EVIDENCE FOR INVOLVEMENT OF SULFHYDRYL OXIDATION IN REGULATION OF FAT-CELL HEXOSE TRANSPORT BY INSULIN
EVIDENCE FOR INVOLVEMENT OF SULFHYDRYL OXIDATION IN REGULATION OF FAT-CELL HEXOSE TRANSPORT BY INSULIN
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DOI：
10.1073/pnas.71.10.4173
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发表时间：
1974-01-01
影响因子：
11.1
通讯作者：
LYNN, WS
中科院分区：
文献类型：
--
作者：
CZECH, MP;LAWRENCE, JC;LYNN, WS
以前的研究表明，氧化剂Cu++，H2 O2，和二酰胺模拟胰岛素对3-O-甲基葡萄糖转运在分离的脂肪细胞的刺激作用。本实验旨在确定巯基氧化是否在葡萄糖转运系统的激活中起关键作用。结果发现，还原剂，如二硫苏糖醇抑制3-O-甲基葡萄糖的运输速率，这种影响是可逆的，当细胞被清洗的还原剂。用1 mM-乙基马来酰亚胺处理细胞5分钟完全阻断胰岛素和氧化剂对己糖转运的作用，而不影响对照转运系统的活性。在这些条件下，125 I标记的胰岛素与脂肪细胞表面受体的结合仅被抑制约50%。在加入N-乙基马来酰亚胺之前，先在脂肪细胞中加入胰岛素或氧化剂10分钟，可完全阻断N-乙基马来酰亚胺对活化转运系统的抑制作用。这种对转运速率的保护作用似乎存在于胰岛素-受体相互作用后被胰岛素改变的部位，因为先前用胰岛素处理脂肪细胞并不能阻止N-乙基马来酰亚胺对胰岛素受体的部分抑制作用。此外，在与胰岛素孵育后用N-乙基马来酰亚胺处理细胞，当细胞被洗涤而不含激素或胰岛素与其受体的结合被胰蛋白酶消化破坏时，可防止升高的转运速率恢复到对照水平。然而，在这些细胞处理N-乙基马来酰亚胺的运输率仍然敏感的细胞松弛素B，根皮苷，还原剂。这些数据表明，在分离的脂肪细胞中的葡萄糖转运系统的一个组件必须保持在其二硫化物状态的表达的运输活动。此外，结果与胰岛素与细胞表面受体的结合触发该组分中的巯基氧化的概念一致，这阻止了其与N-乙基马来酰亚胺的反应。
Previous studies have shown that the oxidants Cu++, H2O2, and diamide mimic the stimulatory effect of insulin on 3-O-methylglucose transport in isolated fat cells. The present experiments were designed to determine whether sulfhydryl oxidation plays a key role in the activation of the glucose transport system. It was found that reductants such as dithiothreitol inhibited 3-O-methylglucose transport rates and that this effect was reversible when cells were washed free of reducing agent. Treatment of cells with 1 mMN-ethylmalcimide for 5 min completely blocked the actions of insulin and oxidants on hexose transport without affecting control transport system activity. Under these conditions, binding of125I-labeled insulin to fat cell surface receptors was inhibited by only about 50%. Addition of insulin or oxidants to fat cells for 10 min before addition ofN-ethylmaleimide completely prevented the inhibitory effect ofN-ethylmaleimide on the activated transport system. This protective effect on transport rates appears to reside at a site that is altered by insulin subsequent to hormone-receptor interaction, since prior treatment of fat cells with insulin did not prevent the partial inhibitory effect ofN-ethylmaleimide on insulin receptors. Furthermore, treatment of cells withN-ethylmaleimide after incubation with insulin prevented the elevated transport rates from returning to control levels when either the cells were washed free of hormone or insulin binding to its receptors was disrupted by trypsin digestion. However, transport rates in these cells treated withN-ethylmaleimide remained sensitive to cytochalasin B, phlorizin, and reductants. These data suggest that a component of the glucose transport system in isolated fat cells must be maintained in its disulfide state for expression of transport activity. Further, the results are consistent with the concept that the binding of insulin to cell surface receptors triggers sulfhydryl oxidation in this component, which prevents its reaction withN-ethylmaleimide.