Dietary caloric restriction is the most potent intervention prolonging life span, as well as reducing age-related diseases, such as obesity and diabetes. Although the mechanism of caloric restriction is not clear, it might be due to the reduction of reactive oxygen species (ROS) by caloric restriction. Sir2, which possesses the NAD- dependent deacetylase and ADP-ribosyltransferase activities, mediates the effect of caloric restriction in yeast, C.elegans and fruit flies. We have demonstrated that the expression of SIRT3, one of the seven mammalian Sir2 homologs, is increased by caloric restriction and cold exposure in brown adipose. We have also shown that murine SIRT3 is localized to the mitochondria inner membrane. Forced expression of SIRT3 elevates cytoplasmic calcium and CREB phosphorylation, leading to an up-regulation of the expressions of PPARgamma coactivator PGC-1 alpha and UCP-1 in brown adipocytes or UCP3 in myocytes. The expression of SIRT3 decreases cellular ROS levels by 40% while increasing oxygen consumption. Mutations of either SIRT3 deacetylase or ADP-ribosylase activities abolish these effects. Interestingly, SIRT3 is down regulated in the brown adipose tissue of several genetically obese mice. Our yeast two- hybrid screening of SIRT3 identified an interaction with a subunit of the complex I of the electron transport chain, which is one of the major sites for ROS production. We hypothesize that SIRT3 regulates mitochondrial function and superoxide production through two pathways: direct interaction with mitochondria! complex I and regulation of a calcium signaling pathway leading to the expression of PGC-1 alpha and uncoupling proteins. Thus, SIRT3 might offer protection against mitochondrial oxidative stress under obesity and diabetes. Our long-term goal is to elucidate the molecular mechanism of SIRT3 action and establish SIRT3 as a drug target for treatments that would mimic caloric restriction's beneficial effects on obesity and diabetes, even aging. The following specific aims will be pursued. 1) To confirm and characterize the interaction between SIRT3 and mitochondrial complex I. 2) To investigate how mitochondrial SIRT3 regulates intracellular calcium signaling. 3) To characterize the effect of SIRT3 on mitochondrial superoxide production and hyperglycemia-induced oxidative stress. 4) To investigate the effect of SIRT3 on adipose oxidative stress during obesity and diabetes in vivo.
