Nicotinamide adenine dinucleotide (NAD+) is found in every single living cell, where it serves as an essential cofactor — a substance whose presence is essential for the activity of molecular machines (enzymes) — involved in fundamental biological processes. Since its discovery in 1906, research continues to pile up showing that NAD+ is critical for maintaining the health of our cells, tissues, and bodies.
But as we age, we see a gradual decline in tissue and cellular NAD+ levels. This decline in NAD+ levels is linked causally to numerous aging-associated diseases, including cognitive decline, cancer, metabolic disease, sarcopenia (progressive and generalized loss of skeletal muscle mass and strength), and frailty. Many of these aging-associated diseases can be slowed down and even reversed by restoring NAD+ levels.
So, what exactly is NAD+, how is it made, and how is it used by cells? Understanding these concepts will help us have a better grasp on the roles of NAD+ in maintaining healthy aging, what happens when we lose our balance of NAD+, and whether and how this deficiency can be treated.
Where is NAD+ found in the cell?
NAD+ is compartmentalized into different structures within each cell. It’s found in the gelatinous liquid that fills the cell (cytoplasm), the cell’s battery packs (mitochondria), and where the cell houses genetic information (nucleus). These subcellular pools of NAD+ are regulated independently of each other, and consistent with this, the enzymes involved in the biosynthesis or degradation of NAD+ are highly compartmentalized as well.
How do cells make NAD+?
NAD+ mediates multiple major biological processes and is always in high demand. NAD+ is constantly synthesized, metabolized, and recycled in the cell to maintain stable intracellular NAD+ levels. Some cells, mostly in the liver, can synthesize NAD+ from scratch (de novo) using multiple dietary sources. NAD+ can be made de novo from L-tryptophan or vitamin precursors, such as nicotinic acid (NA).
Outside the liver, most cells do not express the full array of enzymes necessary to convert tryptophan to NAD+. But it can also be recycled or salvaged from nicotinamide (NAM), a by-product of when enzymes use NAD+ as a cofactor. Most tryptophan is metabolized to NAM in the liver, where it is released into the serum, taken up by peripheral cells, and eventually converted to NAD+. In this pathway, NAM is converted to nicotinamide mononucleotide (NMN) by an enzyme called NAMPT, which can be converted to NAD+. NMN also forms from nicotinamide riboside (NR).
(Rajman et al., 2018 | Cell Metab.) Representation of NAD+ biosynthesis pathways. Biosynthesis pathways and cellular metabolism of NAD+. NAD+ precursors such as NR, NA, NAM, and tryptophan (Trp) provided by diet can be converted into NAD+ via three pathways: (1) NA is converted into NAMN, NAMN is converted into NAAD, and NAAD is converted into NAD+; (2) Trp is converted into QA in a series of steps, which is then converted into NAD+; (3) NR and NAM provided by diet are converted into NAD+ by forming NMN by enzymes NAMPT and NRK. Additionally, NAD+-consuming enzymes like PARPs and sirtuins catalyze NAM production in subcellular compartments, which can be used for NAD+ synthesis.
What enzymes consume NAD+?
NAD+ is also continually turned over by three classes of NAD+-consuming enzymes: sirtuins, PARPs, and the NAD+ glycohydrolases — also referred to as NADases (CD38, CD157, and SARM1).
Sirtuins have emerged as key actors in the efforts to understand and characterize how NAD+ levels affect cellular homeostasis in a wide variety of cellular processes that impact aging and boosting their activity is a key focus of therapies aimed at counteracting aging. Since their discovery, the sirtuins have received much attention because they regulate key metabolic processes, stress responses, and aging biology. Overall, sirtuins have emerged as key actors in the efforts to understand and characterize how NAD+ levels affect cellular homeostasis in a wide variety of cellular processes that impact aging and boosting their activity is a key focus of therapies aimed at counteracting aging.
The human PARP protein family is composed of 17 proteins. Briefly, the PARP-mediated cleavage of NAD+ produces NAM and ADP-ribose as by-products. Overall, targeting PARPs, in particular PARP1, is a promising therapeutic strategy in the aging field. However, more studies are needed to fully understand the contribution of PARPs to the age-related decline in NAD+ levels.
CD38 and CD157 are multifunctional ectoenzymes — enzymes found outside or on the outer membrane of a cell. CD38 modulates essential cell processes, such as immune cell activation, survival, and metabolism. Importantly, other than NAD+, NMN is emerging as an alternative substrate of CD38, whereas CD157 consumes NR as an alternative substrate. Very little is known about the purpose of CD157 enzymatic functions in cellular biology and aging. However, recent evidence shows that, like CD38, CD157 is upregulated in aging tissues and may have a role in aging-related diseases, such as rheumatoid arthritis and cancer.
