Decline of NAD+ during Aging, Age-Related Diseases, and Cancer
Several evidences suggest a decline in NAD+ levels while we age, connecting NAD+ deficits to age-related diseases and cancer.
Inflammation increases during the aging process possibly due to the presence of senescent cells .
CD38 and bone marrow stromal cell antigen-1 (BST- 1) may provide explanations to NAD+ decline during aging.
CD38 is a membrane-bound hydrolase implicated in immune responses and metabolism. NAD+ can be degraded through its hydrolysis, deacetylation, or by NAD+ nucleosidases (also called NAD+ hydrolases or NADases) such as CD38.
Expression and activity of CD38 increase in older mice, promoting NMN degradation in vivo, responsible for NAD+ decline and mitochondrial dysfunctions .
Interestingly, loss of CD38 inhibits glioma progression and extends the survival of glioma- bearing mice.
Targeting CD38 in the tumor microenvironment may clearly serve as a novel therapeutic approach to treat glioma .
Daratumumab, a CD38 monoclonal antibody, rep- resents a first-in-class drug for the treatment of multiple myeloma. It promotes T cell expansion through inhibition of CD38+ immunosuppressive cells, improving patients’ responses .
These findings suggest that NAD+ boosters should be combined with CD38 inhibitors for a more efficient antiaging therapy.
NAD+ Biosynthesis Decreases during Aging, Age-Related Diseases, and Cancer
NAD+ increases can also occur independently of the Preiss–Handler route. NAM and NR are important NAD+ precursors first converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT) and NR kinase (NRK), respectively. NMN is then transformed into NAD+ by NMN adenylyltransferase .
As we age, our bodies undergo changes in metabolism, and a key part of these processes may affect de novo NAD+ synthesis, also called the L-tryptophan/kynurenine pathway (see Figure IB in Box 1). In mammals, the use of the de novo NAD+ biosynthetic pathway is limited to a few specific organs.
Finally, dysregulation of the kynurenine pathway is also linked to genetic disorders and age-related diseases such as obesity and cancer [14,15]. These age-associated changes in de novo NAD+ biosynthesis may have the potential to impact several biological processes, and thus contribute to age-related diseases and cancer in the elderly.
Animal models mimicking downregulation of NAD+ biosyn-thesis are needed to modulate its activity and understand its pathophysiological relevance in age-related pathologies and cancer.
Boosting NAD+ with Niacin in Age-Related Diseases and Cancer
In humans, a lack of nicotinic acid (NA, also called niacin) in the diet causes the vitamin B3 deficiency disease pellagra, characterized by changes in the skin with very characteristic pigmented sunburn-like rashes developing in areas that are exposed to sunlight. Likewise, people with chronic L-tryptophan-poor diets or malnutrition develop pellagra.
Furthermore, several epidemiologic studies in human reported an association between incidence of certain types of cancers and niacin deficiency .
In this regard, low dietary niacin has also been associated with an increased frequency of oral, gastric, and colon cancers, as well as esophageal dysplasia.
In some populations, it was shown that daily supplementation of niacin decreased esophageal cancer incidence and mortality. Although the molecular mechanisms of niacin deprivation and cancer incidence are not well understood, it has been recently reported that NAD+ depletion leads to DNA damage and increased tumorigenesis, and boosting NAD+ levels is shown to play a role in the prevention of liver and pancreatic cancers in mice [19,28,29].
Thus, malnutrition through inadequate amounts and/or diversity of food may affect the intra- cellular pools of nicotinamide and NAD+ thereby influencing cellular responses to genotoxic damage, which can lead to mutagenesis and cancer formation [19,27]. NAD+ boosters are therefore essential in patients at risk of exposure to genotoxic and mutagenic agents, including ionizing or UV radiations or, DNA damaging chemicals.
In addition, niacin deficiency in combination with carcinogenic agents was described to induce and increase tumorigenesis in rats and mice.
For instance, in rats, the lack of niacin together with carcinogen treatment increased tumorigenesis and death of rats [30,31]. Additionally, in mice, the incidence of skin tumours induced by UV was significantly reduced by local application of NAM or by niacin supplementation in the diet .
Boosting NAD+ with NAM in Age-Related Diseases and Cancer
Recent research has focused on uncovering the consequences of a decrease in NAD+ during aging using age-related disease models. In PGC1a knockout mouse, a model of kidney failure, NAD+ levels are reportedly decreased, and boosting NAD+ by NAM improves kidney function .
NAM injections during four days re-establish local NAD+ levels via nicotinamide phos- phoribosyltransferase (NAmPRTase or NAMPT) activation and improve renal function in postischaemic PGC1a knockout mice .
Surgical resection of small renal tumors can induce kidney ischemia severely affecting the renal function. Therefore, NAD+ boosters can be beneficial to protect the organ from severe injury.
