biol. Chem.
PUBLISHED: 08-18-2009
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SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a corepressor of the glucocorticoid receptor, constitutive androstane receptor, and hepatocyte nuclear factor 4alpha. Here we show that SMILE also represses estrogen receptor-related receptor gamma (ERRgamma) transactivation. Knockdown of SMILE gene expression increases ERRgamma activity. SMILE directly interacts with ERRgamma in vitro and in vivo. Domain mapping analysis showed that SMILE binds to the AF2 domain of ERRgamma. SMILE represses ERRgamma transactivation partially through competition with coactivators PGC-1alpha, PGC-1beta, and GRIP1. Interestingly, the repression of SMILE on ERRgamma is released by SIRT1 inhibitors, a catalytically inactive SIRT1 mutant, and SIRT1 small interfering RNA but not by histone protein deacetylase inhibitor. In vivo glutathione S-transferase pulldown and coimmunoprecipitation assays validated that SMILE physically interacts with SIRT1. Furthermore, the ERRgamma inverse agonist GSK5182 enhances the interaction of SMILE with ERRgamma and SMILE-mediated repression. Knockdown of SMILE or SIRT1 blocks the repressive effect of GSK5182. Moreover, chromatin immunoprecipitation assays revealed that GSK5182 augments the association of SMILE and SIRT1 on the promoter of the ERRgamma target PDK4. GSK5182 and adenoviral overexpression of SMILE cooperate to repress ERRgamma-induced PDK4 gene expression, and this repression is released by overexpression of a catalytically defective SIRT1 mutant. Finally, we demonstrated that ERRgamma regulates SMILE gene expression, which in turn inhibits ERRgamma. Overall, these findings implicate SMILE as a novel corepressor of ERRgamma and recruitment of SIRT1 as a novel repressive mechanism for SMILE and ERRgamma inverse agonist.
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DAX-1 acts as a novel corepressor of orphan nuclear receptor HNF4alpha and negatively regulates gluconeogenic enzyme gene expression.
J. Biol. Chem.
PUBLISHED: 08-03-2009
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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.
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The orphan nuclear receptor SHP attenuates renal fibrosis.
J. Am. Soc. Nephrol.
PUBLISHED: 07-30-2009
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The accumulation of extracellular matrix proteins is a common feature of fibrotic kidney diseases. Accumulating evidence suggests that TGF-beta and plasminogen activator inhibitor type 1 (PAI-1) promote the development of renal fibrosis by stimulating the generation and inhibiting the removal of matrix proteins. The small heterodimer partner (SHP) represses PAI-1 expression in the liver by inhibiting TGF-beta signaling, but whether SHP inhibits renal fibrosis is unknown. Here, unilateral ureteral obstruction (UUO) markedly increased the expression of PAI-1, type I collagen, and fibronectin but decreased SHP gene expression. Moreover, in kidneys of SHP-/- mice, the expression of PAI-1, type I collagen, fibronectin and alpha-smooth muscle actin (alpha-SMA) were higher compared with those in kidneys of wild-type mice. In addition, loss of SHP accelerated renal fibrosis after UUO. Adenovirus-mediated overexpression of SHP in cultured rat mesangial cells and renal tubular epithelial cells inhibited TGF-beta-stimulated expression of PAI-1, type I collagen, and fibronectin. SHP inhibited TGF-beta- and Smad3-stimulated PAI-1 promoter activities as well as TGF-beta-stimulated binding of Smad3 to its consensus response element on the PAI-1 promoter. Similarly, in vivo, adenovirus-mediated overexpression of SHP in the kidney inhibited the expression of UUO-induced PAI-1, type I collagen, fibronectin, and alpha-SMA. In summary, SHP attenuates renal fibrosis in obstructive nephropathy, making its pathway a possible therapeutic target for chronic kidney disease.
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Fenofibrate differentially regulates plasminogen activator inhibitor-1 gene expression via adenosine monophosphate-activated protein kinase-dependent induction of orphan nuclear receptor small heterodimer partner.
