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.
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