NR is incorporated into NAAD
Appearance of hepatic NAAD after gavage of Nam or NR, and of hepatic NAMN after gavage of NR suggested that there is an NAD+ and/or NMN deamidating activity when NAD+ and NADP+ levels are high. Alternatively, high levels of NAD+ metabolites might inhibit glutamine-dependent NAD+ synthetase, thereby resulting in accumulation of NAMN and NAAD derived from tryptophan. To test whether NR is incorporated into the peak of NAAD that appears after NR gavage, we synthesized NR with incorporation of deuterium at the ribosyl C2 and 13C into the carbonyl of the Nam moiety. This double-labelled NR was provided to 15 mice by oral gavage at an effective dose of 185 mg kg−1 with the same experimental design used in pharmacokinetic analysis of the three vitamins. The effect of labelled oral NR on the hepatic NAD+ metabolome was first assessed at 2 h after gavage—a time point before the rise in the steady-state level of NAD+ (Fig. 5b).
As shown in Fig. 7a,b, at 2 h, 54% of the NAD+ and 32% of the NADP+ contained at least one heavy atom while 5% of the NAD+ and 6% of the NADP+ incorporated both heavy atoms. Because >50% of hepatic NAD+ incorporates label before a rise in NAD+ accumulation, it is clear that the NAD+ pool is dynamic. As shown in Fig. 7c,d, the majority of hepatic Nam and MeNam following gavage of double-labelled NR incorporated a heavy atom, necessarily the 13C in Nam. Because NR drives increased NAD+ synthesis and ADPR production (Fig. 5), the liberated singly labelled Nam becomes incorporated into NMN and NAD+ in competition with double labelled NR, thereby limiting subsequent incorporation of both labels into the NAD+ pool.