Nicotinamide adenine dinucleotide (NAD+) is synthesized via two major pathways in both prokaryotic and eukaryotic systems (Anderson et al., 2002; Grubmeyer et al., 1999; Panozzo et al., 2002; Sandmeier et al., 2002; Smith and Boeke, 1997) (Figure 1). In one pathway, NAD+ is synthesized from tryptophan (the de novo pathway). In the other, NAD+ is generated by recycling NAD+ degradation products such as nicotinamide (the salvage pathway). In the salvage pathway, nicotinic acid can be taken up by the yeast from the extracellular environment via the nicotinic acid permease transporter (Llorente and Dujon, 2000). Other points of entry into the salvage pathway are by de novo synthesis of NAD+ from tryptophan (Kucharcyzk et al., 1998).
Calorie restriction (CR) extends lifespan in a wide spectrum of organisms, although the mechanism by which this regimen slows ageing is unknown. CR can be imposed in yeast by reducing the glucose concentration in the medium from 2% to 0.5% (Lin et al., 2000). As cells continue to feed on other culture materials, which are rich in amino acids, nucleotides, and vitamins, the growth rate remains strong with the lower glucose levels but imposes a state of partial energy (ATP) limitation. Under these CR conditions, mother cells divide ∼30% more prior to senescence. This increase in replicative lifespan does not occur in strains in which NAD+ synthesis is reduced by removal of salvage pathway function linking an increase in yeast replicative lifespan with the salvage pathway for NAD+ biosynthesis (Lin et al., 2000). Deletion of NPT1, the gene responsible for the conversion of nicotinic acid into nicotinic acid mononucleotide, a precursor to NAD+ in the salvage pathway, prevented an increase in yeast life extension under calorie-restricted conditions. Deletion of QPT1, the gene responsible for conversion of tryptophan products into NAD+ through the de novo biosynthesis pathway, failed to affect lifespan increases under CR. Overexpression of Npt1, a salvage pathway protein responsible for the conversion of nicotinic acid into nicotinic acid mononucleotide, has also been shown to extend lifespan in a manner similar to calorie restriction (Anderson et al., 2002). Increases in lifespan have also been observed when Pnc1, Nma1 and Nma2, all proteins involved in the NAD+ salvage pathway, were overexpressed, further supporting a role for salvage pathway activity in lifespan extension (Anderson et al., 2002). However, the relative contribution of each pathway to NAD+ biosynthesis under both normal and CR conditions is not known. Here we report on the relative contribution of the salvage pathway to NAD+ biosynthesis under both normal and CR conditions.
Materials and methods
Yeast Saccharomyces cerevisiae BY4742 strain MATα his3Δ 1 leu2Δ 1 lys2Δ 0 ura3Δ 0 was acquired from Open Biosystems. The salvage and de novo knockout strains, npt1Δ and qpt1Δ, respectively, were generated by replacing the wild-type (WT) genes with the Kanr marker (G418 marker), as described (Lin et al., 2004). Both wild-type and knockout strains were cultured in synthetic complete medium with 20 g/l glucose (to simulate normal growth conditions) or 5 g/l glucose (to simulate CR conditions) (Sigma-Aldrich, St Louis, MO, USA), as previously described (Sporty et al., 2008). The nicotinic acid content of the synthetic complete medium was determined, using the protocol of Hengen et al. (1978), to be 570 µg/l. The unlabelled medium was supplemented with (54 mCi/mM) carboxy-14C-nicotinic acid (Moravek Biochemicals, Brea, CA, USA), so that the final activity of the medium was 45 pCi/25 ml culture, corresponding to a total additional nicotinic acid supplement of 103 pg and 6.34 µmol 14C labelled nicotinic acid/mol of unlabelled nicotinic acid in the growth medium. Cultures were maintained until they had undergone ∼10 doublings and contained ∼7 × 106 cells/ml, corresponding to mid-log-phase growth.
