Steroid receptor coactivator-1 modulates the function of Pomc neurons and energy homeostasis.

Hypothalamic neurons expressing the anorectic peptide Pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here, we show that Steroid Receptor Coactivator-1 (SRC-1) interacts with a target of leptin receptor activation, phosphorylated STAT3, to potentiate Pomc transcription. Deletion of SRC-1 in Pomc neurons in mice attenuates their depolarization by leptin, decreases Pomc expression and increases food intake leading to high-fat diet-induced obesity. In humans, fifteen rare heterozygous variants in SRC-1 found in severely obese individuals impair leptin-mediated Pomc reporter activity in cells, whilst four variants found in non-obese controls do not. In a knock-in mouse model of a loss of function human variant (SRC-1L1376P), leptin-induced depolarization of Pomc neurons and Pomc expression are significantly reduced, and food intake and body weight are increased. In summary, we demonstrate that SRC-1 modulates the function of hypothalamic Pomc neurons, and suggest that targeting SRC-1 may represent a useful therapeutic strategy for weight loss.

Yin Yang 1 promotes hepatic gluconeogenesis through upregulation of glucocorticoid receptor.

Gluconeogenesis is critical in maintaining blood glucose levels in a normal range during fasting. In this study, we investigated the role of Yin Yang 1 (YY1), a key transcription factor involved in cell proliferation and differentiation, in the regulation of hepatic gluconeogenesis. Our data showed that hepatic YY1 expression levels were induced in mice during fasting conditions and in a state of insulin resistance. Overexpression of YY1 in livers augmented gluconeogenesis, raising fasting blood glucose levels in C57BL/6 mice, whereas liver-specific ablation of YY1 using adenoviral shRNA ameliorated hyperglycemia in wild-type and diabetic db/db mice. At the molecular level, we further demonstrated that the major mechanism of YY1 in the regulation of hepatic glucose production is to modulate the expression of glucocorticoid receptor. Therefore, our study uncovered for the first time that YY1 participates in the regulation of hepatic gluconeogenesis, which implies that YY1 might serve as a potential therapeutic target for hyperglycemia in diabetes.

Steroid receptor coactivator-1 mediates estrogenic actions to prevent body weight gain in female mice.

Estrogen receptor-α (ERα) expressed by hypothalamic proopiomelanocortin and steroidogenic factor-1 neurons largely mediates the antiobesity effects of estrogens in females. However, the critical molecular events that are coupled to ERα and mediate estrogenic effects on energy balance remain unknown. In the current study, we demonstrated that steroid receptor coactivator-1 (SRC1), a nuclear receptor coactivator, is abundantly expressed by both proopiomelanocortin and steroidogenic factor-1 neurons. We further showed that central administration of an ERα agonist, propyl pyrazole triol, acutely increases physical interaction between SRC1 and ERα in the hypothalamus. Finally, we demonstrated that the effects of estrogens on energy homeostasis are significantly blunted in female mice lacking SRC1 globally. Collectively our results indicate that SRC1 is functionally required to mediate the antiobesity effects of estrogen-ERα signals.

[Effects of daidzein on steroid receptor coactivator-1 expression in MC3T3-E1 cells and the mechanism].

OBJECTIVE: To investigate the roles of daidzein in the expressions of steroid receptor coactivator-1 (SRC-1) and nuclear receptor corepressor (NcoR) in MC3T3-E1 osteoblastic cells. METHODS: MC3T3-E1 cells were cultured in α-minimal essential medium (α-MEM) containing 2% fetal bovine serum and treated with various concentrations of daidzein (10(-9), 10(-7) and 10(-5) mol/L) or 17ß-estradiol at 10(-8) mol/L for 3 d. The protein levels of SRC-1 and NcoR in MC3T3-E1 cells were determined by Western blotting. Estrogen receptor (ER) antagonist ICI182780 at 10(-7) mol/L or specific ERα antagonist methyl-piperidino-pyrazole (MPP) at 10(-6) mol/L were used to block the corresponding receptors, and then MC3T3-E1 cells were treated with daidzein at 10(-7) mol/L or 10(-5) mol/L for 3 d. SRC-1 and NcoR protein levels were detected by Western blotting. RESULTS: The protein levels of SRC-1 increased by 2.5 fold (P<0.05) and 2 fold (P<0.05) by 10(-7) and 10(-5) mol/L of daidzein respectively, while the NcoR levels were not significantly altered. 17ß-Estradiol at dose of 10(-8) mol/L did not affect the expression of SRC-1 but decreased NcoR protein expression by 35% (P<0.05). Compared with the control, daidzein at 10(-7) and 10(-5) mol/L did not increase SRC-1 expression when ERs were blocked by antagonist ICI182780. Daidzein at 10(-7) and 10(-5) mol/L up-regulated SRC-1 by 1.8 fold (P<0.05) and 2.4 fold (P<0.05) respectively while ERα was blocked by MPP. CONCLUSION: Daidzein increases protein level of SRC-1 and the ratio of SRC-1/NcoR. ERß, instead of ERα, participates in the action of daidzein in regulating SRC-1 expression. Up-regulation of SRC-1 and increase of SRC-1/NcoR are part of the mechanism of the estrogenic effect of daidzein in improving osteogenesis.

