Factors that modify insulin action

描述（由申请人提供）：“葡萄糖毒性”导致未控制的1型糖尿病患者的胰岛素抵抗，并导致2型糖尿病患者的胰岛素抵抗。血管并发症是糖尿病患者发病和死亡的主要原因。细胞持续暴露于高葡萄糖增加了通过己糖胺合成途径的通量，增强了O-GlcNAc转移酶（OGT）底物UDP-N-乙酰葡糖胺（UDP-GlcNAc）的产生。OGT催化O-GlcNAc与特定Ser/Thr残基的可逆单加成。O-GlcNAc酰化和O-磷酸化通常是相互的。我们最近首次确定了四个 IRS-1（以及11个新的Ser/Thr磷酸化位点）上的O-GlcNAc化。初步数据表明，O-GlcNAc可能影响IRS-1信号转导。我们在Spec. Aim 1中的主要目标是确定O-GlcNAc修饰是否改变IRS-1信号传导。在体外研究中，Ser将在四个O-GlcNAc位点突变为Ala，单独和组合，野生型和突变的IRS-1将通过腺病毒在HepG 2细胞和完整肝脏中表达，并且研究了它们响应胰岛素或IGF-1与IRS-1结合配偶体的相互作用。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 IRS-1表达腺病毒。将研究这些操作对葡萄糖和胰岛素耐量试验以及肝促炎酶表达的影响。在Spec Aim 2中，将在3 T3-L1脂肪细胞中葡萄糖转运和Akt活化的高葡萄糖/低剂量胰岛素诱导的胰岛素抵抗模型中继续进行研究。胰岛素信号PI（3）K在很大程度上得到维持，但Akt活化明显受损。最近的数据表明，PTEN蛋白表达增加，胰岛素刺激的PtdIns（3，4，5）P3减少。雷帕霉素抑制PTEN表达的刺激。mTORC-1活化明显起作用，cPKC可能起作用，但JNK不起作用。将研究增强的PTEN表达、mTORC-1作用和cPKC的作用的机制。几种可能与mTOR协同作用或由mTOR介导的机制将得到解决，包括肌动蛋白动力学的失调和磷蛋白磷酸酶的可能激活。在该模型中的操作方式的分析将与通过将细胞暴露于FFA引起的葡萄糖转运的胰岛素抵抗进行对比。从这个模型中获得的见解将被应用到L-6肌管和完整的大鼠胰岛素抵抗的慢性高血糖症。了解不同的过量营养素如何改变胰岛素的信号传导可能会导致新的治疗靶点的合理开发。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;其患病率正在以流行病的比例增加，这与肥胖症患病率的爆炸性增加相平行。长期提供过量的营养、葡萄糖或脂肪会改变细胞代谢和胰岛素信号传导，并促进糖尿病的血管并发症，这是发病率和死亡率的主要原因。更好地理解不同的营养物质，当长期过量提供时，如何改变细胞对胰岛素的反应，将导致开发新的治疗靶点来预防或减轻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.