Moreover, in a model of muscular dystrophy in zebrafish, NAD+ increases, which functions as an agonist of muscle fiber–extracellular matrix adhesion, and corrects dystrophic phenotype recovering muscle architecture .
Boosting NAD+ with NR in Age-Related Diseases and Cancer
Further research has extensively used NR to ameliorate the effects of NAD+ deficits in pleiotropic disorders. NR naturally occurs in milk [35,36]. NR is converted to NAD+ in two step reactions by nicotinamide riboside kinases (NRKs)-dependent phosphorylation and adenylylation by nicotinamide mononucleotide adenylyl transferases (NMNATs) .
It is considered to be a relevant NAD+ precursor in vivo. Evidences demonstrate the beneficial effect of NR in skeletal muscle aging [37,38] and mitochondrial-associated disorders, such as myopathies [39,40] or those characterized by impaired cytochrome c oxidase biogenesis affecting the respiratory chain .
In line of these findings, a mouse model of Duchenne muscular dystrophy present significant reductions in muscle NAD+ levels accompanied with increased poly-ADP-ribose polymerases (PARP) activity, and reduced expression of NAMPT .
Replenishing NAD+ stores with dietary NR supplementation improved muscle function in these mice through better mitochondrial function .
Additionally, enhanced NAD+ concen- trations by NR are apparently beneficial for some neurodegenerative diseases , as well as in noise-induced hearing loss .
NR-mediated NAD+ repletion is also protective, and even therapeutic, in certain metabolic disorders associated with cancer, such as fatty liver disease [28,45] and type 2 diabetes [28,46]. Metabolic disorders characterized by defective mitochon- drial function could also benefit from an increase in NAD+ levels.
Indeed, stimulation of the oxidative metabolism in liver, muscle, and brown adipose tissue potentially protects against obesity . Interestingly, NAMPT protein levels are not affected in chow- and high fat diet (HFD)-treated mice fed with NR, arguing that in models of obesity, NR directly increases NAD+ levels without affecting other salvage reactions .
Recently, diabetic mice with insulin resistance and sensory neuropathy treated with NR reportedly show a better glucose toler- ance, reduced weight gain and liver damage, and protection against hepatic steatosis and sensory and diabetic neuropathy .
Boosting NAD+ with NMN in Age-Related Diseases and Cancer
NMN is also a key biosynthetic intermediate enhancing NAD+ synthesis and ameliorates various pathologies in mouse disease models [49,50].
Very recent research demonstrate that a 12- month-long NMN administration to regular chow-fed wild-type C57BL/6 mice during normal aging rapidly increases NAD+ levels in numerous tissues and blunts age-associated physio- logical decline in treated mice without any toxic effects . NMN is also beneficial in treating age- and diet-induced diabetes, and vascular dysfunction associated with aging in mice [51,52].
Administration of NMN also protects the heart of mice from ischemia-reperfusion injury  and restores mitochondrial function in muscles of aged mice [37,54].
It has been speculated that NMN is a circulating NAD+ precursor, due to the extracellular activity of NAMPT . However, the mechanisms by which extracellular NMN is converted to cellular NAD+ still remain elusive.
On the one hand, it is reported that NMN is directly trans- ported into hepatocytes . On the other hand, NMN can be dephosphorylated to NR to support elevated NAD+ synthesis [56–59].
It is recently shown that NAM can be metabolized extracellularly into NMN by extracellular NAMPT. NMN is then converted into NR by CD73 . Hence, NR is taken up by the cells and intracellularly phosphorylated firstly into NMN by NRKs and then, converted into NAD+ by NMNATs  (Figure 3).
Thus, mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD+ synthesis. Consistent with these findings, in murine skeletal muscle specifically depleted for NAMPT, administration of NR rapidly restored muscle mass by entering the muscles and replenishing the pools of NAD+ through its conversion to NMN .
Interestingly, mice injected with NMN had increased NAM in their plasma that may come after initial conversion of NMN into NR . However, degradation of NR into NAM could only be observed when cells were cultured in media supplementing with 10% FBS .
Finally, it is important to note that NR is stably associated with protein fractions in milk with a lifetime of weeks .
Notably, as reported above, NMN may be degraded by CD38 in older mice promoting NAD+ decline and mitochondrial dysfunctions , suggesting that NR may be more efficient than NMN in elderly.
Yet, the beneficial synergistic activation of sirtuins and metabolic pathways to replenish NAD+ pools cannot be excluded. However, given its efficient assimilation and high tolerance, NR represents still the most convenient and efficient NAD+ booster.
Overall, these findings suggest that NAD+ decrease in disease models and NAD+ precursors (NAM, NR or NMN) may circumvent NAD+ decline to generate adequate levels of NAD+ during aging and thus be used as preventive and therapeutic antiaging supplements.
NMN and NR supplementations may be equivalent strategies to enhance NAD+ biosynthesis with their own limitations.
Side-Effects of Some NAD+ Boosters
Clearly, several intermediates of the salvage pathway can be considered to boost NAD+ levels but some have contraindications. High doses of NA given to rats are needed to robustly increase NAD+ levels .
