SirT1 and Caloric Restriction Figure 7. Whole body and organ weights following CR. The weights of whole body (A), brain (B), inguinal fat pad (C), and interscapular BAT (D) were obtained from 13–15 months old mice, AL-fed or CR for 25 to 28 weeks. Percentages above bars represent the percent reduction in weight compared to AL-fed mice. doi:10.1371/journal.pone.0001759.g007 level of lipid oxidation under AL feeding conditions. Because SIRT1 is expressed in all tissues it is difficult to extrapolate information obtained from isolated cells and organs to accurately make predictions regarding the intact animal. The means by which CR extends lifespan is not yet clear. In yeast and D. melanogaster, Sir2 has been shown to be required for CR to increase lifespan [6,22]. Here we show that SirT1 is also required in mammals for some responses to CR. First, we confirmed that the CR-mediated increase in physical activity is not observed in SirT1-null mice [27]. Second, we show that, whereas normal mice maintain their metabolic rate when subject to CR, that of the mutant mice drops dramatically. Finally, SirT1 appears to be required for CR to increase lifespan. The beneficial effects of CR on lifespan of normal mice is typically observed after 20 months of age (e.g. [31]). Because our CR experiment was terminated before animals reached this age, we saw no difference in survival between our CR and AL groups of normal mice. However, we had already lost two-thirds of our SirT1-null mice by the end of this experiment and CR had no obvious beneficial effect on lifespan of these mice. In fact, CR seemed to augment the early demise of SirT1-null mice. We interpret our data to indicate that SirT1-null mice are not capable of adapting to CR conditions. Lifespan is thought to be determined by the accumulation of cellular damage arising from ROS although recent evidence from C. elegans suggests that ROS might in fact be responsible for extending lifespan (Schulz et al., 2007). It is perhaps relevant that SirT1-null mice have liver mitochondria that produce less ROS than normal. In regards to mitochondrial uncoupling, this observation is consistent with increased proton leak in these mitochondria; however, the increased leak is not due to a derepression of ucp2, as one could infer from studies in pancreatic beta-cells [15]. Nevertheless, there are a number of other regulators of mitochondrial activity yet to be investigated [32,33]. In conclusion, our study indicates that the absence of SIRT1 results in a metabolically inefficient animal that fails to adapt to CR conditions. Given that there are over 30 SirT1 substrates to date [10], it is remarkable that SirT1-null mice are viable and can sometimes reach 2 years of age. At this point, it is not yet clear which of the many SIRT1 substrates is/are responsible for the phenotype or which tissue(s) share the metabolic defect. A better understanding of the role of SirT1 in energy metabolism may help in designing strategies to provide the health benefits of CR without curtailing dietary energy intake. Materials and Methods Animals All animal experiments were performed according to the Guidelines for the Care and Use of Animals established by the Canadian Council on Animal Care. The mutant mice used in this study carried the sirt1-null allele previously described [11] main- tained on a mixed genetic background derived from intercrosses between the CD1 out bred strain and 129/J. SirT1-null animals were created by crossing heterozygotes and were identified at PLoS ONE | www.plosone.org 8 2008 | Volume 3 | Issue 3 | e1759