ROLE AND FUNCTION OF INSULIN RECEPTOR SULFHYDRYLS

工作总结：在肥胖相关的胰岛素抵抗、慢性暴露中 葡萄糖浓度升高可改变硫醇氧化还原状态 通过一种涉及产生活性氧的机制 物种。胰岛素受体含有反应性巯基(S) 保留功能的受体β亚基的细胞质结构域 不清楚。该受体硫醇基团(S)反应性的变化 用硫醇生物素化试剂进行评估。将细胞培养在 在5.7 mM或25 mM葡萄糖存在不同的时间。另外， 过氧化氢单独或与钒酸盐一起作用的效果是 评估过了。胰岛素受体的免疫沉淀 链霉亲和素结合琼脂糖免疫印迹表明 高糖对受体硫醇反应性的时间依赖性降低。 钒酸盐本身并不改变IR硫醇的反应性(S)，但极大地改变了它的反应性 增强了过氧化氢的氧化作用。这些结果表明 细胞质区存在一个关键的硫醇基团(S)。 对葡萄糖和细胞内敏感的胰岛素受体 氧化剂。这是受体硫醇改变的第一个证据。 在涉及产生低水平的 活性氧物种，这表明这种变化可能 调节受体的功能和/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物 酶，包括环氧合酶、细胞色素P-450、一氧化氮 正在使用合成酶、NADPH氧化酶、黄嘌呤氧化酶和脂氧合酶 为了评估这些酶在葡萄糖介导的体内还原中的作用 受体硫醇反应性和信号转导。正在取得进展 在鉴定反应性受体硫醇基团方面(S)。

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).