tract as bomb calorimetry of fecal material from SirT1-null and normal mice were equally depleted of calories (Figure 1D). The low body mass and hyperphagia of the SirT1-null mice might be a consequence of enhanced activity but we found that young SirT1-null mice are much less active than their normal littermates, particularly during the dark period (Figure 2A). As these mice got older, the difference between the genotypes decreased primarily because the activity of normal animals declined (Figure 2B). Thus the small and hyperphagic SirT1-null mice are also lethargic compared to their normal littermates suggesting that there is inefficient usage of ingested calories in these animals. Hormonal profile of SirT1-null mice Systemic energy metabolism is under the control of hormones. Thyroxine (T4) levels were slightly lower in the SirT1-null serum (Figure 3A) whereas peak corticosterone levels were normal (Figure 3B). Fasted glucose levels of SirT1-null mice were higher than those of normal littermates (Figure 3C) but insulin concentrations were not different (Figure 3D). Three hours after re-feeding, the insulin levels of normal animals rose by nearly 30 times whereas the rise in SirT1-null mice was increased only 10 fold (Figure 3D). The postprandial blood glucose concentration was similar or lower in SirT1-null compared to normal mice (Figure 3C), suggesting that SirT1-null mice might be insulin sensitive. A similar result was observed previously [15]. However, insulin tolerance tests did not reveal a difference between SirT1- and normal animals (data not shown). Blood lactate Figure 2. SirT1-null mice are less active than controls. Activity during 60-minute intervals of individually caged SirT1-null and normal littermates 3–6 months (A) and 9–12 months (B) of age. Insets show histograms of total activity in a 24 hour period. Grey and black lines above the X-axis indicate light and dark periods, respectively. Means and standard errors are represented. Unpaired T-tests were performed to assess statistical significance. doi:10.1371/journal.pone.0001759.g002 SirT1 and Caloric Restriction concentrations were also not different between normal and SirT1-null mice suggesting that there was no loss of food energy through excessive glycolysis (data not shown). Indirect calorimetry To estimate metabolic rate and substrate utilization, we measured oxygen consumption and carbon dioxide production from whole animals. When normalized to body weight, oxygen consumption of SirT1-null mice was higher than that of controls, indicating that they are hypermetabolic (Figure 4A, B), a conclusion consistent with their normal food ingestion and lower body mass. The largest difference in oxygen consumption occurred during the light period when both SirT1-null and normal mice are relatively inactive. Respiratory exchange ratio (RER = VCO2/VO2) indicated that SirT1-null animals rely more on lipid substrates (RER values close to 0.7) during the 6–8 hours before the onset of the dark period, the time when they will have their main meal (Figure 4C). This increased fatty acid oxidation in SirT1-null mice is also apparent when data are plotted as percent relative cumulative frequencies (PRCF), as indicated by the shoulder of the mutants’ curve around RER values of 0.7–0.8 (Figure 4D). We measured free fatty acids in the serum of mice fasted overnight and found that the concentrations tended to be lower in SirT1-null mice than in normal mice; however, this difference was not statistically significant (data not shown). It has been proposed that SirT1 is required for the induction and maintenance of fatty acid oxidation in response to low glucose concentration [19]. However, SirT1-null mice appear to readily switch between glucose to lipid utilization for oxidative phosphor- ylation (Figure 4C). To further test their ability to switch substrates from glucose to lipids, we performed fasting/refeeding experi- ments. When mice are fasted, RER drops to 0.7 as lipids are used as oxidation substrates and following refeeding, RER climbs to 1.0 as glucose becomes the predominant energy source. SirT1-null mice switch readily between lipid and glucose substrates as evidenced by the efficient changes in RER (Figure 4E). Interest- ingly, the day after refeeding, RER in SirT1-null animals started to decline much faster than that in normal animals suggesting limited glucose utilization even when food is freely available. The observation that SirT1-null mice have higher rates of lipid oxidation than normal is consistent with previous observations that in older SirT1-null mice the lipid content of cells in white adipose tissue (WAT) are smaller than those of normal tissues [23]. We compared the total weight of some lipid storage tissues in young and old mice. In young animals, the inguinal fat pads from SirT1- mice were smaller than those of their littermates (Figure 5A) and this tissue increased in mass with age much more slowly than normal in SirT1-null mice (Figure 5D). The interscapular BAT depot was similar in size in young and in older mice (Figure 5B, E) perhaps reflecting its role in energy dissipation. The brain weight of SirT1-null mice is about 20% smaller than normal mice and in both young and older animals (Figure 5 C, E). These results coupled with observations noted above [23] are consistent with the notion that SirT1-null mice burn stored lipids at rates in excess of those in normal animals. Liver mitochondria from SirT1-null mice are less efficient than normal SirT1-null mice are hypermetabolic but lethargic, suggesting that their energy generation system might be defective. We investigated the state 3 (maximal phosphorylating) respiration rate as well as proton conductance in isolated mitochondria from liver and skeletal muscle. We detected no significant differences in the mitochondria from skeletal muscle (data not shown). However, the PLoS ONE | www.plosone.org 3 2008 | Volume 3 | Issue 3 | e1759