Insulin and Receptor-Modulated Pathways of eNOS Regulation

首席调查员/项目主任(最后、第一、中间)：米歇尔·托马斯 项目1：“内皮型一氧化氮合酶调节的胰岛素和受体调节途径”。 托马斯·米歇尔，项目负责人 摘要： 该项目将探索胰岛素和相关受体途径调节血管生成的机制。 血管内皮细胞内皮型一氧化氮合酶及其相关信号蛋白 细胞。该项目与本计划中的每个其他项目在多个层面上进行互动，不仅是因为 内皮来源的NO和胰岛素信号在正常血管内稳态中的中心作用，但也在 说明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。作为对.的回应 包括胰岛素受体NO在内的多种细胞表面受体的激活是由 伊诺斯。本研究的基本假设是内皮型一氧化氮合酶及其相关基因的紊乱 内皮通路参与了NO依赖信号的异常，这些信号已被发现在 糖尿病。我们已经发现eNOS在内皮细胞中经历可逆的亚硝化；Aim 1将 确定亚硝化作用在内皮型一氧化氮合酶调节中的作用。我们计划对eNOS中的半胱氨酸残基(S)进行鉴定 可逆亚硝化；表征参与eNOS亚硝化的受体介导的通路和 反硝化；阐明eNOS亚硝化对酶活性的影响；探索eNOS的后果 酶亚硝化的磷酸化和亚细胞靶向性；并确定两者之间的关系 糖尿病、胰岛素信号转导和eNOS亚硝化。本项目的目标2将探讨脚手架的作用 蛋白小窝蛋白-1在胰岛素介导的内皮细胞信号转导中的作用。遵循siRNA介导的 抑制培养的内皮细胞小窝蛋白，胰岛素依赖的多能蛋白激酶的磷酸化 糖原合成酶激酶3-P(GSK-3-P)明显增强。相比之下，小窝蛋白-1的废除 SiRNA表达显著减弱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-3p。目标3的研究将探索胰岛素增强鞘氨醇的机制。 血管内皮细胞中的1-磷酸(S1P)信号转导。我们将确定胰岛素诱导的途径 合成S1P！受体，并将把这些研究扩展到分析糖尿病患者的动脉制剂 动物。我们胰岛素调节eNOS及相关基因的细胞和分子机制的研究 信号通路可能为糖尿病的药物干预找到新的切入点 动脉病。

Principal Investigator/Program Director (Last, First, Middle): Michel, Thomas PROJECT 1: "Insulin and Receptor-Modulated Pathways of eNOS Regulation." Thomas Michel, Project Leader Abstract: 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.