Foster L J, Li D, Randhawa V K, Klip A
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
J Biol Chem. 2001 Nov 23;276(47):44212-21. doi: 10.1074/jbc.M102964200. Epub 2001 Sep 17.
Insulin enhances plasmalemmal-directed traffic of glucose transporter-4 (GLUT4), but it is unknown whether insulin regulates GLUT4 traffic through endosomal compartments. In L6 myoblasts expressing Myc-tagged GLUT4, insulin markedly stimulated the rate of GLUT4myc recycling. In myoblasts stimulated with insulin to maximize surface GLUT4myc levels, we followed the rates of surface-labeled GLUT4myc endocytosis and chased its intracellular distribution in space and time using confocal immunofluorescence microscopy. Surface-labeled GLUT4myc internalized rapidly (t(12) 3 min), reaching the early endosome by 2 min and the transferrin receptor-rich, perinuclear recycling endosome by 20 min. Upon re-addition of insulin, the t(12) of GLUT4 disappearance from the plasma membrane was unchanged (3 min), but strikingly, GLUT4myc reached the recycling endosome by 10 and left by 20 min. This effect of insulin was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002 or by transiently transfected dominant-negative phosphatidylinositol 3-kinase and protein kinase B mutants. In contrast, insulin did not alter the rate of arrival of rhodamine-labeled transferrin at the recycling endosome. These results reveal a heretofore unknown effect of insulin to accelerate inter-endosomal travel rates of GLUT4 and identify the recycling endosome as an obligatory stage in insulin-dependent GLUT4 recycling.
胰岛素可增强葡萄糖转运蛋白4（GLUT4）向质膜的转运，但胰岛素是否通过内体区室调节GLUT4的转运尚不清楚。在表达Myc标签的GLUT4的L6成肌细胞中，胰岛素显著刺激了GLUT4myc的循环速率。在用胰岛素刺激成肌细胞以使表面GLUT4myc水平最大化后，我们追踪了表面标记的GLUT4myc的内吞速率，并使用共聚焦免疫荧光显微镜在空间和时间上追踪其细胞内分布。表面标记的GLUT4myc迅速内化（t(1/2)为3分钟），2分钟时到达早期内体，20分钟时到达富含转铁蛋白受体的核周循环内体。重新添加胰岛素后，GLUT4从质膜消失的t(1/2)不变（3分钟），但引人注目的是，GLUT4myc在10分钟时到达循环内体，并在20分钟时离开。胰岛素的这种作用被磷脂酰肌醇3激酶抑制剂LY294002或瞬时转染的显性负性磷脂酰肌醇3激酶和蛋白激酶B突变体阻断。相比之下，胰岛素并未改变罗丹明标记的转铁蛋白到达循环内体的速率。这些结果揭示了胰岛素此前未知的加速GLUT4在内体间转运速率的作用，并确定循环内体是胰岛素依赖性GLUT4循环中的一个必经阶段。
