SirT1 and Caloric Restriction Figure 5. Organ weights of SirT1-null mice. Organs from 3–5 month (A–C) and 13–26 month old mice (D–F) were weighed and plotted. Inguinal fat pad (A, D), interscapular BAT (B, E) and brain (C, F) are plotted as means and standard errors. Unpaired T-tests were performed to assess statistical significance. doi:10.1371/journal.pone.0001759.g005 physical activity of SirT1-null mice did not increase with CR but remained similar to that of AL fed animals (Figure 8B). Despite a 40% reduction in caloric intake, body weight- normalized oxygen consumption of normal mice subjected to CR was the same or slightly higher than AL fed animals (Figure 9A, D). The situation with SirT1-null animals was different. Oxygen consumption of SirT1-null mice was significantly lower in CR animals compared to AL fed mice (Figure 9B, E). We interpret this to indicate that the SirT1-null mice are unable to adapt to the reduced calorie intake and consequently their metabolic rate is reduced. RER values of SirT1-null and control mice under CR were similarly low with values close to 0.7 (,70% of all values), indicating a preferential oxidation of lipids (Figure 9C, F). The SirT1-null animals are less fit than normal and many fail to survive the first year following birth. As CR is a well-established means of prolonging the lifespan of mice, we looked at the survival of SirT1-null and normal mice during our CR experiments. These experiments involved relatively few animals but the data seem to indicate that the decreased viability of SirT1-null mice was exacerbated during CR (Figure 10B). Normal animals were too young to suffer significant loss of viability during the course of this experiment so we have no evidence for an effect of CR on their lifespan (Figure 10A). Discussion + Since its catalytic activity is dependent on NAD , SirT1 deacetylase activity has been postulated to be controlled by the metabolic state of the cell [1,2]. In fact, our observations suggest that SirT1 is an important regulator of metabolic activity because SirT1-null mice utilize ingested calories inefficiently and because SirT1-null mice do not adapt normally to CR or to fasting. SirT1-null mice are smaller and lethargic compared to their normal littermates but, per gram of body weight, they consume more food and oxygen. Thus, the SirT1-null animal is metabolically inefficient compared to normal. Liver mitochondria appear to have a lower capacity to produce ATP because of lower state 3 respiration capacity and higher proton leak through the inner mitochondrial membrane. Perhaps to compensate for their reduced oxidative phosphorylation capacity, mitochondria from SirT1-null liver have increased capacity for TCA cycle and beta- oxidation. This compensation seems to be successful in maintain- ing the level of ATP but overnight fasting resulted in much higher levels of phospho-AMPK suggesting that ATP levels are not sustained in SirT1-null mice in the face of food deprivation. SirT1 is known to modulate the activities of several regulators of metabolism but an examination of the characteristics of the SirT1- mouse and the expectations based on published studies yields a number of unexpected contradictions. As a negative regulator of PPARc, SirT1 decreases transcription of genes involved in fat storage [18] so the SirT1-null mice would be expected to have increased WAT depots; in fact WAT is reduced in these mice. SirT1 is a positive regulator of PGC1a [21,28], so one might expect that tissues from SirT1-null mice would have fewer mitochondria. This does not seem to be the case as determined by measurements of the mitochondrial DNA copy number. Recent reports have indicated that SirT1 regulates insulin signaling either directly by deacetylation of IRS2 [29] or indirectly by derepressing PTP1B [30]. Both mechanisms would predict that SirT1-null mice should be insulin resistant. We found that the SirT1-null animals showed normal insulin-dependent glucose regulation. SirT1 is reported to be required for the switch from glucose to lipid oxidation [19] but SirT1-null mice had no difficulty in making this switch; in fact, these animals had a higher PLoS ONE | www.plosone.org 6 2008 | Volume 3 | Issue 3 | e1759