SirT1 and Caloric Restriction Figure 3. Serum thyroxin (T4), corticosterone, glucose and insulin levels. A. T4 levels were measured from serum samples of SirT1-null and normal mice using an EIA kit. B. Corticosterone levels were measured from plasma samples of SirT1-null and normal mice at 4 hours intervals during the diurnal cycle using an EIA kit. The peak values are plotted. Unpaired T-tests were performed to assess statistical significance. Blood glucose (C) and insulin (D) levels of 2–3 months old mutant and wild type mice were measured after 24 hour fasting and again 3 hours after refeeding using a standard glucometer (C) and an ELISA kit (D). Means and standard errors are represented. Two-way ANOVAs was performed to assess statistical significance of interaction between genotype and dietary condition (p*-values on top of panel), and unpaired T-test to assess difference between genotypes within dietary condition groups (p-value over bars). doi:10.1371/journal.pone.0001759.g003 rate of respiration under state 3 conditions was lower in SirT1-null liver mitochondria suggesting that these mitochondria would produce less ATP at full capacity than those from normal mice (Figure 6A). Measurement of proton motive force under state 4 (non-phosphorylating) respiration conditions (Figure 6B) revealed that it was lower in SirT1-null liver mitochondria. This suggests that the inner membranes of the SirT1-null mitochondria are relatively permeable to protons and that less proton motive force would be generated to produce ATP. Uncoupling proteins such as UCP2 can diminish proton motive force and SirT1 is known to inhibit the expression of ucp2 in pancreatic beta-cells [15]. The level of ucp2 mRNA was measured in liver from SirT1-null and normal mice but no difference in abundance was detected (data not shown). Western blots (Figure 6C) indicated that no UCP2 protein was detectable in mitochondria from either normal or SirT1-null liver. In addition to ATP synthesis, mitochondria are the major site for the generation of reactive oxygen species (ROS). We measured the capacity of liver mitochondria to produce H2O2 under several conditions. In the presence of succinate or palmitoylcarnitine, liver mitochondria from SirT1-null animals produced less H2O2 than normal (Figure 6D), consistent with the observation that these mitochondria have a higher proton leak (see Figure 6B and [24,25]). We also took advantage of this H2O2 production assay as an indirect method to probe the capacity of pathways upstream of the electron transport chain (ETC) to provide reducing equivalents (RE) to the ETC. Mitochondria were subjected to three different substrates (succinate, pyruvate+malate or palmitoylcarnitine) in the presence of antimycin so that H2O2 would be produced predominantly from complex III in all conditions. Succinate feeds into complex II directly, so the H2O2 produced using this substrate is not affected by the capacity of any pathways upstream of the ETC to provide RE. Mitochondria from both normal and SirT1- liver produced similar levels of H2O2 under this condition (Figure 6E). On the other hand, pyruvate+malate and palmitoyl- carnitine first need to be oxidized through beta-oxidation and/or the TCA cycle, so the H2O2 produced using these substrates is affected by the capacity of these upstream pathways to provide reducing equivalents to the ETC. SirT1-null mitochondria produced higher levels of H2O2 when using pyruvate+malate or palmitoylcarnitine (Figure 6E), suggesting that SirT1-null mito- chondria have higher than normal beta-oxidation and TCA cycle capacities. These results are consistent with an increased lipid utilization in SirT1-null mice. AMP-activated protein kinase (AMPK) is a sensor for the availability of energy in cells. Our observations with SirT1-null liver mitochondria suggest that they are less efficient than normal in producing ATP. We measured the levels of the active form of AMPK, phopho(Thr172)-AMPKa (pAMPK), in liver from mice fed AL or fasted for 24 hours. Similar levels of activated pAMPK were observed in liver from AL-fed mice, regardless of the genotype (Figure 6F–G). Normal mice had reduced pAMPK after 24 hour fasting while SirT1-null liver had a 2-fold increase in pAMPK signal (Figure 6F–G). These data suggest that liver from PLoS ONE | www.plosone.org 4 2008 | Volume 3 | Issue 3 | e1759