PI 3 KINASE ISOFORMS AND INSULIN ACTION

PI 3 激酶异构体和胰岛素作用

• 批准号: 6635142
• 负责人: C RONALD KAHN
• 金额: $ 49.31万
• 依托单位: JOSLIN DIABETES CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6635142
• 关键词: 3T3 cells; CHO cells; adipocytes; cell line; diabetes mellitus; disease /disorder model; enzyme reconstitution; gene targeting; genetically modified animals; hormone regulation /control mechanism; insulin; insulin receptor; isozymes; laboratory mouse; laboratory rat; phosphatidylinositol 3 kinase; receptor coupling; transfection /expression vector; yeast two hybrid system

A central pathway in this insulin stimulated network is the enzyme PI 3-kinase. Inhibition of this enzyme results in a blockade of almost all of insulin's metabolic actions, as well as its effects on cell growth and differentiation. Classically, PI 3-kinase is a heterodimer consisting of a regulatory subunit of 85 kDa (p85alpha) and a catalytic subunit of 110 kDa (p110alpha). Our work and that of others, however, has demonstrated that there are actually as many as seven possible regulatory subunits of PI 3-kinase, six of which are the result of three different alternative splicing events of the p85 gene. Both PI 3-kinase activity and the alternative splicing are regulated in animal models of diabetes. The major goal of this grant is to dissect the roles of the various alternatively spliced forms of PI 3-kinase in insulin action, to explore how these components interact with each other and with other signaling molecules in the different compartments within the cell and assess their alterations in pathophysiologic states. To achieve this we will: (1) Study the similarities, differences and potentially complementary roles of various isoforms of PI 3-kinase in coupling the insulin receptor to down-stream effector systems in insulin action in vivo by creation and characterization of mice in which specific isoforms of PI 3-kinase have been knocked out using both global and conditional/tissue specific strategies of gene targeting. (2) Determine the actions of different isoforms of PI 3-kinase in differential insulin signaling by transfection of standard cell lines such as NIH 3T3 and CHO cells, more insulin responsive cells such as 3T3-L1 adipocytes and L6 myotubes, as well as cells lacking specific isoforms of PI 3-kinase derived from knockout animal models. These cells will be studied before and after reconstitution with different isoforms of PI 3-kinase, and in the case of the mouse embryo fibroblast, before and after conversion to adipocyte-like cell lines with PPARgamma2. These studies will make use of both retroviral and adenoviral vectors. (3) Determine the subcellular compartmentalization, differential partnering and differences in regulation of the various PI 3-kinase isoforms in cells in culture and tissues from intact animals, including identification and characterization of potential interacting molecules of the alternatively splice forms of PI 3-kinase using the yeast 2-hybrid system. (4) Study of the effects of hormonal treatment and disease states on differential regulation of PI 3-kinase isoforms and characterize two recently cloned phosphatidylinositol phosphate 5-phosphatases and cytoplasmic extracts of diabetic animals as possible negative regulators of the PI 3-kinase system. Together these studies should provide important insights into the role of PI 3-kinase isoforms in insulin action and diabetes.

该胰岛素刺激网络中的中心途径是PI 3-激酶。 对这种酶的抑制会导致几乎所有胰岛素的代谢作用及其对细胞生长和分化的影响。 通常，PI 3-激酶是由85 kDa（P85Alpha）的调节亚基和110 kDa（P110Alpha）的催化亚基组成的异二聚体。然而，我们的工作和其他工作表明，实际上有多达七个可能的PI 3-激酶的调节亚基，其中六个是p85基因的三个不同替代剪接事件的结果。 PI 3-激酶活性和替代剪接都受到糖尿病动物模型的调节。 该赠款的主要目的是剖析PI 3-激酶在胰岛素作用中的各种剪接形式的作用，以探索这些成分如何相互相互作用，并与细胞内不同隔室中其他信号分子相互作用并评估其病理生理状态的改变。 为此，我们将：（1）研究PI 3-激酶各种同工型在将胰岛素受体耦合到体内胰岛素作用中胰岛素作用中的相似性，差异和潜在的互补作用，这是通过创建和对PI 3-激酶的特定策略的特定策略来构建和表征的体内胰岛素作用的。 （2）通过转染标准细胞系（如NIH 3T3和CHO细胞），胰岛素反应性细胞，例如3T3-L1脂肪细胞和L6肌管等标准细胞系，确定PI 3-激酶在差异胰岛素信号传导中的不同同工型的作用，以及缺乏来自PI 3-KINSTIME ocking Ountsove ocking Ountsody Ountsod Onskit Outsive offience ockity Ountsod Outtine Ounts Outsive firctive offer Onspient Onskit Outsive offience。 这些细胞将在重建前后使用不同的PI 3-激酶的同工型进行研究，在小鼠胚胎成纤维细胞的情况下，转化为带有PPARGAMMA2的脂肪细胞样细胞系之前和之后。 这些研究将利用逆转录病毒和腺病毒载体。 （3）确定来自完整动物的细胞和组织中各种PI 3-激酶同工型调节的亚细胞隔室化，差异伴侣的差异和差异，包括使用酵母2-乳腺2-乳腺2-乳化体系统的PI 3-基因酶替代拼接形式的潜在相互作用分子的鉴定和表征。 （4）研究激素治疗和疾病状态对PI 3-激酶同工型差异调节的影响，并表征了两种最近克隆的磷脂酰肌醇5-磷酸酶和糖尿病动物的胞质提取物的影响，是PI 3-激酶系统的糖尿病动物的细胞质提取物。 这些研究共同提供了对PI 3-激酶同工型在胰岛素作用和糖尿病中的作用的重要见解。