a. Mitochondria were isolated from livers of fed or fasted (6-48 h) wt mice and analyzed for SIRT3 expression by western blot analysis, electron transfer flavoprotein (ETF) was used as a reference; b. Mitochondria isolated from livers of fed or fasted wt and SIRT3-/- mice were analyzed by western blotting analysis with an antiserum specific for anti-acetyllysine, ATP synthase alpha was used as a reference. The arrows identify candidate SIRT3 target proteins: deacetylated during fasting in wt mice, relatively hyperacetylated and not deacetylated during fasting in SIRT3-/- mice.

a, b. Fatty acid oxidation was measured by incubation of liver extract from wt and SIRT3-/- mice with ¹⁴C-palmitate, acid-soluble metabolites [(ASM), panel a] and captured CO₂ (panel b), n=10/genotype; c. Mitochondrial fatty acid oxidation was measured in other oxidizing tissues, including heart, liver, mixed gastrocnemius and soleus skeletal muscle (SKM), and brown adipose tissue (BAT) (CO₂, n=7/genotype, 100 μM substrate), d. Fatty acid oxidation was measured by incubation of ¹⁴C-palmitate (100 μM) in liver extract from wt and SIRT3-/- mice one week after injection with adenovirus expression vectors for GFP or SIRT3 (ASM, n=3-4/category); *p<0.05, **p<0.01.

a. Hepatic ATP levels were measured in fed and fasted wt and SIRT3-/- mice (n=5/genotype/condition) b, c. Core temperature (b) and blood glucose (c) were measured in fed and fasted wt and SIRT3-/- mice exposed to cold (4°C) for 6 h (n=5/genotype/condition); *p<0.05, **p<0.01

a, b. Metabolomic analyses were conducted on mouse liver tissue (a) and plasma (b), data obtained in SIRT3-/- mice are shown relative to wt mice (n=5/genotype, fasted 24 h); c. Livers extracts from SIRT3-/- and wt mice were analyzed for total phospholipids, triglycerides and cholesterol esters (n=5/genotype, fed or fasted 24 h), *p<0.05

a. Liver extracts from wt and SIRT3-/- mice (fed or fasted) were immunoprecipitated with an anti-acetyllysine antiserum and analyzed with anti-LCAD antiserum; b. Expression vectors for wt SIRT3, SIRT3-H248Y (catalytically-inactive SIRT3 mutant), SIRT4, or SIRT5 were co-transfected with expression vectors for FLAG-tagged LCAD and the level of LCAD acetylation was assessed; c. Recombinant LCAD expressed in E. coli was incubated in vitro with recombinant SIRT3 or SIRT3-H248Y, and LCAD acetylation status was assessed; d. Expression vectors for wt SIRT3, SIRT3-H248Y, SIRT4 and SIRT5 (HA-tagged) were co-transfected with expression vectors for FLAG-tagged LCAD and assessed for interaction by co-immunoprecipitation; e. LCAD was expressed and purified with SIRT3 or SIRT3-H248Y and its enzymatic activity measured in vitro using 2, 6 dimethylheptanoyl-CoA as a substrate (n=4 independent assays); f. Recombinant LCAD was expressed in E. coli in the absence (Control) or presence of nicotinamide (NAM, 50 mM), purified and its enzymatic activity measured in vitro using 2, 6 dimethylheptanoyl-CoA as a substrate (n=4 independent assays); g. Expression vectors for wt LCAD, LCAD single acetylation point mutant (LCAD-K42R), or LCAD eight acetylation point mutant (LCAD-8KR) were co-transfected with expression vectors for wt SIRT3 or SIRT3-H248Y, and the level of acetylation was assessed; h. Wild-type LCAD, LCAD-K42R, or LCAD-8KR were expressed, and measured for enzymatic activity in vitro using 2, 6 dimethylheptanoyl-CoA as a substrate (n=5 measurement/sample, error bars represent data two independent protein purifications); *p<0.05, **p<0.01.