Ang II and Aldosterone Effects on Insulin Resistance in Cardiovascular Tissue

描述（由申请人提供）：胰岛素敏感性降低是各种病理条件的基本特征，如2型糖尿病和高血压。最近的研究表明，肾素-血管紧张素-醛固酮系统（RAAS）升高和相关的氧化应激增强与糖尿病和高血压患者胰岛素抵抗的发病机制和心血管并发症有关。我们假设丝氨酸（Ser）激酶信号通路的激活，与活性氧（ROS）的过量产生相结合，构成血管紧张素II （Ang II）和醛固酮促进心血管组织胰岛素抵抗的机制之一。在我们的初步研究以及其他实验室的工作中，氧化还原敏感的丝氨酸激酶Rho激酶（ROK）被认为是Ang II和醛固酮诱导的胰岛素抵抗的潜在重要介质。主要在脂肪和骨骼肌组织中进行的研究表明，包括ROK在内的几种丝氨酸激酶可能通过诱导关键对接蛋白胰岛素受体底物-1 （IRS-1）的位点特异性丝氨酸磷酸化来抑制胰岛素代谢信号。因此，我们假设RAAS激活氧化还原敏感的ROK通过引起心血管组织中IRS-1 Ser位点定向磷酸化来抑制胰岛素介导的IRS-1 /PI3-K/Akt信号。这一假设的推论是，长期暴露于过量的RAAS激活ROS/ROK通路，导致血管舒张、心肌葡萄糖利用和舒张舒张受损，因为ROK诱导了IRS-1位点特异性丝氨酸磷酸化。在我们提出的研究中，我们计划使用质谱和其他新技术来研究IRS-1位点特异性丝氨酸和酪氨酸（Tyr）磷酸化与Ang II和醛固酮诱导的胰岛素抵抗的关系。ROK在介导胰岛素抵抗中的作用将在原代培养细胞和体内/离体研究中进行研究，研究对象是长期过量暴露于Ang II和/或醛固酮、有或没有Ang II和矿化皮质激素受体阻断和ROK抑制的啮齿动物心血管组织。为了解决Specific Aim 1，我们将在原代培养的大鼠内皮细胞、血管平滑肌细胞和心肌细胞对胰岛素的代谢和功能反应之前，在用Ang II、醛固酮或两者预处理的细胞中，对两种ROK亚型以及ROS/ROK信号的化学抑制剂使用siRNA。在Specific Aim 2中，我们将在体内和体外测定慢性Ang II和/或醛固酮暴露对心脏、脉管系统和骨骼肌中胰岛素/IRS-1通路代谢信号的影响。我们将利用我们最先进的啮齿动物成像中心进行关键的体内研究，使用正电子发射断层扫描（PET）来评估胰岛素刺激下的心脏葡萄糖摄取，并使用电影磁共振成像来评估胰岛素敏感的心脏舒张舒张。我们还将使用视频显微镜直接可视化骨骼肌小动脉来评估胰岛素诱导的一氧化氮依赖性血管舒张。共同研究人员包括一名成像物理学家、一名血管生物学家和一名质谱专家，以评估IRS-1对接蛋白的位点特异性Ser和Tyr磷酸化。我们提出的研究将提供新的机制，通过Ang II和/或醛固酮，共同或单独作用，促进胰岛素代谢信号受损和心血管功能受损的胰岛素抵抗条件下，如高血压和糖尿病。这项研究应该揭示新的治疗策略，可以防止与高血压和糖尿病患者RAAS升高相关的IRS-1过度丝氨酸磷酸化。胰岛素对正常的心血管功能和维持正常的血糖水平至关重要。组织对胰岛素正常代谢作用的抵抗通常存在于高血压患者中，是糖尿病和心血管疾病的前兆。心血管组织和骨骼肌中胰岛素抵抗的基本机制尚不清楚，我们提出的工作旨在阐明这些机制。更好地了解胰岛素抵抗的相关因素将有助于开发治疗靶点，以帮助预防糖尿病和心血管疾病。

DESCRIPTION (provided by applicant): Decreased insulin sensitivity is a cardinal feature of various pathological conditions such as type 2 diabetes and hypertension. Recent research has implicated a heightened renin-angiotensin-aldosterone system (RAAS) and associated enhanced oxidative stress in the pathogenesis and cardiovascular complications of insulin resistance in people with diabetes and hypertension. We posit that activation of serine (Ser) kinase signaling pathways, in conjunction with excess generation of reactive oxygen species (ROS), constitute one of the mechanisms whereby angiotensin II (Ang II) and aldosterone contribute to insulin resistance in cardiovascular tissue. In our preliminary investigation, as well as in work from other laboratories, the redox-sensitive Ser kinase Rho kinase (ROK), has surfaced as a potentially important mediator of Ang II and aldosterone induced insulin resistance. Research, primarily conducted in fat and skeletal muscle tissue, indicates that several Ser kinases, including ROK, may inhibit insulin metabolic signaling by inducing site specific Ser phosphorylation of the critical docking protein insulin receptor substrate-1 (IRS-1). Accordingly, we hypothesize that RAAS activation of redox sensitive ROK inhibits insulin mediated IRS- 1/PI3-K/Akt signaling by causing site directed IRS-1 Ser phosphorylation in cardiovascular tissue. A corollary to this hypothesis is that chronic exposure to excess RAAS activates ROS/ROK pathways leading to impairment of vasorelaxation, myocardial glucose utilization and