Role of Leucine Metabolism in Leucine Signaling

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。含蛋白质的膳食后血浆亮氨酸浓度上升，刺激蛋白质合成和胰岛素使用的细胞信号传导途径，例如哺乳动物雷帕霉素靶点 (mTOR) 途径。 mTOR 信号通路对于细胞周期进展、肥大生长和许多关键脂肪组织功能非常重要。因此，了解亮氨酸信号如何终止以及如何调节该终止非常重要。拟议的研究将重点关注由支链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（一种细胞外调节受体）如何刺激线粒体基质酶 BCKD 的活性？我们将在四个具体目标中解决这些问题。首先，将开发一种快速简单的测试系统，用于测量 BCKD 的急性调节（活性状态），该系统基于特异性识别磷酸化 BCKD（非活性，pS293）的新抗体。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：将使用时间过程、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 BCKD 的胰岛素调节所需的胞质信号传导途径。还将研究 BCKD 激酶和已知受胰岛素作用影响的代谢物的潜在作用。最后，将检验胰岛素对 BCKD 的激活是由 BCKD 磷酸酶的激活引起的假设。 BCKD磷酸酶亚基将被克隆和测序，以制备抗体和过表达重组蛋白。这些抗体将用于检查胰岛素对 BCKD 磷酸酶及其抑制剂蛋白的潜在影响。重组磷酸酶将用于测试胰岛素作用的潜在机制以及物理和动力学表征。这项研究的结果将深入了解碳水化合物和蛋白质代谢整合的机制。

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.