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 (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.

NAD+（烟酰胺腺嘌呤二核苷酸，Nicotinamide Adenine Dinucleotide）在哺乳动物体内主要由烟酰胺（nicotinamide, Nam）经限速酶烟酰胺磷酸核糖转移酶（Nam phosphoribosyltransferase, Nampt）合成，并可被NAD+降解酶催化分解为Nam。多项研究表明，哺乳动物组织内的NAD+水平会随衰老及衰老相关疾病进程发生下降，且已有研究提示补充NAD+有助于促进健康衰老。尽管提高Nampt的表达或许是实现健康衰老的潜在干预靶点，但已有研究显示，强制过表达Nampt基因可使该酶的蛋白水平提升10倍以上，却仅能使哺乳动物细胞内的NAD+水平提升40%~60%。导致NAD+水平提升有限的具体分子机制仍有待阐明。本研究发现，细胞内Nampt的活性受到抑制，且过表达Nampt会激活NAD+的分解代谢通路。结合对细胞体积的测定，我们利用以氘（²H, D）标记的Nam进行的通量实验，测定了生理条件下各类哺乳动物细胞（包括原代培养心肌细胞）内NAD+合成速率（RS）与分解速率（RB）的绝对值（单位为μM/h），同时测定了细胞内NAD+的绝对浓度，并探讨了细胞总Nampt活性、RS、RB与NAD+浓度之间的关联。实验结果显示，细胞内的NAD+浓度维持在400~700 μM的狭窄区间内。NAD+合成速率（RS）远低于细胞总Nampt活性，这表明细胞内以Nam为底物的NAD+合成通路受到了抑制。过表达Nampt使细胞总Nampt活性提升6倍，却仅能使细胞内NAD+浓度提升1.6倍。在此实验条件下，RS提升了2倍，同时RB也同步提升了2倍。细胞内NAD+浓度的小幅提升，可能是由于NAD+合成的增幅受到抑制，同时其分解代谢也被激活所致。本研究结果表明，细胞内NAD+浓度并不会因Nampt表达的生理性波动出现显著变化，同时也揭示了NAD+合成与分解代谢之间的紧密关联。