Ming-Dao Chen

Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes

A series of clinical trials and animal experiments have demonstrated that ginseng and its major active constituent, ginsenosides, possess glucose-lowering action. In our previous study, ginsenoside Rb(1) has been shown to regulate peroxisome proliferator-activated receptor gamma activity to facilitate adipogenesis of 3T3-L1 cells. However, the effect of Rb(1) on glucose transport in insulin-sensitive cells and its molecular mechanism need further elucidation. In this study, Rb(1) significantly stimulated basal and insulin-mediated glucose uptake in a time- and dose-dependent manner in 3T3-L1 adipocytes and C2C12 myotubes; the maximal effect was achieved at a concentration of 1 microM and a time of 3 h. In adipocytes, Rb(1) promoted GLUT1 and GLUT4 translocations to the cell surface, which was examined by analyzing their distribution in subcellular membrane fractions, and enhanced translocation of GLUT4 was confirmed using the transfection of GLUT4-green fluorescence protein in Chinese Hamster Ovary cells. Meanwhile, Rb(1) increased the phosphorylation of insulin receptor substrate-1 and protein kinase B (PKB), and stimulated phosphatidylinositol 3-kinase (PI3K) activity in the absence of the activation of the insulin receptor. Rb(1)-induced glucose uptake as well as GLUT1 and GLUT4 translocations was inhibited by the PI3K inhibitor. These results suggest that ginsenoside Rb(1) stimulates glucose transport in insulin-sensitive cells by promoting translocations of GLUT1 and GLUT4 by partially activating the insulin signaling pathway. These findings are useful in understanding the hypoglycemic and anti-diabetic properties of ginseng and ginsenosides.

Serum visfatin concentrations in obese adolescents and its correlation with age and high-density lipoprotein cholesterol

Visfatin was recently identified as an adipocytokine and has insulin mimetic properties, but its role in adolescents remains largely unknown. In this study, we examined the impact of adolescent obesity on circulating visfatin levels and the relationship between visfatin and anthropometric indices, insulin sensitivity, and blood lipids in Chinese adolescents (11-18 years). Serum visfatin, adiponectin, leptin, and blood lipids were measured in 76 non-obese and 72 obese adolescents. The medians of serum visfatin levels were significantly higher in obese subjects of 34.68ng/ml than in non-obese subjects of 28.67ng/ml (P=0.002). There were no significant correlations in the non-obese group between the serum visfatin concentration and the anthropometric indices or the lipid parameters. However, visfatin levels were negatively correlated with age, early insulin secretion index (EISI), Tanner stage, and positively correlated with HDL-c in the obese adolescents. These relationships, except that for EISI and Tanner stage, remained significant (P<0.05) after adjusting for age, gender, and body mass index. Moreover, unlike adiponectin and leptin, visfatin concentration did not correlate with testosterone in non-obese and obese boys. In conclusion, visfatin levels may decrease with age and be related to the HDL metabolism in obese adolescents.

Berberine Acutely Inhibits Insulin Secretion from β-Cells through 3′,5′-Cyclic Adenosine 5′-Monophosphate Signaling Pathway

Berberine, a hypoglycemic agent, has recently been shown to activate AMP-activated protein kinase (AMPK) contributing to its beneficial metabolic effects in peripheral tissues. However, whether berberine exerts a regulatory effect on beta-cells via AMPK or other signaling pathways and counteracts glucolipotoxicity remains uncertain. In the present study, the impact of berberine on beta-cell function was investigated in vivo and in vitro. In high-fat-fed rats, berberine treatment for 6 wk significantly decreased plasma glucose and insulin levels before and after an oral glucose challenge along with the reduction of body weight and improvement of blood lipid profile. In accordance with the in vivo results, berberine acutely decreased glucose-stimulated insulin secretion (GSIS) and palmitate-potentiated insulin secretion in MIN6 cells and rat islets. However, pretreated with berberine for 24 h augmented the response of MIN6 cells and rat islets to glucose and attenuated the glucolipotoxicity. Berberine acutely increased AMPK activity in MIN6 cells. However, compound C, an AMPK inhibitor, completely reversed troglitazone-suppressed GSIS, not berberine-suppressed GSIS. Otherwise, berberine decreased cAMP-raising agent-potentiated insulin secretion in MIN6 cells and rat islets. These results suggest that the activation of AMPK is required for troglitazone-suppressed GSIS, whereas cAMP signaling pathway contributes, at least in part, to the regulatory effect of berberine on insulin secretion.

C-reactive protein inhibits adiponectin gene expression and secretion in 3T3-L1 adipocytes.

C-reactive protein (CRP) is considered as one of the most sensitive markers of inflammation. The aim of the present study is to investigate the effects of CRP on the production of adiponectin in 3T3-L1 adipocytes. Northern and western blot analysis revealed that CRP treatment inhibited adiponectin mRNA expression and secretion in a dose- and time-dependent manner. Co-incubation of adipocytes with rosiglitazone and CRP decreased induction of adiponectin gene expression by rosiglitazone. However, luciferase reporter assays did not show that CRP affected the activity of approximately 2.1 kb adiponectin gene promoter, which was increased by rosiglitazone alone. Pharmacological inhibition of phosphatidylinositol (PI)-3 kinase by LY294002 partially reversed inhibition of adiponectin gene expression by CRP. These results collectively suggest that CRP suppresses adiponectin gene expression partially through the PI-3 kinase pathway, and that decreased production of adiponectin might represent a mechanism by which CRP regulates insulin sensitivity.

Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes ☆

Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of (3)[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.

