“NAD+ is the closest we’ve gotten to a fountain of youth”
according to David Sinclair, director for the Institute of Aging at Harvard Medical School.
As we age, levels of NAD+ in humans and animals decrease to about 1/2 of what they are in youth. Researchers like Dr Sinclair have found that restoring levels of NAD+ in mice make them look and behave like they did when young and extends their lifespan significantly.
WHAT IS NAD+
Scientists have now confirmed a direct link between falling NAD+ levels and aging in both animal and in human subjects.
Read more about NAD+
NAD+ DECLINES WITH AGE
Read more about NAD+
NAD+ METABOLISM IN HUMANS
NAD+ can be synthesized in humans from several different molecules (precursors), thru the De Novo and Salvage Pathways.
Nampt is the rate-limiting step in the salvage process (97).
SUBLINGUAL NMN AND NAD+ BYPASS THE LIVER and NAMPT BOTTLENECKS
All NAD+ supplements can restore NAD+ in the Liver but does not solve NAD+ deficiency throughout the body. This research shows that NAD+ in the liver is metabolized by CD38 to NAM, and ONLY NAM is excreted to the rest of the body.
Sublingual administration of NMN or NAD+ supplements bypass the 2 bottlenecks – the liver and NAMPT processing of NAM.
Providing NAD+ or its immediate precursor, NMN, directly to the bloodstream is much more effective than dropping large quantities of NAM or NR into the liver and trying to force it to produce more NMN and NAD to the bloodstream.
In mammals, the entire NAD+ pool is consumed 2-4 times a day and recycled thru the “salvage pathway”, which is responsible for 85% of our NAD+ (14).
NMN is more correctly referred to as a NAD+ intermediate because
NMN is the last step before conversion to NAD+
In 2004 Dr Charles Brenner discovered that NR can be phosphorylated to NMN in the body, to increase NAD+ without going thru NAM. This allows it to bypass the “NAMPT bottleneck”.
Chromadex has licensed methods to produce NR and has been selling NR commercially since 2014. Dr Brenner is now the chief scientific advisor for Chromadex.
NMN was prohibitively expensive until 2017, but there are no patents controlling the production of NMN so the prices have been dropping rapidly, and is now about the same as NR. Dr Sinclair is the leading researcher in use of NMN.
The NAD+ molecule itself is too large to make it through the digestive system intact when taken as capsules, which is why NR and NMN have been used as supplements to increase NAD+ levels in the body.
Recently however, research has shown NMN and NR are also mostly digested by the stomach and liver. Sublingual delivery of NAD+ and NMN allows them to bypass the GI tract.
Poor Bioavailability from Capsules
NAD+ not effective in drinking water or in capsule form
The NAD+ molecule is twice as large as NR or NMN, and is totally degraded in the Gastro-Intestinal tract, so researchers do not use NAD+ in drinking water of mice and it is not sold in capsule form for humans.
NAD+ in IV or IP injections
Research has been successful using NAD+ injections in mice.
In humans, clinics that provide NAD+ by IV are exploding in popularity, even though they charge over $1,000 a day and require the patient to be hooked up to a drip IV for 8 hours.
NMN and NR poor bioavailability in capsules
NMN and NR capsules are only partially digested in the stomach, but are almost totally metabolized in the liver and excreted as NAM (Liu, 2018).
Sublingual delivery solves the bioavailability problem
Molecules like NMN, NR and NAD+ that have low molecular weight and are hydrophilic can be absorbed through the capillaries under the tongue directly into the bloodstream. This is called Sublingual (under the tongue) delivery.
This solves the bioavailability problem of capsules that get digested in the stomach, so NAD+ can be used instead of NAD+ precursors. It also greatly improves the effectiveness of NMN.
THE PROBLEM with CAPSULES – DIGESTED TO NAM
This research published in 2018 confirms that most oral supplements of NMN and NR are digested to NAM in the GI tract or the liver.
At 50Mg/Kg of body weight, NO NR or NMN made it out of the liver intact.
Future pharmacological and nutraceutical efforts to boost NAD will need to take into account the minimal oral bioavailability of NR and NMN (R)
Unlike in cell culture where NR and NMN are readily incorporated into NAD, oral administration fails to deliver NR or NMN to tissues (R)
Interestingly, we found that neither compound was able to enter the circulation intact in substantial quantities when delivered orally (R)
That question is totally irrelevant if a molecule NEVER REACHES THE BLOODSTREAM.
This study used a small dose – 50 Mg/Kg of bodyweight (equivalent to 250 Mg for a 70 kg human), vs the 300-400 Mg/Kg commonly tested in other research. Perhaps higher dosages allow NAD+ precursors to make it past the Liver to other tissues.
It is clear that for Oral Supplements (Capsules), the bioavailability of any NAD+ precursor is very poor outside of the Liver.
SUBLINGUAL DELIVERY BYPASSES THE STOMACH AND LIVER
The absorption of the different molecules delivered through the sublingual route can be 3 to 10 times greater than oral route and is only surpassed by direct IV injection (r).
SUBLINGUAL CAN BE MORE BIOAVAILABLE THAN IP INJECTION !
With intraperitoneal injection, the primary route of absorption is via the mesenteric vessels, which drain into the portal vein and pass through the liver before reaching the bloodstream.
This means, IP avoids the GI tract, but is still sent directly to the Liver, where much of it is converted to NAD+. Elevated NAD+ in the liver is good, but its far better to reach the bloodstream with intact NMN.
