Malide D, Cushman S W
Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
J Cell Sci. 1997 Nov;110 ( Pt 22):2795-806. doi: 10.1242/jcs.110.22.2795.
Studies using functional and pharmacological approaches have implicated PI 3-kinase as a key intermediate in the glucose transport and GLUT4 translocation responses to insulin. Confocal microscopy was used to investigate the effects of the PI 3-kinase inhibitor wortmannin in isolated rat adipose cells. Independent of insulin, wortmannin induces the appearance of phase-lucent vacuoles containing the endosomal markers TfR, Rab4, M6PR, and cellubrevin. When added before or with insulin, wortmannin blocks insulin-stimulated GLUT4 translocation, but does not influence the basal VAMP2-containing GLUT4 compartment. These results substantiate the concept of a specialized basal GLUT4 compartment mostly distinct from that of the recycling receptors. However, when added after insulin, wortmannin induces a rapid redistribution of GLUT4 from the cell surface into those endosomal-derived vacuoles where the GLUT4 co-localize with TfR, Rab4, cellubrevin, and VAMP2, but not with clathrin, M6PR, Golgi complex markers TGN38-mannosidase II and gamma-adaptin, and lysosomal marker lgp-120. Therefore, wortmannin also disrupts insulin-stimulated GLUT4 traffic in the recycling endosomal pathway, at a step distal to the sorting of recycling proteins from late endosomal and TGN markers; wortmannin does not appear to affect internalization from the plasma membrane, and delivery from early to late endosomes or from late endosomes to the TGN. In combination with previous kinetic biochemical studies, these results suggest that: (i) insulin stimulates the exocytosis of GLUT4 through a direct pathway from a specialized basal compartment to the plasma membrane, (ii) during endocytosis in the presence of insulin, GLUT4 is sorted out of the TfR compartment into a separate recycling pathway back to the plasma membrane, and (iii) both of these pathways involve wortmannin sensitive enzymes.
运用功能和药理学方法进行的研究表明，磷脂酰肌醇3-激酶（PI 3-激酶）是胰岛素介导的葡萄糖转运及葡萄糖转运蛋白4（GLUT4）转位反应中的关键中间体。共聚焦显微镜用于研究PI 3-激酶抑制剂渥曼青霉素对分离的大鼠脂肪细胞的影响。与胰岛素无关，渥曼青霉素可诱导出现含有内体标志物转铁蛋白受体（TfR）、Rab4、甘露糖6-磷酸受体（M6PR）和细胞ubrevin的透明空泡。在胰岛素之前或与胰岛素同时添加时，渥曼青霉素可阻断胰岛素刺激的GLUT4转位，但不影响含VAMP2的基础GLUT4区室。这些结果证实了一个专门的基础GLUT4区室在很大程度上不同于循环受体区室的概念。然而，在胰岛素之后添加时，渥曼青霉素可诱导GLUT4从细胞表面迅速重新分布到那些内体来源的空泡中，在这些空泡中GLUT4与TfR、Rab4、细胞ubrevin和VAMP2共定位，但不与网格蛋白、M6PR、高尔基体复合体标志物TGN38-甘露糖苷酶II和γ-衔接蛋白以及溶酶体标志物lgp-120共定位。因此，渥曼青霉素也会破坏回收内体途径中胰岛素刺激的GLUT4运输过程，该过程发生在从晚期内体和TGN标志物中分拣回收蛋白的步骤的远端；渥曼青霉素似乎不影响从质膜的内化以及从早期内体到晚期内体或从晚期内体到TGN的转运。结合先前的动力学生化研究，这些结果表明：（i）胰岛素通过一条从专门的基础区室到质膜的直接途径刺激GLUT4的胞吐作用，（ii）在胰岛素存在的情况下进行内吞作用期间，GLUT4从TfR区室中分拣出来进入一条回到质膜的单独回收途径，以及（iii）这两条途径均涉及对渥曼青霉素敏感的酶。
