Factors that modify insulin action

描述(申请人提供)：“葡萄糖毒性”是1型糖尿病患者胰岛素抵抗的原因，也是2型糖尿病患者胰岛素抵抗的原因。糖尿病血管并发症是糖尿病患者发病和死亡的主要原因。细胞持续暴露在高糖环境中，通过己糖胺合成途径增加通量，促进O-GlcNAc转移酶(OGT)底物UDP-N-乙酰氨基葡萄糖(UDP-GlcNAc)的产生。OGT催化O-GlcNAc与特定的Ser/Thr残基的可逆单加成反应。O-GlcN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地点 IRS-1上的O-GlcN酰化(以及11个新的Ser/Thr磷酸化位点)的质谱分析。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将在四个O-GlcNAc位点突变为Ala，野生型和突变的IRS-1将通过腺病毒在HepG2细胞和完整肝脏中表达，并研究它们与IRS-1结合伙伴对胰岛素或IGF-1的反应。在体内研究中，内源性小鼠IRS-1将被shRNA腺病毒敲除，并被野生型或突变体取代 表达IRS-1的腺病毒。这些操作对葡萄糖和胰岛素耐量试验以及肝脏糖异生酶的表达的影响将被研究。在Spec Aim中，将继续在高糖/低剂量胰岛素诱导的3T3-L1脂肪细胞葡萄糖转运和Akt激活的胰岛素抵抗模型中进行研究。胰岛素对PI(3)K的信号在很大程度上保持不变，但Akt的激活明显受损。最近的研究表明，PTEN蛋白表达增加，胰岛素刺激的PtdIns(3，4，5)P3减少。雷帕霉素可抑制PTEN的表达。MTORC-1的激活明显起作用，cPKC可能起作用，但JNK不起作用。我们将探讨PTEN表达增强的机制、mTORC-1的作用以及cPKC的作用。几个可能与mTOR协同或由mTOR介导的机制将被解决，包括肌动蛋白动力学的失调和可能的磷酸化蛋白磷酸酶的激活(S)。在这个模型中，对工作方式的分析将与将细胞暴露于FFA而引起的葡萄糖转运的胰岛素抵抗进行对比。从该模型中获得的见解将应用于L-6肌管和因慢性高血糖而产生胰岛素抵抗的完整大鼠。了解不同的过量营养素是如何改变胰岛素信号的，可能会导致合理开发新的治疗靶点。公共卫生相关性：糖尿病是美国发病率和死亡率的主要原因；其流行率正在以流行的比例增加，这与肥胖症流行率的爆炸性增长相平行。长期供应过量的营养、葡萄糖或脂肪会改变细胞代谢和胰岛素信号，并促进糖尿病的血管并发症，糖尿病是发病率和死亡率的主要原因。更好地理解长期过量提供不同的营养物质如何改变细胞对胰岛素的反应，将导致开发新的治疗目标来预防或减轻2型糖尿病。

DESCRIPTION (provided by applicant): "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. O-GlcNAcylation 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 HepG2 cells and in intact liver via adenovirus, 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. Recent data indicate that PTEN protein expression is increased and insulin stimulated PtdIns(3,4,5)P3 is diminished. Stimulation of PTEN expression is inhibited by rapamycin. mTORC-1 activation clearly plays a role, cPKC may contribute, but JNK does not. The mechanism of enhanced PTEN expression, mTORC-1 action and the role of cPKC will be investigated. Several mechanisms which may synergize with or be mediated by mTOR will be addressed, including dysregulation of actin dynamics and possible activation of a phosphoprotein phosphatase(s). The analysis of the modus operandi in this model will be contrasted with the insulin resistance of glucose transport elicited by exposing cells to FFA. Insights gained from this model will be applied to L-6 myotubes and to intact rats made insulin resistant by chronic hyperglycemia. Understanding how different excess nutrients modify insulin's signaling may lead to the rational development of novel therapeutic targets. PUBLIC HEALTH RELEVANCE: Diabetes mellitus is a major cause of morbidity and mortality in the US; its prevalence is increasing in epidemic proportions, which parallels the explosive increase in the prevalence of obesity. Chronic provision of excess nutrients, glucose or fat alters cell metabolism and insulin signaling and promotes the vascular complications of diabetes, the major causes of morbidity and mortality. Improved understanding of how different nutrients, when provided chronically in excess, alter the cell's response to insulin, will lead to the development of novel therapeutic targets to prevent or mitigate type 2 diabetes.