Factors that modify insulin action

描述（由申请人提供）：“葡萄糖毒性”导致不受控制的1型糖尿病的胰岛素抵抗，并促成2型糖尿病的胰岛素抵抗。在3 T3-L1脂肪细胞中，在高糖+0.6 nM胰岛素中预孵育协同下调胰岛素对葡萄糖转运的刺激和Akt/PKB活化，而Akt/PKB活化远离磷脂酰肌醇-3-激酶（PI（3）K）活化。己糖胺合成途径与胰岛素抵抗有关;其主要产物是O-GlcNAc-转移酶（OGT）的底物UDP-N-乙酰葡糖胺（UDP-GlcNAc）。OGT催化将单个O-GlcNAc添加到特定的Ser/Thr残基上。O-GlcNAc酰化和O-磷酸化通常是相互的。体内慢性葡萄糖流量增加可增强细胞和肌肉中的蛋白质O-GlcNAc化。将在3 T3-L1脂肪细胞、L-6肌管和胰岛素抵抗小鼠模型的骨骼肌中研究增强的O-GlcNAc化在胰岛素抵抗中的作用。葡萄糖/胰岛素响应性O-GlcNAc酰化蛋白将通过质谱结合免疫学方法进行鉴定。特别感兴趣的是参与GLUT 4运输的蛋白质。将鉴定O-GlcNAc酰化位点，并评价修饰的功能意义。将研究O-GlcNAc-ase过表达的影响，以检测预防过度O-GlcNAc酰化是否减轻或预防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，包括葡萄糖转运。将研究葡萄糖诱导的胰岛素抵抗中Akt活化受损的机制。如果质膜（PM）上胰岛素刺激的3-磷酸肌醇减少，它们的去磷酸化可能会加速，例如通过SHIP 2或PTEN，或PI（3）K可能会被错误启动。如果胰岛素正常刺激胰岛素抵抗细胞中PM处的3-磷酸肌醇，则磷酸肌醇依赖性激酶-1（PDK-1）的活性或对Akt本身的作用（例如，加速的去磷酸化或蛋白质相互作用）。将评估O-GlcNAc酰化改变在已识别缺陷中的可能作用。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.