Cellular NAD concentrations can influence NAD cap levels. (A) The synthesis of NAD from NAM or NR. NAM is converted to NMN by the rate-limiting NAMPT, using PRPP as a cosubstrate. NMN is also the product of NR phosphorylation by NR kinases. NMN is converted to NAD by the NMNAT enzyme. FK866 is a small molecule inhibitor of NAMPT. (NAMPT) Nicotinamide phosphoribosyltransferase; (NMNAT) NMN adenylyltransferase; (NRK) NR kinase; (PRPP) phosphoribosyl pyrophosphate. (B) Quantitation of cellular total NAD following 48 h treatment with 5 nM FK866 or 5 mM NAM to decrease and increase cellular NAD levels, respectively. (C) Quantification of NAD-caps, detected with NAD-CapQ following 48 h treatment with 5 nM FK866 or 5 mM NAM. Mean values are plotted. Error bars represent ±SD. Statistical significance was calculated by one-way ANOVA with a Dunnett's multiple comparison post-test. P-values are denoted by asterisks. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.
