ROLE AND FUNCTION OF INSULIN RECEPTOR SULFHYDRYLS

工作总结：在肥胖相关的胰岛素抵抗中， 葡萄糖浓度升高可以改变巯基氧化还原状态， 通过一种机制，包括产生活性氧， 物种 胰岛素受体中含有反应性巯基， 受体β亚单位的胞质结构域，其功能保留 不清楚这种受体巯基的反应性变化已经被证实是 用硫醇-生物素化剂评价。 将细胞孵育在 在5.7mM或25 mM葡萄糖的存在下持续不同量的时间。 还有， 过氧化氢单独或与钒酸盐一起的作用是 评估。 胰岛素受体的免疫沉淀， 用链霉亲和素结合的琼脂糖进行免疫印迹表明， 高葡萄糖导致的受体巯基反应性的时间依赖性降低。 单独的钒酸盐不改变IR硫醇的反应性，但显著地改变IR硫醇的反应性。 增强过氧化氢的氧化作用。这些结果表明 在细胞质结构域中有一个关键的巯基， 胰岛素受体，对葡萄糖敏感， 氧化剂 这是第一个改变受体巯基的证据 在涉及产生低水平的 活性氧，这表明这种变化可能 调节受体功能和/或其与其他受体的相互作用模式 细胞成分 几种氧化剂生成的特异性抑制剂 酶，包括环氧合酶、细胞色素P-450、一氧化氮 合成酶、NADPH氧化酶、黄嘌呤氧化酶和脂氧合酶正在被使用 为了评估这些酶在葡萄糖介导的减少中的功能， 受体巯基反应性和信号转导。 正在取得进展 在反应性受体巯基的鉴定中。

Summary of work: In obesity-related insulin resistance, chronic exposure to elevated concentrations of glucose can alter the thiol redox state of the cells by a mechanism that involves generation of reactive oxygen species. The insulin receptor contains reactive sulfhydryl group(s) in the cytoplasmic domain of the receptor beta-subunit whose function remain unclear. Changes in reactivity of this receptor thiol group(s) has been evaluated with a thiol-biotinylating agent. Cells were incubated in the presence of 5.7 mM or 25 mM glucose for varying amounts of time. Also, the effect of hydrogen peroxide alone or together with vanadate was assessed. Immunoprecipitation of the insulin receptor followed by immunoblotting with streptavidin-bound agarose has indicated a time-dependent reduction in receptor thiol reactivity by high glucose. Vanadate alone did not alter the reactivity of IR thiol(s) but greatly enhanced the oxidative effect of hydrogen peroxide. These results indicate that there is a critical thiol group(s) in the cytoplasmic domain of the insulin receptor that is susceptible to glucose and intracellular oxidants. This is the first evidence for alteration of receptor thiol reactivity under conditions that involve generation of low levels of reactive oxygen species, which suggests that this alteration might regulate the receptor function and/or its mode of interaction with other cellular constituents. Specific inhibitors of several oxidant-generating enzymes, including cyclo-oxygenase, cytochrome P-450, nitric oxide synthase, NADPH oxidase, xanthine oxidase, and lipoxygenase are being used to assess the function of these enzymes in glucose-mediated reduction in receptor thiol reactivity and signal transduction. Progress is being made in the identification of the reactive receptor thiol group(s).