Hepatology
PUBLISHED: 07-14-2009
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Plasminogen activator inhibitor type I (PAI-1) is a marker of the fibrinolytic system and serves as a possible predictor for hepatic metabolic syndromes. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARalpha) agonist, is a drug used for treatment of hyperlipidemia. Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of crucial genes involved in various metabolic pathways. In this study, we show that fenofibrate increased SHP gene expression in cultured liver cells and in the normal and diabetic mouse liver by activating the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway in a PPARalpha-independent manner. Administration of transforming growth factor beta (TGF-beta) or a methionine-deficient and choline-deficient (MCD) diet to induce the progressive fibrosing steatohepatitis model in C57BL/6 mice was significantly reversed by fenofibrate via AMPK-mediated induction of SHP gene expression with a dramatic decrease in PAI-1 messenger RNA (mRNA) and protein expression along with other fibrotic marker genes. No reversal was observed in SHP null mice treated with fenofibrate. Treatment with another PPARalpha agonist, WY14643, showed contrasting effects on these marker gene expressions in wild-type and SHP null mice, demonstrating the specificity of fenofibrate in activating AMPK signaling. Fenofibrate exhibited a differential inhibitory pattern on PAI-1 gene expression depending on the transcription factors inhibited by SHP. Conclusion: By demonstrating that a PPARalpha-independent fenofibrate-AMPK-SHP regulatory cascade can play a key role in PAI-1 gene down-regulation and reversal of fibrosis, our study suggests that various AMPK activators regulating SHP might provide a novel pharmacologic option in ameliorating hepatic metabolic syndromes.
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Cilostazol inhibits high glucose- and angiotensin II-induced type 1 plasminogen activator inhibitor expression in artery wall and neointimal region after vascular injury.
Atherosclerosis
PUBLISHED: 06-05-2009
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Increased expression of plasminogen activator inhibitor-1 (PAI-1) in vascular tissues is a potential factor linking diabetes to restenosis after percutaneous coronary intervention. Recent studies have shown that cilostazol, a selective type 3 phosphodiesterase inhibitor, prevents neointimal hyperplasia and in-stent thrombosis in patients with diabetes after coronary angioplasty and stent implantation. However, the molecular mechanism of this drug has not been fully elucidated. We examined whether cilostazol inhibits PAI-1 expression in vascular smooth muscle cells (VSMCs) and neointimal hyperplasia. We found that cilostazol effectively inhibits angiotensin II-, high glucose- and TGF-beta-stimulated PAI-1 expression in vivo and in vitro. Cilostazol attenuated PAI-1 expression in neointimal regions and inhibited neointimal hyperplasia after balloon injury. Cilostazol inhibited PAI-1 expression by multiple mechanisms including downregulation of TGF-beta, JNK and p38 signaling pathways. Cilostazol also inhibited transactivating activity at the PAI-1 promoter by Smad3, leading to a suppression of PAI-1 gene transcription. Taken together with its antiproliferative effect on VSMCs, this may explain how cilostazol exerts its antithrombogenic effects after angioplasty and stent implantation.
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Molecular characterization of SMILE as a novel corepressor of nuclear receptors.
Nucleic Acids Res.
PUBLISHED: 05-08-2009
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SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a coregulator in ER signaling. In this study, we have examined the effects of SMILE on other NRs (nuclear receptors). SMILE inhibits GR, CAR and HNF4 alpha-mediated transactivation. Knockdown of SMILE gene expression increases the transactivation of the NRs. SMILE interacts with GR, CAR and HNF4 alpha in vitro and in vivo. SMILE and these NRs colocalize in the nucleus. SMILE binds to the ligand-binding domain or AF2 domain of the NRs. Competitions between SMILE and the coactivators GRIP1 or PGC-1 alpha have been demonstrated in vitro and in vivo. Furthermore, an intrinsic repressive activity of SMILE is observed in Gal4-fusion system, and the intrinsic repressive domain is mapped to the C-terminus of SMILE, spanning residues 203-354. Moreover, SMILE interacts with specific HDACs (histone deacetylases) and SMILE-mediated repression is released by HDAC inhibitor trichostatin A, in a NR-specific manner. Finally, ChIP (chromatin immunoprecipitation) assays reveal that SMILE associates with the NRs on the target gene promoters. Adenoviral overexpression of SMILE represses GR-, CAR- and HNF4 alpha-mediated target gene expression. Overall, these results suggest that SMILE functions as a novel corepressor of NRs via competition with coactivators and the recruitment of HDACs.
