Role of PI 3-Kinase isoforms in insulin action

描述(由申请人提供)：这是美国国立卫生研究院授予的DK55545的竞争性更新，重点是PI 3-激酶在胰岛素作用中的作用。PI3K是胰岛素作用于代谢调控的关键节点，也是胰岛素信号转导的关键点。在这项资助下使用体外和体内方法的先前工作中，包括建立和鉴定特定异构体PI 3-激酶调节和催化亚基缺失的小鼠和细胞系，我们已经证明这种酶以积极和消极的方式控制胰岛素信号，并不仅仅参与PIP3的产生。这包括PI 3-激酶不同调节亚基(P85)和催化亚基(P110)的活性和性质的差异；调节亚基和催化亚基之间的化学计量比对胰岛素作用的重要影响；PI 3-激酶允许Akt和非典型PKC之间下游信号分化的能力；以及疾病状态下PI 3-K活性的变化。此外，我们还发现了PI3-激酶通路和其他信号通路之间的几个未知的联系，包括p110a和p110b催化亚基之间支持下游胰岛素作用的差异，以及p85调节亚基与几个参与胰岛素抵抗的通路之间的重要联系，包括应激蛋白激酶JNK和p38；细胞和组织中主要的PIP3磷酸酶PTEN；以及PI3-激酶、内质网(ER)应激和未折叠蛋白反应之间的新联系。后者通过p85a与转录因子XBP-1的相互作用而发生，可以改变参与胰岛素抵抗的内质网应激反应。这导致我们提出了新的假设，即不同的PI3-K催化和调节亚基既是胰岛素信号通路的分歧部位，也是生理和病理状态下正、负调控的部位，以及与其他信号系统的串扰部位。在接下来的五年里，我们将在分子和生理水平上扩展这些观察，通过定义不同的PI 3-激酶调节和催化亚基如何产生不同的信号，涉及的特定信号复合体，以及这个系统在体内胰岛素抵抗和糖尿病状态中的作用。具体地说，我们将通过确定体内和肌肉中与P85调节亚基相互作用的特定途径和分子，以及确定P85是否在糖尿病和其他胰岛素分泌改变状态下胰腺b细胞的ER应激反应中发挥作用，进一步阐明PI 3-K调节亚基与ER应激诱导之间的联系。这将在体外和体内通过创造b细胞特异性p85a ko小鼠与分泌突变胰岛素分子的小鼠或患有肥胖和高胰岛素血症的小鼠杂交来完成。我们还将确定与严重胰岛素抵抗相关的第一个人类P85突变的特征，并确定PI3K催化亚基p110a和p110b在PI3K途径中不同的胰岛素信号转导中的不同作用。

DESCRIPTION (provided by applicant): This is a competitive renewal of NIH grant DK55545 which is focused on the role of PI 3-kinase in insulin action. PI 3-kinase is a critical node in insulin action in control of metabolism and a key point of divergence of insulin signaling. In previous work under this grant using both in vitro and in vivo approaches, including creation and characterization of mice and cell lines in which specific isoforms of PI 3-kinase regulatory and catalytic subunits have been deleted, we have demonstrated that this enzyme controls insulin signaling in both positive and negative ways, and is involved in much more than simply generation of PIP3. This includes differences in the activity and properties of the different regulatory (p85) and catalytic (p110) subunits of PI 3-kinase; important effects of stoichiometry between regulatory and catalytic subunits on insulin action; the ability of PI 3-kinase to allow divergence of the downstream signal between Akt and atypical PKCs; and alterations in PI 3-kinase activity in disease states. In addition, we have identified several previously unrecognized links between the PI 3-kinase pathway and other signaling pathways, including differences between the p110a and p110b catalytic subunits in support of downstream insulin actions and important links between the p85 regulatory subunits and several pathways involved in insulin resistance, including the stress kinases JNK and p38; the major PIP3 phosphatase in cells and tissues PTEN; and a novel link between PI 3-kinase, endoplasmic reticulum (ER) stress and the unfolded protein response. The latter occurs through interaction of p85a with the transcription factor XBP-1 and can modify the ER stress response involved in insulin resistance. This has led us to new hypotheses about the important role of the different PI 3-kinase catalytic and regulator subunits as both sites of divergence in the insulin signaling pathway and sites of both positive and negative regulation in physiological and pathological states, as well as sites for cross-talk with other signaling systems. In the next five years, we will expand upon these observations at both the molecular and physiological levels by defining how different signals are generated by different regulatory and catalytic subunits of PI 3- kinase, the specific signaling complexes involved, and the role of this system in vivo in insulin resistant and diabetic states. Specificall we will further elucidate the link between PI 3-kinase regulatory subunits and induction of ER stress by defining the specific pathways and molecules interacting with the p85 regulatory subunits in vivo and muscle and determining if p85 plays a role in the ER stress response in pancreatic b-cells in diabetes and other states with altered insulin secretion. This will be done i vitro and in vivo through creation of b-cell specific p85a KO mice crossed with a mouse that secretes a mutant insulin molecule or mice with states of obesity and hyperinsulinemia. We will also characterize the first human mutation in p85 associated with severe insulin resistance and determine the differential roles of the PI 3-kinase catalytic subunits p110a and p110b in divergent insulin signaling in the PI 3-kinase pathway.