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.
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 this abnormity. A better understanding of factors involved in insulin resistance should help in development of therapeutic targets to help prevent diabetes and cardiovascular disease.
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