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Bile acids inhibit duodenal secretin expression via orphan nuclear receptor small heterodimer partner (SHP).
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 04-16-2009
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Small heterodimer partner (SHP) is an orphan nuclear receptor in which gene expression can be upregulated by bile acids. It regulates its target genes by repressing the transcriptional activities of other nuclear receptors including NeuroD, which has been shown to regulate secretin gene expression. Here, we evaluated the regulation on duodenal secretin gene expression by SHP and selected bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). In vitro treatment of CDCA or fexaramine elevated the SHP transcript level and occupancy on secretin promoter. The increase in the SHP level, induced by bile acid treatment or overexpression, reduced secretin gene expression, whereas this gene inhibitory effect was reversed by silencing of endogenous SHP. In in vivo studies, double-immunofluorescence staining demonstrated the coexpression of secretin and SHP in mouse duodenum. Feeding mice with 1% CA-enriched rodent chow resulted in upregulation of SHP and a concomitant decrease in secretin transcript and protein levels in duodenum compared with the control group fed with normal chow. A diet enriched with 5% cholestyramine led to a decrease in SHP level and a corresponding increase in secretin expression. Overall, this study showed that bile acids via SHP inhibit duodenal secretin gene expression. Because secretin is a key hormone that stimulates bile flow in cholangiocytes, this pathway thus provides a novel means to modulate secretin-stimulated choleresis in response to intraduodenal bile acids.
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Conserved properties of a urochordate estrogen receptor-related receptor (ERR) with mammalian ERRalpha.
Biochim. Biophys. Acta
PUBLISHED: 04-01-2009
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Estrogen receptor-related receptors (ERRs) were the first orphan nuclear receptors identified on the basis of their sequence similarity to the estrogen receptors. Although unique ERRs were found in some marine invertebrates, the molecular functions of these receptors are not well understood. In the present study, we identified three transcript variants of the tunicate Halocynthia roretzi ERR (Hr-ERR), varying in their 3 untranslated regions, and putatively encoding a unique receptor deriving from an ancestor protein common to vertebrate ERRalpha/beta/gamma. Maternal mRNA of Hr-ERR was detected throughout the entire egg cytoplasm and early embryos. Zygotic Hr-ERR was predominantly expressed in the heart, and at lower levels in muscle, stomach, gonad and digestive glands. Electrophoretic mobility shift assay demonstrated that Hr-ERR directly binds to the estrogen-response element (ERE) and ERR-response element (ERRE). Gene reporter assays also showed that Hr-ERR activates transcription through ERE and ERRE. Hr-ERR-mediated transactivation was modulated by various cofactors for mammalian ERRs, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha and small heterodimer partner. In addition, the ERR antagonists 4-hydroxytamoxifen and diethylstilbestrol inhibited the Hr-ERR-mediated transactivation, whereas Hr-ERR activity on ERE was further induced by genistein, an ERRalpha agonist. Taken together, our results show that Hr-ERR is an unduplicated ERR that however, possesses functional properties common to ERRalpha and not to ERRbeta/gamma.
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The orphan nuclear receptor estrogen receptor-related receptor gamma negatively regulates BMP2-induced osteoblast differentiation and bone formation.