Novel PPARγ partial agonists with weak activity and no cytotoxicity; identified by a simple PPARγ ligand screening system.

Peroxisome proliferator-activated receptors (PPARs) are the transcriptional factor that regulate glucose and lipid homeostasis and widely well-known as molecular targets for improvement of metabolic disorder. Because major transcriptional activity of PPARs depends on their proper ligands, the studies for PPAR ligands have been continuously developed. We previously reported the simple enzyme-linked immunosorbent assay (ELISA) systems to screen PPAR ligands and a chemical library including flavonoid derivatives have applied to these systems. In this study, we introduce two compounds (KU16476 and KU28843) identified as PPARγ partial agonists by a screening ELISA for PPARγ ligand. KU16476 and KU28843 significantly increased binding between PPARγ and SRC-1 in a simple ELISA system. Co-activator recruiting-induced abilities of two compounds were less than that of indomethacin, a well-known PPARγ agonist. To determine whether these compounds would be PPARγ partial agonists, each candidate with indomethacin were applied to a simple ELISA based on binding between PPARγ and SRC-1. Cotreatment with indomethacin significantly increased binding between PPARγ and SRC-1 than treatment of indomethacin or candidate alone. Two compounds had no considerable cytotoxicities, induced partial adipogenesis, and accumulated lipid droplets in 3T3-L1 fibroblast. Also, these two compounds enhanced expression of PPARγ-mediated genes such as aP2 and UCP-2. By docking study, we confirmed that two compounds bound well to the active site of PPARγ with hydrophobic interactions. We suggest that two compounds identified by a simple ELISA system can be PPARγ partial agonists. These PPARγ partial agonists and these studies to find out novel PPARγ agonists may contribute to drug development against metabolic disorders.

Human pregnane X receptor agonism by Ginkgo biloba extract: assessment of the role of individual ginkgolides.

Ginkgo biloba extract activates pregnane X receptor (PXR), but how this occurs is not known. Therefore, we investigated the mechanism of PXR activation by the extract and the role of five individual terpene trilactones in the activation. In a cell-based reporter gene assay, G. biloba extract activated human PXR (hPXR), and at a concentration present in the extract, ginkgolide A, but not ginkgolide B, ginkgolide C, ginkgolide J, or bilobalide was partially responsible for the increase in hPXR activity of the extract. Likewise, in cultured human hepatocytes, only ginkgolide A contributed to the increase in hPXR target gene expression (CYP3A4 mRNA and CYP3A-mediated testosterone 6ß-hydroxylation). The extract, but none of the terpene trilactones, bound to hPXR ligand-binding domain, as analyzed by a time-resolved fluorescence resonance energy transfer competitive binding assay. Only the extract and ginkgolide A recruited steroid receptor coactivator-1, as determined by a mammalian two-hybrid assay. Compared with hPXR, rat PXR (rPXR) was activated to a lesser extent by G. biloba extract. Similar to hPXR, only ginkgolide A contributed to rPXR activation by the extract. In contrast to the effect of G. biloba extract on PXR function, it did not affect hPXR expression. Overall, the main conclusions are that G. biloba extract is an hPXR agonist, and among the five terpene trilactones investigated, only ginkgolide A contributes to the actions of the extract. Our findings provide insights into the biological and chemical mechanisms of hPXR activation by G. biloba extract.

Differential binding with ERá and ERâ of the phytoestrogen-rich plant Pueraria mirifica

Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERá and hERâ and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the â-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the â-galactosidase activity (EC50) of the tuber extracts in relation to 17â-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERá and hERâ, respectively, with a higher relative estrogenic potency with hERâ than with hERá. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17â-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.

Differential binding with ERalpha and ERbeta of the phytoestrogen-rich plant Pueraria mirifica.

Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERalpha and hERbeta and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the beta-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the beta-galactosidase activity (EC(50)) of the tuber extracts in relation to 17beta-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERalpha and hERbeta, respectively, with a higher relative estrogenic potency with hERbeta than with hERalpha. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17beta-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.