SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation

Sirtuins are NAD-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting. Proteomic analysis of mitochondrial proteins revealed the acetylation levels of numerous mitochondrial proteins change during fasting. The dependence of SIRT3 enzymatic activity on NAD suggests that SIRT3 serves as a metabolic sensor and couples the energy status of the cell with the level of mitochondrial protein acetylation. To explore further a possible role of SIRT3 in regulating metabolism, we monitored the protein expression level of hepatic SIRT3 during fasting in wild-type mice. Although hepatic SIRT3 expression was low under basal conditions, its expression was induced during fasting (Fig. 1a). The increase in SIRT3 protein expression during fasting was concomitant with a relative decrease in the acetylation levels of some mitochondrial proteins (two indicated by arrows in Fig. 1b), suggesting that SIRT3 mediated their deacetylation. In agreement with this model, the same two proteins were hyperacetylated in Sirt3 mice under basal conditions, and their acetylation levels did not change with fasting when SIRT3 was missing (Fig. 1b). Interestingly, the level of acetylation of other mitochondrial proteins did not change during fasting or in the absence of SIRT3, demonstrating the selectivity of SIRT3-mediated deacetylation (Fig. 1b). SIRT3 expression was also upregulated in response to fasting in brown adipose tissue but not in the brain, heart or kidney (Supplementary Fig. 2). Because the liver is an important site of metabolic regulation under fasting conditions, we used a metabolomic approach to screen multiple metabolic pathways. No differences were observed in the levels of 15 amino acids and eight organic acids in livers between fasted