J. Biol. Chem.
PUBLISHED: 03-26-2009
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Estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERRalpha is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERRgamma in osteoblast differentiation. Here, we showed that ERRgamma is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERRgamma reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERRgamma expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERRgamma plays an important role in osteoblast differentiation. In addition, ERRgamma significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERRgamma physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERRgamma strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERRgamma is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity.
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Pharmacological stimulation of NADH oxidation ameliorates obesity and related phenotypes in mice.
Diabetes
PUBLISHED: 01-09-2009
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Nicotinamide adenine dinucleotides (NAD+ and NADH) play a crucial role in cellular energy metabolism, and a dysregulated NAD+-to-NADH ratio is implicated in metabolic syndrome. However, it is still unknown whether a modulating intracellular NAD+-to-NADH ratio is beneficial in treating metabolic syndrome. We tried to determine whether pharmacological stimulation of NADH oxidation provides therapeutic effects in rodent models of metabolic syndrome.
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An indole derivative protects against acetaminophen-induced liver injury by directly binding to N-acetyl-p-benzoquinone imine in mice.
Antioxid. Redox Signal.
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Acetaminophen (APAP)-induced liver injury is mainly due to the excessive formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) through the formation of a reactive intermediate, N-acetyl-p-benzoquinone imine (NAPQI), in both humans and rodents. Here, we show that the indole-derived synthetic compound has a protective effect against APAP-induced liver injury in C57Bl/6 mice model.
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Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are required for steroidogenesis in testicular Leydig cells.
J. Biol. Chem.
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Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.
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Hypoxia induces PDK4 gene expression through induction of the orphan nuclear receptor ERR?.
PLoS ONE
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Multiple cellular signaling pathways that control metabolism and survival are activated when cell are incubated under hypoxic conditions. Activation of the hypoxia inducible factor (HIF)-1 promotes expression of genes that increase the capacity to cope with the stress imposed by a reduced oxygen environment. Here we show that the orphan nuclear receptor estrogen related receptor ? (ERR?) plays a critical role in hypoxia-mediated activation of pyruvate dehydrogenase kinase 4 (PDK4) gene expression. ERR? mRNA and protein levels were increased by hypoxia or desferrioxamine (DFO) treatment in hepatoma cell lines. Co-expression of HIF-1? and ? increased ERR? promoter activity as well as mRNA expression, while knockdown of endogenous HIF-1? reduced the hypoxia-mediated induction of ERR?. In addition, hypoxia also increased the promoter activity and mRNA level of PDK4 in HepG2 cells. Adenovirus mediated-overexpression of ERR? specifically increased PDK4 gene expression, while ablation of endogenous ERR? significantly decreased hypoxia-mediated induction of PDK4 gene expression. Finally, GSK5182, an inverse agonist of ERR?, strongly inhibited the hypoxia-mediated induction of PDK4 protein and promoter activity. Regulation of the transcriptional activity of ERR? may provide a therapeutic approach for the regulation of PDK4 gene expression under hypoxia.
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Estrogen-related receptor ? controls hepatic CB1 receptor-mediated CYP2E1 expression and oxidative liver injury by alcohol.
Gut
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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 ? (ERR?) is a constitutively active transcriptional activator regulating gene expression.
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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.
J. Biol. Chem.
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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 C? (PKC?). 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 PKC? 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.
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Orphan nuclear receptor small heterodimer partner negatively regulates growth hormone-mediated induction of hepatic gluconeogenesis through inhibition of signal transducer and activator of transcription 5 (STAT5) transactivation.
