p2 in pancreatic beta-cells. The level of ucp2 mRNA was measured in liver from SirT1-null and normal mice but no difference in abundance was detected. Western blots 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 MedChemExpress 405169-16-6 species. 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, consistent with the observation that these mitochondria have a higher proton leak. 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 to provide reducing equivalents to the ETC. Mitochondria were subjected to three different substrates 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 SirT1null liver produced similar levels of H2O2 under this condition. On the other hand, pyruvate+malate and palmitoylcarnitine 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, suggesting that SirT1-null mitochondria 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 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-AMPKa, in liver from mice fed AL or fasted for 24 hours. Similar levels of activated pAMPK were observed in 1828342 liver from AL-fed mice, regardless of the genotype. Normal mice had reduced pAMPK after 24 hour fasting while SirT1-null liver had a 2-fold increase in pAMPK signal. These data suggest that liver from SirT1 and Caloric Restriction 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 and Drosophila melanogaster when exposed to caloric restriction. 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 16103101 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. Brain did not show any decrease in weight with CR but the inguinal fat pad weight was reduced more dramatically in the SirT1-null mice than in normal animals. Brown fat was reduced to similar degrees in SirT1-null and normal mice. 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-