Robinson L J, Pang S, Harris D S, Heuser J, James D E
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Cell Biol. 1992 Jun;117(6):1181-96. doi: 10.1083/jcb.117.6.1181.
Insulin stimulates the movement of two glucose transporter isoforms (GLUT1 and GLUT4) to the plasma membrane (PM) in adipocytes. To study this process we have prepared highly purified PM fragments by gently sonicating 3T3-L1 adipocytes grown on glass coverslips. Using confocal laser immunofluorescence microscopy we observed increased PM labeling for GLUT1 (2.3-fold) and GLUT4 (eightfold) after insulin treatment in intact cells. EM immunolabeling of PM fragments indicated that in the nonstimulated state GLUT4 was mainly localized to flat clathrin lattices. Whereas GLUT4 labeling of clathrin lattices was only slightly increased after insulin treatment, labeling of uncoated PM regions was markedly increased with insulin. These data suggest that GLUT4 recycles from the cell surface both in the presence and absence of insulin. In streptolysin-O permeabilized adipocytes, insulin, and GTP gamma S increased PM levels of GLUT4 to a similar extent as observed with insulin in intact cells. In the absence of an exogenous ATP source the magnitude of these effects was considerably reduced. Removal of ATP per se caused a significant increase in cell surface levels of GLUT4 suggesting that ATP may be required for intracellular sequestration of these transporters. When insulin and GTP gamma S were added together, in the presence of ATP, PM GLUT4 levels were similar to levels observed when either insulin or GTP gamma S was added individually. Addition of GTP gamma S was able to overcome this ATP dependence of insulin-stimulated GLUT4 movement. GTP gamma S had no effect on constitutive secretion of adipsin in permeabilized cells. In addition, there was no effect of insulin or GTP gamma S on GLUT4 movement to the PM in noninsulin sensitive streptolysin-O-permeabilized 3T3-L1 fibroblasts overexpressing GLUT4. We conclude that the insulin-stimulated movement of GLUT4 to the cell surface in adipocytes may require ATP early in the insulin signaling pathway and a GTP-binding protein(s) at a later step(s). We propose that the association of GLUT4 with clathrin lattices may be important in maintaining the exclusive intracellular location of this transporter in the absence of insulin.
胰岛素可刺激脂肪细胞中两种葡萄糖转运体亚型（GLUT1和GLUT4）向质膜（PM）移动。为研究此过程，我们通过轻柔超声处理生长在玻璃盖玻片上的3T3-L1脂肪细胞，制备了高度纯化的质膜片段。利用共聚焦激光免疫荧光显微镜，我们观察到完整细胞经胰岛素处理后，GLUT1的质膜标记增加了2.3倍，GLUT4的质膜标记增加了8倍。质膜片段的电子显微镜免疫标记表明，在未受刺激状态下，GLUT4主要定位于扁平网格蛋白晶格。胰岛素处理后，网格蛋白晶格上的GLUT4标记仅略有增加，而无包被质膜区域的标记则因胰岛素而显著增加。这些数据表明，无论有无胰岛素，GLUT4都可从细胞表面循环利用。在经链球菌溶血素-O通透处理的脂肪细胞中，胰岛素和GTPγS使GLUT4的质膜水平升高，其程度与完整细胞中胰岛素作用时相似。在无外源ATP来源时，这些效应的幅度显著降低。去除ATP本身会导致GLUT4细胞表面水平显著升高，这表明ATP可能是这些转运体在细胞内隔离所必需的。当胰岛素和GTPγS在ATP存在下一起添加时，质膜GLUT4水平与单独添加胰岛素或GTPγS时观察到的水平相似。添加GTPγS能够克服胰岛素刺激的GLUT4移动对ATP的依赖性。GTPγS对通透细胞中脂肪分化相关蛋白的组成性分泌没有影响。此外，在过表达GLUT4的非胰岛素敏感的经链球菌溶血素-O通透处理的3T3-L1成纤维细胞中，胰岛素或GTPγS对GLUT4向质膜的移动没有影响。我们得出结论，脂肪细胞中胰岛素刺激的GLUT4向细胞表面的移动在胰岛素信号通路早期可能需要ATP，在后期步骤可能需要一种GTP结合蛋白。我们提出，在无胰岛素时，GLUT4与网格蛋白晶格的结合可能对维持该转运体在细胞内的独特定位很重要。