J. Biol. Chem.
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Growth hormone (GH) is a key metabolic regulator mediating glucose and lipid metabolism. Ataxia telangiectasia mutated (ATM) is a member of the phosphatidylinositol 3-kinase superfamily and regulates cell cycle progression. The orphan nuclear receptor small heterodimer partner (SHP: NR0B2) plays a pivotal role in regulating metabolic processes. Here, we studied the role of ATM on GH-dependent regulation of hepatic gluconeogenesis in the liver. GH induced phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase gene expression in primary hepatocytes. GH treatment and adenovirus-mediated STAT5 overexpression in hepatocytes increased glucose production, which was blocked by a JAK2 inhibitor, AG490, dominant negative STAT5, and STAT5 knockdown. We identified a STAT5 binding site on the PEPCK gene promoter using reporter assays and point mutation analysis. Up-regulation of SHP by metformin-mediated activation of the ATM-AMP-activated protein kinase pathway led to inhibition of GH-mediated induction of hepatic gluconeogenesis, which was abolished by an ATM inhibitor, KU-55933. Immunoprecipitation studies showed that SHP physically interacted with STAT5 and inhibited STAT5 recruitment on the PEPCK gene promoter. GH-induced hepatic gluconeogenesis was decreased by either metformin or Ad-SHP, whereas the inhibition by metformin was abolished by SHP knockdown. Finally, the increase of hepatic gluconeogenesis following GH treatment was significantly higher in the liver of SHP null mice compared with that of wild-type mice. Overall, our results suggest that the ATM-AMP-activated protein kinase-SHP network, as a novel mechanism for regulating hepatic glucose homeostasis via a GH-dependent pathway, may be a potential therapeutic target for insulin resistance.
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The estrogen-related receptor alpha upregulates secretin expressions in response to hypertonicity and angiotensin II stimulation.
PLoS ONE
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Osmoregulation via maintenance of water and salt homeostasis is a vital process. In the brain, a functional secretin (SCT) and secretin receptor (SCTR) axis has recently been shown to mediate central actions of angiotensin II (ANGII), including initiation of water intake and stimulation of vasopressin (VP) expression and release. In this report, we provide evidence that estrogen-related receptor ? (ERR?, NR3B1), a transcription factor mainly involved in metabolism, acts as an upstream activator of the SCT gene. In vitro studies using mouse hypothalamic cell line N-42 show that ERR? upregulates SCT promoter and gene expression. More importantly, knockdown of endogenous ERR? abolishes SCT promoter activation in response to hypertonic and ANGII stimulations. In mouse brain, ERR? coexpresses with SCT in various osmoregulatory brain regions, including the lamina terminalis and the paraventricular nucleus of the hypothalamus, and its expression is induced by hyperosmotic and ANGII treatments. Based on our data, we propose that both the upregulation of ERR? and/or the increased binding of ERR? to the mouse SCT promoter are two possible mechanisms for the elevated SCT expression upon hyperosmolality and central ANGII stimulation.
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Metformin inhibits growth hormone-mediated hepatic PDK4 gene expression through induction of orphan nuclear receptor small heterodimer partner.
Diabetes
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Growth hormone (GH) is a counter-regulatory hormone that plays an important role in preventing hypoglycemia during fasting. Because inhibition of the pyruvate dehydrogenase complex (PDC) by pyruvate dehydrogenase kinase 4 (PDK4) conserves substrates for gluconeogenesis, we tested whether GH increases PDK4 expression in liver by a signaling pathway sensitive to inhibition by metformin. The effects of GH and metformin were determined in the liver of wild-type, small heterodimer partner (SHP)-, PDK4-, and signal transducer and activator of transcription 5 (STAT5)-null mice. Administration of GH in vivo increased PDK4 expression via a pathway dependent on STAT5 phosphorylation. Metformin inhibited the induction of PDK4 expression by GH via a pathway dependent on AMP-activated protein kinase (AMPK) and SHP induction. The increase in PDK4 expression and PDC phosphorylation by GH was reduced in STAT5-null mice. Metformin decreased GH-mediated induction of PDK4 expression and metabolites in wild-type but not in SHP-null mice. In primary hepatocytes, dominant-negative mutant-AMPK and SHP knockdown prevented the inhibitory effect of metformin on GH-stimulated PDK4 expression. SHP directly inhibited STAT5 association on the PDK4 gene promoter. Metformin inhibits GH-induced PDK4 expression and metabolites via an AMPK-SHP-dependent pathway. The metformin-AMPK-SHP network may provide a novel therapeutic approach for the treatment of hepatic metabolic disorders induced by the GH-mediated pathway.
