Much excitement arose from the idea that sirtuins regulate health and life span in many different organisms in accord with diet. In particular, it was shown that NAD+ and NADH could vary with the availability of dietary energy and nutrients. For example, an increase in NAD+ (or decrease in NADH) was proposed to mediate the extension of life and health span by dietary restriction (DR) 6. This study challenged the dogma arising from earlier studies, which found that NAD+ was present in excess to NADH in cells and did not vary much with diet 7. Reciprocally, many recent studies have provided evidence that defects in maintaining NAD+ levels and the accompanying decline in activity of sirtuins may help drive normal aging 8, 9. These latter studies are additionally exciting because they also demonstrate that NAD+ deficiency and associated pathologies may be normalized by supplementation with NAD+ precursors and intermediates. This review expands on this new framework, considering aging and diseases, and discusses the emergence of approaches to counter effects of aging by small molecules that can rescue defects in NAD+ and sirtuin activity.

NAD+ plays a key role in regulating metabolism and circadian rhythm

The canonical role of NAD+, mentioned above, is to facilitate hydrogen transfer in key metabolic pathways (Figure 1a). For example, NAD+ is converted to NADH in the glyceraldehyde-3-phosphate dehydrogenase step of glycolysis, a pathway in which glucose is converted to pyruvate. Conversion of NAD+ to NADH is also important in mitochondrial metabolism. In that compartment, NAD+ is converted to NADH in four steps of the mitochondrial TCA cycle, in which acetyl-CoA is oxidized to carbon dioxide. NAD+ is also converted to NADH during the oxidation of fatty acids and amino acids in mitochondria. In these mitochondrial pathways, the NADH generated is an electron donor for oxidative phosphorylation and ATP synthesis.
In addition to these canonical uses of NAD+ and NADH, PARPs transfer ADP-ribose from NAD+ to itself, histones, and other proteins at sites of DNA damage to facilitate repair and maintenance of genomic integrity (Figure 1b). Damaged DNA recruits PARP and activates its poly-ADP-ribosylation activity in situ. Thus, acute DNA damage, for example by ionizing radiation, can trigger a sudden depletion of NAD+ due to PARP activation. PARP inhibitors are in clinical trials as anti-cancer agents 10, because they can sensitize tumor cells to apoptotic killing by genotoxic agents through the prevention of DNA repair.
Sirtuins are NAD+-dependent deacylases, which play key roles in responding to nutritional and environmental perturbations, such as fasting, DR, DNA damage, and oxidative stress (Figure 1c). In general, their activation triggers nuclear transcriptional programs that enhance metabolic efficiency and also upregulate mitochondrial oxidative metabolism and the accompanying resistance to oxidative stress 11. Sirtuins foster this resistance by increasing anti-oxidant pathways (e.g. SOD2 and IDE2 in mitochondria) and by facilitating DNA damage repair through deacetylation or ADP-ribosylation of repair proteins 12. Accordingly, many studies have shown that sirtuins promote longevity in yeast, worms, flies, and mice, and can mitigate many diseases of aging in murine models, such as type 2 diabetes, cancer, cardiovascular diseases, neurodegenerative diseases, and pro-inflammatory diseases 11, 13, 14. Although a challenge was raised to the proposed conserved role of sirtuins in aging/longevity control 15 (Box 1), many recent studies have upheld the original claims 16–23.
The role of sirtuins in aging and longevity control
Early studies have demonstrated that Sir2 and its orthologs play an important role in aging/longevity control in diverse model organisms including yeast, worms, and flies (24–26). In those organisms, it has also been shown that Sir2 and its orthologs mediate caloric restriction-induced lifespan extension in certain genetic backgrounds (25, 27–30). Although many studies have reported that SIRT1, the mammalian ortholog of Sir2, mediates anti-aging effects of caloric restriction in mice (13), mice overexpressing SIRT1 in the whole body failed to show lifespan extension (31). Furthermore, previous results showing lifespan extension by Sir2 orthologs in worms and flies were called into question (15), bringing considerable debate around the importance of sirtuins in aging/longevity control to the field of aging research. However, more recently, an increasing number of studies have reconfirmed the original claims (16–23). In mammals, it has been reported that whole-body Sirt6 transgenic mice show lifespan extension, in males (17). Most recently, it has been demonstrated that increasing SIRT1 specifically in the brain, particularly in the dorsomedial and lateral hypothalamic nuclei, delays aging and extends lifespan in both male and female mice (20). These new studies have thus put the controversy to rest, and provide a firmer foundation for the importance of sirtuins as an evolutionarily conserved aging/longevity regulator.
Among the many ways sirtuins influence metabolism is by regulating the circadian clock machinery. SIRT1, the most studied member of mammalian sirtuins, deacetylates central clock components in the liver 32, 33, and also amplifies the expression of the circadian transcription factors BMAL and CLOCK in the suprachiasmatic nucleus (SCN) of the hypothalamus via deacetylation of PGC-1α 34. In the latter case, a loss of SIRT1 function occurs with aging, which results in damped levels of the clock components and deterioration of central circadian control. Defects in central circadian control have been associated with disease and premature aging, underscoring the metabolic importance of circadian function 35.
Reciprocally, NAD+ synthesis is regulated by the circadian machinery to provide a critical link from the clock oscillator to metabolic pathways 36. In this regard, one must remember that NAD+ synthesis encompasses both de novo and salvage pathways, with some differences between lower organisms and mammals (Figure 2). Importantly, one of the key target genes of BMAL and CLOCK is the rate-limiting enzyme for NAD+ biosynthesis from nicotinamide, nicotinamide phosphoribosyltransferase or NAMPT 37, 38. NAD+ is synthesized in a circadian oscillatory fashion systemically, leading to a circadian schedule of sirtuin activation and mitochondrial metabolism, such as oxidation of fatty acids 39. Any decline in central and peripheral circadian function with aging would thus degrade the temporal order of metabolism, which may contribute to a deterioration in health.