Factors that modify insulin action

摘要 葡萄糖毒性是未控制的1型糖尿病患者胰岛素抵抗的原因，并对 对2型糖尿病患者的影响。它导致血管并发症，是发病率和死亡率的主要原因。 在糖尿病患者中。细胞持续暴露在高糖环境中会增加通过氨基己糖合成的通量 途径，促进O-GlcNAc底物UDP-N-乙酰氨基葡萄糖(UDP-GlcNAc)的产生 转移酶(OGT)。OGT催化O-GlcNAc与特定的Ser/Thr残基的可逆单加成反应。OGlcN酰化 和O-磷酸化通常是相互作用的。我们最近首次确定了四个 IRS-1上O-GlcN酰化位点(以及11个新的丝氨酸/苏氨酸磷酸化位点)的质谱学研究。 初步数据提示O-GlcNAc可能影响IRS-1信号转导。我们在Spec的主要目标。 目的1是确定O-GlcNAc修饰是否改变了IRS-1信号。在体外研究中，Ser Will 在O-GlcNAc的四个位点上分别突变为Ala，野生型和突变的IRS-1将是 胰岛素和胰岛素样生长因子-1在Hek-293细胞中的表达及其与IRS-1结合伙伴的相互作用 学习。在体内研究中，内源性小鼠IRS-1将被shRNA腺病毒敲除并 用野生型或突变型表达IRS-1的腺病毒替代。这些操作的影响对 将研究葡萄糖和胰岛素耐量试验以及肝脏糖异生酶的表达。在……里面 SPEC Aim 2将在高糖/低剂量胰岛素诱导的胰岛素抵抗模型中继续进行研究 3T3-L1脂肪细胞葡萄糖转运与Akt激活胰岛素对PI(3)K的信号在很大程度上保持不变，但 AKT激活明显受损。初步数据显示，PTEN蛋白表达增加， 胰岛素刺激的PtdIns(3，4，5)P3减少。MTORC-1激活和cPKC起作用，而JNK不起作用。 将讨论几种可能与mTOR协同的机制，包括肌动蛋白的失调。 一种磷酸蛋白磷酸酶的动力学和可能的激活。作案手法分析 这一模型将与细胞暴露在葡萄糖转运过程中引起的胰岛素抵抗进行对比。 FFA。从这个模型中获得的见解随后将应用于L-6肌管和正常大鼠。理解 不同营养过剩如何修饰胰岛素S信号可能导致小说的理性发展 治疗靶点。

Abstract ¿Glucose toxicity¿ accounts for insulin resistance in patients with uncontrolled type 1 diabetes and contributes to it in type 2 diabetes. It contributes to the vascular complications, the major causes of morbidity and mortality in diabetic patients. Sustained exposure of cells to high glucose increases flux via the hexosamine synthesis pathway, enhancing the production of UDP-N-acetyl glucosamine (UDP-GlcNAc), the substrate of O-GlcNAc transferase (OGT). OGT catalyzes the reversible, single addition of O-GlcNAc to specific Ser/Thr residues. OGlcNAcylation and O-phosphorylation are often reciprocal. We have recently identified, for the first time, four sites of O-GlcNAcylation on IRS-1 (as well as 11 novel Ser/Thr phosphorylation sites) by mass spectrometry. Preliminary data suggest that O-GlcNAc may affect IRS-1 signal transduction. Our major objective in Spec. Aim 1 is to firmly establish whether the O-GlcNAc modification alters IRS-1 signaling. In in vitro studies Ser will be mutated to Ala at the four O-GlcNAc sites, singly and in combination, wild type and mutated IRS-1 will be expressed in Hek-293 cells and their interactions with IRS-1 binding partners in response to insulin or to IGF-1 studied. In in vivo studies endogenous mouse IRS-1 will be knocked out with shRNA adenovirus and substituted with wild type or mutant IRS-1-expressing adenovirus. The effect of these manipulations on glucose and insulin tolerance tests and the expression of hepatic gluconeogenic enzymes will be studied. In Spec Aim 2 studies will be continued in a model of high glucose/ low dose insulin-induced insulin resistance of glucose transport and Akt activation in 3T3-L1 adipocytes. Insulin signaling to PI(3)K is largely maintained, but Akt activation is markedly impaired. Preliminary data suggest that PTEN protein expression is increased and insulin stimulated PtdIns(3,4,5)P3 is diminished. mTORC-1 activation and cPKC play a role, but JNK does not. Several mechanisms which may synergize with mTOR will be addressed, including dysregulation of actin dynamics and possible activation of a phosphoprotein phosphatase. The analysis of the modus operandi in this model will be contrasted with the insulin resistance of glucose transport elicited by exposing the cells to FFA. Insights gained from this model will then be applied to L-6 myotubes and to intact rats. Understanding how different excess nutrients modify insulin¿s signalling may lead to the rational development of novel therapeutic targets.