Hayes G R, Lockwood D H
Department of Medicine, University of Rochester School of Medicine and Dentistry, NY 14642.
Proc Natl Acad Sci U S A. 1987 Nov;84(22):8115-9. doi: 10.1073/pnas.84.22.8115.
The oxidant H2O2 has many insulin-like effects in rat adipocytes. To determine whether these effects could be mediated by the tyrosine kinase activity of the insulin receptor, the ability of H2O2 to stimulate receptor phosphorylation in intact adipocytes and partially purified insulin receptors has been examined. Phosphorylation of the beta subunit of the insulin receptor was increased approximately 2-fold by treatment of intact cells with 3 mM H2O2, a concentration that maximally stimulates 2-deoxyglucose uptake. Stimulation of receptor phosphorylation was rapid, reaching maximal levels within 5 min, and preceded activation of glucose transport. Phosphoamino acid analysis of insulin receptors from H2O2-treated adipocytes showed that 32P incorporation into phosphotyrosine and phosphoserine residues of the beta subunit was enhanced. Furthermore, partially purified receptors from H2O2-treated cells exhibit increased tyrosine kinase activity, as measured by phosphorylation of the peptide Glu80Tyr20. In contrast, the direct addition of H2O2 to partially purified insulin receptors did not stimulate tyrosine kinase activity or insulin receptor autophosphorylation. This was not due to breakdown of H2O2 or oxidation of ATP or the required divalent cations. To define the factors involved in H2O2's effect, we have examined receptor phosphorylation in fat cell homogenates and purified plasma membranes. Although insulin stimulated receptor phosphorylation in both of these systems, H2O2 was only effective in the cell homogenates. These data demonstrate that, under certain conditions, H2O2 stimulates insulin receptor phosphorylation and tyrosine kinase activity, suggesting that the insulin-like effects of H2O2 may be mediated by stimulation of insulin receptor phosphorylation. This does not appear to be a direct effect of H2O2 on the insulin receptor and requires nonplasma membrane cellular constituents.
氧化剂过氧化氢在大鼠脂肪细胞中具有许多类似胰岛素的作用。为了确定这些作用是否可由胰岛素受体的酪氨酸激酶活性介导，研究了过氧化氢刺激完整脂肪细胞和部分纯化的胰岛素受体中受体磷酸化的能力。用3 mM过氧化氢处理完整细胞可使胰岛素受体β亚基的磷酸化增加约2倍，该浓度能最大程度地刺激2-脱氧葡萄糖摄取。受体磷酸化的刺激迅速，在5分钟内达到最高水平，并先于葡萄糖转运的激活。对用过氧化氢处理的脂肪细胞中的胰岛素受体进行磷酸氨基酸分析表明，β亚基的磷酸酪氨酸和磷酸丝氨酸残基中32P的掺入增加。此外，用过氧化氢处理的细胞中部分纯化的受体表现出酪氨酸激酶活性增加，这通过肽Glu80Tyr20的磷酸化来测定。相比之下，将过氧化氢直接添加到部分纯化的胰岛素受体中不会刺激酪氨酸激酶活性或胰岛素受体自身磷酸化。这不是由于过氧化氢的分解、ATP或所需二价阳离子的氧化所致。为了确定参与过氧化氢作用的因素，我们研究了脂肪细胞匀浆和纯化质膜中的受体磷酸化。尽管胰岛素在这两个系统中均刺激受体磷酸化，但过氧化氢仅在细胞匀浆中有效。这些数据表明，在某些条件下，过氧化氢刺激胰岛素受体磷酸化和酪氨酸激酶活性，提示过氧化氢的胰岛素样作用可能由胰岛素受体磷酸化的刺激介导。这似乎不是过氧化氢对胰岛素受体的直接作用，并且需要非质膜细胞成分。