diastolic relaxation because of ROK induced site-specific Ser phosphorylation of IRS-1. In our proposed research, we plan to use mass spectroscopy and other novel techniques to investigate site specific Ser and tyrosine (Tyr) phosphorylation of IRS-1 in relation to Ang II and aldosterone induced insulin resistance. The role of ROK in mediating insulin resistance will be investigated in primary cultured cells and in vivo/ex vivo studies in cardiovascular tissues of rodents with excess long-term exposure to Ang II and/or aldosterone, with and without Ang II and mineralocorticoid receptor blockade, and ROK inhibition. To address Specific Aim 1, we will employ siRNA for the two ROK isoforms as well as chemical inhibitors of ROS/ROK signaling in cells pretreated with Ang II, aldosterone, or both, before measuring metabolic and functional responses to insulin in primary cultured rat endothelial, vascular smooth muscle and cardiomyocyte cells. In Specific Aim 2, we will conduct both in vivo and ex vivo determination of the impact of chronic Ang II and/or aldosterone exposure on metabolic signaling through the insulin/IRS-1 pathway in heart, vasculature and skeletal muscle. We will utilize our state of the art rodent imaging center to conduct critical in vivo investigations, employ positron emission tomography (PET) scanning to evaluate insulin stimulated glucose uptake in the heart, and cine-magnetic resonance imaging to evaluate insulin sensitive cardiac diastolic relaxation. We will also employ direct visualization of skeletal muscle arterioles using video microscopy to evaluate insulin induced nitric oxide dependent vasodilation. Co-investigators include an imaging physicist, a vascular biologist and an expert in mass spectroscopy to evaluate site specific Ser and Tyr phosphorylation of the IRS-1 docking protein. Our proposed investigation should provide novel information on the mechanisms by which Ang II and/or aldosterone, acting collectively and individually, contribute to impaired insulin metabolic signaling and compromised cardiovascular function in conditions of insulin resistance such as hypertension and diabetes. This research should uncover new therapeutic strategies that can prevent excessive Ser phosphorylation of IRS-1 associated with a heightened RAAS in persons with hypertension and diabetes. Lay Summary Insulin is critical for normal cardiovascular function as well as for maintaining normal blood glucose levels. Tissue resistance to the normal metabolic actions of insulin is often present in persons with hypertension, and is a precursor for diabetes and cardiovascular disease. The fundamental mechanisms underlying insulin resistance in cardiovascular tissue, as well as skeletal muscle, are not well understood, and our proposed work is directed at elucidation of these mechanisms. A better understanding of factors involved in insulin resistance should enable development of therapeutic targets to help prevent diabetes and cardiovascular disease.