Targeted NAD+ metabolomics8,9 allows simultaneous assessment of functionally important metabolites such as NAD+ and NADP+ along with metabolites that could serve as biomarkers of biosynthetic processes, such as NA, NAR, NAMN, NR, NMN and NAAD. In addition, quantification of increases in ADPR, Nam, MeNam, Me2PY and N-methyl-4-pyridone-5-carboxamide (Me4PY) on a common absolute scale with NAD+ permits assessment of increased NAD+-consuming activities associated with NAD+ precursor vitamin supplementation.
Hepatic concentrations of 13 NAD+ metabolites were quantified in three to four mice at seven time points after gavage of saline and each vitamin. In addition, on each experimental day, three mice were gavaged with saline and euthanized to serve as time zero samples. Each vitamin produced a temporally distinct pattern of hepatic NAD+ metabolites. Consistent with rapid phosphorylation of NR and NAR by NR kinases41, the only NAD+ metabolites that do not produce hepatic peaks as a function of gavage of NAD+ precursor vitamins are NR and NAR (Supplementary Fig. 1a,b). The accumulation curves of some metabolites as a function of each vitamin are strikingly similar. For example, the accumulation of NMN (Fig. 5a) is nearly identical to that of NAD+ (Fig. 5b) and NADP+ (Fig. 5c), though at a scale of ∼1:400:40, respectively. In addition, the accumulation of Me4PY (Fig. 5f) is nearly identical to that of Me2PY (Supplementary Fig. 1c).
As shown in Fig. 5b, NA produced the least increase in hepatic NAD+ and also was 4–6 h faster than NR and Nam in kinetics of hepatic NAD+ accumulation. When NA was provided by oral gavage, liver NA peaked (340±30 pmol mg−1) in 15 min (Fig. 5g). Hepatic NA appearance was followed by an expected peak of 220±29 NAAD at 1 h post gavage (Fig. 5i) and a rise in hepatic NAD+ from 990±25 baseline to 2,200±150 at 2 h (Fig. 5b). Hepatic NADP+ due to NA (Fig. 5c) rose in parallel to that of hepatic NAD+. In the hours after gavage of NA, as hepatic NAD+ and NADP+ fell, there was clear evidence of enhanced NAD+-consuming activities with significant rises in ADPR (Fig. 5j), Nam (Fig. 5d), MeNam (Fig. 5e), Me2PY (Supplementary Fig. 1c) and Me4PY (Fig. 5f). Thus, oral administration of NA doubled hepatic NAD+ from ∼1 to ∼2 mM through expected intermediates and produced an increase in NAD+ consumption and methylated products, MeNam, Me2PY and Me4PY. Net conversion by the liver of NA to Nam has been documented for decades30,37,38. Essentially, the liver transiently elevates NAD+ biosynthetic capacity so long as NA is available while increasing NAD+-consuming activities, thereby making Nam available to other tissues. Expression of hepatic NNMT results in net production of MeNam from NAD+ precursors, which stabilizes SIRT1 protein in liver and is associated with better lipid parameters in mice and some human populations42,43.
As shown in Fig. 5g and consistent with radioactive experiments39, oral Nam was not used by the liver as NA because it did not produce a peak of NA at any time after gavage. Though there was a clear increase in hepatic NAD+ 2 h after Nam gavage, the Nam gavage drove increased hepatic NAD+ accumulation from 2 to 8 h with a peak at 8 h (Fig. 5b). Nam gavage produced two peaks of Nam in the liver (Fig. 5d), the first at 15 min, consistent with simple transport of Nam to liver. The second broad peak was coincident with elevation of NAD+ and NADP+ (Fig. 5c,d) and elevation of the NAD+-consuming metabolomic signature of ADPR (Fig. 5j), MeNam, Me4PY and Me2PY (Fig. 5e,f and Supplementary Fig. 1c).
