Proteomics Studies of Type 2 Diabetes Using Knockout Mouse Models

描述(申请人提供)：本申请的长期目标是获得促进胰腺细胞再生的机制和因素的基本知识，以改进内源性细胞的保存或再生，或在体外产生移植细胞的治疗策略，这对预防和治疗糖尿病至关重要。这个为期五年的项目的具体目标是应用全球定量蛋白质组和基因组技术来研究独特的胰岛素抵抗基因敲除小鼠模型，以确定调节细胞复制和生存的新的胰岛蛋白和循环因子，并对关键的新蛋白成员进行功能研究，以更好地了解调节胰岛生长的机制和因素。在糖尿病和胰岛素抵抗的啮齿动物模型、肥胖和/或2型糖尿病患者以及其他与胰岛素抵抗相关的常见疾病中，已观察到胰岛素抵抗引起的细胞团/胰岛生长增加；然而，导致胰岛素抵抗状态下细胞增殖的因素仍未确定。蛋白质组和基因组技术应用于独特的胰岛素抵抗基因敲除模型，包括肝脏特异性胰岛素受体基因敲除(LIRKO)和胰岛素受体底物-1(IRS1KO)基因敲除模型，将为识别参与这一重要反馈机制的新蛋白质和因子提供独特的机会。计划的具体目标是：(1)确定与胰岛生长有关的新的调节蛋白；(2)发现刺激细胞再生的新的血液循环因子；(3)对选定的新调节蛋白和因子进行功能研究。整个方法包括蛋白质组范围的稳定同位素16O/18O标记，准确的质量和时间(AMT)标记策略，以及高分辨率LC分离和高灵敏度傅立叶变换离子回旋共振(FTICR)质谱仪，以提供广泛和高通量的差异蛋白质丰度的定量测量。组织的mRNA水平将使用定量RT-PCR技术进行测量，以补充蛋白质丰度数据。这项研究产生的大量数据有望成为糖尿病研究界的宝贵资源，这项工作将为更广泛的机制研究奠定基础。公共卫生相关性：在肥胖和/或2型糖尿病患者中，通常可以观察到胰岛素抵抗引起的胰岛生长增加；然而，导致胰岛增生的因素尚不清楚。这项应用的目的是获得促进胰岛再生的机制和因素的基础知识，以便通过应用先进的蛋白质组技术来改进糖尿病的预防和治疗策略。

DESCRIPTION (provided by applicant): The long term objective of this application is to gain fundamental knowledge of mechanisms and factors that promote pancreatic ¿-cell regeneration to enable improved therapeutic strategies for either preserving or regenerating of endogenous ¿-cells or for generating ¿-cells in vitro for transplantation, which are essential for the prevention and cure of diabetes. The specific goals of this five-year project are to apply global quantitative proteomic and genomic technologies to study unique insulin-resistant knockout mouse models in order to identify novel islet proteins and circulating factors that regulate ¿-cell replication and survival, and to perform functional studies on key novel protein players to gain better understanding of the mechanisms and factors that regulate islet growth. Increased ¿-cell mass/islet growth in response to insulin resistance has been observed in rodent models of diabetes and insulin resistance, human patients with obesity and/or type 2 diabetes, and other common disorders associated with insulin resistance; however, the factors contributing to ¿-cell hyperplasia in insulin-resistant states remain to be characterized. Proteomic and genomic technologies applied to unique insulin-resistant knockout models, including the liver specific insulin receptor knockout (LIRKO) and insulin receptor substrate-1 (IRS-1) knockout (IRS1KO) models, will provide unique opportunity for the identification of novel proteins and factors that are involved in this important feedback mechanism. The planned Specific Aims are: (1) to identify novel regulatory islet proteins involved in islet growth, (2) to discover new blood circulatory factors that stimulate ¿-cell regeneration, and (3) to perform functional studies for selected novel regulatory proteins and factors. The overall approach incorporates proteome-wide stable isotope 16O/18O labeling, the accurate mass and time (AMT) tag strategy, and high resolution LC separations coupled with high sensitivity Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to provide broad and high throughput quantitative measurements of differential protein abundances. The tissue mRNA levels will be measured using quantitative RT-PCR technology to complement the protein abundance data. The large scale of data generated from this study is anticipated to be a valuable resource for the diabetes research community and this work will form a basis for more extensive mechanistic studies. PUBLIC HEALTH RELEVANCE: Increased pancreatic islet growth in response to insulin resistance has been commonly observed in human patients with obesity and/or type 2 diabetes; however, the factors contributing to islets hyperplasia are poorly understood. The objective of this application is to gain fundamental knowledge of mechanisms and factors that promote pancreatic islets regeneration to enable improved therapeutic strategies for the prevention and cure of diabetes by applying advanced proteomic technologies.