Calorie restriction decreases intracellular NADH levels. Measurements of intracellular levels of NAD (A,C) and NADH (B,D) in various yeast strains grown in 2% or 0.5% glucose. Results show average of three independent experiments, each conducted in duplicate. Error bars denote standard deviations. NAD and NADH levels are shown as levels of NAD or NADH (in micromolar) normalized to the concentrations of proteins from the extracts (in micrograms) present in the reaction. Reactions contain cell extracts from 10⁵ cells. (WT) Wild-type yeast strain PSY316; (+Hap4-oe) wild-type cells overexpressing Hap4; (2%) cells grown in 2% glucose; (0.5%) cells grown in 0.5% glucose. npt1Δ, cyt1Δ, and sir2Δ hmr1Δ are isogenic derivatives of PSY316.

NADH inhibits Sir2 NAD-dependent histone deacetylase activity. (A) Kinetic analysis of the Sir2 NAD-dependent histone deacetylase activities in the presence of 0 μM (filled squares), 50 μM (open squares); 100 μM (filled circles), or 250 μM (open circles) NADH. Here 150 ng of recombinant GST-tagged yeast Sir2 protein was assayed with various concentrations of NAD and NADH for 2.5 h at 30°C. Data are shown as a Lineweaver-Burk double reciprocal plot of 1/V (CPM/h) versus 1/[NAD] (μM). The results show the average of three independent experiments, each measured in duplicate. (B) Kinetic analysis of the human SIRT1 NAD-dependent histone deacetylase activities in the presence of 0 μM (filled squares), 50 μM (open squares), 150 μM (filled circles), or 300 μM (open circles) NADH. Experiments were carried out as in A, except that 50 ng of recombinant GST-tagged human SIRT1 protein (Takata and Ishikawa 2003) was used, and the reaction was carried out for 2 h at 37°C.

Overexpressing NADH dehydrogenase increases the intracellular NAD/NADH ratio and life span. (A) Measurements of NADH (closed bars, left panel) and NAD (open bars, right panel) levels in cells overexpressing Nde1 and Nde2. The results show the average of four independent experiments, each conducted in duplicate. Error bars denote standard deviations. (B) Life-span analysis of cells overexpressing Nde1 and Nde2 grown in 2% and 0.5% glucose. Average life spans on 2% glucose: +vectors, 19.4; +Nde1-oe, +Nde2-oe: 27.2. Average life spans on 0.5% glucose: +vectors, 25.8; +Nde1-oe, +Nde2-oe: 27.1. (+ vectors) Cells carrying control vectors ppp35 and ppp81; (+Nde1-oe, +Nde2-oe) cells overexpressing Nde1 and Nde2.

Calorie restriction in pnc1Δ mutants. (A) Calorie restriction slightly extends life span in a pnc1Δ mutant. (B) Overexpressing Nnt1 restores the full life-span extension by calorie restriction in a pnc1Δ mutant. Average life spans on 2% glucose: pnc1Δ + vector, 20.9; + vector, 20.16; Δpnc1 +Nnt1-oe, 21.6. Average life spans on 0.5% glucose: pnc1Δ + vector, 23.9; + vector, 27.3; pnc1Δ +Nnt1-oe, 28. (+ vector) cells carrying a control vector ppp35; (+Nnt1-oe) cells overexpressing Nnt1. pnc1Δ was derived from PSY316.