SirT1 and Caloric Restriction Figure 4. SirT1-null mice are hypermetabolic. Whole animal indirect calorimetry (IC) was used to assess oxygen consumption normalized to body weight (VO2/BWT) plotted (panel A) at 30 minute intervals during a 24 hour period or (panel B) as the percent relative cumulative frequency (PRCF) of VO2/BWT. The respiratory exchange ratio (RER = VO2/VCO2) was calculated from VO2 and VCO2 data and plotted at 30 minute intervals during a 24 hour period (panel C) or as PRCF (panel D). In a fasting-refeeding experiment (panel E), food was either removed or added at the indicated times (arrows) and the interval RER plotted. Grey and black lines above the X-axis (panels A, C, and E) indicate light and dark periods, respectively. An RER of 1.0 is expected for glucose oxidation and an RER of 0.7 occurs during lipid oxidation. Means and standard errors are represented. An unpaired T-test using medians was performed to assess statistical significance (B). doi:10.1371/journal.pone.0001759.g004 SirT1-null mice maintains normal ATP levels under AL conditions but fails to maintain ATP levels during fasting. SirT1 is required for CR response in mammals Sir2 is required for lifespan extension in Saccharomyces cerevisiae [22] and Drosophila melanogaster [6] when exposed to caloric restriction (CR). To determine whether SirT1 is also required for CR response in mammals, we subjected 5 to 7 month old SirT1-null mice to a diet with 40% reduced calorie content for up to 44 weeks and compared their response with that of their normal littermates. CR resulted in a reduced body weight that was similar in both normal and SirT1-null animals (Figure 7A). Brain did not show any decrease in weight with CR (Figure 7B) but the inguinal fat pad weight was reduced more dramatically in the SirT1-null mice than in normal animals (Figure 7C). Brown fat was reduced to similar degrees in SirT1-null and normal mice (Figure 7D). These data suggest that SirT1-null mice rely more on lipid mobilized from WAT than normal mice, an observation consistent with the higher lipid utilization of SirT1-null animals under AL conditions. SirT1 protein in heart, liver, BAT and WAT of CR treated normal mice was only marginally, but not significantly, increased compared to AL-fed mice (data not shown), a result different from that previously reported [26]. The daily cumulative physical activity of normal animals increased with CR, as previously reported [27], while that of AL fed normal mice declined with age (Figure 8A). In contrast, the PLoS ONE | www.plosone.org 5 2008 | Volume 3 | Issue 3 | e1759