胰高糖素样肽-1受体激动剂通过促进血管内皮产生NO进而改善慢性胰岛素抵抗的机制研究

Insulin resistance and endothelial dysfunction are cardinal features of many metabolic disorders such as type 2 diabetes（T2DM）. Insulin vascular and metabolic actions are tightly coupled. To exert its action in muscle, insulin has to be first delivered to the microvasculature nourishing the muscle, and then be transported through the vascular endothelium into the muscle interstitium. Strong evidence has confirmed that altered muscle microvascular perfusion profoundly affects insulin delivery and action in muscle. As such, incretin-based therapy has emerged as a major treatment option for T2DM. Beyond enhancing insulin secretion, incretins have multiple extra-pancreatic actions. Glucagon-like peptide-1 (GLP-1), a major incretin, causes vasodilation, improves endothelial function, and stimulates muscle glucose uptake independent of insulin.We have recently proved that GLP-1, when given acutely, increases muscle glucose uptake by recruiting muscle microvasculature and increasing insulin delivery to muscle. Whether sustained GLP1R activation, using GLP-1 receptor agonist (GLP1RA), also improves muscle microvascular insulin responsiveness and thus metabolic actions of insulin in chronic insulin resistance status has not been studied. In the proposed study, we will examine the effects of sustained GLP1R activation on muscle microvascular blood volume and glucose uptake using a combined contrast-enhanced ultrasound (CEU) and insulin clamp approach in high fat diet (HFD) rats; and we will also examine the direct stimulatory effect of GLP1RA on NO production and cAMP/PKA/eNOS signaling pathway in cultured palmitate induced insulin resistant endothelial cells. These findings will significantly advance our understanding of the physiology and pathophysiology of GLP-1RA and the regulation of muscle microvasculature in chronic insulin resistance. It will also facilitate the design of future studies to prevent and/or treat insulin resistance related diseases .

以胰岛素抵抗（IR）为主要特征的各种代谢性疾病已成为全球性健康威胁。依赖NO的微循环开放是改善骨骼肌IR的关键环节。我们前期研究发现胰高糖素样肽-1受体激动剂（GLP1RA）除促胰岛素分泌外，还可促进IR状态下血管内皮产生NO，提示其在IR时仍可发挥血管舒张作用，结合GLP1RA对高脂喂养的大鼠骨骼肌微循环的灌注改善，由此推测GLP1RA可能通过激活血管内皮GLP1R产生NO，增加微循环灌注，改善或逆转饮食诱导和/或遗传性慢性IR。本研究拟用对照增强超声,胰岛素钳夹,siRNA等技术，观察GLP1RA在体对IR大鼠骨骼肌微血管开放，胰岛素跨膜转运及葡萄糖的摄取，离体对IR内皮cAMP/PKA/eNOS通路的影响，研究创新性从血管扩张和胰岛素的代谢作用耦联的角度揭示GLP1RA增敏胰岛素的新机制，为该药延伸用于广大IR人群提供理论和实验依据。

以胰岛素抵抗（IR）为主要特征的各种代谢性疾病已成为全球性健康威胁。依赖NO的微循环开放是改善骨骼肌IR的关键环节。我们前期研究发现胰高糖素样肽-1受体激动剂（GLP1RA）除了能够促进胰岛素分泌外，还可促进IR状态下血管内皮产生NO，提示NO在IR时仍可发挥血管舒张作用，结合利拉鲁肽对高脂喂养的小鼠下肢动脉循环的灌注改善，由此推测利拉鲁肽可能通过激活血管内皮细胞产生NO，增加微循环灌注，改善或逆转饮食诱导和/或遗传性慢性IR。. 本研究结合体内体外实验，体内实验建立T2DM和T1DM小鼠模型，利拉鲁肽给药后对下肢动脉缺血小鼠的表征影响，通过激光多普勒超声检测后肢血流灌注量。免疫荧光染色、WB检测腓肠肌CD31表达量。体外分离培养内皮祖细胞EPCs，通过成管和划痕实验评估EPCs管样结构形成、迁移能力。WB检测AKT、p-AKT、eNOS、p-eNOS蛋白表达变化。试剂盒测定NO水平。利拉鲁肽可改善糖尿病后肢缺血小鼠下肢血流灌注量且不依赖于降糖作用，其机制可能是通过PI3K/AKT/eNOS/NO通路改善EPCs成管、迁移功能。同时，体外实验进一步发现，利拉鲁肽能抑制高糖作用下人脐静脉内皮细胞（HUVECs）的线粒体自噬，减少线粒体功能损伤，恢复内皮细胞功能。. 本研究从PI3K-Akt-eNOS信号通路与胰岛素抵抗的关系入手，建立了稳定的胰岛素抵抗的小鼠和内皮细胞模型，探究GLP-1RA通过改善内皮细胞NO释放后对改善骨骼肌胰岛素抵抗的作用。我们的结果表明，NO释放的增加有利于改善内皮功能障碍及血管舒张能力，可能为以胰岛素抵抗为特点的代谢和心血管疾病提供新的治疗策略。