sto-609 and Insulin-Resistance

This study investigated the molecular mechanism of saponarin, a flavone glucoside, in the regulation of insulin sensitivity. Saponarin suppressed the rate of gluconeogenesis and increased cellular glucose uptake in HepG2 and TE671 cells by regulating AMPK. Using an in vitro kinase assay, we showed that saponarin did not directly interact with the AMPK protein. Instead, saponarin increased intracellular calcium levels and induced AMPK phosphorylation, which was diminished by co-stimulation with STO-609, an inhibitor of CAMKKβ. Transcription of hepatic gluconeogenesis genes was upregulated by nuclear translocation of CRTC2 and HDAC5, coactivators of CREB and FoxO1 transcription factors, respectively. This nuclear translocation was inhibited by increased phosphorylation of CRTC2 and HDAC5 by saponarin-induced AMPK in HepG2 cells and suppression of CREB and FoxO1 transactivation activities in cells stimulated by saponarin. The results from a chromatin immunoprecipitation assay confirmed the reduced binding of CRTC2 on the PEPCK and G6Pase promoters. In TE671 cells, AMPK phosphorylated HDAC5, which suppressed nuclear penetration and upregulated GLUT4 transcription, leading to enhanced glucose uptake. Collectively, these results suggest that saponarin activates AMPK in a calcium-dependent manner, thus regulating gluconeogenesis and glucose uptake.

Topics: AMP-Activated Protein Kinases; Apigenin; Benzimidazoles; Biphenyl Compounds; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cyclic AMP Response Element-Binding Protein; Enzyme Activators; Forkhead Box Protein O1; Forkhead Transcription Factors; Gluconeogenesis; Glucose; Glucose Transporter Type 4; Glucosides; Hep G2 Cells; Histone Deacetylases; Humans; Insulin Resistance; Metformin; Naphthalimides; Phosphorylation; Pyrones; Thiophenes; Transcription Factors

This study aims to investigate the effects of ramipril (RPL) on endothelial dysfunction associated with diabetes mellitus using cultured human aortic endothelial cells (HAECs) and a type 2 diabetic animal model. The effect of RPL on vasodilatory function in fat-fed, streptozotocin-treated rats was assessed. RPL treatment of 8 weeks alleviated insulin resistance and inhibited the decrease in endothelium-dependent vasodilation in diabetic rats. RPL treatment also reduced serum advanced glycation end products (AGE) concentration and rat aorta reactive oxygen species formation and increased aorta endothelium heme oxygenase-1 (HO-1) expression. Exposure of HAECs to high concentrations of glucose induced prolonged oxidative stress, apoptosis, and accumulation of AGEs. These effects were abolished by incubation of ramiprilat (RPT), the active metabolite of RPL. However, treatment of HAECs with STO-609, a CaMKKβ (Ca(2+)/calmodulin-dependent protein kinase kinase-β) inhibitor; compound C, an AMPK (AMP-activated protein kinase) inhibitor; and Zn(II)PPIX, a selective HO-1 inhibitor, blocked these beneficial effects of RPT. In addition, RPT increased nuclear factor erythroid 2-related factor-2 (Nrf-2) nuclear translocation and activation in a CaMKKβ/AMPK pathway-dependent manner, leading to increased expression of the Nrf-2-regulated antioxidant enzyme, HO-1. The inhibition of CaMKKβ or AMPK by pharmaceutical approach ablated RPT-induced HO-1 expression. Taken together, RPL ameliorates insulin resistance and endothelial dysfunction in diabetes via reducing oxidative stress. These effects are mediated by RPL activation of CaMKK-β, which in turn activates the AMPK-Nrf-2-HO-1 pathway for enhanced endothelial function.

Topics: AMP-Activated Protein Kinases; Animals; Aorta; Apoptosis; Benzimidazoles; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cells, Cultured; Diabetes Mellitus, Experimental; Endothelial Cells; Enzyme Activation; Glucose; Glycation End Products, Advanced; Heme Oxygenase (Decyclizing); Humans; Insulin Resistance; Male; Naphthalimides; NF-E2-Related Factor 2; Oxidative Stress; Phosphorylation; Protein Kinase Inhibitors; Protoporphyrins; Pyrazoles; Pyrimidines; Ramipril; Rats; Reactive Oxygen Species; Signal Transduction; Vasodilation

Detailed knowledge of the pathways by which ghrelin and leptin signal to AMPK in hypothalamic neurons and lead to regulation of appetite and glucose homeostasis is central to the development of effective means to combat obesity. Here we identify CaMKK2 as a component of one of these pathways, show that it regulates hypothalamic production of the orexigenic hormone NPY, provide evidence that it functions as an AMPKalpha kinase in the hypothalamus, and demonstrate that it forms a unique signaling complex with AMPKalpha and beta. Acute pharmacologic inhibition of CaMKK2 in wild-type mice, but not CaMKK2 null mice, inhibits appetite and promotes weight loss consistent with decreased NPY and AgRP mRNAs. Moreover, the loss of CaMKK2 protects mice from high-fat diet-induced obesity, insulin resistance, and glucose intolerance. These data underscore the potential of targeting CaMKK2 as a therapeutic intervention.

Topics: Acetyl-CoA Carboxylase; Agouti-Related Protein; AMP-Activated Protein Kinase Kinases; Animals; Appetite Regulation; Benzimidazoles; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cells, Cultured; Diet, Atherogenic; Energy Metabolism; Female; Glucose Intolerance; Glucose Tolerance Test; Hypothalamus; Immunoblotting; Immunoenzyme Techniques; Immunoprecipitation; In Situ Hybridization; Insulin; Insulin Resistance; Integrases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Naphthalimides; Neuropeptide Y; Protein Kinases; RNA, Messenger; Transfection; Weight Loss