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2型糖尿病和高血压等各种病理状况的主要特征。最近的研究表明，在糖尿病和高血压患者的胰岛素抵抗的发病机制和心血管并发症中，肾素-血管紧张素-醛固酮系统（RAAS）和相关的氧化应激增强。我们认为，丝氨酸（Ser）激酶信号通路的激活，与活性氧（ROS）的过量产生，构成了血管紧张素II（Ang II）和醛固酮在心血管组织中的胰岛素抵抗的机制之一。在我们的初步研究中，以及在其他实验室的工作中，氧化还原敏感性丝氨酸激酶Rho激酶（ROK），已浮出水面，作为一个潜在的重要调解人血管紧张素II和醛固酮诱导的胰岛素抵抗。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Ser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Ser磷酸化来抑制胰岛素代谢信号传导。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的位点定向IRS-1 Ser磷酸化来抑制胰岛素介导的IRS- 1/PI 3-K/Akt信号传导。这一假说的推论是，长期暴露于过量的RAAS激活ROS/ROK途径，导致血管舒张功能受损，心肌葡萄糖利用和舒张松弛，因为ROK诱导IRS-1的位点特异性Ser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的位点特异性Ser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。将在原代培养细胞中研究ROK在介导胰岛素抵抗中的作用，并在长期过量暴露于Ang II和/或醛固酮的啮齿动物的心血管组织中进行体内/离体研究，使用和不使用Ang II和盐皮质激素受体阻断剂以及ROK抑制剂。为了解决具体目标1，我们将采用siRNA的两个ROK亚型，以及ROS/ROK信号的化学抑制剂在细胞预处理与血管紧张素II，醛固酮，或两者，在原代培养的大鼠内皮细胞，血管平滑肌细胞和心肌细胞的胰岛素的代谢和功能反应之前。在具体目标2中，我们将进行体内和离体测定慢性Ang II和/或醛固酮暴露对通过心脏、血管系统和骨骼肌中的胰岛素/IRS-1途径的代谢信号传导的影响。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，采用正电子发射断层扫描（PET）扫描来评估心脏中胰岛素刺激的葡萄糖摄取，并采用电影磁共振成像来评估胰岛素敏感的心脏舒张松弛。我们还将采用视频显微镜直接观察骨骼肌小动脉来评价胰岛素诱导的一氧化氮依赖性血管舒张。共同研究者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。我们的研究提供了新的信息，血管紧张素II和/或醛固酮，共同和单独作用的机制，有助于受损的胰岛素代谢信号和受损的心血管功能的条件下，胰岛素抵抗，如高血压和糖尿病。这项研究应该发现新的治疗策略，可以防止IRS-1的过度Ser磷酸化与高血压和糖尿病患者RAAS升高相关。胰岛素对正常的心血管功能以及维持正常的血糖水平至关重要。对胰岛素正常代谢作用的组织抵抗通常存在于高血压患者中，并且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应该能够开发治疗靶点，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.