SirT1 and Caloric Restriction Figure 8. Activity of mice during CR. Total activity of normal (A) and SirT1-null (B) mice was monitored using a Micromax system for 24- hour periods at the indicated time after the start of CR. Animals were 5– 7 months old at the beginning of CR. Means and standard errors are represented. The activity of CR normal mice increased with time whereas that of AL controls decreased. CR did not alter the activity of SirT1-null mice. doi:10.1371/journal.pone.0001759.g008 weaning by a characteristic eyelid defect. The genotypes of animals were determined by a PCR-based test carried out on DNA isolated from tail tip biopsy. The primers TTCACATTGCATGTG- TGTGG and TAGCCTGCGTAGTGTTGGTG amplify a 423 bp fragment from the normal sirt1 allele while a 526 bp fragment from the allele is amplified from the first primer and Figure 10. CR had no beneficial effect on survival of SirT1-null mice. Survival curve for normal (A) and SirT1-null (B) mice that were fed the CR of AL diets. Mutant mice that were euthanized because of eyelid inflammation were excluded from this graph. Grey rectangles represent the period when CR started (5 to 7 months old). doi:10.1371/journal.pone.0001759.g010 ATTTGGTAGGGACCCAAAGG, a sequence derived from the pgk-1 gene inserted to create the allele by homologous recombination. sirt1-null mice were normally housed in cages with littermates of the same sex. Feces and bomb calorimetry Two to four months old mice were caged individually in metabolic chambers and feces were collected after 48 to 72 hours. Feces were dehydrated in a speedvac at 50uC overnight and Figure 9. Whole animal oxygen consumption during CR. Oxygen consumption normalized to whole body weight (VO2/BWT) for normal (A, D) and SirT1-null (B, E) mice was measured after 2–4 weeks (A–C) and 21–26 weeks (D–F) of CR or AL diet as indicated. RER from the same animals at 2– 4 weeks (C) or 21–26 weeks (F) of CR were also obtained. Data were plotted as PRCF as in Figure 4. The normal mice adapted to CR by maintaining their levels of oxygen consumption but the oxygen consumption of SirT1-null animals was higher in AL than in CR. This difference was particularly evident in older SirT1-null mice (panel E) when AL animals’ oxygen consumption was very high. doi:10.1371/journal.pone.0001759.g009 PLoS ONE | www.plosone.org 9 2008 | Volume 3 | Issue 3 | e1759