1,25-二羟维生素D3改善骨骼肌胰岛素抵抗新作用及机制研究

Insulin resistance (IR) constitutes a common and broadly prevalent metabolic disorder, which seems to govern the pathophysiology of diabetes mellitus, metabolic syndrome, and obesity.Evidence has showed that IR causes endothelial dysfunction and impairs insulin-mediated increases in total muscle blood flow.1,25-dihydroxy-vitamin D3 （1,25(OH)2D3）is the most active metabolite of vitamin D and plays a pivotal role in reducing IR and improving endothelium function. However,the underlying mechanisms remain poorly understood.This proposal will focus on the effect of 1,25(OH)2D3 on skeletal muscle(SM) microvasculature.The hypothesis is that 1,25(OH)2D3 improves IR by increase SM microvascular volume and glucose uptake. 1,25(OH)2D3 binding the receptor (VDR) may activate IRS/PI3-K/Akt/eNOS pathway,lead to increased endothelial nitric oxide (NO) production,increased SM capillary recruitment,insulin transmembrane transportation , insulin-mediated GLUT4 plasma membrane translocation and glucose uptake. By employing a series of biochemical, molecular and cellular methodologies, and new state-of-the-art technologies including RNA interference, hyperinsulinemic-euglycemic clamp,contrast-enhanced ultrasound and isotope tracer technology,the hypothesis will be tested by pursuing two specific aims.Aim 1 is to assess the effects of 1,25(OH)2D3 on basal and insulin-stimulated muscular microvascular perfusion,glucose transport, glycogen synthesis, insulin signalling in SM from high-fat induced IR rats.Aim 2 is to access the roles of VDR in the regulation of insulin signaling and NO production in endothelium pretreated with palmitate. The protein expression of IRS/PI3K/Akt/eNOS will be evaluated in cultured HUVEC when VDR is stimulated or silenced. The results will provide new insights into 1,25(OH)2D3 signaling in regulating muscular microvascular perfusion and may translate into potential therapeutic approaches to delay, prevent and treat IR related diseases.

近年来维生素D的骨外作用研究备受重视。临床试验和基础研究证实1,25-二羟维生素D3（1,25(OH)2D3）能改善机体胰岛素抵抗（IR）状态，但机制不明。针对骨骼肌是外周IR的主要靶器官，本研究将在前期工作的基础上，采用对照增强超声，胰岛素钳夹实验和RNA干扰技术，从骨骼肌微循环的角度来探讨1,25(OH)2D3作用IR大鼠后骨骼肌微血管开放的程度，胰岛素跨内皮细胞转运，骨骼肌胰岛素信号通路及对葡萄糖的摄取能力等方面的影响，明确1,25(OH)2D3对骨骼肌IR的改善作用，同时在细胞水平探讨1,25(OH)2D3对血管内皮PI3K/Akt/eNOS通路及NO合成的影响，明确1,25(OH)2D3改善骨骼肌IR的直接靶细胞。通过这一原创性研究，将有助于进一步揭示骨骼肌IR的发病机制，为1,25(OH)2D3治疗IR及相关疾病提供崭新视角，理论基础和实验依据。

以胰岛素抵抗为主要特征的2型糖尿病（T2DM）已成为全球性的健康威胁，由此引起的心血管并发症是目前老龄人群致死致残的首要原因。内皮胰岛素抵抗是整体胰岛素抵抗的启动和初始环节。促炎性信号、氧化应激、蛋白激酶C、脂质毒性等是导致内皮细胞胰岛素抵抗的主要机制。大量的临床证据表明维生素D特别是其活性形式1,25-二羟维生素D3与IR密切相关，补充1,25（OH）2D3可明显提高机体对胰岛素的敏感性。.建立高脂喂养的IR大鼠模型，采用CEU技术观察正常SD大鼠胰岛素输注下不同时相股内侧肌群的微血管血流速度和血流量；使用CEU测量了大鼠后肢MBV和用多普勒超声测量股动脉的血流量（FBF）响应低频等长收缩（0.1赫兹x 10分钟）；使用CEU检测肌肉MBV，MFV和MBF观察高脂饮食中（HFD）微血管胰岛素抵抗是否先于代谢胰岛素抵抗；检测餐后水平血浆游离脂肪酸提高对胰岛素介导的肌肉微血管扩张的影响；观察1，25-（OH）2D3输注对肌微血管和肱动脉血流的影响。0.6 mM palmitate干预人脐静脉内皮细胞（HUVEC）18h后形成内皮IR状态。观察1,25(OH)2D3对血管内皮IR状态下PI3K/Akt/eNOS通路及NO合成的影响，明确1,25(OH)2D3其对骨骼肌IR的改善作用依赖血管内皮细胞VDR介导的PI3K/Akt/eNOS通路的激活。.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大鼠和内皮细胞模型，研究发现微血管胰岛素抵抗在代谢胰岛素抵抗发生前就已经存在，1，25-（OH）2D3能够显著改善肌微循环从而有助于增加胰岛素的肌肉输送从而控制血糖。在细胞方面，1,25(OH)2D3可以通过促进内皮细胞产生NO，减少ET-1，改善内皮胰岛素抵抗。我们的结果表明，循环维生素D水平增加有利于改善内皮功能障碍，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。