CYCLOHEXIMIDE DECREASES GLUCOSE TRANSPORTERS IN RAT ADIPOCYTE PLASMA-MEMBRANES WITHOUT AFFECTING INSULIN-STIMULATED GLUCOSE-TRANSPORT
CYCLOHEXIMIDE DECREASES GLUCOSE TRANSPORTERS IN RAT ADIPOCYTE PLASMA-MEMBRANES WITHOUT AFFECTING INSULIN-STIMULATED GLUCOSE-TRANSPORT
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DOI：
10.1042/bj2510491
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发表时间：
1988-04-15
影响因子：
4.1
通讯作者：
KARNIELI, E
中科院分区：
文献类型：
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作者：
MATTHAEI, S;OLEFSKY, JM;KARNIELI, E
本研究探讨了胰岛素刺激的葡萄糖转运和胰岛素诱导的葡萄糖转运蛋白易位在离体大鼠脂肪细胞之间的关系。将脂肪细胞与放线菌酮（一种有效的蛋白质合成抑制剂）一起孵育或不孵育60分钟，然后与胰岛素一起孵育或不孵育30分钟。孵育后，我们测量了3-O-甲基葡萄糖在脂肪细胞中的转运，并制备了亚细胞膜组分。通过细胞松弛素B结合试验测定各种膜组分中葡萄糖转运蛋白的数量。放线菌酮不影响基础和胰岛素刺激的3-O-甲基葡萄糖摄取。此外，放线菌酮既不影响胰岛素刺激的3-O-甲基葡萄糖转运的Vmax也不影响Km。在Hexaast中，与单独用胰岛素孵育的细胞相比，来自用放线菌酮和胰岛素预孵育的细胞的质膜中的葡萄糖转运蛋白的数量显著减少（10.5 ± 0.01）。0.8和22.2 .+-。1.8 pmol/mg蛋白; P < 0.005）。与对照细胞相比，单独与环己酰亚胺孵育的细胞中葡萄糖转运蛋白的数量没有显着差异。SDS/聚丙烯酰胺凝胶电泳分析的[3 H]细胞松弛素-B-光标记的血浆膜馏分显示，放线菌酮减少胰岛素刺激膜标记的葡萄糖转运蛋白的量。然而，蛋白质的表观分子量没有改变放线菌酮处理。放线菌酮对胰岛素刺激的低密度微粒体膜中葡萄糖转运蛋白的二维电泳图谱的影响显示pI-6.4葡萄糖转运蛋白亚型减少，而胰岛素可转位亚型（pI 5.6）减少。因此，观察到的胰岛素刺激的葡萄糖转运和胰岛素诱导的葡萄糖转运蛋白易位之间的差异强烈表明，一个仍然未知的蛋白质合成依赖性机制参与胰岛素激活葡萄糖转运。
This study examines the relationship between insulin-stimulated glucose transport and insulin-induced translocation of glucose trasnporters in isolated rat adipocytes. Adipose cells were incubated with or without cycloheximide, a potent inhibitor of protein synthesis, for 60 min and then for an additional 30 min with or without insulin. After the incubation we measured 3-O-methylglucose transport in the adipose cells, and subcellular membrane fractions were prepared. The numbers of glucose transporters in the various membrane fractions were determined by the cytochalasin B binding assay. Basal and insulin-stimulated 3-O-methylglucose uptakes were not affected by cycloheximide. Furthermore, cycloheximide affected neither Vmax nor Km of insulin-stimulated 3-O-methylglucose transport. In constrast, the number of glucose transporters in plasma membranes derived from cells preincubated with cycloheximide and insulin was markedly decreased compared with those from cells incubated with insulin alone (10.5 .+-. 0.8 and 22.2 .+-. 1.8 pmol/mg of protein respectively; P < 0.005). The number of glucose transporter in cells incubated with cycloheximide alone was not significantly different compared with control cells. SDS/polyacrylamide-gel-electrophoretic analysis of [3H]cytochalasin-B-photolabeled plasma-membrane fractions revealed that cycloheximide decreases the amount of labelled glucose transporters in insulin-stimulated membranes. However, the apparent molecular mass of the protein was not changed by cycloheximide treatment. The effect of cycloheximide on the two-dimensional electrophoretic profile of the glucose transporter in insulin-stimulated low-density microsomal membranes revealed a decrease in the pI-6.4 glucose-transporter isoform, whereas the insulin-translocatable isoform (pI 5.6) was decreased. Thus the observed discrepancy between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters strongly suggests that a still unknown protein-synthesis-dependent mechanism is involved in insulin activation of glucose transport.