Of the metabolites associated with NAD+-consuming activities, ADPR is the only one that must be formed from NAD+ because Nam, MeNam and the oxidized forms of MeNam could appear in liver from the gavaged Nam without conversion to NAD+. Interestingly, of three NAD+ precursor vitamins provided in bolus at equivalent oral doses, Nam provided the least increase in ADPR (Fig. 5j). Whereas the area under the curve (AUC) of the Nam-driven rise in hepatic NAD+ indicated a ∼50% advantage of Nam over NA (Fig. 5b), there was a >50% deficit in Nam-driven ADPR accumulation versus NA (Fig. 5j). This is consistent with the idea that high-dose NA, though not an ideal hepatic NAD+ precursor, is effective as a cholesterol agent whereas Nam is not44 because high-dose Nam inhibits sirtuins1. Notably, NR is active as a cholesterol-lowering agent in overfed mice30.
As shown in Fig. 1, Nam is expected to proceed through NMN but not NR, NAR, NaMN or NAAD en route to forming NAD+. Though there was no elevation of hepatic NR or NAR with oral Nam, there was also little elevation of hepatic NMN—this metabolite never reached a mean value of 5 pmol mg−1 at any time after Nam administration (Fig. 5a). Surprisingly, as shown in Fig. 5i, 2–4 h after oral Nam, NAAD was elevated to nearly 200 from a baseline of <2 pmol mg−1. Elevated NAAD occurred during the broad peak of elevated hepatic NAD+ and NADP+ (Fig. 5b,c). These data suggest that the rise in NAAD is a biomarker of increased NAD+ synthesis and does not depend on the conventionally described precursors of NAAD, namely NA and tryptophan.
As shown in Fig. 5b, NR elevated hepatic NAD+ by more than fourfold with a peak at 6 h post gavage. NR also produced the greatest elevation of NMN (Fig. 5a), NADP+ (Fig. 5c), Nam (Fig. 5d), NAMN (Fig. 5h), NAAD (Fig. 5i) and ADPR (Fig. 5j) in terms of peak height and AUC. Importantly, although gavage of Nam produces a peak of Nam in the liver at 15 min, the peak of Nam from NR gavage corresponds to the peak of NAD+, NMN, NADP+ and ADPR. These data establish that oral NR has clearly different hepatic pharmacokinetics than oral Nam. More NAD+ and NADP+ were produced from NR than from Nam. In addition, there was three times as much accumulation of ADPR, indicating that NR drives greater NAD+-consuming activities in liver than mole equivalent doses of Nam and NA. Though it has been speculated that NR would be a more potent NAD+ and sirtuin-boosting vitamin than conventional niacins45, these are the first in vivo data in support of this hypothesis.
As was seen in the n=1 human blood experiment, at time points in which the abundant NAD+ metabolites, NAD+ and NADP+, were elevated by NR by ∼twofold or more, NAAD rose from undetectable levels to ∼10% of the level of NAD+, thereby becoming a highly sensitive biomarker of increased NAD+ metabolism. Though compounds such as MeNam, Me2PY and Me4PY are also correlated with increased NAD+ synthesis, they are waste products that can be produced without NAD+ synthesis, whereas NAAD is functional NAD+ precursor.
To test whether NAAD is also elevated in other tissues and through other routes of administration, we euthanized mice after 6 days of NR or saline by IP administration and analysed hepatic and cardiac NAD+ metabolomes. As shown in Fig. 6, steady-state levels of hepatic NAD+ and NADP+ are much more responsive to NR than are steady state levels of cardiac NAD+ and NADP+. However, cardiac NAD+ metabolism was clearly elevated on the basis of statistically significant elevation of NMN, Nam, MeNam and Me4PY. Among these metabolites, only NMN, which was elevated in the heart by approximately twofold, could be considered diagnostic for increased NAD+ formation. In addition, NAMN and NAAD were increased by about ∼100-fold in heart and liver with NAAD rising to ∼10% of the concentration of heart and liver NAD+ in supplemented animals. These data validate NAAD as a metabolite that sensitively and reliably marks increased NAD+ metabolism even in tissues in which steady-state levels of NAD+ are little changed.