CHROMIUM ENHANCEMENT OF INSULIN SIGNALING

说明(申请人提供)：铬是一种微量营养素，可增强胰岛素的作用，是40多年前发现的“葡萄糖耐量因子”的基本成分。铬可能对美国1500万2型糖尿病患者有益，许多美国人已经单独或在复合维生素配方中将铬(III)作为日常膳食补充剂。然而，关于铬(III)的生物学，包括吸收、生物有效性、作用靶标，甚至是金属离子本身或某些有机金属络合物是否是生物活性物种，目前还缺乏信息和持续的争议。本项目的目标是明确铬增强胰岛素作用的生化基础。在初步研究中，各种有机和无机形式的铬(III)被发现在增强胰岛素触发的初始信号事件方面同样有效。在培养的完整活细胞中添加纳摩尔浓度的铬，可以在低于最佳剂量的胰岛素下增加胰岛素刺激的Tyr磷酸化。这种作用归因于胰岛素受体(IR)去磷酸化受损，通过向纯化的膜中添加铬来检测。该项目将扩展这些研究，以检验铬(III)抑制蛋白质酪氨酸磷酸酶(PTP)对激活的IR的去磷酸化的假设。这可以通过铬(III)与PTP的相互作用而发生，通过与活性部位半胱氨酸的相互作用来阻断酶的活性，或者通过与底物Tyr磷酸化IR的相互作用来阻止酶的去磷酸化。实验将使用去磷酸化试验来鉴定目标蛋白(PTP或IR)。对反应产物的分析将显示铬是否对IR中不同的Tyr磷酸化位点具有选择性，这将增强特定的下游信号通路。与铬相互作用的结构决定因素将通过突变和与重组目标蛋白的铬(III)结合试验来确定。这一结果将为了解铬(III)的分子作用提供新的知识，为了解膳食铬的生物学效应提供基础，并为防治2型糖尿病的干预打开新的机会。

DESCRIPTION (provided by applicant): Chromium is a micronutrient that potentiates the action of insulin and is an essential component of "glucose tolerance factor" discovered over 40 years ago. Chromium could have beneficial effects for the >15 million type-2 diabetics in the USA, and many Americans already take Cr (III) as a daily dietary supplement, alone or in multivitamin formulations. Nonetheless, there is scant information and continuing controversy regarding the biology of Cr (III), including uptake, bioavailability, target of action and even whether the metal ion itself or some organo-metallic complex is the bioactive species. The goal of this project is to define the biochemical basis for chromium enhancement of insulin action. In preliminary studies various organic and inorganic forms of Cr (III) were found to be equally efficacious in potentiating initial signaling events triggered by insulin. Chromium added at nanomolar concentrations to intact living cells in culture increased insulin-stimulated Tyr phosphorylation at sub-optimal doses of insulin. The effect was attributed to impaired insulin receptor (IR) dephosphorylation, assayed by addition of chromium to purified membranes. This project will extend these studies to test the hypothesis that Cr (III) inhibits the dephosphorylation of the activated IR by protein Tyr phosphatases (PTP). This could occur by interaction of the Cr (III) either with PTP, blocking the enzyme activity by interacting with an active site cysteine, or with the substrate, the Tyr phosphorylated IR, protecting it from dephosphorylation. Experiments will use dephosphorylation assays to identify the target protein (PTP or IR). Analysis of reaction products will show whether chromium action is selective for different Tyr phosphorylation sites in the IR, which would enhance specific downstream signaling pathways. Structural determinants for interaction with chromium will be determined by mutagenesis and Cr(III) binding assays with recombinant target protein. The results will provide new knowledge of the molecular actions of Cr (III), provide a basis for understanding the biological effects of dietary chromium and open new opportunities for interventions to combat type-2 diabetes.