NAD+ and NADH extraction and quantitation
NAD+ and NADH were extracted using a single sample extraction and HPLC processing procedure that enabled the isolation and quantitation of total cellular NAD+ and NADH from pools of yeast (Sporty et al., 2008). UV absorbance was monitored at 260 nm for NAD+ and NADH and at 340 nm for NADH only, and pertinent peak areas were integrated using area-under-the-curve algorithms. Peak identification and calibration of NAD+ and NADH areas for quantitation were determined using standard solutions of authentic NAD+ and NADH (Sigma-Aldrich).
HPLC fractions were collected, dried and converted to graphite as previously described (Buchholz et al., 2000; Getachew et al., 2006; Miyashita et al., 2001). 14C contents in the graphite samples were quantified by AMS (Ognibene et al., 2002). 14C contents [units, attomole (amol) 14C] for fractions that contributed to either the NAD+ or NADH peaks were integrated, corrected for recovery efficiency (Sporty et al., 2008) and converted to salvage pathway NAD+ or NADH contents, assuming that 14C-labelled NAD+ and NADH were derived from the 14C-labelled nicotinic acid and using the ratio of moles of 14C labelled nicotinic acid : moles of unlabelled nicotinic acid in the growth medium of 6.34 × 10−6:1.
Lifespan analyses for wild-type yeast, qpt1Δ and npt1Δ mutants grown on 2% and 0.5% glucose were carried out as previously described (Lin et al., 2000). All lifespan analyses in this study were carried out independently at least twice, with >45 cells/strain/experiment.
Conversions and statistics
Metabolite contents in units of amol/cell were calculated from both the measured UV absorbance and the measured 14C content, using the number of cells in the extraction. Metabolite contents (amol/cell) were converted to concentrations (mM) using a derived cell volume for each yeast strain. Mean yeast diameters were determined for all three strains by measuring the diameters of at least 30 individual yeast cells, using a high-magnification optical Zeiss microscope (Carl Zeiss Maple Grove, MN, USA) and assuming that the yeast were spherical. Differences in metabolite contents were assessed by unpaired two-tailed Student's t-tests. A significance level of <0.05 was considered meaningful. A significance level of 0.05–0.10 was considered evidence of a possible trend, while a significance level of >0.10 was considered to indicate no significant difference.
Results and discussion
Total yeast cellular NAD+ and NADH contents (amol/cell) measured by UV detection, and 14C-NAD+ and 14C-NADH yeast contents measured by AMS, under normal and calorie-restricted conditions, are presented in Table 1. Optical microscopy yielded a mean diameter of 5.1 ± 0.3 µm for wild-type yeast. Assuming yeast to be spherical, with a volume of 4/3 × π × R3, where R is the radius of 2.5 µm, yields a mean volume of approximately 70 µm3. Both knockout strains were slightly smaller than the wild-type strain. Optical microscopy of the de novo pathway knockout (qpt1Δ) and salvage pathway knockout (npt1Δ) yeast strains respectively revealed mean diameters of 4.3 ± 0.3 µm and 4.2 ± 0.4 µm, and a mean volume of 40 µm3/cell was derived for each strain. Thus, the amol/cell data in Table 1 were converted to mM concentrations shown in Table 2, using a volume of 70 µm3 for the wild-type yeast and 40 µm3 for the two knockout strains. Total NAD+ and NADH concentrations (mM), NAD+: NADH ratios for the BY4742 wild-type, NAD+ salvage pathway knockout, and NAD+de novo pathway knockout yeast strains are shown in Table 2, together with previously published data on NAD+ and NADH concentrations in wild-type yeast. Lifespan analyses for wild-type yeast, qpt1Δ and npt1Δ mutants grown on 2% and 0.5% glucose are shown in Figure 2.
