Coordinated regulation of signaling events for insulin biosynthesis and secretion

胰岛素生物合成和分泌信号事件的协调调节

• 批准号: 7759636
• 负责人: Gen-Sheng Feng
• 金额: $ 36.71万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7759636
• 关键词: Address; Alleles; Anabolism; Automobile Driving; Beta Cell; Binding; Cell Line; Cell Proliferation; Cell physiology; Cells; Complement; Data; Development; Diabetes Mellitus; Enzymes; Epidermal Growth Factor; Etiology; Event; Failure; Functional disorder; Gene Targeting; Genes; Glucose; Goals; Growth; Growth Factor; Growth Factor Receptors; Hormones; In Vitro; Indium; Insulin; Insulin Receptor; Insulin Signaling Pathway; Islet Cell; Knock-out; Knowledge; Lead; Mediating; Metabolic Diseases; Molecular; Mus; Mutant Strains Mice; Natural regeneration; Non-Insulin-Dependent Diabetes Mellitus; Pancreas; Pathogenesis; Pathway interactions; Physiological; Production; Protein Tyrosine Phosphatase; Proteins; Regulation; Role; Scheme; Science; Signal Pathway; Signal Transduction; Small Interfering RNA; System; Techniques; Testing; Text; Tissues; Work; base; blood glucose regulation; cell type; cytokine; design; impaired glucose tolerance; in vivo; insulin secretion; insulin signaling; insulinoma; mature animal; mouse model; novel; precursor cell; public health relevance; research study; response

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.

描述（由申请人提供）：该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，当前的重点是剖析Shp 2酪氨酸磷酸酶在细胞中协调信号级联中的功能。虽然胰腺细胞衰竭是所有形式糖尿病的关键组成部分，但对细胞功能障碍的分子基础知之甚少。这主要是因为对β-细胞中介导葡萄糖和胰岛素信号的细胞质成分知之甚少。Shp 2是一种具有两个SH 2结构域的细胞质酪氨酸磷酸酶，其涉及信号传导途径的调节和协调。特别是，Shp 2已被证明在体外促进胰岛素刺激的Erk活化，尽管Shp 2在胰岛素信号传导中的生理意义尚不清楚。在最近的研究中，我们已经成功地在小鼠中创建了条件性Shp 2敲除等位基因Shp 2flox，这使我们能够在体内研究特定细胞类型或组织中的特定Shp 2功能。我们已经产生了在成熟β-细胞或Pdx 1+胰腺前体细胞中缺失Shp 2的突变小鼠，并将表征这些新的小鼠模型以测试工作假设，即Shp 2在协调β-细胞中胰岛素生物合成和分泌中起协调和控制几种信号通路的强度的作用。作为体内基因靶向方法的补充，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传导机制。我们的具体目标是：1）确定Shp 2在β-细胞功能和葡萄糖稳态中的生理作用; 2）剖析Shp 2在β-细胞中作用的分子机制;和3）研究Shp 2在胰腺发育和β-细胞再生中的功能。本实验的成功完成将填补我们对β-细胞胞质信号事件协调调控的知识空白，甚至可能为β-细胞在葡萄糖稳态和2型糖尿病发病机制中的功能调控提供新的范例。本项目的目标是了解控制细胞胰岛素生物合成的细胞内信号机制，以及2型糖尿病（世界上最常见的代谢疾病）细胞衰竭的分子基础。2型糖尿病的特征在于响应于葡萄糖的有缺陷的胰腺（细胞）胰岛素释放和受损的胰岛素对其靶组织的作用。β-细胞衰竭可能是由β-细胞团的不充分扩增和/或β-细胞对葡萄糖应答的失败引起的。现在，一个关键的问题是要了解的分子信号方案的β细胞传感葡萄糖和分泌胰岛素。我们最初克隆了鼠Shp 2（Syp）作为蛋白酪氨酸磷酸酶，其含有两个Src同源2（SH 2）结构域（Feng et al.，Science 1993）。Shp 2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传导事件。特别地，Shp 2结合胰岛素受体底物（IRS）和Grb 2相关结合剂（GAB）蛋白。几个研究小组已经证明了Shp 2在体外介导胰岛素刺激的Erk活化中的积极作用。虽然这些研究表明Shp 2在胰岛素作用中的假定作用，但尚未获得体内生理学证据。为了确定Shp 2在β-细胞中的功能，我们已经产生了在胰腺或β-细胞中选择性缺失Shp 2的突变小鼠系。这些突变小鼠表现出葡萄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Shp 2作为一个协调器，微调和整合多个信号的胰岛素生物合成和分泌的β细胞。我们的初步实验数据表明Shp 2确实在促进β-细胞中胰岛素的生物合成和分泌以控制葡萄糖稳态中具有关键作用。成功完成这三个目标中的拟议实验不仅将填补我们在β-细胞中分子信号传导事件方面的知识空白，而且甚至可能刷新我们目前对葡萄糖刺激胰岛素分泌中β-细胞功能以及2型糖尿病病因学的看法。