Ishiki Manabu, Randhawa Varinder K, Poon Vincent, Jebailey Lellean, Klip Amira
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8.
J Biol Chem. 2005 Aug 5;280(31):28792-802. doi: 10.1074/jbc.M500501200. Epub 2005 Jun 13.
Insulin increases glucose uptake into muscle via glucose transporter-4 (GLUT4) translocation to the cell membrane, but the regulated events in GLUT4 traffic are unknown. Here we focus on the role of class IA phosphatidylinositol (PI) 3-kinase and specific phosphoinositides in the steps of GLUT4 arrival and fusion with the membrane, using L6 muscle cells expressing GLUT4myc. To this end, we detected the availability of the myc epitope at the cell surface or intravesicular spaces and of the cytosol-facing C-terminal epitope, in cells and membrane lawns derived from them. We observed the following: (a) Wortmannin and LY294002 at concentrations that inhibit class IA PI 3-kinase reduced but did not abate the C terminus gain, yet the myc epitope was unavailable for detection unless lawns or cells were permeabilized, suggesting the presence of GLUT4myc in docked, unfused vesicles. Accordingly, GLUT4myc-containing vesicles were detected by immunoelectron microscopy of membranes from cells pretreated with wortmannin and insulin, but not insulin or wortmannin alone. (b) Insulin caused greater immunological availability of the C terminus than myc epitopes, suggesting that C terminus unmasking had occurred. Delivering phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) to intact cells significantly increased lawn-associated myc signal without C terminus gain. Conversely, phosphatidylinositol 3-phosphate (PI3P) increased the detection of C terminus epitope without any myc gain. We propose that insulin regulates GLUT4 membrane arrival, fusion, and C terminus unmasking, through distinct phosphoinositides. PI(3,4,5)P(3) causes arrival and fusion without unmasking, whereas PI3P causes arrival and unmasking without fusion.
胰岛素通过葡萄糖转运蛋白4（GLUT4）转位至细胞膜来增加肌肉对葡萄糖的摄取，但GLUT4转运过程中的调控事件尚不清楚。在此，我们利用表达GLUT4myc的L6肌肉细胞，聚焦于I类磷脂酰肌醇（PI）3激酶和特定磷酸肌醇在GLUT4到达细胞膜并与之融合步骤中的作用。为此，我们检测了细胞表面或囊泡内空间中myc表位以及面向胞质的C末端表位在细胞及其衍生的膜片层中的可及性。我们观察到以下情况：（a）渥曼青霉素和LY294002在抑制I类PI 3激酶的浓度下可减少但不会消除C末端的增加，然而除非膜片层或细胞被通透化，否则myc表位无法被检测到，这表明GLUT4myc存在于停靠但未融合的囊泡中。相应地，在用渥曼青霉素和胰岛素预处理的细胞的膜进行免疫电子显微镜检查时可检测到含GLUT4myc的囊泡，但单独使用胰岛素或渥曼青霉素时则检测不到。（b）胰岛素导致C末端的免疫可及性比myc表位更高，这表明发生了C末端的暴露。向完整细胞递送磷脂酰肌醇3,4,5 -三磷酸（PI(3,4,5)P(3)）可显著增加与膜片层相关的myc信号，但不会增加C末端，相反，磷脂酰肌醇3 -磷酸（PI3P）可增加C末端表位的检测，而不会增加任何myc信号。我们提出胰岛素通过不同的磷酸肌醇来调节GLUT4到达细胞膜、融合以及C末端的暴露。PI(3,4,5)P(3)导致到达和融合但不暴露，而PI3P导致到达和暴露但不融合。
