Quantitative Mitochondrial Proteomics of Healthy and Diabetic Mice

描述（由申请人提供）：本申请涉及广泛的挑战领域（03）：生物标志物发现和验证，以及特定的挑战主题03-DK-103：鉴定与NIDDK疾病相关的亚细胞器的正常和患病蛋白质组。线粒体功能障碍已被假定为2型糖尿病（T2 DM）的统一主题，目前仅在美国就影响超过2000万人。线粒体含量和氧化能力降低、脂肪酸氧化改变和胰岛源性活性氧升高与胰岛素抵抗和T2 DM的发生相关。然而，这些观察结果的重要性最近受到了挑战，目前仍不清楚线粒体组成和/或功能的变化是否会导致T2 DM病因。该提案的长期目标是通过阐明伴随该疾病发作的成分变化，以前所未有的分辨率更全面地了解线粒体功能障碍在T2 DM中的重要性。尽管有上述观察结果，但由于至少两个原因，对T2 DM中线粒体功能障碍的分子基础的理解在很大程度上仍然缺乏。首先，直到最近，大部分哺乳动物线粒体蛋白质组本身是不确定的，使得不可能在这种疾病的不同阶段进行线粒体组成的全面比较。其次，用于研究健康和糖尿病患者之间基因表达的总体差异的微阵列分析由于细胞mRNA和蛋白质水平之间的显著不良相关性而受到削弱。在这里，我们建议使用最先进的定量蛋白质组学（AIM 1）建立一个全面的蛋白质组学和磷酸化蛋白质组学变化的地图，发生在肥胖依赖性T2 DM的发病过程中。为此，我们将利用两种小鼠品系：C57 BL/6（B6）leptinob/ob小鼠，其对糖尿病具有抗性，以及BTBR leptinob/ob小鼠，其随着年龄的增长而发展为严重的糖尿病。在这个项目的两年时间里，我们将集中研究骨骼肌的线粒体，这是体内胰岛素介导的葡萄糖处理的主要场所。这种蛋白质组资源也将提供一个机会，探索这些线粒体改变发生的机制。特别是，我们将使用整合基因组学来阐明在线粒体基因表达和蛋白质功能（AIM 2）的控制中发挥作用的转录后和翻译后机制。我们的目标的完成将阐明伴随T2 DM发病的骨骼肌线粒体重组，为糖尿病社区提供丰富的定量蛋白质组学资源，并为识别蛋白质生物标志物和设计针对该细胞器的肌肉特异性疗法以治疗T2 DM奠定基础。 公共卫生相关性：线粒体功能障碍是2型糖尿病（T2 DM）的一个突出特征，但这种功能障碍的潜在基础尚不清楚。我们建议使用最先进的蛋白质组学技术，建立一个地图的线粒体改变发生在骨骼肌在肥胖引起的糖尿病发作。这一目标的完成将有助于确定这种疾病的线粒体生物标志物，并为设计针对这种细胞器的治疗方法以治疗T2 DM提供框架。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (03): Biomarker Discovery and Validation, and specific Challenge Topic 03-DK-103: Identify the normal and diseased proteome of subcellular organelles of relevance to NIDDK diseases. Mitochondrial dysfunction has been postulated as a unifying theme of type 2 diabetes mellitus (T2DM), which currently affects more than 20 million individuals in the US alone. Decreased mitochondrial content and oxidative capacity, altered fatty acid oxidation, and a rise in mitochondria-derived reactive oxygen species have been associated with the development of insulin resistance and T2DM. However, the importance of these observations has recently been challenged, and it remains unclear which, if any, changes to mitochondrial composition and/or function actually contribute to T2DM etiology. The long-term objective of this proposal is to arrive at a fuller understanding of the importance of mitochondrial dysfunction in T2DM by elucidating the compositional changes that accompany the onset of this disease at unprecedented resolution. Despite the observations noted above, an understanding of the molecular basis for mitochondrial dysfunction in T2DM has largely been lacking for at least two reasons. First, until recently, much of the mammalian mitochondria proteome itself was undefined, making it impossible to conduct comprehensive comparisons of mitochondrial composition during different stages of this disease. Second, microarray analyses used to investigate global differences in gene expression between healthy and diabetic patients have been crippled by the markedly poor correlation between cellular mRNA and protein levels. Here, we propose to establish a comprehensive map of the proteomic and phosphoproteomic changes that occur during the onset of obesity-dependent T2DM using state-of-the-art quantitative proteomics (AIM1). To do so, we will take advantage of two mouse strains: C57BL/6 (B6) leptinob/ob mice, which are resistant to diabetes, and BTBR leptinob/ob mice, which develop severe diabetes as they age. During the two-year timeframe of this project, we will focus on mitochondria from skeletal muscle, the primary site of insulin- mediated glucose disposal in the body. This proteomic resource will also provide an opportunity to explore the mechanisms by which these mitochondrial alterations occur. In particular, we will use integrative genomics to elucidate the post-transcriptional and post-translational mechanisms at play in the control of mitochondrial gene expression and protein function (AIM2). Completion of our aims will clarify the mitochondrial restructuring in skeletal muscle that accompanies the onset of T2DM, provide a rich quantitative proteomic resource for the diabetes community, and lay the groundwork for identifying protein biomarkers and designing muscle-specific therapies targeted against this organelle to treat T2DM. PUBLIC HEALTH RELEVANCE: Mitochondrial dysfunction is a prominent feature of type 2 diabetes mellitus (T2DM), but the underlying basis for this dysfunction is not well understood. We propose to use state-of-the-art proteomics technologies to establish a map of mitochondrial alterations that occur in skeletal muscle during the onset of obesity-induced diabetes. Completion of this goal will help identify mitochondrial biomarkers for this disease, and provide a framework for designing therapies targeted against this organelle to treat T2DM.