Nicotinamide adenine dinucleotide (NAD(+)) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD(+) decomposition products. NAD(+) biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD(+) biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD(+) and NADH (the reduced form of NAD(+)) analyses on BY4742 wild-type, NAD(+) salvage pathway knockout (npt1Delta) and NAD(+) de novo pathway knockout (qpt1Delta) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized (14)C labelled nicotinic acid in the culture media combined with HPLC speciation and both UV and (14)C detection to quantitate the total amounts of NAD(+) and NADH and the amounts derived from the salvage pathway. We observed that wild-type and qpt1Delta yeast exclusively utilized extracellular nicotinic acid for NAD(+) and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions, suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observed that NAD(+) concentrations decreased in all three strains under CR. However, unlike the wild-type strain, NADH concentrations did not decrease and NAD(+): NADH ratios did not increase under CR for either knockout strain. Lifespan analyses revealed that CR resulted in a lifespan increase of approximately 25% for the wild-type and qpt1Delta strains, while no increase in lifespan was observed for the npt1Delta strain. In combination, these data suggest that having a functional salvage pathway is required for lifespan extension under CR. Copyright (c) 2009 John Wiley & Sons, Ltd.
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