Coordinated regulation of signaling events for insulin biosynthesis and secretion

描述(申请人提供)：这个项目的目标是破译控制胰岛素生物合成和分泌的分子信号机制，目前的重点是剖析Shp2酪氨酸磷酸酶在(细胞)中协调信号级联反应中的功能。虽然胰腺(细胞衰竭)是所有形式的糖尿病的重要组成部分，但其背后的分子基础(细胞功能障碍)却知之甚少。这主要是因为人们对(-细胞中调节葡萄糖和胰岛素信号的细胞质成分知之甚少)。Shp2是一种含有两个SH2结构域的细胞质酪氨酸磷酸酶，参与信号通路的调节和协调。特别是，Shp2在体外被证明可以促进胰岛素刺激的ERK激活，尽管Shp2在胰岛素信号转导中的生理意义尚不清楚。在最近的研究中，我们成功地在小鼠身上创建了一个条件性Shp2基因敲除等位基因Shp2flx，它使我们能够在体内研究特定细胞类型或组织中特定的Shp2功能。我们已经培育出Shp2在成熟细胞或Pdx1+胰腺前体细胞中缺失的突变小鼠，并将对这些新的小鼠模型进行表征，以检验Shp2在(-细胞中协调和控制几个信号通路的强度)中协调和控制胰岛素生物合成和分泌的工作假说。作为体内基因打靶方法的补充，我们还将使用siRNA介导的基因敲除技术来破译细胞中的分子信号机制。我们的具体目标是：1)确定Shp2在细胞功能和葡萄糖稳态中的生理作用；2)剖析Shp2在细胞中作用的分子机制；3)研究Shp2在胰腺发育和细胞再生中的作用。这些实验的成功完成将填补我们在(-细胞)胞质信号事件协调调控方面的知识空白，甚至可能导致(-细胞在葡萄糖稳态中的功能调节以及在2型糖尿病发病机制中的新范式。公共卫生相关性这个项目的目标是了解细胞内控制胰岛素生物合成的信号机制，以及世界上最常见的代谢性疾病-2型糖尿病细胞衰竭的分子基础。2型糖尿病的特征是胰腺(细胞)胰岛素对葡萄糖的反应有缺陷，胰岛素对靶组织的作用受损。(-细胞衰竭可能是由于(-细胞团的不充分扩张和/或(-细胞对葡萄糖的反应失败)引起的。现在，一个关键的问题是了解细胞感知葡萄糖和胰岛素分泌的分子信号机制。我们最初克隆的小鼠Shp2(SYP)是一种含有两个Src同源2(SH2)结构域的蛋白质酪氨酸磷酸酶(冯等人，1993年)。Shp2是一种细胞质酶，参与调节由生长因子、细胞因子和激素触发的信号事件。特别是，Shp2结合了胰岛素受体底物(IRS)和Grb2相关结合蛋白(GAB)。几个小组已经证明Shp2在体外介导胰岛素刺激的ERK激活方面具有积极作用。虽然这些研究表明Shp2在胰岛素作用中起着推定的作用，但体内的生理证据尚未获得。为了确定Shp2在(-细胞)中的功能，我们建立了Shp2在胰腺或(-细胞中选择性缺失的突变小鼠系。这些突变小鼠表现出糖耐量受损和胰岛素分泌和产生缺陷。我们的中心工作假设是Shp2作为一个协调者，微调和整合胰岛素生物合成和分泌的多种信号。我们的初步实验数据表明，Shp2确实在促进胰岛素的生物合成和分泌以控制葡萄糖稳态方面发挥了关键作用。这三个目标的拟议实验的成功完成不仅将填补我们在(-细胞)分子信号事件方面的知识空白，甚至可能刷新我们目前对(-细胞在葡萄糖刺激的胰岛素分泌中的功能以及对2型糖尿病病因的看法。

DESCRIPTION (provided by applicant): The goal of this project is to decipher molecular signaling mechanisms for control of insulin biosynthesis and secretion, and the immediate focus is on dissecting the function of Shp2 tyrosine phosphatase in orchestrating signaling cascades in ( cells. Although pancreatic ( cell failure is a critical component in all forms of diabetes, the molecular basis underlying ( cell dysfunction is poorly understood. This is mainly because that little is known for the cytoplasmic components mediating glucose and insulin signals in (-cells. Shp2 is a cytoplasmic tyrosine phosphatase with two SH2 domains that is implicated in regulation and coordination of signaling pathways. In particular, Shp2 has been shown to promote insulin-stimulated Erk activation in vitro, although the physiological significance of Shp2 function in insulin signaling is unclear. In recent studies, we have successfully created a conditional Shp2 knockout allele, Shp2flox, in mice, which allows us to investigate specific Shp2 functions in a specific cell type or tissue in vivo. We have generated mutant mice with Shp2 deleted in mature (-cells or in Pdx1+ pancreatic precursor cells, and will characterize these novel mouse models to test the working hypothesis that Shp2 acts to coordinate and control the strength of several signaling pathways in orchestrating insulin biosynthesis and secretion in (-cells. In complement with the gene targeting approach in vivo, we will also use siRNA- mediated gene knockdown technique to decipher the molecular signaling mechanisms in ( cells. Our specific aims are: 1) to determine the physiological role of Shp2 in (-cell function and glucose homeostasis; 2) to dissect the molecular mechanism for Shp2 action in (-cells; and 3) to investigate the Shp2 function in pancreatic development and (-cell regeneration. Successful completion of the proposed experiments will fill in a gap in our knowledge for coordinated regulation of cytoplasmic signaling events in (-cells, and may even lead to a new paradigm on regulation of (-cell functions in glucose homeostasis and also in pathogenesis of type 2 diabetes. PUBLIC HEALTH RELEVANCE The goal of this project is to understand the intracellular signaling mechanisms for control of insulin biosynthesis in ( cells and also the molecular basis for ( cell failure in Type 2 diabetes, the most common metabolic disease in the world. Type 2 diabetes is characterized by defective pancreatic ( cell insulin release in response to glucose and impaired insulin action on its target tissues. (-cell failure is likely caused by inadequate expansion of the (-cell mass and/or failure of the (-cells to respond to glucose. Now, a crucial issue is to understand the molecular signaling scheme for (-cell sensing of glucose and secretion of insulin. We originally cloned murine Shp2 (Syp) as a protein tyrosine phosphatase that contains two Src homology 2 (SH2) domains (Feng et al., Science 1993). Shp2 is a cytoplasmic enzyme and has been implicated in regulation of signaling events triggered by growth factors, cytokines and hormones. In particular, Shp2 binds the insulin receptor substrate (IRS) and Grb2-associated binder (GAB) proteins. Several groups have demonstrated a positive effect of Shp2 in mediating insulin-stimulated Erk activation in vitro. While these studies suggest a putative role of Shp2 in insulin action, the physiological evidence in vivo is yet to be obtained. To determine Shp2 function in (-cells, we have generated mutant mouse lines with Shp2 selectively deleted in the pancreas or (-cells. These mutant mice displayed impaired glucose tolerance and defective insulin secretion and production. Our central working hypothesis is that Shp2 acts as a coordinator to fine-tune and integrate multiple signals for insulin biosynthesis and secretion in (-cells. Our preliminary experimental data suggest that Shp2 indeed has a critical role in promoting insulin biosynthesis and secretion in (-cells for control of glucose homeostasis. Successful completion of the proposed experiments in these three Aims will not only fill in a gap in our knowledge in molecular signaling events in (- cells, but may even refresh our current view on (-cell functions in glucose-stimulated insulin secretion and also on the etiology of type 2 diabetes.