PI 3 KINASE ISOFORMS AND INSULIN ACTION

在这个胰岛素刺激的网络中的中心途径是酶PI 3-激酶。 这种酶的抑制导致几乎所有胰岛素代谢作用的阻断，以及其对细胞生长和分化的影响。 经典地，PI 3-激酶是由85 kDa的调节亚基（p85 α）和110 kDa的催化亚基（p110 α）组成的异源二聚体。然而，我们的工作和其他人的工作表明，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基因的3种不同选择性剪接事件的结果。 在糖尿病动物模型中PI 3-激酶活性和选择性剪接都受到调节。 这项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，探索这些组分如何相互作用以及与细胞内不同隔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改变。 为此，我们将：（1）研究PI 3-激酶的各种异构体在体内胰岛素作用中将胰岛素受体偶联至下游效应子系统中的相似性、差异性和潜在的互补作用，通过创建和表征其中PI 3-激酶的特定异构体已经使用基因靶向的全局和条件/组织特异性策略敲除的小鼠。 (2)通过转染标准细胞系如NIH 3 T3和CHO细胞、更多胰岛素应答细胞如3 T3-L1脂肪细胞和L 6肌管以及来自敲除动物模型的缺乏PI 3-激酶特异性同种型的细胞，确定PI 3-激酶的不同同种型在差异胰岛素信号传导中的作用。 将在用PI 3-激酶的不同亚型重建之前和之后研究这些细胞，并且在小鼠胚胎成纤维细胞的情况下，在用PPARgamma 2转化成脂肪细胞样细胞系之前和之后研究这些细胞。 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-激酶亚型的亚细胞区室化、差异配对和调节差异，包括使用酵母双杂交系统鉴定和表征PI 3-激酶可变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-激酶亚型的差异调节的影响，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。 总之，这些研究应该提供重要的见解PI 3-激酶亚型的作用，胰岛素的作用和糖尿病。

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.