Insulin and Receptor-Modulated Pathways of eNOS Regulation

本项目将探讨胰岛素和相关受体通路调节胰岛素分泌的机制。 内皮型一氧化氮合酶及其相关信号蛋白在血管内皮细胞中的表达 细胞该项目与本计划中的其他项目在多个层面上相互作用，不仅是考虑到 内皮源性NO和胰岛素信号在正常血管稳态中的中心作用， 考虑到它们在动脉功能障碍、氧化应激和糖尿病血管病变中的重要性。响应于 激活不同的细胞表面受体包括胰岛素受体NO是由 eNOS。这项研究计划的基本假设是，eNOS和相关基因的紊乱， 内皮途径导致NO依赖性信号传导异常， 糖尿病我们已经发现eNOS在内皮细胞中发生可逆的亚硝化; Aim 1将 确定亚硝化在eNOS调节中的作用。我们计划鉴定eNOS中的半胱氨酸残基 进行可逆亚硝化;表征参与eNOS亚硝化的受体介导的途径， 反硝化;阐明eNOS亚硝化对酶活性的影响;探索eNOS亚硝化的后果 磷酸化和亚细胞靶向酶亚硝化;并确定之间的关系 糖尿病、胰岛素信号传导和eNOS亚硝化。本项目的目标2将探讨脚手架的作用 caveolin-1蛋白在胰岛素介导的内皮细胞信号转导中的作用。siRNA介导的 在培养的内皮细胞中敲低小窝蛋白，多能激酶的胰岛素依赖性磷酸化 糖原合成酶激酶3-P（GSK- 3-P）显著增强。相比之下，caveolin-1的废除 siRNA表达显著减弱了Rho的基础活性和受体依赖性调节， 内皮细胞这些研究将确定小窝蛋白抑制Pho激活的机制 GTP酶和GSK-3 p。目标3中的研究将探索胰岛素增强鞘氨醇的机制 1-磷酸盐（S1 P）信号在血管内皮细胞。我们将确定胰岛素诱导 合成S1 P！受体，并将这些研究扩展到分析动脉制剂，从糖尿病 动物我们研究了胰岛素调节eNOS及相关的细胞和分子机制， 信号通路可能导致糖尿病药物干预的新点的识别 动脉病

This project will explore the mechanisms by which insulin and related receptor pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) and associated signaling proteins in vascular endothelial cells. This project interacts at multiple levels with each of the other projects in this Program, not only in view of the central role of endothelium-derived NO and insulin signaling in normal vascular homeostasis, but also on account of their importance in arterial dysfunction, oxidative stress, and diabetic vasculopathy. In response to the activation of diverse cell surface receptors? including the insulin receptor? NO is synthesized by the eNOS. The fundamental hypothesis of this research program is that derangements in eNOS and in related endothelial pathways contribute to the abnormalities in NO-dependent signaling that have been identified in diabetes. We have discovered that eNOS undergoes reversible nitrosation in endothelial cells; Aim 1 will determine the roles of nitrosation in the regulation of eNOS. We plan to identify the cysteine residue(s) in eNOS that undergo reversible nitrosation; characterize receptor-mediated pathways involved in eNOS nitrosation and denitrosation; elucidate the effects of eNOS nitrosation on enzyme activity; explore the consequences of eNOS phosphorylation and subcellular targeting on enzyme nitrosation; and determine the relationships between diabetes, insulin signaling and eNOS nitrosation. Aim 2 of this project will explore the role of the scaffolding protein caveolin-1 in insulin-mediated signal transduction in endothelial cells. Following siRNA-mediated knockdown of caveolin in cultured endothelial cells, insulin-dependent phosphorylation of the pluripotent kinase glycogen synthase kinase 3-p (GSK- 3-P) is markedly enhanced. By contrast, the abrogation of caveolin-1 expression by siRNA markedly attenuates basal activity and receptor-dependent regulation of Rho in endothelial cells. These studies will define the mechanisms whereby caveolin inhibits the activation of Pho GTPases and GSK- 3p. Studies in Aim 3 will explore the mechanisms whereby insulin potentiates sphingosine 1-phophate (S1P) signaling in vascular endothelial cells. We will identify the pathways whereby insulin induces synthesis of the S1 P! receptor, and will extend these studies to analyze arterial preparations from diabetic animals. Our studies of the cellular and molecular mechanisms whereby insulin regulates eNOS and related signaling pathways may lead to the identification of new points for pharmacological intervention in diabetic arteriopathy.