Factors that modify insulin action

描述（由申请人提供）：“葡萄糖毒性”可解释未控制的1型糖尿病患者的胰岛素抵抗，并有助于2型糖尿病患者的胰岛素抵抗。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葡萄糖转运和Akt/PKB激活远端磷脂酰肌醇-3-激酶（PI(3)K）激活。己糖胺合成途径与胰岛素抵抗有关；其主要产物是o - gicnac转移酶（OGT）的底物udp - n -乙酰氨基葡萄糖（UDP-GIcNAc）。OGT催化单个O-GIcNAc加成到特定的丝氨酸/苏氨酸残基上。o -氨基酰化和o -磷酸化通常是相互作用的。体内慢性葡萄糖通量增加可增强细胞和肌肉中的蛋白o - gicnac酰化。我们将在胰岛素抵抗小鼠模型的3T3-L1脂肪细胞、L-6肌管和骨骼肌中研究o - gicnac酰化增强在胰岛素抵抗中的作用。葡萄糖/胰岛素应答的o - gic酰化蛋白将通过质谱结合免疫学方法进行鉴定。特别令人感兴趣的是参与GLUT4运输的蛋白质。将鉴定o - gicnac酰化位点，并评估该修饰的功能意义。我们将研究o - gicnac酶过表达的影响，以检验防止过度的o - gicnac化是否能减轻或预防葡萄糖诱导的胰岛素抵抗。Akt是PI(3)K参与胰岛素代谢作用的下游靶点，包括葡萄糖转运。Akt激活受损在葡萄糖诱导的胰岛素抵抗中的机制将被研究。如果胰岛素刺激的质膜（PM）上的3-磷酸肌苷减少，它们的去磷酸化可能会加速，例如通过SHIP2或PTEN，或者PI(3)K可能被错误靶向。如果胰岛素正常刺激胰岛素抵抗细胞中PM处的3-磷酸肌苷，则将检查磷酸肌苷依赖性激酶-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）。将评估改变的o - gicnac酰化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发展。

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.