Additionally, relevant and unpleasant side effects through NA-induced prostaglandin- mediated cutaneous vasodilation (flushing) affecting patient compliance are due to the activation of the G-protein-coupled receptor GPR109A (HM74A) and represent a limitation in the pharma- cological use of NA .
NAM is much less efficient than NA as a lipid lowering agent and has also several side effects; in particular, it causes hepatic toxicity through NAM-mediated inhibition of sirtuins .
The metabolism of these conventional compounds to NAD+ is also different, as NA is converted via the three-step Preiss–Handler pathway, whereas NAM is metabolized into NMN via NAMPT and then to NAD+ by NMNATs 
Manipulating NAD+ by Manipulating Enzyme Activity of Salvage Reactions
Enhancing the activity of enzymes that participate in salvage reactions can also be a strategic intervention to increase NAD+ concentrations. Different studies have addressed the importance of regulating the activity of NAMPT during disease, including metabolic disorders and cancer.
NAMPT is necessary in boosting NAD+ pools via the salvage pathway.
Consequently, NAMPT deletion provokes obesity-related insulin resistance, a phenotype rescued by boosting NAD+ levels in the white adipose tissue by giving NMN in drinking water .
Conversely, in a mouse model for atherosclerosis, NAMPT depletion promotes macro- phage reversal cholesterol transport, a key process for peripheral cholesterol efflux during atherosclerosis reversion .
Other recent reports suggest that NAMPT downregulation could be beneficial in treating pancreatic ductal adenocarcinoma [69,70] and colorectal cancer .
Recent findings show that Duchenne muscular dystrophy was accompanied by reduced levels of NAMPT in mice . Moreover, NAMPT knockout mice exhibit a dramatic decline in intramuscular NAD+ content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance.
NR treatment induced a modest increase in intra- muscular NAD+ pools but sufficient to rapidly restore muscle mass. Importantly, overexpres- sion of NAMPT preserves muscle NAD+ levels and exercise capacity in aged mice .
Inhibitors against NAMPT are being used in several phase II clinical trials as anticancer therapy.
Given that NAMPT activation is important to boost NAD+ levels, therapy involving NAMPT inhibition should be considered with caution. Although levels of NAD+ remain to be determined in models with NAMPT depletion, further investigation on the effects of NAMPT modulation is clearly required.
The specific mechanisms and actual benefits of regulation of NAMPT activity remain elusive, evidencing the need of more specific disease models.
Can Dietary Restriction and Protein Catabolism Maintain NAD+ Levels?
Among the questions that still remain not well understood is why DR profoundly increases lifespan? Can DR affect NAD+ levels?
It is well established that overfeeding and obesity are important risk factors for cancer in humans  and obesity-induced liver and colorectal cancer, among others, can shorten lifespan.
Earlier research has also shown that both increased physical activity and reduction in caloric intake (without suffering malnourishment) can extend lifespan in yeasts, flies, worms, fish, rodents, and primates [3–8].
Furthermore, a recent study pointed to the importance of the ratio of macronutrients more than the caloric intake as the determinant factor in nutrition-mediated health status and lifespan extension .
Although in humans it is difficult to measure the beneficial effects of DR and currently there is no reliable data that describe the consequences of significantly limiting food intake, some studies have assessed how DR affects health status.
People practicing DR seem to be healthier, at least based on risk parameters such as LDL cholesterol, triglycerides, and blood pressure .
Activation of the salvage pathways during DR could be turned on and glucose restriction can stimulate SIRT1 through activation of the AMPK-NAMPT pathway resulting in inhibition of skeletal myoblast differentiation .
Interestingly, effects of NMN supplementation and exercise on glucose tolerance in HFD-treated mice are very similar .
Even though these effects are tissue-specific since exercise predominantly affects muscle, whereas NMN shows major effects in liver, and that mechanism of action can be different, exercise and NMN predominantly affect mitochondrial functions and may both contribute to the boost of NAD+.
It is thus tempting to speculate that L-tryptophan concentrations and thus the de novo NAD+ biosynthesis could fluctuate during DR ameliorating the aging process.
Recent studies in humans and mice suggest that moderate exercise can increase blood NAD+ levels and decrease L-tryptophan levels .
A possible explanation for this phenomenon is that DR, and/or exercise, can induce autophagy and promote the release of several metabolites and essential amino acids .
Aging is proposed to be responsible for diverse pathologies, however, it should be considered as a disease among other diseases that appear in time while individuals age.
Although some questions still remain unclear, NAD+ precursors may present possible therapeutic solutions for the maintenance of NAD+ levels during aging and thus may provide prophylaxis to live longer and better.
Although more research is needed to understand the efficacy as well as potential adverse side effects of NAD+ Replacement Therapies in humans, recent studies already provided some pharmacological properties, showing low toxicity and high effectiveness.