Pharmacological Restoration of Diabetic Vascular Dysfunction

描述(由申请人提供)： 问题：尽管积极调整危险因素，糖尿病(DM)仍会增加心血管疾病(CV)和全因死亡率。我们的长期目标是确定降低糖尿病心血管风险的新目标。血管线粒体是一个潜在的新靶点。线粒体调节内皮功能和平滑肌细胞(SMC)的增殖，线粒体功能障碍是糖尿病的标志。运动引起健康血管中线粒体质量的适应性改善；这种反应在糖尿病中是不存在的。本研究提出的问题是，以GLP-1为靶点的eNOS和SIRT能否恢复线粒体生物发生的信号，改善线粒体动力学和血管功能。GLP-1是一种糖尿病药物，还可以诱导eNOS、SIRT1、线粒体生物发生和自噬21。新的初步数据表明，萨格列汀(saxagliptin，Saxa)，一种增加循环内源性GLP-1的二肽基肽酶-4抑制剂，可以恢复运动诱导的内皮型一氧化氮合酶和线粒体蛋白的表达，并提高运动距离。假说：GLP-1通过eNOS和SIRT1信号通路改善糖尿病血管线粒体功能异常和应激介导的线粒体动力学。SA#1：GLP-1对糖尿病患者血管线粒体适应性的影响是什么？理论基础：8天运动的对照组大鼠表现出线粒体生物发生的激活，线粒体功能的改善以及融合的增加和分裂的减少。我们将研究GLP-1对糖尿病患者血管线粒体功能和周转的影响，并使用靶向缺失eNOS、内皮细胞(EC)SIRT1、SMC SIRT1来测试体内线粒体的动态平衡适应。假设1.1：在高脂诱导的糖尿病中，GLP-1的干预将通过增强血管线粒体对运动的适应(生物发生、融合和自噬)来改善线粒体功能。假设1.2：内皮型一氧化氮合酶和/或SIRT1是GLP-1挽救线粒体适应性所必需的。方法：1.雄性C57BL6在饮食与高脂(HF)饮食对比中持续10周，将被随机分为GLP-1组或运动或不运动组，为期8天。1B.C57eNOS-/-和c57b eNOS+/+将与SA1a一样处理。1C。SIRT1 EC或SMC缺失的C57小鼠和对照组将被视为SA1a。这些实验将阐明eNOS和SIRT1对体内运动中血管线粒体适应的重要性，并确定糖尿病和GLP-1对这些途径的影响。SA#2：糖尿病如何影响体外血管细胞的动态线粒体适应？理论基础：线粒体功能的调节需要线粒体生物遗传和通过分裂、融合和自噬重塑之间的复杂相互作用。假设2.1：DM模型中的内皮细胞和/或平滑肌细胞线粒体动力学受损。假设2.2：GLP-1通过eNOS和/或SIRT改善DM细胞的线粒体功能和动力学。方法：这些实验将在体外检测DM和GLP-1对线粒体质量和动力学的影响，并利用遗传学方法确定eNOS和SIRT对血管细胞线粒体质量的相对贡献。对退伍军人人群的影响：线粒体动力学是降低糖尿病患者过度心血管风险的新目标。针对线粒体ROS的干预在人类研究中一直无效。我们观察到，8天的Saxa治疗恢复了线粒体生物发生的血管诱导，这证明了目前可用的药物可以靶向受损的血管线粒体适应。糖尿病患者线粒体动力学的药理学恢复可能对糖尿病的大血管和微血管并发症产生影响。

DESCRIPTION (provided by applicant): Problem: Diabetes (DM) increases cardiovascular (CV) and all-cause mortality despite aggressive risk factor modification. Our long term goal is to define novel targets for cardiovascular risk reduction in diabetes. Vascular mitochondria are a potential new target. Mitochondria regulate endothelial function and smooth muscle cell (SMC) proliferation and mitochondrial dysfunction is a hallmark of diabetes. Exercise elicits an adaptive improvement in mitochondrial quality in healthy vessels; this response is absent in diabetes. The question addressed in this proposal is whether pharmacological targeting of eNOS and SIRT with glucagon- like peptide 1 (GLP-1) can restore signaling to mitochondrial biogenesis and improve mitochondrial dynamics and vascular function. GLP-1 is a diabetes medication that also induces eNOS, SIRT1, mitochondrial biogenesis and autophagy 21. New preliminary data suggest that saxagliptin (SAXA), a dipeptidyl peptidase-4 inhibitor that increases circulating endogenous GLP-1, restores induction of eNOS and mitochondrial protein expression with exercise in DM and improves running distance. Hypotheses: Abnormal mitochondrial function and impaired stress mediated mitochondrial dynamics in the diabetic vasculature will be improved by GLP-1 via eNOS and SIRT1 signaling. SA #1: What is the impact of GLP-1 on vascular mitochondrial adaptation in DM? Rationale: Control rats subjected to 8 day exercise show increased activation of the mitochondrial biogenesis, improved mitochondrial function plus increased fusion and decreased fission. We will examine the impact of GLP-1 on vascular mitochondrial function and turnover in diabetes and use targeted deletion of eNOS, endothelial cell (EC) SIRT1, SMC SIRT1 with or without exercise to test mitochondrial homeostatic adaptation in vivo. Hypothesis 1.1: Intervention with GLP-1 will improve mitochondrial function through augmentation of vascular mitochondrial adaptation (biogenesis, fusion and autophagy) to exercise in high fat induced diabetes. Hypothesis 1.2: eNOS and/or SIRT1 are required for GLP-1 rescue of mitochondrial adaptation. Approach: 1a. Male C57BL6 on chow versus high fat (HF) diet for 10 weeks will be randomized to GLP-1 or vehicle with or without exercise for 8 days. 1b. c57eNOS -/- and c57b eNOS +/+ will be treated as in SA1a. 1c. c57 mice with EC or SMC deletion of SIRT1 and controls will be treated as in SA1a. These experiments will clarify the importance of eNOS and SIRT1 for vascular mitochondrial adaptation to exercise in vivo and define the impact of diabetes and GLP-1 on these pathways. SA #2: How does diabetes affect dynamic mitochondrial adaptation in vascular cells in vitro? Rationale: Regulation of mitochondrial function requires a complex interplay between mitochondrial biogene- sis and remodeling through fission, fusion and autophagy. Hypothesis 2.1: Endothelial cells and/or smooth muscle cells from DM models will have impaired mitochondrial dynamics. Hypothesis 2.2: GLP-1 will improve mitochondrial function and dynamics in DM cells through eNOS and/or SIRT. Approach: These experiments will examine the impact of DM and GLP-1 on mitochondrial quality and dynamics in vitro and employ genetic approaches to define the relative contribution of eNOS and SIRT for mitochondrial quality in vascular cells. Impact on the Veteran Population: Mitochondrial dynamics are a novel target to decrease excess CV risk in diabetes. Interventions targeting mitochondrial ROS have been consistently ineffective in human studies. Our observation that 8 days of treatment with SAXA restores vascular induction of mitochondrial biogenesis provides proof of concept that impaired vascular mitochondrial adaptation is targetable with a currently available drug. Pharmacological restoration of mitochondrial dynamics in diabetes could have implications for macrovascular and microvascular complications of diabetes.