Long term alcohol consumption causes mitochondrial dysfunction associated with morphological changes and alterations in electron transport and ATP synthase activity in multiple tissues, including liver and heart. The implications of these deficiencies for the physiological function of liver and heart in chronically alcoholic animals are poorly understood. An understanding of the consequences of the changes in oxidative phosphorylation associated with chronic ethanol intake requires an integrated and quantitative approach to the control of energy metabolism in the tissue. Metabolic Control Analysis (MCA) is a type of sensitivity analysis, which provides a frameword to describe the distribution of control in complex metabolic networks. It has been applied extensively to the quantitative analysis of bioenergetic systems, both in isolated mitochondria and intact cells and tissues. The present proposal is designed to use MCA to characterize changes in the control of mitochondria oxidative phosphorylation in isolated mitochondria from heart and liver after chronic ethanol consumption using the top-down approach, a recently developed analytical tool that enables the identification of the control exerted by segments of the oxidative phosphorylation machinery (or other complex enzymatic pathways).
The Specific Aims are: (1) to identify by top-down elasticity analysis the functional units of oxidative phosphorylation that are affected by chronic ethanol consumption in intact mitochondria from heart and liver; (2) to quantify the ethanol-induced changes in the control patterns of heart and liver mitochondria under different conditions of substrate supply and energy utilization. We will use the results of the top-down elasticity analysis, in combination with double inhibitor titrations and titrations with specific inhibitors of select steps of the oxidative phosphorylation system. We will also determine how changes in the control distribution in the system relate to changes in enzymatic activities reported earlier and to changes in mitochondrial membrane properties. The results of this study are expected to provide insights into the effect of chronic ethanol exposure on cellular responses to physiological stress that may affect the organisms susceptibility to the development alcohol-related disorders.
