Lambert, A. J., and B. J. Merry. Effect of caloric restriction on mitochondrial reactive oxygen species production and bioenergetics: reversal by insulin. /ajpregu.00341.2003.-To gain insight into the antiaging mechanisms of caloric restriction (CR), mitochondria from liver tissue of male Brown Norway rats were used to study the effects of CR and insulin on mitochondrial reactive oxygen species production and bioenergetics. As assessed by hydrogen peroxide measurement, CR resulted in a decrease

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... in the production rate of reactive oxygen species. This decrease was attributed to a decrease in protonmotive force in mitochondria from the CR animals. The decrease in protonmotive force resulted from an increase in proton leak activity and a concomitant decrease in substrate oxidation activity. Each of these effects of CR was reversed by subjecting CR animals to 2 wk of insulin treatment. To achieve continuous and stable insulin delivery, animals were placed under temporary halothane anesthesia and miniosmotic pumps were implanted subcutaneously. To gain further insight into how CR and insulin exerted its effects on mitochondrial bioenergetics, the effects of CR and insulin were quantified using modular metabolic control analysis. This analysis revealed that the effects of CR were transmitted through different reaction branches of the bioenergetic system, and insulin reversed the effects of CR by acting through the same branches. These results provide a plausible mechanism by which mitochondrial reactive oxygen species production is lowered by CR and a complete description of the effects of CR on mitochondrial bioenergetics. They also indicate that these changes may be due to lowered insulin concentrations and altered insulin signaling in the CR animal. control analysis; aging BIOLOGICAL AGING IN MAMMALS is characterized by a progressive decline in cellular function that leads to age-related pathology and death. To date, the most effective way to slow down this deleterious process in laboratory rodents is caloric restriction (CR). By restriction of calories throughout life span, the mean, maximum, and 10th decile of survival in rats and mice is increased. The effect of CR feeding on survival is directly proportional to the intensity of CR and its duration (39). In addition to the positive effects on survivorship, the onset and incidence of age-related disease are retarded (46). CR also ameliorates the age-related decline in a wide variety of processes such as DNA repair and protein turnover (8, 33, 44) . As yet the underlying mechanism by which CR brings about these beneficial changes remains to be resolved. An attenuation of the rate of accrual of tissue oxidative damage by decreased generation of oxidants and free radicals provides a plausible explanation of the effect of CR on aging (40). It has been proposed that mitochondria play a pivotal role in the aging process, for these organelles are considered to be the main producers of reactive oxygen species (ROS) and their DNA is particularly susceptible to free radical damage (9, 45). Mitochondria are also central to bioenergetics, as their main function is to use the electrochemical energy in the protonmotive force ⌬p (which is generated by proton pumping coupled to electron transport) to synthesize ATP from ADP. Whether CR brings about retarded aging via changes in mitochondrial metabolism remains uncertain, but it is a provocative idea given the reduced rate of ROS generation in mitochondria isolated from tissues of CR animals (17, 36, 43) . To date, a complete description of the effects of CR on mitochondrial bioenergetics has not been reported nor has the mechanism underlying the reduced ROS generation from these organelles under CR feeding been identified. Metabolic control analysis is an approach used to describe and quantify control in complex systems, and it encompasses control analysis, elasticity analysis, and regulation analysis (24). Control analysis describes and quantifies the distribution of control within a system. Elasticity analysis describes the kinetic responses of reactions to changes in levels of intermediates and is used to identify sites of action of effectors within a system. Regulation analysis quantifies the response of a system to an external effector such as an added inhibitor or hormone. Complex systems can be simplified by grouping related processes and intermediates into reaction blocks or modules, known as the "top-down" or "modular" approach of metabolic control analysis (7) . This method has been applied to gain understanding of control and regulation in a wide variety of systems, particularly energy metabolism in mitochondria and whole cells. Examples of the approach include analysis of control of energy metabolism in isolated rat liver mitochondria (18); identification of sites of action of thyroid hormones on mitochondrial energy metabolism in isolated rat liver mitochondria and hepatocytes (20); analysis of the effects of cadmium on oxidative phosphorylation in isolated potato tuber mitochondria (27-29); characterization of oxidative phosphorylation in pig skeletal muscle mitochondria (35); analysis of calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria (26); analysis of ATP turnover, glycolysis, and oxidative phosphorylation in rat hepatocytes (1, 3); analysis of the effects of epinephrine and glucagon on rat hepatocyte metabolism (2); analysis of the effects of aging on mitochondrial energy metabolism in mouse hepatocytes (21); analysis of signal transduction in lymphocytes (31); and analysis of DNA microarray expression data (11).