NAD+ metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity

Abstract

Background: NAD+ is a coenzyme for hydride transfer enzymes and a substrate for sirtuins and other NAD+-dependent ADPribose transfer enzymes. In wild-type Saccharomyces cerevisiae, calorie restriction accomplished by glucose limitation extends replicative lifespan in a manner that depends on Sir2 and the NAD+ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Though alterations in the NAD+ to nicotinamide ratio and the NAD+ to NADH ratio are anticipated by models to account for the effects of calorie restriction, the nature of a putative change in NAD+ metabolism requires analytical definition and quantification of the key metabolites.

Results: Hydrophilic interaction chromatography followed by tandem electrospray mass spectrometry were used to identify the 12 compounds that constitute the core NAD+ metabolome and 6 related nucleosides and nucleotides. Whereas yeast extract and nicotinic acid increase net NAD+ synthesis in a manner that can account for extended lifespan, glucose restriction does not alter NAD+ or nicotinamide levels in ways that would increase Sir2 activity.

Conclusions: The results constrain the possible mechanisms by which calorie restriction may regulate Sir2 and suggest that provision of vitamins and calorie restriction extend lifespan by different mechanisms.

Figure 1

De novo synthesis begins with tryptophan, which is converted to NaMN in six enzymatic steps. NaMN is then adenylylated by Nma1 and Nma2 to NaAD, which is converted to NAD⁺ by glutamine-dependent NAD⁺ synthetase, Qns1. NaMN is also formed by salvage of Nam, via nicotinamidase, Pnc1, and nicotinic acid phosphoribosyltransferase, Npt1. Environmental NA also generates NaMN via the phosphoribosylation activity of Npt1. NR is converted to NMN via Nrk1, then to NAD⁺ by Nma1 and Nma2. Additionally, NAR can utilize Nrk1-dependent phosphorylation and both NR and NAR can be converted to NAD⁺ in Nrk1-independent pathways initiated by nucleoside splitting activities. The nucleosides NR and NAR are produced intracellularly by the 5'-nucleotidase activities of Sdt1 and Isn1 acting on NMN and NAR. NAD⁺ is broken down by the sirtuins, Sir2 and Hst1-4.

Figure 2

Chromatograms of 18 NAD⁺-related compounds generated using SRM LC-MS. All compounds except quinolinic acid were detected using positive ion mode. Injected amount was 10 pmol for all compounds.