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(申请人提供)：这些研究的长期目标是在亮氨酸作为直接作用的营养信号的背景下了解亮氨酸代谢的调节。蛋白质餐后血浆亮氨酸浓度升高，刺激蛋白质合成和胰岛素使用的细胞信号通路，如哺乳动物的雷帕霉素靶点(MTOR)通路。MTOR信号通路对细胞周期进程、肥大生长和许多关键的脂肪组织功能具有重要作用。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由支链α-酮酸脱氢酶(BCKD)催化的亮氨酸代谢的主要限速步骤，该酶由特定的线粒体内激酶和磷酸酶调节。两个基本问题推动了这项拟议的研究。1)亮氨酸的α-酮酸(KIC)激活BCKD是否是亮氨酸激活mTOR的一个步骤？2)胰岛素，一个细胞外调节的受体，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，将开发一种快速、简单的检测系统，用于基于一种新的抗体来测量BCKD的急性调节(活动状态)，该抗体可以识别磷酸化的BCKD(失活的，pS293)。其次，将使用三种方法来确定BCKD激活在mTOR亮氨酸调节中的作用：时间过程、结构-活性和分子方法。第三，胰岛素调节BCKD的机制将被研究。BCKD的胰岛素调节所需的胞浆信号通路将被确定。BCKD激酶和已知受胰岛素作用影响的代谢物的潜在作用也将被研究。最后，BCKD的胰岛素激活是BCKD磷酸酶激活的假设将得到验证。为了制备抗体和高效表达重组蛋白，我们将对BCKD磷酸酶亚基进行克隆和测序。这些抗体将用于检测胰岛素对BCKD磷酸酶及其抑制蛋白的潜在影响。重组磷酸酶将用于测试胰岛素作用的潜在机制，并用于物理和动力学表征。这项研究的结果将为深入了解碳水化合物和蛋白质代谢的整合机制提供依据。