PI 3 KINASE ISOFORMS AND INSULIN ACTION

在这个胰岛素刺激的网络的中心途径是酶PI 3-激酶。抑制这种酶会导致几乎所有胰岛素的代谢作用被阻断，以及它对细胞生长和分化的影响。典型地，PI 3-激酶是一个异源二聚体，由一个85 kDa的调控亚基（p85 α）和一个110 kDa的催化亚基（p110 α）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7个可能的调节亚基，其中6个是p85基因三种不同的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都受到调节。这项资助的主要目标是剖析PI 3-激酶的各种可选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下的变化。为了实现这一目标，我们将：(1)通过创建和表征使用全局和条件/组织特异性基因靶向策略敲除PI 3-激酶特定异构体的小鼠，研究PI 3-激酶各种异构体在体内胰岛素受体与下游效应系统耦合中的异构体和潜在互补作用。(2)通过转染NIH 3T3和CHO细胞等标准细胞系、3T3- l1脂肪细胞和L6肌管等胰岛素反应性更强的细胞，以及敲除动物模型中缺乏PI 3-激酶特异性同工型的细胞，确定PI 3-激酶不同同工型在差异胰岛素信号传导中的作用。我们将研究这些细胞在用不同的PI 3-激酶同工异构体重组前后，以及在小鼠胚胎成纤维细胞中用PPARgamma2转化为脂肪细胞样细胞系前后的情况。这些研究将利用逆转录病毒和腺病毒载体。(3)确定培养细胞和完整动物组织中各种PI - 3激酶同种异构体的亚细胞区隔化、差异配对和调节差异，包括使用酵母2-杂交系统鉴定和表征PI - 3激酶可选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-激酶异构体的差异调控的影响，并表征最近克隆的两种磷脂酰肌醇磷酸5-磷酸酶和糖尿病动物的细胞质提取物可能是PI 3-激酶系统的负调控因子。总之，这些研究应该为PI 3-激酶同工型在胰岛素作用和糖尿病中的作用提供重要的见解。

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.