A major enzymatic loop that operates in most cells is the NAD+ salvage pathway. Enzymes such as SIRT1 and poly(ADP-ribose) polymerase-1 (PARP-1) heavily use NAD+ as coenzyme, risking depletion of the intracellular stores, which can lead to cell death. Thus, levels of NAD+ need to be controlled even in the absence of de novo biosynthesis through nutritional pathways. The NAD+ salvage pathway allows nicotinamide (NAM), the by-product of enzymes that use NAD+ as coenzyme, to be reconverted into NAD+ via the use of a group of nicotinamide mononucleotide adenylyltransferases (NMNATs) and the nicotinamide phosphoribosyltransferase enzyme NAMPT. Importantly, NAMPT is the rate-limiting step enzyme within the NAD+ salvage pathway (Nakahata et al. 2009; Ramsey et al. 2009). Thus, changes in NAMPT activity will directly dictate the levels of intracellular NAD+.
The rhythmicity of NAD+ levels parallels the antiphasic oscillation in NAM, both oscillations being abolished in cells with a mutation in the circadian machinery (Nakahata et al. 2009). Thus, the clock directs the oscillation of critical metabolites. Because SIRT1 associates with and modulates CLOCK–BMAL1 (Nakahata et al. 2008), this suggested the presence of an enzymatic feedback loop, in which CLOCK–BMAL1 would control its own activity by directing the oscillatory synthesis of NAD+. This was demonstrated to indeed be the case. The regulatory region of the Nampt gene contains two E-box promoter elements, known to bind CLOCK–BMAL1. Additional experiments demonstrated that the expression of the Nampt gene is indeed controlled by CLOCK–BMAL1 in a complex that contains SIRT1. Thus, SIRT1 is present in both the transcriptional regulatory loop of the Nampt gene and the NAD+ enzymatic salvage pathway. This two-ways control results in the circadian expression of the Nampt gene, a circadian function of the NAD+ salvage pathway and thereby in a circadian synthesis of NAD+. Importantly, the use of FK866, a highly specific NAMPT pharmacological inhibitor, abolished NAD+ circadian oscillations and thereby SIRT1 cyclic activity (Nakahata et al. 2009). This finding is of interest because FK866 is used to control cell death in human cancer tissues. Thus, in addition to revealing a critical enzymatic circadian cycle, these results suggest that a direct molecular coupling exists among circadian clock, energy metabolism, and cell survival.
Thus, the circadian clock is directly implicated in controlling the intracellular levels of critical metabolites, generating an interlocking of the transcriptional feedback clock loop with the enzymatic feedback loop of the NAD+ salvage pathway (Fig. 5). This view is confirmed by recent results using mice deficient in CD38, a NAD+ hydrolase, which display NAD+ levels elevated during most of the circadian cycle. CD38-null mice show altered circadian rhythmicity, CCG expression, and aberrant metabolism ((Sahar et al. 2011). The oscillation of NAD+ could have important consequences for cellular physiology, including changes in chromatin remodeling and downstream molecular pathways. Thus, a direct link between circadian control and metabolic regulation exists, revealing that SIRT1 is implicated in controlling the cellular levels of its own coenzyme. It would be critical to understand how other sirtuins may contribute to circadian rhythms.