Li D, Randhawa V K, Patel N, Hayashi M, Klip A
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8 and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1A8, Canada.
J Biol Chem. 2001 Jun 22;276(25):22883-91. doi: 10.1074/jbc.M010143200. Epub 2001 Apr 10.
The intracellular traffic of the glucose transporter 4 (GLUT4) in muscle cells remains largely unexplored. Here we make use of L6 myoblasts stably expressing GLUT4 with an exofacially directed Myc-tag (GLUT4myc) to determine the exocytic and endocytic rates of the transporter. Insulin caused a rapid (t(12) = 4 min) gain, whereas hyperosmolarity (0.45 m sucrose) caused a slow (t(12) = 20 min) gain in surface GLUT4myc molecules. With prior insulin stimulation followed by addition of hypertonic sucrose, the increase in surface GLUT4myc was partly additive. Unlike the effect of insulin, the GLUT4myc gain caused by hyperosmolarity was insensitive to wortmannin or to tetanus toxin cleavage of VAMP2 and VAMP3. Disappearance of GLUT4myc from the cell surface was rapid (t(12) = 1.5 min). Insulin had no effect on the initial rate of GLUT4myc internalization. In contrast, hyperosmolarity almost completely abolished GLUT4myc internalization. Surface GLUT4myc accumulation in response to hyperosmolarity was only partially blocked by inhibition of tyrosine kinases with erbstatin analog (erbstatin A) and genistein. However, neither inhibitor interfered with the ability of hyperosmolarity to block GLUT4myc internalization. We propose that hyperosmolarity increases surface GLUT4myc by preventing GLUT4 endocytosis and stimulating its exocytosis via a pathway independent of phosphatidylinositol 3-kinase activity and of VAMP2 or VAMP3. A tetanus toxin-insensitive v-SNARE such as TI-VAMP detected in these cells, might mediate membrane fusion of the hyperosmolarity-sensitive pool.
肌肉细胞中葡萄糖转运蛋白4（GLUT4）的细胞内运输过程在很大程度上仍未被探索。在此，我们利用稳定表达带有面向细胞外的Myc标签的GLUT4（GLUT4myc）的L6成肌细胞来确定该转运蛋白的胞吐和内吞速率。胰岛素导致表面GLUT4myc分子快速增加（t(1/2) = 4分钟），而高渗（0.45 M蔗糖）则导致表面GLUT4myc分子缓慢增加（t(1/2) = 20分钟）。在预先进行胰岛素刺激后再添加高渗蔗糖，表面GLUT4myc的增加部分具有加和性。与胰岛素的作用不同，高渗引起的GLUT4myc增加对渥曼青霉素或破伤风毒素切割VAMP2和VAMP3不敏感。GLUT4myc从细胞表面消失很快（t(1/2) = 1.5分钟）。胰岛素对GLUT4myc内化的初始速率没有影响。相反，高渗几乎完全消除了GLUT4myc的内化。用厄波他汀类似物（厄波他汀A）和染料木黄酮抑制酪氨酸激酶只能部分阻断高渗引起的表面GLUT4myc积累。然而，这两种抑制剂都不影响高渗阻断GLUT4myc内化的能力。我们提出，高渗通过阻止GLUT4内吞并通过一条独立于磷脂酰肌醇3激酶活性以及VAMP2或VAMP-3的途径刺激其胞吐作用，从而增加表面GLUT4myc。在这些细胞中检测到的一种对破伤风毒素不敏感的v-SNARE，如TI-VAMP，可能介导高渗敏感池的膜融合。
