Azizi Paymon M, Zyla Roman E, Guan Sha, Wang Changsen, Liu Jun, Bolz Steffen-Sebastian, Heit Bryan, Klip Amira, Lee Warren L
Institute of Medical Sciences, University of Toronto, Toronto, ON M5S 1A8, Canada Keenan Research Centre, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada Programme in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
Keenan Research Centre, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada.
Mol Biol Cell. 2015 Feb 15;26(4):740-50. doi: 10.1091/mbc.E14-08-1307. Epub 2014 Dec 24.
Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel-derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal colocalization. Insulin transcytosis required dynamin but was unaffected by caveolin-1 knockdown or cholesterol depletion. Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion. Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1. This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent.
胰岛素穿过微血管的转运对于到达其靶器官（如脂肪和肌肉组织）是必要的，并且是胰岛素作用的限速环节。形态学证据表明胰岛素进入微血管的内皮细胞，对源自大血管的内皮细胞的研究显示有胰岛素摄取；然而，关于胰岛素的实际转胞吞作用以及这在相关微血管内皮细胞中如何发生，人们了解甚少。我们报告了一种研究胰岛素穿过单个原代人脂肪微血管内皮细胞（HAMECs）的转胞吞作用的方法，该方法涉及胰岛素摄取，随后通过全内反射荧光显微镜观察囊泡介导的胞吐作用。在这种情况下，荧光团偶联胰岛素的胞吐作用取决于其初始结合和摄取，这是可饱和的，且远大于在肌肉细胞中的情况。与在肌肉细胞内被降解不同，胰岛素在HAMECs内是稳定的，并且未与溶酶体共定位。胰岛素转胞吞作用需要发动蛋白，但不受小窝蛋白 -1基因敲低或胆固醇耗竭的影响。相反，网格蛋白介导的内吞作用抑制剂Pitstop 2或siRNA介导的网格蛋白耗竭显著抑制了胰岛素转胞吞作用。因此，在HAMECs中内化1分钟的胰岛素与网格蛋白的共定位远多于与小窝蛋白 -1的共定位。这项研究构成了囊泡介导的胰岛素转胞吞作用的首个证据，并突出表明其初始摄取是网格蛋白依赖性的且独立于小窝。
