grounded to powder. Gross energy (GE) of feces was determined using an automatic bomb calorimeter (Parr, 1271, Parr Instru- ments, Moline, IL, USA). Activity monitoring Mice were caged individually and activity was recorded for 24- hour periods in a MicroMax activity monitoring system with 16 infrared beams per cage (AccuScan Instruments, Columbus, OH, USA). Total activity data were used for analyses. Lighting was on a normal 12 h light/dark cycle. Indirect calorimetry Mice were caged individually and oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured using a four- chamber Oxymax system with automatic temperature and light controls (Columbus Instruments, Columbus, OH). Temperature was maintained at 24uC, and lighting was on a normal 12 h light/dark cycle. System settings included a flow rate of 0.5 L/min, a sample line-purge time of 2 min, and a measurement period of 60 s every 12 minutes. The respiratory exchange ratio (RER) was calculated as the ratio of VCO2 produced/VO2 consumed. Isolation of mitochondria Four to 5 month old mice were euthanized by decapitation for isolation of liver and skeletal muscle mitochondria. All media were ice-cold, and the procedures done on ice or at 4uC. Isolation of skeletal muscle mitochondria was performed using a modified method of Chappell and Perry [34]. Briefly, the skeletal muscles (pectoral, forelimb and hind limb muscles) were quickly dissected and placed in basic medium for muscle (BMM: 140 mM KCl, 20 mM HEPES, 5 mM MgCl2, 1 mM EGTA; pH 7.0). Muscle was cleaned of visible connective tissue and fat, minced by razor blade and placed in 15 volumes of homogenizing medium (HMM: BMM supplemented with 1 mM ATP and 1% BSA (w/v)). One unit of protease (Subtilisin A, Sigma, Oakville, ON, Canada) per g tissue wet weight was added to the muscle mixture. Tissue was homogenized using a glass/Teflon Potter-Elvehjem tissue grinder and fractionated by centrifugation at 800 g (10 min). The supernatant was collected and respun at 12,000 g (10 min). The resulting pellet was resuspended in BMM and incubated on ice for 3 min to allow myofibrillar repolymerization. Samples were spun at 800 g (10 min), the supernatant collected then spun at 12,000 g (10 min). The final pellet was resuspended in 220 ml of BM. Liver mitochondria were isolated as described [35]. Briefly, the liver was rapidly dissected and immersed in ice-cold basic medium for liver (BML: 250 mM sucrose; 10 mM Tris-HCl; 1 mM EGTA; pH 7.0), minced, then homogenized in HML (BML plus 0.5% de-fatted BSA) using a glass/Teflon Potter-Elvehjem tissue grinder. Liver homog- enate was centrifuged at 800 g (4 min), followed by a centrifugation of the supernatant at 12,000 g for 7 min to pellet mitochondria. The pellet was washed twice in BML. The final pellet was resuspended in 250–400 ul of BML. Protein concentration was determined using a modified Lowry method with BSA as the standard. Mitochondrial respiration Oxygen consumption was measured in isolated mitochondria (0.5 mg/ml) at 37uC using a Clark-type oxygen electrode (Hansatech, Norfolk, UK) and incubated in standard incubation medium (IM: 120 mM KCl, 1 mM EGTA, 5 mM KH2PO4, 5 mM MgCl2 and 5 mM HEPES; pH 7.4) containing 0.3% defatted BSA and assumed to contain 406 nmol O/ml at 37uC [36]. State 3 (maximum phosphorylating) respiration was determined using 10 mM succinate as substrate, and 250 mM SirT1 and Caloric Restriction ADP and 5 mM rotenone. State 4 (non-phosphorylating, or maximal leak-dependent respiration) was determined following addition of oligomycin (12 mg/mg protein). All measurements were done in duplicate. Mitochondrial protonmotive force A methyl-triphenyl-phosphonium (TPMP+)-sensitive electrode was used to assess mitochondrial protonmotive force (Dp) [37–39] in non- phosphorylating skeletal muscle and liver mitochondria (see above). The TPMP+ electrode was calibrated by sequential 1 mM additions of TPMP+. Nigericin (80 mn/ml) was added to convert the pH component of Dp into mV units. The kinetics of proton conductance was assessed by incremental addition of malonate (up to 5 mM). After each run, 0.2 mM FCCP was added to release TPMP+ for baseline correction. TPMP+ measurements were done in triplicate and simultaneous with oxygen consumption determinations. Mitochondrial H2O2 production capacity Mitochondrial H2O2 production rate was determined in freshly isolated mitochondria from liver using the p-hydroxyphenylacetate (PHPA) fluorometric assay [35,40]. Mitochondria (0.6 mg/ml) were incubated in IM supplemented with 0.3% defatted BSA. H2O2 production was determined using pyruvate/malate (10 mM/5 mM; complex I-linked substrate), succinate (10 mM; complex II-linked substrate), or palmitoylcarnitine (20 mM; lipid-derived substrate that feeds into both complex I and II), under various conditions: 1) succinate, to assess H2O2 production generated by reverse electron flow through complex I, which is highly sensitive to uncoupling of oxygen consumption from oxidative phosphorylation, e.g. through the activation of uncoupling proteins [24]; 2) palmitoylcarnitine in the presence of oligomycin, to assess H2O2 production in resting mitochondria; 3) succinate in the presence of rotenone (5 mM) and antimycin (10 mM) to assess H2O2 production with full reduction of complexes I and III; 4) pyruvate/malate in the presence of antimycin (10 mM) to assess H2O2 production with full reduction of complex III and 5) palmitoylcarnitine in the presence of antimycin (10 mM) to assess H2O2 production with full reduction of complex III. All measurements were performed in the presence of superoxide dismutase (100 U/ml) to convert extra-mitochondrially-released superoxide to H2O2. H2O2 production was monitored for up to 25 min using a temperature-controlled fluorimeter (BioTek, FLx800) at 37uC. Fluorescence readings were converted to H2O2 production rates by use of a standard curve. Data were normalized to amount of proteins. To control for day-to-day variability, paired T-tests were used to determine statistical significance (1 SirT1-null and 1 WT were used simultaneously on the same day). Immunoblots For the evaluation of UCP2 levels, liver mitochondria from SirT1 mutant and WT mice, as well as spleen mitochondria from WT mice, were isolated (see ‘‘Isolation of mitochondria’’ section above) and 30 mg of protein was loaded on a NuPAGE Bis-Tris 4– 12% gradient precast polyacylamide gel (Invitrogen, Burlington, ON, Canada), electrophoresed, blotted on a nitrocellulose membrane and probed with an antibody to UCP2 (C20) (cat. #: sc-6525, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Spleen was used as a positive control for UCP2 expression. Loading was verified by Ponceau Red staining. For evaluation of phospho-AMPKa and AMPKa levels in liver tissue, SirT1 mutant and WT mice were euthanized by cervical dislocation, and pieces of liver were harvested and flash frozen within 1 min. Tissues were homogenized in ice-cold RIPA-phosphatase inhibitor buffer (Tris 20 mM pH 8.0, NP-40 1%, sodium deoxy- cholate 0.25%, NaCl 150 mM, EDTA 1 mM, Complete protease PLoS ONE | www.plosone.org 10 2008 | Volume 3 | Issue 3 | e1759