Table 1. Quantitation of NAD+ and NADH contents (amol/cell) via HPLC with UV detection (UV) and HPLC with 14C detection (AMS) for wild-type, qpt1Δ and npt1Δ yeast under both normal and CR growth conditions
Normal (2% glucose)
Calorie-restricted (0.5% glucose)
AMS : UV
AMS : UV
AMS : UV
AMS : UV
The ratio of AMS- to UV-quantitated values under the two growth conditions is also shown (AMS : UV). Each value is the mean and standard deviation of at least three replicate experiments (n = 3–6).
Table 2. Previously reported total cellular NAD+, NADH and NAD+ + NADH concentrations (mM) and NAD+ : NADH ratios, together with those from this study for wild-type yeast, npt1Δ and qpt1Δ strains under normal (2% glucose) and calorie-restricted (0.5% glucose) growth conditions
NAD+ + NADH (mM)
NAD+ : NADH
Reported values were converted to concentrations (mM) as previously described (Lin et al., 2004).
p < 0.05 compared to present study wild-type.
p > 0.1 compared to present study wild-type.
0.05 < p < 0.10 compared to present study wild-type.
p < 0.05 compared to NAD+ under normal growth.
p < 0.05 compared to NADH under normal growth.
0.05 < p < 0.10 compared to qpt1Δ NADH levels under normal growth.
p > 0.10 compared to npt1Δ NADH levels under normal growth.
p < 0.05 compared to NAD+ : NADH ratio under normal growth conditions.
p > 0.1 compared to NAD+ : NADH ratio under normal growth.
Here, we have used 14C-nicotinic acid to specifically label NAD+ and NADH through the salvage pathway. The sensitivity provided by AMS allows for low levels of nicotinic acid, about 103 pg, to be added to 25 ml medium that has an endogenous content of 14.25 µg, yielding a molar ratio of 14C-labelled nicotinic acid : total nicotinic acid in the growth medium of 6.34 × 10−6:1. Consequently, addition of the labelled nicotinic acid does not significantly perturb media conditions and the growth medium remains physiologically relevant. UV quantitation provides total NAD+ and NADH concentrations in cells, while AMS quantitates 14C-labelled NAD+ and NADH contents. Only NAD+ and NADH derived from the importation of 14C-nicotinic acid into the salvage pathway should contain a 14C label. The AMS : UV ratios for NAD+ and NADH in Table 1 indicate the fraction of total NAD+ or NADH that is synthesized from the salvage pathway.
Table 1 reveals that UV-quantified NAD+ and NADH levels were statistically similar to AMS-quantified 14C-NAD+ and 14C-NADH levels for both normal and CR conditions in the qpt1Δ strain. Furthermore, the AMS : UV ratios in Table 1 for the qpt1Δ strain indicate that all cellular NAD+ and NADH is derived from extracellular nicotinic acid via the salvage pathway, under both normal growth and CR conditions. This is not surprising, since deletion of QPT1 should prevent conversion of tryptophan products into NAD+ through the de novo biosynthesis pathway. Under such a scenario, all cellular NAD+ and NADH must come from the salvage pathway.
Conversely, the UV and AMS data and AMS : UV ratios in Table 1 for npt1Δ cells indicate that 14C-labelled NAD+ and NADH are completely absent under both normal growth and CR conditions. 14C-labelled NAD+ and NADH contents quantified by AMS were not statistically significant from zero for all npt1Δ samples grown under either normal or CR conditions. Again, such a finding is not surprising, since deletion of NPT1 should disrupt the salvage pathway and prevent conversion of recycled and imported nicotinic acid into NAD+ through the salvage biosynthesis pathway. All cellular NAD+ and NADH must come from the de novo pathway in that situation. The AMS data and AMS : UV ratios for NAD+ and NADH in Table 1 from the qpt1Δ and npt1Δ strains indicate that both our experimental design and extraction/purification/labelling strategies appear robust.
