Carnitine and coenzyme A are cofactors in cellular intermediary metabolism. Multiple disease states are associated with cellular acyl-CoA accumulation which interferes with short-term metabolic regulation. The current proposal is designed to test the hypothesis that the status of the coenzyme A pool is a key factor in the long-term regulation of cellular energy homeostasis. Specifically, it is hypothesized that 1) cellular coenzyme A content is regulated via both biosynthesis and degradation based on the coenzyme A and acyl-CoA contents in the cytosol and mitochondria, and 2) the status of the coenzyme A pool, either directly or through secondary metabolic alterations, contributes to the regulation of cellular mitochondrial content. Hydroxycobalamin[c-lactam) (HCCL) treatment in rats has been developed as a facile model of acyl-CoA buildup secondary to decreased L-methylmalonyl CoA mutase activity. Studies will be performed to characterize the regulation of coenzyme A biosynthesis, degradation, and subcellular distribution in response to acyl-CoA accretion. The acute effects of acyl-CoA accumulation on redistribution of cellular CoA between the cytosolic and mitochondrial compartments will be defined and the consequences of this redistribution on coenzyme A homeostasis assessed. A feature of HCCL treatment is an increased hepatic mitochondrial content. mtDNA content and levels of mitochondrial mRNAs will be quantified in liver from control and HCCL-treated rats. Using isolated mitochondria and in vitro cell culture, kinetics of mitochondrial protein synthesis will be quantified in the absence and presence of HCCL treatment. Early molecular events in the initiation of mitochondrial proliferation will be identified and used to define the metabolic conditions required for the enhanced mitochondrial content. Acyl-CoA accumulation will be induced acutely and the initiation of mitochondrial proliferation monitored. Previous work has identified decreased cytochrome b content in mitochondria from HCCL-treated rats. Formation, processing and translation of cytochrome b mRNA will be quantified to define the basis for the HCCL-induced complex III defect. The current proposal thus utilizes a well-defined model of metabolic disease to define the metabolic regulation of cellular mitochondrial and coenzyme A homeostasis. The project will provide new insights into the cellular mechanism of disease, will contribute to the development of new therapeutic strategies, and will provide information on the fundamental cell biology of mitochondria.