SARM1 was only very recently assigned to the NADase family along with CD38 and CD157. SARM1-mediated NAD+ degradation plays a key role in axonal degeneration after axonal injury. SARM1 is primarily found in neurons and promotes neuronal morphogenesis and inflammation., but it is also found in certain immune cells, such as macrophages and T lymphocytes, and regulates their functions. SARM1 is emerging as a therapeutic target to prevent or ameliorate neurodegenerative diseases and traumatic brain injuries.
How else is NAD+ used in the cell?
Beyond the main groups of enzymes that consume NAD+ discussed so far, NAD+ is widely used as a cofactor or substrate for biochemical reactions. Over 300 enzymes are known to rely on NAD+ for their activity. Accordingly, NAD+ is a mediator of key cellular functions and adaptation to metabolic needs. Some of these critical cellular processes include metabolic pathways, maintenance and repair of DNA to safeguard genomic stability, and autophagy — the cell’s recycling mechanism. Collectively, these functions are important for maintaining systemic health and balance. However, during aging, declining NAD+ levels can impinge on these processes and exacerbate aging-related diseases.
How is NAD+ related to aging?
During aging, NAD+ levels decline, and many enzymes associated with NAD+ degradation and biosynthesis are altered. Many of the cellular processes that influence or are influenced by aging — such as metabolic dysfunction, DNA repair failure, genomic instability, inflammation, cellular senescence, and neurodegeneration — are regulated by NAD+ levels. Also, this decline in NAD+ levels during aging has been linked to the development and progression of aging-related diseases, including atherosclerosis, arthritis, hypertension, cognitive decline, diabetes, and cancer.
(Amjad et al., 2021 | Mol Metab.) NAD+ decline at the core of hallmarks of aging. The age-dependent decline in NAD+ levels contribute to many hallmarks of aging. This includes DNA damage, epigenetic alteration, deregulated nutrient-sensing, loss of proteostasis, altered cellular communication, cellular senescence, stem cell exhaustion, mitochondrial dysfunction, compromised autophagy, and possibly telomere attrition.
How can NAD+ levels be restored?
NAD+ levels can be somewhat restored with dietary precursors like NMN, NR, and NAM. Also, The cellular level of NAD+ can be increased either by stimulation or activation of enzymes involved in NAD+ biosynthesis like NAD+ precursors or by inhibition of enzymes that consume or degrade NAD+. For example, targeting CD38 and CD157 with small-molecule inhibitors may make these commonly used NAD+ precursor metabolites more efficient in restoring NAD+ levels in aging individuals.
Some potential strategies that boost NAD+ levels include lifestyle changes, such as increasing exercise, reducing caloric intake, eating a healthy diet, and following a consistent daily circadian rhythm pattern by conforming to healthy sleeping habits and mealtimes.
All of these approaches promote tissue NAD+ levels and are beneficial for health. These include improved tissue and organ function, protection from cognitive decline, improved metabolic health, reduced inflammation, and increased physiological benefits, such as increased physical activity, which may collectively extend patient healthspan and potentially lifespan.
(Covarrubias et al., 2021 | Nat Rev Mol Cell Biol.) Therapeutic approaches to restore NAD+ levels and their impact on health. Aging is linked to decreased NAD+ levels that promote or exacerbate aging-related diseases. Thus, restoring NAD+ levels has emerged as a therapeutic approach to prevent and treat aging-related diseases and to restore health and vigor during the aging process. Some potential strategies that boost NAD+ levels include lifestyle changes, the use of small-molecule inhibitors or activators to boost NAD+ biosynthesis, and the use of dietary supplements, including NAD+ precursors, such as NMN and NR.
Outstanding questions and future directions
Aging processes and age-related disorders are likely to benefit from NAD+ repletion. There is evidence from experiments in model organisms, such as rodents, of NAD+ replenishment, potentially delaying and preventing metabolic conditions, hearing loss, muscle atrophy, and cognitive decline are encouraging for future perspectives. Moreover, NAD+ precursors are safely administered in people, thus implicating a possible translational aspect of preclinical benefits of NAD+ supplementation — an exciting prospect that opens avenues for future studies to test the impact of elevated NAD+ biosynthesis in aging and age-associated diseases in human.
Targeting NAD+ metabolism has emerged as a potential therapeutic approach to ameliorate aging-related disease and extend the human healthspan and lifespan. However, much remains to be learned about how NAD+ influences human health and aging biology. This includes a deeper understanding of the molecular mechanisms that regulate NAD+ levels, how to effectively restore NAD+ levels during aging, whether doing so is safe and whether NAD+ repletion will have beneficial effects in aging humans.