The AMS : UV ratio data in Table 1 indicate that UV-quantified NAD+ and NADH levels were statistically similar to AMS-quantified 14C-NAD+ and 14C-NADH levels in wild-type yeast. The AMS : UV ratios in Table 1 for the wild-type yeast indicate that, like the qpt1Δ strain, all cellular NAD+ and NADH is derived from extracellular nicotinic acid under both normal growth and CR conditions. Because disruption of the salvage pathway in the npt1Δ strain prevents incorporation of 14C from extracellular labelled nicotinic acid into NAD+ and NADH, it is clear that NAD+ and NADH in the wild-type strain are derived solely from the salvage pathway. These findings are consistent with the absence of salvage pathway activity in npt1Δ yeast grown under CR (Anderson et al., 2002). Furthermore, the salvage pathway is exclusively used under both normal and CR conditions. Under normal growth conditions in which extracellular nicotinic acid concentrations are not growth-limiting, yeast preferentially use the salvage pathway over the de novo synthesis pathway, likely conserving tryptophan, the NAD+ precursor for de novo biosynthesis, for protein building, ultimately resulting in exponential cell growth.
Table 1 also reveals that both knockout strains have lower NAD+ and NADH contents (units of amol/cell) than the wild-type strain. Such a finding is not surprising, since the knockout strains had smaller sizes and smaller volumes (40 µm3) than the wild-type strain (70 µm3). The calculated volume of 70 µm3/cell for the wild-type yeast strain is identical to that assumed previously (based on the work of Sherman, 2002) in calculating yeast metabolite concentrations (Lin et al., 2004; Sporty et al., 2008) and indicates that our methodology for calculating yeast cell volumes is appropriate. Other investigators have also noted that yeast knockout strains can have significantly smaller volumes than wild-type yeast (Jorgensen et al., 2002). Converting NAD+ and NADH contents (amol/cell) from Table 1 to NAD+ and NADH concentrations (mM) shown in Table 2 allows us to make more meaningful comparisons between the three strains and also to compare our data with those from previously reported studies.
Table 2 reveals that NAD+ concentrations from wild-type yeast grown under normal or CR conditions are comparable to those found in previously published reports (Anderson et al., 2002; Lin et al., 2004; Sporty et al., 2008). Table 2 also reveals that NAD+ concentrations from the qpt1Δ strain are statistically similar to NAD+ concentrations from wild-type yeast for both normal and CR growth conditions. However, the npt1Δ strain possesses a NAD+ concentration that is only three-quarters of that found in the wild-type strain during normal growth conditions and possesses an NAD+ concentration that is only two-thirds of that found in the wild-type strain during CR. These differences in NAD+ concentration are both significant (p < 0.5). This finding is consistent with previous studies that found that deleting the NPT1 gene decreases the NAD+ level, whereas deleting the QPT1 gene had no effect on NAD+ level (Sandmeier et al., 2002; Smith and Boeke, 1997).
Lin et al., 2004 have suggested that CR extends yeast lifespan by lowering the level of NADH resulting in increased NAD+: NADH ratios. However, they have only reported NAD+ and NADH measurements in wild-type yeast. Our NAD+ and NADH concentrations and NAD+: NADH ratios (Table 2) for wild-type yeast under normal and CR conditions are comparable to those of Lin et al. (2004) and our previous data (Sporty et al., 2008). However, under normal growth conditions total NAD+: NADH ratios for the knockout strains were significantly higher than those observed in wild-type cells and NADH levels were significantly depressed. Low NADH levels for the knockout strains under normal growth could be associated with smaller cell sizes for the npt1Δ and qpt1Δ strains. The citric acid cycle, which reduces NAD+ to generate NADH, is coupled to oxidative phosphorylation by the oxidation of NADH, yielding ATP that is required for cell growth, reproduction and survival. Such low levels of NADH may inhibit cellular anabolic processes and, plausibly, cell growth.
Our metabolite data for CR reveal that, similar to the work of Lin et al., (2004) and Sporty et al. (2008), NAD+: NADH ratios are observed to increase for the wild-type strain (Table 2). However, NAD+: NADH ratios do not increase under CR conditions for either of the knockout strains. Furthermore, unlike wild-type yeast, where NADH concentrations decrease, NADH levels show evidence of a possible increase in the qpt1Δ mutant and do not decrease in the npt1Δ mutant during CR. In contrast to previous studies (Lin et al., 2004; Sporty et al., 2008), we also found that NAD+ concentrations decrease in all three strains under CR.
