Ang II and Overnutrition and Insulin resistance in Cardiovascular Tissue

描述(申请人提供)：胰岛素敏感性降低是肥胖、高血压和糖尿病等疾病状态的常见特征。营养过剩(特别是以摄入过多脂肪和碳水化合物为特征的营养过剩)是高血压和糖尿病患病率增加的主要因素。这些共病可能是由INS介导的心血管(CV)和骨骼肌组织中血管松弛和葡萄糖运输减少所致。除了过度营养外，其他几种机制，如肾素-血管紧张素系统(RAS)的增强激活、炎症以及相关的INS代谢信号异常，可能有助于解释INS抵抗与高血压之间的联系。越来越多的证据表明，过度营养和血管紧张素II(Ang II)可能通过哺乳动物靶标雷帕霉素(MTOR)/S6激酶1(S6K1)信号通路促进胰岛素抵抗。MTOR是一种高度保守的营养感受器，它通过S6K1的磷酸化(P)来调节INS代谢信号。S6K1是一种进化上保守的丝氨酸激酶。越来越多的证据表明，S6K1的慢性激活，通过过度营养，通过增加关键的INS信号/对接分子，INS受体底物蛋白1(IRS-1)的Ser(P)，导致磷酸肌醇3激酶(PI3-K)的参与和蛋白激酶B(Akt)的刺激，促进脂肪、肝脏和骨骼肌组织的INS抵抗。我们最近的工作表明，血管紧张素转换酶II激活了S6K1，导致INS代谢信号和生物学后果的减弱，如一氧化氮(NO)介导的血管松弛受损。这项建议旨在研究血管紧张素转换酶II和过度营养单独和共同促进心血管和骨骼肌组织胰岛素抵抗的新分子机制。为了评估INS代谢信号对心血管功能的影响，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，心肌和骨骼肌的葡萄糖摄取和代谢受损，导致舒张期功能障碍，心肌和骨骼肌血流减弱，缺血再适应受损。我们已经证明，肥胖模型中INS刺激的葡萄糖摄取受损和舒张期功能障碍都与全身和心肌INS代谢信号受损以及组织RAS表达增加有关。对于这项提议，我们将利用新的基因敲除和基因敲除策略，以及创新的啮齿动物成像工具，来评估增加的S6K1信号(Ang II和/或过量营养)对心肌功能以及冠状动脉和骨骼微血管血流对INS代谢信号的影响。为了达到目标1，我们将研究原代培养的内皮细胞、血管平滑肌细胞和心肌细胞中Ang II和S6K1的激活与INS信号的关系。代谢信号的结果将与包括一氧化氮产生、心肌细胞葡萄糖运输和舒张期松弛在内的功能指标相关联。为了进一步探索Ang II和过度营养在S6K1中的集体和独立作用，Aim 2将重点研究Ang II对S6K1-/-和C57BL/6小鼠的体内/体外影响，Ang II产生缓慢的升压反应和/或高脂肪(60%)和高蔗糖(20%)饮食。一组动物将接受AT1R阻滞剂(奥美沙坦)的治疗，剂量为0.5 mg/kg/天，通过遥测确定的剂量对db/db小鼠的血压没有影响。INS抵抗将通过高胰岛素血症、正常血糖钳夹、心脏PET扫描、体外IRS-1(P)和INS代谢信号以及心肌和骨骼肌葡萄糖摄取来评估。最后，体内INS介导的骨骼肌小动脉和体外冠状动脉小动脉，NO诱导的松弛，以及体内心肌葡萄糖摄取和舒张期松弛将与体外S6K1活性和IRS-1位点特异性Servs.Tyr(P)以及由此产生的IRS-1/PI3-K/Akt信号有关。

DESCRIPTION (provided by applicant): Diminished insulin (INS) sensitivity is a common feature of disease states such as obesity, hypertension and diabetes. Over-nutrition (especially that characterized by excess intake of fat 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 over-nutrition, several other mechanisms, such as enhanced activation of the renin- angiotensin-system (RAS), inflammation, and associated abnormalities in INS metabolic signaling, may help explain the linkage between INS resistance and hypertension. There is emerging evidence that over-nutrition and angiotensin II (ANG II) 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 ANG II 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 and over-nutrition 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 RAS 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 and/or excess nutrients) on myocardial function and coronary and skeletal microvascular blood flow responses to INS metabolic signaling. To address Aim 1, we will examine the relationship between ANG II 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 and over-nutrition on S6K1, Aim 2 will focus on in vivo/ex vivo effects in the S6K1-/- and C57BL/6 mice treated with ANG II that produces a slow pressor response and/or a high fat (60%) and high sucrose (20%) diet. A cohort of animals will be treated with an AT1R blocker (olmesartan) at a dose of 0.5 mg/kg/day, a dose determined by telemetry to have no effect on blood pressure in db/db mice. INS resistance will be assessed by hyperinsulinemic, 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 Ser vs. Tyr (P) and the resultant downstream IRS-1/PI3-K/Akt signaling.