ANG II OPPOSES INS MEDIATED VASORELAXATION & GLU UTILZA

ANG II 反对 INS 介导的血管舒张

• 批准号: 7014014
• 负责人: James Russell Sowers
• 金额: $ 28.08万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-10 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7014014
• 关键词: angiotensin II; biological signal transduction; electrospray ionization mass spectrometry; free radical oxygen; glucose; glucose transport; guanine nucleotide binding protein; hormone regulation /control mechanism; hypertension; inhibitor /antagonist; insulin; insulin sensitivity /resistance; insulinlike growth factor; laboratory rat; liquid chromatography; nitric oxide; noninsulin dependent diabetes mellitus; phosphatidylinositol 3 kinase; phosphorylation; posttranslational modifications; tissue /cell culture; transcription factor; transfection; vascular endothelium; vascular smooth muscle; vasomotion

Insulin (INS) resistance and hypertension often coexist, and frequently progress to diabetes and cardiovascular disease. INS and its structurally and functionally similar peptide, insulin-like growth factor (IGF-1), both have been shown to promote vasorelaxation, as well as glucose (GLU) disposal. Recent studies in our laboratory suggest that these actions of INS/ IGF-1 are mediated through the activation of phosphatidylinositol- 3-kinase (PI3-K) and the downstream, protein kinase B (Akt) signaling cascade. This signaling pathway increases: nitric oxide (NO) production by endothelial cells (EC), vascular smooth muscle cell (VSMC)- sodium pump (Na +, K+-ATPase) and myosin bound phosphatase (MBP) activity and GLU transport in skeletal muscle and adipocytes. Thus, functional alterations in these INS/ IGF-1 signaling pathways are likely to play an important role in these pathologies. In this regard, there is emerging evidence that angiotensin II (Ang II) interferes with this signaling, resulting in a state of resistance to INS/ IGF-1 mediated vasorelaxation and GLU transport; however, the role of intermediary signaling molecules are unclear. We hypothesize that Ang II exerts antagonistic effects on INS / IGF-l-stimulated PI3-K/Akt signaling through the generation of reactive oxygen species (ROS) and activation of RhoA. Further, it is proposed that in states of lNS/ IGF-1 resistance and hypertension there is exaggerated tissue Ang II mediated generation of ROS, and activation of RhoA. To test the main hypothesis and its corollary, we will address the following 2 specific aims: 1. To determine the role of RhoA and ROS in mediating the inhibitory effects of Ang II on the Akt-mediated actions of INS and IGF-1 involved in NO metabolism in EC; VSMC Na+,K+-ATPase; MBP activation; and also in the potentiation of GLU transport in skeletal muscle and adipose tissue. 2. To ascertain the role or Ang H-mediated RhoA/ROS stimulation in the impairment of GLU disposal and vasorelaxation in INS-resistant and Ang II overexpressing rodent models of hypertension. Using primary cultures of EC and VSMC, we will examine the role of Ang II-stimulated RhoA and ROS on NO metabolism in EC and VSMC Na +, K+-ATPase and MBP activity. To delineate the role of Akt, RhoA, and ROS signaling in mediating the counterregulatory actions of Ang II and INS/ IGF-1, we will interrupt each of these signaling pathways by transfecting VSMC with dominant negative constructs of Akt and RhoA, and antisense expression of p22phox, one of the critical components of NAD(P)H oxidase. We will profile and quantify site-specific phosphorylation and ROS-induced posttranslational modifications of the signaling molecules (i.e., Akt, RhoA, phoxes) and enzymes (i.e., eNOS, sodium pump, MBP) using liquid chromatography-tandem mass spectrometry. These studies will be complemented with in vivo and ex vivo studies of vasculature and skeletal muscle/adipocytes in rodent models of hypertension and INS resistance as well as normotensive controls. This proposed investigation should significantly enhance our knowledge regarding the role of Ang II in the pathogenesis of INS resistance and hypertension

胰岛素抵抗和高血压常同时存在，并常发展为糖尿病和心血管疾病。INS及其结构和功能相似的肽，胰岛素样生长因子（IGF-1），都已被证明可以促进血管舒张，以及葡萄糖（GLU）的处置。我们实验室最近的研究表明，INS/ IGF-1的这些作用是通过激活磷脂酰肌醇-3-激酶（PI 3-K）和下游蛋白激酶B（Akt）信号级联介导的。这种信号通路增加：内皮细胞（EC）产生一氧化氮（NO）、血管平滑肌细胞（VSMC）钠泵（Na +，K+-ATPase）和肌球蛋白结合磷酸酶（MBP）活性以及骨骼肌和脂肪细胞的葡萄糖转运。因此，这些INS/ IGF-1信号通路的功能改变可能在这些病理中发挥重要作用。在这方面，有新的证据表明，血管紧张素II（Ang II）干扰这种信号传导，导致对INS/ IGF-1介导的血管舒张和GLU转运的抵抗状态;然而，中间信号分子的作用尚不清楚。我们推测Ang II通过产生活性氧（ROS）和激活RhoA对INS /IGF-1刺激的PI 3-K/Akt信号通路发挥拮抗作用。此外，提出在INS/ IGF-1抗性和高血压状态下，存在过度的组织Ang II介导的ROS产生和RhoA活化。为了检验主要假设及其推论，我们将解决以下两个具体目标：1。确定RhoA和ROS在介导Ang II对Akt介导的INS和IGF-1参与EC中NO代谢、VSMC Na+，K+-ATP酶、MBP活化以及骨骼肌和脂肪组织中GLU转运增强的抑制作用中的作用。2.在INS抵抗和Ang II过表达的高血压啮齿动物模型中，确定Ang H介导的RhoA/ROS刺激在GLU处理和血管舒张受损中的作用。利用原代培养的EC和VSMC，我们将研究Ang II刺激的RhoA和ROS对EC和VSMC中NO代谢的作用Na +，K+-ATP酶和MBP活性。为了阐明Akt、RhoA和ROS信号在介导Ang II和INS/ IGF-1的反调节作用中的作用，我们将通过用Akt和RhoA的显性负性结构和NAD（P）H氧化酶的关键组分之一p22 phox的反义表达来阻断VSMC的这些信号通路中的每一个。我们将分析和定量信号分子的位点特异性磷酸化和ROS诱导的翻译后修饰（即，Akt、RhoA、phoxes）和酶（即，eNOS、钠泵、MBP）。这些研究将通过高血压和INS抵抗啮齿动物模型以及血压正常对照中血管系统和骨骼肌/脂肪细胞的体内和离体研究进行补充。这项研究将大大提高我们对血管紧张素Ⅱ在胰岛素抵抗和高血压发病机制中作用的认识