Our lifespan data (Figure 2) for wild-type yeast, qpt1Δ and npt1Δ mutants grown on 2% glucose and 0.5% glucose are consistent with those previous reports (Lin et al., 2000). CR results in an average increase in lifespan of approximately 25% in the wild-type yeast and qpt1Δ mutant, while no increase in lifespan is observed for the npt1Δ mutant.
Studies of ageing in S. cerevisiae have also led to the finding that the SIR2 gene regulates the lifespan in yeast mother cells (Kaeberlein et al., 1999). This increase in replicative lifespan does not occur in a sir2 mutant or in strains in which NAD synthesis is reduced (Lin et al., 2000). The silencing activity of Sir2p increases in CR cells (Lin et al., 2000) and this increased activity is required for increased lifespan in CR. One possibility is that the activation of respiration converts more NADH to NAD+ (Lin et al., 2004) and the resulting increase in the NAD/NADH ratio activates Sir2p. It has also been suggested that nicotinamide, which is generated during the deacetylation reaction and can inhibit Sir2p in vitro, is a negative regulator of Sir2p in vivo (Bitterman et al., 2002). Other work has suggested that increased expression of the PNC1 gene, which encodes an enzyme that deaminates nicotinamide, is necessary for lifespan expansion by CR (Anderson et al., 2003), and that nicotinamide depletion is sufficient to activate Sir2p. However, because PNC1 converts nicotinamide to nicotinic acid as part of the NAD salvage pathway, it is plausible that these mechanisms are not mutually exclusive. Our data, together with those of others, show that rDNA silencing and lifespan extension under CR are lost in the npt1Δ mutant (Lin et al., 2000; Smith and Boeke, 1997). For the npt1Δ mutant, it is possible that, although our data indicate that the NAD+: NADH ratio is high, the NAD+ level is too low to activate selected NAD+-dependent enzymes. For the qpt1Δ mutant, our data and those of others show that deleting QPT1 has no effect on total NAD+ levels or rDNA silencing or lifespan (Lin et al., 2000; Sandmeier et al., 2002; Smith and Boeke, 1997). Since lifespan extension for both wild-type and qpt1Δ yeast occurs under CR, while the NAD+: NADH ratio significantly decreases for the mutant but increases for the wild-type strain, our data suggest that a high NAD+: NADH ratio under CR alone is not sufficient to extend lifespan. This suggests that perhaps a threshold level of NAD and/or NADH is required for CR-induced beneficial effects, and increased metabolic activity may also play an important role in CR. Our data also suggest that QPT1 plays essentially no role in NAD+ production in wild-type yeast, yet the qpt1Δ strain has a phenotype of smaller NADH levels and cell size, suggesting that there may be another role for QPT1 in cellular metabolism.
In summary, we have shown that both wild-type BY4742 and qpt1Δ yeast exclusively utilize extracellular nicotinic acid for NAD+ biosynthesis during exponential growth under both normal and CR conditions, suggesting that the de novo pathway plays little role if a functional salvage pathway is present. Under CR conditions, cells do not alter biosynthesis pathway activity and continue to synthesize NAD+ from extracellular nicotinic acid. We also observe that NAD+ concentrations decrease in all three strains under CR. However, unlike the wild-type strain, NADH concentrations do not decrease and NAD+: NADH ratios do not increase under CR for either knockout strain. Since lifespan analyses reveal that CR results in an average increase in lifespan of approximately 25% in the wild-type yeast and qpt1Δ mutant, while no increase in lifespan is observed for the npt1Δ mutant, these data suggest that having a functional salvage pathway is required for lifespan extension under CR.
This work was performed under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344 and was supported by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program (P41 RR013461).