Role of Leucine Metabolism in Leucine Signaling

亮氨酸代谢在亮氨酸信号转导中的作用

• 批准号: 6748429
• 负责人: CHRISTOPHER JOHN LYNCH
• 金额: $ 37.43万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-20 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6748429
• 关键词: adipocytes; aminoacid metabolism; antibody; biological signal transduction; carbohydrate metabolism; chemical kinetics; enzyme activity; enzyme inhibitors; hormone regulation /control mechanism; immunoprecipitation; insulin; laboratory rabbit; laboratory rat; leucine; mitochondria; phosphorylation; protein kinase; protein sequence; recombinant proteins

DESCRIPTION (provided by applicant): The long-term goal of these studies is to understand the regulation of leucine metabolism in the context of leucine's role as a direct acting nutrient signal. Rises in plasma leucine concentration after a protein-containing meal stimulate protein synthesis and cell signaling pathways used by insulin such as the mammalian Target of Rapamycin (mTOR) pathway. The mTOR signaling pathway is important for cell cycle progression, hypertrophic growth and a number of key adipose tissue functions. Therefore, it is important to understand how the leucine signal is terminated and how that termination is regulated. The proposed studies will focus on the major rate-limiting step in leucine metabolism catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKD), which is regulated by a specific intramitochondrial kinase and phosphatase. Two fundamental questions drive the proposed studies. 1) Is activation of BCKD by the alpha-keto acid of leucine (KIC) a step in the activation of mTOR by leucine? 2) How does insulin, an extracellularly regulated receptor, stimulate the activity of a mitochondrial matrix enzyme, BCKD? In the four specific aims we will address these questions. First a rapid simple test system to measure acute regulation (activity state) of BCKD based on a new antibody that specifically recognizes phosphorylated BCKD (inactive, pS293) will be developed. Second, three approaches will be used to determine the role of BCKD activation in leucine regulation of mTOR: time course, structure-activity and molecular approaches will be used. Third, the mechanism whereby insulin regulates BCKD will be investigated. The cytosolic signaling pathway required for insulin regulation of BCKD will be determined. The potential role of the BCKD kinase and metabolites known to be affected by insulin action will also be investigated. Finally, the hypothesis that insulin activation of BCKD results from activation of BCKD phosphatase will be tested. Subunits of BCKD phosphatase will be cloned and sequenced in order to prepared antibodies and over express recombinant protein. The antibodies will be used to examine the potential effects of insulin on BCKD phosphatase and its inhibitor protein. The recombinant phosphatase will be used to test potential mechanisms of insulin action and for physical and kinetic characterization. The results from this study will provide insight into mechanisms involved in integration of carbohydrate and protein metabolism.

描述（由申请人提供）：这些研究的长期目标是了解亮氨酸作为直接作用营养信号的作用背景下亮氨酸代谢的调节。在含蛋白质的膳食后血浆亮氨酸浓度的升高刺激蛋白质合成和胰岛素所使用的细胞信号传导途径，例如哺乳动物雷帕霉素靶（mTOR）途径。mTOR信号通路对于细胞周期进展、肥大性生长和许多关键脂肪组织功能是重要的。因此，重要的是要了解亮氨酸信号是如何终止的，以及这种终止是如何调节的。拟议的研究将集中在亮氨酸代谢的主要限速步骤催化的支链α-酮酸脱氢酶（BCKD），这是由一个特定的线粒体内激酶和磷酸酶调节。两个基本问题驱动了拟议的研究。1)亮氨酸的α-酮酸（KIC）激活BCKD是亮氨酸激活mTOR的一个步骤吗？2)胰岛素是一种细胞外调节受体，它如何刺激线粒体基质酶BCKD的活性？在四个具体目标中，我们将解决这些问题。首先，将开发基于特异性识别磷酸化BCKD（失活，pS293）的新抗体的快速简单测试系统，以测量BCKD的急性调节（活性状态）。其次，将使用三种方法来确定BCKD激活在mTOR的亮氨酸调节中的作用：时间过程、结构-活性和分子方法。第三，研究胰岛素调节BCKD的机制。将确定胰岛素调节BCKD所需的胞质信号传导途径。还将研究已知受胰岛素作用影响的BCKD激酶和代谢物的潜在作用。最后，将检验BCKD的胰岛素活化是由BCKD磷酸酶活化引起的假设。为了制备抗体和过表达重组蛋白，将对BCKD磷酸酶亚基进行克隆和测序。这些抗体将用于检查胰岛素对BCKD磷酸酶及其抑制蛋白的潜在影响。重组磷酸酶将用于检测胰岛素作用的潜在机制以及物理和动力学表征。这项研究的结果将提供深入了解碳水化合物和蛋白质代谢的整合机制。