Astragaloside IV attenuates lipolysis and improves insulin resistance induced by TNFα in 3T3-L1 adipocytes

Increased circulating free fatty acid (FFA) concentrations have been demonstrated to potentially link obesity, insulin resistance and cardiovascular diseases. Astragaloside IV (AS‐IV) is a saponin which is widely used in traditional Chinese medicine to treat type 2 diabetes and cardiovascular diseases. The purpose of the present study was to examine the effects of AS‐IV on the lipolysis and insulin resistance induced by tumor necrosis factor‐α (TNFα) in cultured 3T3‐L1 adipocytes. TNFα promotes lipolysis in mammal adipocytes via the mitogen activated protein kinase (MAPK) family resulting in reduced expression/function of perilipin. Application of AS‐IV inhibited TNFα‐induced accelerated lipolysis in a dose‐dependent manner, which was compatible with suppressed phosphorylation of ERK1/2 and reversed the downregulation of perilipin. Moreover, TNFα induced downregulation of key enzymes in lipogenesis, including LPL, FAS and GPAT, were also attenuated by AS‐IV. Further studies showed that AS‐IV improved TNFα‐induced insulin resistance in 3T3‐L1 adipocytes. This study provides the first direct evidence of the antilipolytic action of AS‐IV in adipocytes, which may allow this agent to decrease the circulating FFA levels, thus increase insulin sensitivity and treat cardiovascular diseases. Copyright

Expression of adiponectin receptors in mouse adrenal glands and the adrenocortical Y-1 cell line: adiponectin regulates steroidogenesis.

Obesity is frequently associated with malfunctions of the hypothalamus-pituitary-adrenal (HPA) axis and hyperaldosteronism, but the mechanism underlying this association remains unclear. Since the adrenal glands are embedded in adipose tissue, direct cross-talk between adipose tissue and the adrenal gland has been proposed. A previous study found that adiponectin receptor mRNA was expressed in human adrenal glands and aldosterone-producing adenoma (APA). However, the expression of adiponectin receptors in adrenal glands has not been confirmed at the protein level or in other species. Furthermore, it is unclear whether adiponectin receptors expressed in adrenal cells are functional. We found, for the first time, that adiponectin receptor (AdipoR1 and AdipoR2) mRNA and protein were expressed in mouse adrenal and adrenocortical Y-1 cells. However, adiponectin itself was not expressed in mouse adrenal or Y-1 cells. Furthermore, adiponectin acutely reduced basal levels of corticosterone and aldosterone secretion. ACTH-induced steroid secretion was also inhibited by adiponectin, and this was accompanied by a parallel change in the expression of the key genes involved in steroidogenesis. These findings indicate that adiponectin may take part in the modulation of steroidogenesis. Thus, adiponectin is likely to have physiological and/or pathophysiological significance as an endocrine regulator of adrenocortical function.

Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion

AbstractAim:To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion.Methods:Receptors ofbombesin3, cholecystokinin (CCK)-A, CCK-B, glucagon-like peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexin1, orexin2, neuromedin-B, neuropeptide Y (NPY) 1 and NPY5, neurotensin (NT) 1, NT2, NT3, and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin, orexin A and orexin B at a cohort of concentrations for different lengths of time, and GnRH in medium was determined by radioimmunoassay (RIA).Results:Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexin1, neuromedin-B, NPY1, NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7 cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min.Conclusion:Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism.

Leydig cell hypoplasia (LCH) is a rare form of male pseudohermaphroditism caused by inactivating mutations in the luteinizing hormone receptor gene (LHCGR). The majority of LHCGR mutations are located in the coding sequence, resulting in impairment of either LH/CG binding or signal transduction. We report a Chinese family with two siblings (46, XY and 46, XX) carrying a missense mutation (c. 455 T>C, p. Ile152Thr) and a splice site mutation (c. 537‐3 C>A). Computational analysis of the missense mutation in the three‐dimensional structural model predicted it might influence the distribution of hydrogen bonds and intermolecular contacts between the hormone and receptor. Consistent with these findings, in vitro mutant analysis revealed a marked impairment of human chorionic gonadotropin binding and signal transduction. The splice‐acceptor mutation (c. 537‐3 C>A) resulted in abnormal splicing of LHCGR mRNA, skipping exon 7. This report expands the genotypic spectrum of LHCGR mutations, with relevant implications for the molecular analysis of this gene.

Effects of C-reactive protein on adipokines genes expression in 3T3-L1 adipocytes

Adipose tissue is now recognized to be an important endocrine organ, secreting a variety of adipokines that are involved in the regulation of energy metabolism, insulin resistance and metabolic syndrome. C-reactive protein (CRP) is considered as one of the most sensitive markers of inflammation. A number of studies have shown that elevation of CRP concentrations is an independent predictive parameter of type 2 diabetes mellitus, which is also strongly associated with various components of the metabolic syndrome. The aim of the present study is to investigate the effects of CRP on adipokines genes expression in 3T3-L1 adipocytes. Quantitative real-time PCR analysis revealed that CRP inhibited adiponectin, leptin and peroxisome proliferator-activated receptor-gamma (PPAR-γ) genes expression and raised tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA levels in matured 3T3-L1 adipocytes in a dose and time-dependent manner. Pharmacological inhibition of phosphatidylinositol (PI)-3 kinase by wortmannin partially reversed the effects of CRP on adiponectin, TNF-α and leptin genes expression. These results collectively suggest that CRP regulates adiponectin, TNF-α, leptin, IL-6 and PPAR-γ genes expression, and that might represent a mechanism by which CRP regulates insulin resistance, obesity and metabolic syndrome.