Czech M P, Lawrence J C, Lynn W S
Proc Natl Acad Sci U S A. 1974 Oct;71(10):4173-7. doi: 10.1073/pnas.71.10.4173.
Previous studies have shown that the oxidants Cu(++), H(2)O(2), 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 mM N-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 of (125)I-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 of N-ethylmaleimide completely prevented the inhibitory effect of N-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 of N-ethylmaleimide on insulin receptors. Furthermore, treatment of cells with N-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 with N-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 with N-ethylmaleimide.
先前的研究表明，氧化剂铜离子（Cu(++)）、过氧化氢（H₂O₂）和二酰胺可模拟胰岛素对分离脂肪细胞中3 - O - 甲基葡萄糖转运的刺激作用。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。研究发现，诸如二硫苏糖醇等还原剂会抑制3 - O - 甲基葡萄糖的转运速率，并且当细胞用还原剂冲洗干净后，这种作用是可逆的。用1 mM N - 乙基马来酰亚胺处理细胞5分钟，可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，¹²⁵I标记的胰岛素与脂肪细胞表面受体的结合仅被抑制约50%。在添加N - 乙基马来酰亚胺之前，先将胰岛素或氧化剂添加到脂肪细胞中10分钟，可完全防止N - 乙基马来酰亚胺对激活的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激素 - 受体相互作用后被胰岛素改变的位点，因为预先用胰岛素处理脂肪细胞并不能防止N - 乙基马来酰亚胺对胰岛素受体的部分抑制作用。此外，在用胰岛素孵育细胞后，用N - 乙基马来酰亚胺处理细胞，当细胞用激素冲洗干净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，用N - 乙基马来酰亚胺处理的这些细胞中的转运速率对细胞松弛素B、根皮苷和还原剂仍敏感。这些数据表明，分离脂肪细胞中葡萄糖转运系统的一个组分必须保持其二硫键状态才能表达转运活性。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这一概念一致，这可防止其与N - 乙基马来酰亚胺反应。
