Interactions of the RAAS and a Western Diet on Insulin Metabolic Actions

描述（由申请人提供）： 胰岛素（INS）敏感性受损是肥胖、高血压和糖尿病等疾病的常见特征。西方饮食（WD），特别是以过量摄入高脂肪、高糖和碳水化合物为特征，是高血压和糖尿病患病率增加的主要因素。这些合并症可能是由INS介导的血管舒张和心血管（CV）和骨骼肌组织中葡萄糖转运的减少引起的。除了我们的WD，其他几种机制，如增强激活的肾素-血管紧张素-醛固酮系统（RAAS）和相关的异常INS代谢信号，可能有助于解释INS抵抗和高血压之间的联系。 有新的证据表明，RAAS的增强激活可能通过哺乳动物雷帕霉素靶蛋白（mTOR）/S6激酶1（S6 K1）信号通路促进INS抗性。mTOR是一种高度保守的营养传感器，通过其S6 K1（一种进化上保守的丝氨酸（Ser）激酶）的磷酸化[（p）]调节INS代谢信号。越来越多的证据表明，通过过量营养素慢性激活S6 K1，通过增加关键INS信号传导/对接分子INS受体底物蛋白1（IRS-1）的Ser（p），促进脂肪、肝脏和骨骼肌组织中的INS抗性，导致磷酸肌醇3激酶（PI 3-K）参与和蛋白激酶B（Akt）刺激受损。我们最近的工作表明，S6 K1被CV组织中的血管紧张素II（Ang II）和醛固酮激活，导致INS代谢信号和生物学后果减少，例如受损的一氧化氮（NO）介导的血管舒张。该建议旨在研究血管紧张素II、醛固酮和WD单独和共同促进CV和骨骼肌组织中INS抵抗的新分子机制。 为了评估INS代谢信号传导的CV功能效应，我们将利用我们最先进的啮齿动物成像中心。在INS抵抗状态下，心肌和骨骼肌葡萄糖摄取和代谢受损，导致舒张功能障碍、心肌和骨骼肌血流减弱以及缺血性再适应受损。我们已经表明，INS刺激的葡萄糖摄取受损和舒张功能障碍与肥胖模型中全身和心肌INS代谢信号受损和组织RAAS表达增加有关。对于这项提议，我们将利用新的敲除和敲低策略，以及创新的啮齿动物成像工具，以评估增加的S6 K1信号（Ang II/醛固酮和/或WD）对心肌功能和冠状动脉和骨骼微血管血流对INS代谢信号的反应的影响。 为了解决目标1，我们将在原代培养的内皮细胞、血管平滑肌细胞和心肌细胞中检查Ang II/醛固酮/WD与S6 K1活化和INS信号传导之间的关系。 代谢信号结果将与功能测量相关，包括NO产生、心肌细胞葡萄糖转运和舒张。 为了进一步探索Ang II/醛固酮和WD对S6 K1的集体以及独立作用，目的2将集中于用产生缓慢升压反应和/或WD的Ang II/醛固酮处理的S6 K1-/-和C57 BL/6小鼠中的体内/离体效应。一组动物将接受AT 1 R阻滞剂或盐皮质激素受体治疗，剂量通过遥测确定，对小鼠血压无影响。 将通过高胰岛素和正葡萄糖钳夹、心脏PET扫描、离体IRS-1（p）和INS代谢信号传导以及心脏和骨骼肌中的葡萄糖摄取来评估INS抵抗。最后，体内INS介导的骨骼肌小动脉和离体冠状小动脉、NO诱导的舒张以及体内心脏葡萄糖摄取和舒张将与离体S6 K1活性和IRS-1位点特异性Se vs.酪氨酸（p）以及所得下游IRS-1/PI 3-K/Akt信号传导相关。

DESCRIPTION (provided by applicant): Project Summary/Abstract Impaired insulin (INS) sensitivity is a common feature of disease states such as obesity, hypertension and diabetes. A western diet (WD), especially characterized by excess intake of high fat, high sucrose and carbohydrates, is a major factor in the increased prevalence of hypertension and diabetes. These co- morbidities may be driven by a decrease in INS-mediated vasorelaxation and glucose transport in cardiovascular (CV) and skeletal muscle tissue. In addition to our WD, several other mechanisms, such as enhanced activation of the renin-angiotensin-aldosterone-system (RAAS) and associated abnormalities in INS metabolic signaling, may help explain the linkage between INS resistance and hypertension. There is emerging evidence that enhanced activation of the RAAS may promote INS resistance through the mammalian target of rapamycin (mTOR)/S6 kinase 1 (S6K1) signaling pathway. mTOR, a highly conserved nutrient sensor, modulates INS metabolic signaling through its phosphorylation [(p)] of S6K1, an evolutionarily conserved serine (Ser) kinase. Evidence is mounting that chronic activation of S6K1, by excessive nutrients, promotes INS resistance in fat, liver and skeletal muscle tissue through increased Ser (p) of the critical INS signaling/docking molecule, INS receptor substrate protein 1 (IRS-1), leading to impaired phosphoinositol 3 kinase (PI3-K) engagement and protein kinase B (Akt) stimulation. Our recent work indicates that S6K1 is activated by angiotensin II (Ang II) and aldosterone in CV tissue leading to diminished INS metabolic signaling and biological consequences, such as impaired nitric oxide (NO)-mediated vascular relaxation. This proposal seeks to investigate novel molecular mechanisms by which Ang II, aldosterone and a WD individually and collectively promote INS resistance in CV and skeletal muscle tissue. To evaluate the CV functional effects of INS metabolic signaling, we will utilize our state-of-the-art rodent imaging center. In the INS resistant state, myocardial and skeletal muscle glucose uptake and metabolism is impaired, leading to diastolic dysfunction, attenuated myocardial and skeletal muscle blood flow, and impaired ischemic reconditioning. We have shown that both impaired INS stimulated glucose uptake and diastolic dysfunction are related to impaired systemic and myocardial INS metabolic signaling in models of obesity and increased tissue RAAS expression. For this proposal, we will utilize novel knockout and knockdown strategies, as well as innovative rodent imaging tools, to evaluate the impact of increased S6K1 signaling (Ang II/aldosterone and/or WD) on myocardial function and coronary and skeletal microvascular blood flow responses to INS metabolic signaling. To address Objective 1, we will examine the relationship between Ang II/aldosterone/WD and S6K1 activation and INS signaling in primary cultured endothelial cells, vascular smooth muscle cells and cardiomyocytes. Metabolic signaling results will be correlated to functional measures including NO production, cardiomyocyte glucose transport and diastolic relaxation. To further explore the collective, as well as the independent, roles of Ang II/aldosterone and a WD on S6K1, Objective 2 will focus on in vivo/ex vivo effects in the S6K1-/- and C57BL/6 mice treated with Ang II/aldosterone that produces a slow pressor response and/or a WD. A cohort of animals will be treated with an AT1R blocker or a mineralocortiod receptor in doses determined by telemetry to have no effect on blood pressure in mice. INS resistance will be assessed by hyperinsulinemic and euglycemic clamp, cardiac PET scanning, ex vivo IRS-1 (p) and INS metabolic signaling, and glucose uptake in heart and skeletal muscle. Finally, in vivo INS mediated skeletal muscle arteriolar and ex vivo coronary arteriolar, NO induced relaxation, and in vivo cardiac glucose uptake and diastolic relaxation will be related to ex vivo S6K1 activity and IRS-1 site specific Se vs. tyrosine (p) and the resultant downstream IRS-1/PI3-K/Akt signaling.