Nicotinamide is an inhibitor of SIRT1 in vitro, but can be a stimulator in cells

Abstract

Nicotinamide (NAM), a form of vitamin B₃, plays essential roles in cell physiology through facilitating NAD⁺ redox homeostasis and providing NAD⁺ as a substrate to a class of enzymes that catalyze non-redox reactions. These non-redox enzymes include the sirtuin family proteins which deacetylate target proteins while cleaving NAD⁺ to yield NAM. Since the finding that NAM exerts feedback inhibition to the sirtuin reactions, NAM has been widely used as an inhibitor in the studies where SIRT1, a key member of sirtuins, may have a role in certain cell physiology. However, once administered to cells, NAM is rapidly converted to NAD⁺ and, therefore, the cellular concentration of NAM decreases rapidly while that of NAD⁺ increases. The result would be an inhibition of SIRT1 for a limited duration, followed by an increase in the activity. This possibility raises a concern on the validity of the interpretation of the results in the studies that use NAM as a SIRT1 inhibitor. To understand better the effects of cellular administration of NAM, we reviewed published literature in which treatment with NAM was used to inhibit SIRT1 and found that the expected inhibitory effect of NAM was either unreliable or muted in many cases. In addition, studies demonstrated NAM administration stimulates SIRT1 activity and improves the functions of cells and organs. To determine if NAM administration can generate conditions in cells and tissues that are stimulatory to SIRT1, the changes in the cellular levels of NAM and NAD⁺ reported in the literature were examined and the factors that are involved in the availability of NAD⁺ to SIRT1 were evaluated. We conclude that NAM treatment can hypothetically be stimulatory to SIRT1.

Keywords: NAD+; NAMPT; Nicotinamide (NAM); SIRT1; Salvage pathway; Sirtuin.

Fig. 1

Conversion of NAM to NAD⁺ and other metabolites in cytosol and facilitated NAD⁺ production in putative microdomains in the nucleus. Upon administration, NAM transported to the cytosol reacts with PRPP to produce NMN which is, in turn, adenylated to become NAD⁺ through the activities of NAMT and NMNAT-1. A small fraction of NAD⁺ is reduced to NADH or phosphorylated to NADP⁺ which can be reduced to NADPH. NAD⁺ is also degraded to NAM and ADP-ribose by non-reducing enzymes such as SIRT1 and ARTs. NAM also inhibits the activities of these NAD⁺-consuming enzymes. ADP-ribose is removed through poly(ADP-ribosylation) and mono(ADP-ribosylation) (not shown). A limited amount of NAM is removed through conversion to methyl-NAM (MeNAM) which is further metabolized to 2-PY and 4-PY, but these conversions are quite inefficient. Meanwhile, in the nucleus (brown circle), NMNAT-1 was found to co-localize with SIRT1 on target chromosomes and facilitate NAD⁺ supply to SIRT1 [96]. NAMPT was also found to activate SIRT1 in target gene expression [96], and is expected to localize near SIRT1 forming a microdomain (beige circle) in which local concentration of NAM is lowered. These hypothetical microdomains create a condition where SIRT1 is activated within target chromosomes even in the presence of high cellular NAM. In addition, cells were found to secrete eNAMPT which converts NAM in media or body fluids to NMN. This would lower cellular levels of NAM, but the production of NAD⁺ may be maintained by the influx of NMN instead of NAM, and thus may facilitate SIRT1 activation. eNAMPT extracellular NAMPT, NMNAT-1 NMN adenyl transferase, ARTs mono-ADP-ribosyl transferases, 2-PY N-methyl-2-pyridone-5-carboxamide, 4-PY N-methyl-4-pyridone-5-carboxamide, NAMPT NMNAT-1, acetyl- acetylated transcription factor, a SIRT1 target

Fig. 2

Observed change in the levels of NAM and NAD⁺ in the tissues administered with high-dose NAM. The data reported by Collins and Chaykin (in Table 1, Fig. 3, and Fig. 4 of [52]) were adopted to show representative changes in the levels of NAM and NAD⁺ after NAM administration in mice. The numbers in the y-axes of the graphs are the radioactivity in NAM or NAD⁺, which were measured in the tissues isolated at intervals from 5 to 60 min (x-axis) after intraperitoneal administration of radioactive NAM (0.9 μmol). In all the organs, the levels of NAM increased rapidly during the initial 10 min and then declined with varying rates for the 50 min of chase. During this chase period, radioactive NAD⁺ increased linearly in all the organs but with a wide variation in the rates. As a consequence, the degree of NAD⁺ conversion at the final 60-min point appeared quite different from tissue to tissue. A large amount of NAM was converted to NAD⁺ in the kidneys and heart while only a small amount was in the spleen and skeletal muscles. This difference in the conversion rates appears to be independent of the amount of NAM-entered cells (note that the radioactivity in the y-axis is different in all four graphs). In the report, the conversion that occurred in other tissues fell between the two extremes of the cases of the kidneys and skeletal muscles