Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD(+) synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry

NAD(+) is mainly synthesized from nicotinamide (Nam) by the rate-limiting enzyme Nam phosphoribosyltransferase (Nampt) and degraded to Nam by NAD(+)-degrading enzymes in mammals. Numerous studies report that tissue NAD(+) levels decrease during aging and age-related diseases and suggest that NAD(+) replenishment promotes healthy aging. Although increased expression of Nampt might be a promising intervention for healthy aging, forced expression of Nampt gene, inducing more than 10-fold increases in the enzyme protein level, has been reported to elevate NAD(+) levels only 40–60% in mammalian cells. Mechanisms underlying the limited increases in NAD(+) levels remain to be determined. Here we show that Nampt is inhibited in cells and that enhanced expression of Nampt activates NAD(+) breakdown. Combined with the measurement of each cell’s volume, we determined absolute values (μM/h) of the rates of NAD(+) synthesis (R(S)) and breakdown (R(B)) using a flux assay with a (2)H (D)-labeled Nam, together with the absolute NAD(+) concentrations in various mammalian cells including primary cultured cardiomyocytes under the physiological conditions and investigated the relations among total cellular Nampt activity, R(S), R(B), and the NAD(+) concentration. NAD(+) concentration was maintained within a narrow range (400–700 μM) in the cells. R(S) was much smaller than the total Nampt activity, indicating that NAD(+) synthesis from Nam in the cells is suppressed. Forced expression of Nampt leading to 6-fold increase in total Nampt activity induced only a 1.6-fold increase in cellular NAD(+) concentration. Under the conditions, R(S) increased by 2-fold, while 2-fold increase in R(B) was also observed. The small increase in cellular NAD(+) concentration is likely due to both inhibited increase in the NAD(+) synthesis and the activation of its breakdown. Our findings suggest that cellular NAD(+) concentrations do not vary dramatically by the physiological fluctuation of Nampt expression and show the tight link between the NAD(+) synthesis and its breakdown.