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。在3T3-L1脂肪细胞中，高糖+0.6 nM胰岛素预孵育协同下调胰岛素刺激的葡萄糖转运和磷脂酰肌醇-3-激酶(PI(3)K)末端Akt/PKB的激活。氨基己糖合成途径与胰岛素抵抗有关，其主要产物是O-GIcNAc-转移酶(OGT)底物UDP-N-乙酰氨基葡萄糖(UDP-GIcNAc)。OGT催化单个O-GIcNAc加成到特定的Ser/Thr残基上。O-GIcN酰化和O-磷酸化通常是相互作用的。在体内，慢性增加的葡萄糖通量增强了细胞和肌肉中的蛋白质O-GIcN酰化。增强的O-GIcN酰化在胰岛素抵抗中的作用将在3T3-L1脂肪细胞、L-6肌管和胰岛素抵抗小鼠模型的骨骼肌中进行研究。葡萄糖/胰岛素响应性O-GIcN酰化蛋白将通过质谱学和免疫学方法相结合进行鉴定。特别令人感兴趣的是参与GLUT4运输的蛋白质。将确定O-GIcN酰化位点(S)，并评估该修饰的功能意义。将研究O-GIcNAc-ase过度表达的影响，以测试防止过度的O-GIcN酰化是否减轻或防止葡萄糖诱导的胰岛素抵抗。AKT是PI(3)K的下游靶点，参与胰岛素的代谢效应，包括葡萄糖运输。在葡萄糖诱导的胰岛素抵抗中Akt活性受损的机制将被研究。如果质膜上胰岛素刺激的3-磷酸肌醇减少，它们的去磷酸化可能被SHIP2或PTEN加速，或者PI(3)K被错误定位。如果胰岛素正常刺激胰岛素抵抗细胞质膜上的3-磷酸肌醇，则将检测磷酸肌醇依赖激酶-1(PDK-1)的活性或对Akt本身的影响(例如，加速去磷酸化或蛋白质相互作用)。将评估O-GIcN酰化改变在已识别缺陷中的可能作用(S)。明确胰岛素抵抗的机制可能会导致新的治疗靶点的开发。

DESCRIPTION (provided by applicant): "Glucose toxicity" accounts for insulin resistance in uncontrolled Type 1 diabetes and contributes to it in Type 2 diabetes. In 3T3-L1 adipocytes, preincubation in high glucose + 0.6 nM insulin synergistically down-regulate insulin stimulation of glucose transport and Akt/PKB activation distal to phosphatidyl inositol-3- kinase (PI(3)K) activation. The hexosamine synthesis pathway has been implicated in insulin resistance; its major product is UDP-N-acetylglucosamine (UDP-GIcNAc), the substrate of O-GIcNAc-transferase (OGT). OGT catalyzes the addition of single O-GIcNAc to specific Ser/Thr residues. O-GIcNAcylation and O-phosphorylation are often reciprocal. Chronic increased glucose flux enhances protein O-GIcNAcylation in cells and in muscle, in vivo. The role of enhanced O-GIcNAcylation in insulin resistance will be studied in 3T3-L1 adipocytes, L-6 myotubes and in skeletal muscle of insulin-resistant mouse models. Glucose/insulin-responsive O-GIcNAcylated proteins will be identified by mass spectrometry combined with immunological methods. Of special interest are proteins involved in GLUT4 trafficking. O-GIcNAcylation site(s) will be identified and the functional significance of the modification evaluated. The effect of O-GIcNAc-ase overexpression will be studied to test whether preventing excessive O-GIcNAcylation mitigates or prevents glucose-induced insulin resistance. Akt is a downstream target of PI(3)K involved in insulin's metabolic effects, including glucose transport. The mechanism of impaired Akt activation in glucose-induced insulin resistance will be studied. If insulin-stimulated 3-phosphoinositides at the plasma membrane (PM) are decreased, their dephosphorylation may be accelerated, e.g. by SHIP2 or PTEN, or PI(3)K may be mistargeted. If insulin normally stimulates 3-phosphoinositides at the PM in insulin-resistant cells, the activity of phosphoinositide dependent kinase-1 (PDK-1) or effects on Akt itself (e.g., accelerated dephosphorylation or protein interactions) will be examined. The possible role of altered O-GIcNAcylation in the identified defect(s) will be assessed. Defining mechanisms of insulin resistance may lead to the development of novel therapeutic targets.