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Orphan nuclear receptor estrogen-related receptor ? (ERR?) is key regulator of hepatic gluconeogenesis.
J. Biol. Chem.
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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 ? (ERR?) is a novel downstream mediator of glucagon action in hepatic gluconeogenesis and demonstrate a beneficial impact of the inverse agonist GSK5182. Hepatic ERR? expression was increased by fasting-dependent activation of the cAMP-response element-binding protein-CRTC2 pathway. Overexpression of ERR? induced Pck1 and G6PC gene expression and glucose production in primary hepatocytes, whereas abolition of ERR? gene expression attenuated forskolin-mediated induction of gluconeogenic gene expression. Deletion and mutation analyses of the Pck1 promoter showed that ERR? 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 ERR?, specifically inhibits the transcriptional activity of ERR? in a PGC-1? dependent manner. Finally, the ERR? inverse agonist ameliorated hyperglycemia through inhibition of hepatic gluconeogenesis in db/db mice. Control of hepatic glucose production by an ERR?-specific inverse agonist is a new potential therapeutic approach for the treatment of type 2 diabetes.
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Mediation of glucolipotoxicity in INS-1 rat insulinoma cells by small heterodimer partner interacting leucine zipper protein (SMILE).
Biochem. Biophys. Res. Commun.
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Sustained elevations of glucose and free fatty acid concentration have deleterious effects on pancreatic beta cell function. One of the hallmarks of such glucolipotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Sterol regulatory element binding protein-1c (SREBP-1c), a lipogenic transcription factor, is related to the development of beta cell dysfunction caused by elevated concentrations of glucose and free fatty acid. Small heterodimer partner (SHP) interacting leucine zipper protein (SMILE), also known as Zhangfei, is a novel protein which interacts with SHP that mediates glucotoxicity in INS-1 rat insulinoma cells. Treatment of INS-1 cells with high concentrations of glucose and palmitate increased SREBP-1c and SMILE expression, and decreased insulin gene expression. Adenovirus-mediated overexpression of SREBP-1c in INS-1 cells induced SMILE expression. Moreover, adenovirus-mediated overexpression of SMILE (Ad-SMILE) in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. Ad-SMILE overexpression also inhibited the expression of beta-cell enriched transcription factors including pancreatic duodenal homeobox factor-1, beta cell E box transactivator 2 and RIPE3b1/MafA, in INS-1 cells. Finally, in COS-1 cells, expression of SMILE inhibited the insulin promoter activity induced by these same beta-cell enriched transcription factors. These results collectively suggest that SMILE plays an important role in the development of beta cell dysfunction induced by glucolipotoxicity.
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Enhanced activation of NAD(P)H: quinone oxidoreductase 1 attenuates spontaneous hypertension by improvement of endothelial nitric oxide synthase coupling via tumor suppressor kinase liver kinase B1/adenosine 5-monophosphate-activated protein kinase-mediat
J. Hypertens.
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Guanosine 5-triphosphate cyclohydrolase-1 (GTPCH-1) is a rate-limiting enzyme in de-novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial nitric oxide synthase (eNOS) coupling. Adenosine 5-monophosphate-activated protein kinase (AMPK) is crucial for GTPCH-1 preservation, and tumor suppressor kinase liver kinase B1 (LKB1), an upstream kinase of AMPK, is activated by NAD-dependent class III histone deacetylase sirtuin 1 (SIRT1)-mediated deacetylation. ?-Lapachone has been shown to increase cellular NAD/NADH ratio via NAD(P)H:quinone oxidoreductase 1 (NQO1) activation. In this study, we have evaluated whether ?-lapachone-induced NQO1 activation modulates blood pressure (BP) through preservation of GTPCH-1 in a hypertensive animal model.
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