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

摘要

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 (RS) and breakdown (RB) using a flux assay with a ²H (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, RS, RB, and the NAD⁺ concentration. NAD⁺ concentration was maintained within a narrow range (400–700 μM) in the cells. RS 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, RS increased by 2-fold, while 2-fold increase in RB 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.