Effects of insulin on the microvasculature

描述（由申请人提供）：本更新申请的统一假设是2型糖尿病和胰岛素抵抗糖尿病前期状态存在微血管内皮功能障碍，其特征为胰岛素和底物向毛细血管的输送受损（毛细血管募集受损）以及毛细血管内皮的营养物质/激素交换减少。在目前的奖项中，我们证明了胰岛素诱导的骨骼肌微血管募集比总血流量增加更快，胰岛素浓度更低。我们还证明了在诱导的胰岛素抵抗（FFA或TNF-α输注或肥胖）状态下骨骼肌内毛细血管募集受损。这种毛细血管募集的损害伴随着胰岛素介导的葡萄糖摄取减少。在目标1中，我们将测试生理相关浓度的胰岛素是否直接作用于内皮细胞以激活信号通路：a）增加一氧化氮的产生; B）选择性扩张毛细血管前小动脉; c）促进葡萄糖和胰岛素的跨内皮运动。在目标2中，我们将检验以下假设：胰岛素抵抗，无论是遗传性的还是由饮食控制引起的，都伴随着微血管胰岛素抵抗和微血管炎症的证据，并且对胰岛素介导的毛细血管募集的抵抗伴随着胰岛素向肌肉平滑肌的转运减少。在目标3下提出的研究中，我们将检验以下假设：生理相关浓度的胰岛素在外周和中枢（CMS内）均起作用，以募集骨骼肌中的微血管。为了量化微血管募集，研究将利用测量微血管体积（超声造影）和毛细血管表面交换面积（1-MX提取）的方法（在我们位于弗吉尼亚州和塔斯马尼亚州霍巴特的实验室联合开发）。为了定量跨内皮胰岛素转运，我们将在体内和体外测量荧光或放射性标记的胰岛素的跨内皮通量。我们将使用共聚焦显微镜和蛋白质印迹法的组合来定义胰岛素信号传导和炎症途径的活性。这些研究，当成功完成，应提供一个更全面的了解微血管作为一个网站的调节胰岛素作用在健康和胰岛素抵抗状态。

DESCRIPTION (provided by applicant): The unifying hypothesis of this renewal application is that microvascular endothelial dysfunction is present in type 2 diabetes and in insulin-resistant pre-diabetic states and is characterized by impaired insulin and substrate delivery to capillaries (impaired capillary recruitment) and by decreased nutrients/hormone exchange across the capillary endothelium. During the current award, we demonstrated that insulin-induced microvascular recruitment in skeletal muscle occurs more rapidly and at lower insulin concentrations than increases in total blood flow. We also demonstrated that capillary recruitment within skeletal muscle is impaired in states of induced insulin resistance (FFA or TNF-a infusion, or obesity). This impairment in capillary recruitment was accompanied by diminished insulin-mediated glucose uptake. In Aim 1, we will test whether insulin at physiologically relevant concentrations acts directly on endothelial cells to activate signaling pathways that: a) increase nitric oxide production; b) selectively dilate precapillary arterioles; c) facilitate the trans-endothelial movement of glucose and insulin. In the Aim 2, we will test the hypothesis that insulin resistance, either genetic or provoked by dietary manipulation, is accompanied by microvascular insulin resistance and evidence of microvascular inflammation and that resistance to insulin-mediated capillary recruitment is accompanied by reduced transport of insulin into muscle interstitium. In studies proposed under Aim 3, we will test the hypotheses that: insulin at physiologic relevant concentrations acts both peripherally and centrally (within the CMS) to recruit microvasculature in skeletal muscle. To quantify microvascular recruitment, studies will utilize methods (developed jointly in our laboratories in Virginia and Hobart, Tasmania) for measurement of microvascular volume (contrast-enhanced ultrasound) and capillary surface exchange area (1-MX extraction). To quantify trans-endothelial insulin transport, we will measure both in vivo and in vitro the trans-endothelial flux of fluorescent or radio-labeled insulin. We will define the activity of insulin-signaling and inflammatory pathways using a combination of confocal microscopy and Western blotting methods. These studies, when successfully completed, should provide a more comprehensive understanding of the role of the microvasculature as a site of regulation of insulin action in health and insulin resistant states.