CHROMIUM ENHANCEMENT OF INSULIN SIGNALING

描述（由申请人提供）：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”的重要组成部分。铬可能对美国1500万2型糖尿病患者有益，许多美国人已经将铬（III）作为每日膳食补充剂，单独服用或与多种维生素制剂一起服用。然而，关于Cr （III）的生物学，包括摄取、生物利用度、作用靶点，甚至是金属离子本身或某些有机金属配合物是否是生物活性物质，信息很少，争议仍在继续。该项目的目标是确定铬增强胰岛素作用的生化基础。在初步研究中，发现各种有机和无机形式的Cr （III）在胰岛素触发的初始信号事件中同样有效。在培养的完整活细胞中以纳摩尔浓度添加铬，在次优剂量的胰岛素下增加胰岛素刺激的Tyr磷酸化。该效应归因于胰岛素受体（IR）去磷酸化受损，通过在纯化膜中添加铬来测定。本项目将扩展这些研究，以验证Cr （III）抑制酪氨酸蛋白磷酸酶（PTP）对激活IR的去磷酸化的假设。这可能通过Cr （III）与PTP相互作用发生，通过与活性位点半胱氨酸相互作用阻断酶活性，或与底物（Tyr磷酸化IR，保护其免于去磷酸化）相互作用。实验将使用去磷酸化测定来确定目标蛋白（PTP或IR）。对反应产物的分析将显示铬的作用是否对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诱变和Cr（III）与重组靶蛋白的结合试验来确定。该结果将为Cr （III）的分子作用提供新的知识，为理解膳食铬的生物学效应提供基础，并为干预2型糖尿病提供新的机会。

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.