Carvalho Eugenia, Schellhorn Sarah E, Zabolotny Janice M, Martin Sally, Tozzo Effie, Peroni Odile D, Houseknecht Karen L, Mundt Adrian, James David E, Kahn Barbara B
Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, 99 Brookline Avenue, Boston, MA 02215, USA.
J Biol Chem. 2004 May 14;279(20):21598-605. doi: 10.1074/jbc.M312269200. Epub 2004 Feb 24.
The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2-GLUT4-Tg) mice were studied. GLUT4 amount was reduced by 80-95% in aP2-GLUT4-/- adipocytes and increased approximately 10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase (IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2-GLUT4-Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.
在缺乏胰岛素的情况下，大多数葡萄糖转运蛋白4（GLUT4）被隔离在独特的细胞内囊泡中。在胰岛素刺激下，GLUT4囊泡转位并与质膜融合。为了确定GLUT4含量对GLUT4以及其他囊泡蛋白的分布和亚细胞运输的影响，我们研究了脂肪特异性GLUT4缺陷（aP2-GLUT4-/-）小鼠和脂肪特异性GLUT4过表达（aP2-GLUT4-Tg）小鼠的脂肪细胞。与对照相比，aP2-GLUT4-/-脂肪细胞中的GLUT4量减少了80 - 95%，而aP2-GLUT4-Tg脂肪细胞中的GLUT4量增加了约10倍。胰岛素反应性氨肽酶（IRAP）蛋白量在aP2-GLUT4-/-脂肪细胞中减少了35%，在aP2-GLUT4-Tg脂肪细胞中增加了45%。囊泡相关膜蛋白2（VAMP2）蛋白在aP2-GLUT4-/-脂肪细胞中也减少了60%，在aP2-GLUT4-Tg脂肪细胞中增加了2倍。在aP2-GLUT4-Tg中，IRAP和VAMP2的mRNA水平未受影响，这表明GLUT4的过表达影响IRAP和VAMP2蛋白的稳定性。在aP2-GLUT4-/-或aP2-GLUT4-Tg脂肪细胞中， Syntaxin4、SNAP23、Munc-18c和GLUT1的量及亚细胞分布均未改变，但转铁蛋白受体在aP2-GLUT4-Tg脂肪细胞中部分重新分布到质膜。免疫金电子显微镜显示，脂肪细胞中GLUT4的过表达使每个囊泡中GLUT4分子的数量增加了近2倍，每个细胞中含有GLUT4和IRAP的囊泡数量增加了3倍。此外，在未受刺激的aP2-GLUT4-Tg脂肪细胞中，质膜上细胞GLUT4和IRAP的比例分别增加了4倍和2倍，这表明GLUT4和IRAP的隔离是可饱和的。我们的结果表明，GLUT4的过表达或缺陷会影响包括IRAP和VAMP2在内的其他GLUT4囊泡蛋白的量，并且GLUT4的隔离是可饱和的。
