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

• Published in: PloS one 2019-03, Vol.14 (3), p.e0214000-e0214000
• Main Authors: Hara, Nobumasa, Osago, Harumi, Hiyoshi, Mineyoshi, Kobayashi-Miura, Mikiko, Tsuchiya, Mikako
• Format: Article
• Language: English
• Subjects: Age; Age related diseases; Aging; Animals; Biochemistry; Biology and Life Sciences; Breakdown; Cancer; Cardiomyocytes; Cell cycle; Cell Size; Cells, Cultured; Enzymes; Gene expression; Humans; Inhibition; Isotope Labeling; Kinetics; Mammalian cells; Mammals; Mass spectrometry; Mass spectroscopy; Medicine; Medicine and Health Sciences; Metabolism; Mice; Myocytes, Cardiac - metabolism; NAD; NAD - biosynthesis; NAD - metabolism; Neurodegeneration; Nicotinamide; Nicotinamide phosphoribosyltransferase; Nicotinamide Phosphoribosyltransferase - metabolism; Oxidative stress; Phosphoribosyltransferase; Physical Sciences; Physiology; Protein folding; Proteins; Quantitative analysis; Rats; Replenishment; Research and analysis methods; Stable isotopes; Synthesis; Tandem Mass Spectrometry; University faculty
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0214000

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.