Pharmacological Restoration of Diabetic Vascular Dysfunction

描述（由申请人提供）： 问题：糖尿病（DM）增加心血管（CV）和全因死亡率，尽管积极的风险因素修改。我们的长期目标是确定降低糖尿病心血管风险的新靶点。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）增殖，并且线粒体功能障碍是糖尿病的标志。运动可以使健康血管的线粒体质量得到适应性改善;这种反应在糖尿病患者中是不存在的。该提案中解决的问题是用胰高血糖素样肽1（GLP-1）药理学靶向eNOS和SIRT是否可以恢复线粒体生物发生的信号传导并改善线粒体动力学和血管功能。GLP-1是一种糖尿病药物，也可诱导eNOS、SIRT 1、线粒体生物合成和自噬21。新的初步数据表明，沙格列汀（SAXA），一种二肽基肽酶-4抑制剂，增加循环内源性GLP-1，恢复诱导eNOS和线粒体蛋白表达与运动DM和改善跑步距离。 假设：GLP-1通过eNOS和SIRT 1信号传导改善糖尿病血管系统中异常的线粒体功能和受损的应激介导的线粒体动力学。 SA #1：GLP-1对DM患者血管线粒体适应性的影响是什么？基本原理：进行8天运动的对照大鼠显示线粒体生物发生的活化增加，线粒体功能改善加上融合增加和分裂减少。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eNOS、内皮细胞（EC）SIRT 1、SMC SIRT 1的靶向缺失，在运动或不运动的情况下测试体内线粒体稳态适应。假设1.1：GLP-1的干预将通过增强血管线粒体适应（生物发生、融合和自噬）来改善线粒体功能，以在高脂肪诱导的糖尿病中进行运动。假设1.2：eNOS和/或SIRT 1是GLP-1拯救线粒体适应所必需的。方法：1a.将接受普通饲料与高脂肪（HF）饮食10周的雄性C57 BL 6随机分配至GLP-1或溶媒组，伴或不伴运动8天。1b. c57 eNOS-/-和c57 b eNOS +/+将按照SA 1a进行处理。1c.具有SIRT 1的EC或SMC缺失的c57小鼠和对照将如SA 1a中那样处理。这些实验将阐明eNOS和SIRT 1对血管线粒体适应体内运动的重要性，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？基本原理：线粒体功能的调节需要线粒体生物合成与通过分裂、融合和自噬的重塑之间的复杂相互作用。假设2.1：来自DM模型的内皮细胞和/或平滑肌细胞将具有受损的线粒体动力学。假设2.2：GLP-1将通过eNOS和/或SIRT改善DM细胞中的线粒体功能和动力学。方法：这些实验将检查DM和GLP-1对体外线粒体质量和动力学的影响，并采用遗传方法来确定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CV风险的新靶点。针对线粒体ROS的干预措施在人类研究中一直无效。我们观察到，用SAXA治疗8天恢复了线粒体生物发生的血管诱导，这提供了概念证据，即受损的血管线粒体适应是目前可用药物的靶向。糖尿病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有影响。

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.