PI 3 KINASE ISOFORMS AND INSULIN ACTION

PI 3 激酶异构体和胰岛素作用

• 批准号: 6177439
• 负责人: C RONALD KAHN
• 金额: $ 45.45万
• 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/6177439
• 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.

该胰岛素刺激网络的中心途径是 PI 3-激酶。 抑制这种酶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被阻断。 传统上，PI 3-激酶是一种异二聚体，由 85 kDa 的调节亚基 (p85alpha) 和 110 kDa 的催化亚基 (p110alpha) 组成。然而，我们和其他人的工作已经证明，PI 3-激酶实际上有多达七个可能的调节亚基，其中六个是 p85 基因的三个不同选择性剪接事件的结果。 PI 3 激酶活性和选择性剪接在糖尿病动物模型中均受到调节。 该资助的主要目标是剖析 PI 3 激酶的各种选择性剪接形式在胰岛素作用中的作用，探索这些成分如何相互作用以及如何与细胞内不同区室中的其他信号分子相互作用，并评估它们在病理生理状态中的变化。 为了实现这一目标，我们将：（1）通过使用全局和条件/组织特异性基因靶向策略敲除特定 PI 3-激酶同工型小鼠并对其进行表征，研究各种 PI 3-激酶同工型在体内胰岛素作用中将胰岛素受体与下游效应器系统偶联的相似性、差异和潜在互补作用。 (2) 通过转染标准细胞系（例如 NIH 3T3 和 CHO 细胞）、胰岛素敏感细胞（例如 3T3-L1 脂肪细胞和 L6 肌管）以及来自敲除动物模型的缺乏特定 PI 3-激酶同工型的细胞，确定不同 PI 3-激酶亚型在差异胰岛素信号传导中的作用。 这些细胞将在用不同的 PI 3-激酶亚型重建之前和之后进行研究，对于小鼠胚胎成纤维细胞来说，将在使用 PPARgamma2 转化为脂肪细胞样细胞系之前和之后进行研究。 这些研究将利用逆转录病毒和腺病毒载体。 (3) 确定完整动物培养细胞和组织中各种 PI 3-激酶亚型的亚细胞区室化、差异伙伴关系和调节差异，包括使用酵母 2-杂交系统鉴定和表征 PI 3-激酶选择性剪接形式的潜在相互作用分子。 (4) 研究激素治疗和疾病状态对 PI 3-激酶亚型差异调节的影响，并表征两种最近克隆的磷脂酰肌醇磷酸 5-磷酸酶和糖尿病动物的细胞质提取物作为 PI 3-激酶系统的可能负调节剂。 这些研究共同为 PI 3 激酶亚型在胰岛素作用和糖尿病中的作用提供了重要的见解。