Sublingual delivery is not filtered by the Liver and can reach systemic circulation intact, so can actually result in greater bioavailability that direct injection! Some examples are:
- A sublingual formulation of zol… exhibited a faster rate of absorption and higher drug exposure as compared to subcutaneous injection (r)
- sublingually administered epin… results in more rapid absorption and a higher peak plasma concentration compared to injected epin… .(r)
- 40mg of sublingually administered pir.. was found to be as effective as a 75 mg intramuscular injection of dicl… (r)
NAD+ METABOLISM IN HUMANS
NAD+ can be synthesized in humans from several different molecules (precursors), thru the De Novo and Salvage Pathways.
Nampt is the rate-limiting step in the salvage process (97).
As we age, Nampt enzyme activity is lower, resulting in less NAM recycling, less NAD+, more disease and aging (97,101).
SUBLINGUAL NMN AND NAD+ BYPASSES THE NAMPT BOTTLENECK
Restoring NAD+ in the Liver does not solve NAD+ deficiency throughout the body.
In the Liver, the CD38 enzyme metabolizes NAD+ to NAM, which is excreted to the rest of the body (r).
Sublingual delivery of NMN or NAD+ directly to the bloodstream bypasses the liver and the Nampt bottleneck that is the root cause of NAD+ deficiency in many tissues.
Why no sublingual NR?
NR is not stable by itself (see below) , so Chromadex adds Chloride to make it stable.
ALL NR sold is actually Nicotinamide Riboside CHLORIDE
Unfortunately, the taste is not acceptable for sublingual use.
So NR is only available in capsule form, which much pass through the stomach and liver where it is metabolized to the less effective Nicotinamide (Liu, 2018) .
ADVANTAGES OF NAD+ SUPPLEMENTATION:
NAD+ crosses the blood brain barrier to increase NAD+ in the hypothalamus – NMN and NR can not.
Exogenous NAD+ directly increases NAD+ levels in the hypothalamus to increase energy expenditure and decrease hunger. The hypothalamus is the master regulator of energy metabolism which impacts the entire body (more below).
NMN and NR do raise NAD+ in the blood which can then reach the hypothalamus, but the increase in NAD+ is limited by homeostasis (below).
NAD+ supplied direct to the blood is not dependent on the liver, so is not limited by homeostasis as NMN and NR.
NAD+ also influences serotonin and other neurotransmitters which have proven useful in treating addictions and other neurological conditions.
Below are some of the disease and illnesses that we believe NAD+ supplementation is more effective for than NMN.
- Chronic fatigue
- Weight control
- Mood disorders
- Alcohol and drug addiction
Research has also shown NAD+ supplementation to be effective for the following conditions (more here).
- Protects against liver damage
- Multiple Sclerosis autoimmune-related neurodegeneration
- Heart disease
- Heart damage from stroke
- Brain damage from injury
NAD+ REACHES THE BRAIN MORE THAN NMN OR NR
Once in the bloodstream NAD+ was thought to be too large to cross the cell membrane, making it ineffective at restoring the NAD+ contents inside the cells of many tissues. In this article we show that is not true for heart and brain, and perhaps other tissues.
In fact, this research published in March 2018 shows NAD+ is able to cross the blood brain barrier and quickly increases levels of NAD+ in the hypothalamus, while NR and NMN do not.
Administration of 1 mg/kg of NAD+ reduced hunger and weight gain, and increases energy expenditure and fat burning in mice (r).
Elevating NAD+ levels the hypothalamus has great impact throughout the body, as it regulates hunger and energy expenditure.
Restoring NAD+ levels in the hypothalamus to those of a young animal is very likely to have a positive impact on organs and tissues throughout the body.
Even more tantalizing are the possible implications for aging itself.
That the hypothalamus as master aging clock, is a credible theory on aging.
ADVANTAGES OF NMN SUPPLEMENTATION
NMN shows a particular ability to restore vascular growth and benefit tissues such as muscle and heart that haven’t been replicated in studies with NR or NAD+.
Below are the three studies that made the biggest splash’s about the potential for reversing aging by restoring NAD+ to youthful levels that have ALL been accomplished using NMN
After 6 days of NMN, 22 month old mice had the muscle capacity, endurance and metabolism of 6 month old mice (2013 Sinclair study)
NMN effectively mitigates age-associated physiological decline in mice (2016 Mills Long Term study)
“The old mice became as fit and strong as young mice” (Sinclair, 2018)
The third study identifies the key cellular mechanisms behind vascular aging and the critical role it plays on muscle health.
Dr Sinclairs team fed NMN to old mice. After two months, the mice had increased muscular blood flow, enhanced physical performance and endurance and the old mice became as fit and strong as young mice.
NEW BLOOD VESSELS sprouted within the skeletal muscles, capillary density increased and matched the capillary growth of young mice.
- NMN restored the vascular system of old mice to that of young mice.
- Mice treated with NMN had had nearly 100% increased endurance.
Renewed capillary growth and increased blood flow may help reverse heart and neurological problems in addition to sarcopenia.
According to Dr. Sinclair, the same mechanism could spur the creation of blood vessels in the brain, where “the lack of oxygen and buildup of waste products sets off a downward spiral of disease and disability,” such as Parkinson’s and Alzheimer’s.
COMBATTING ALZHEIMERS DISEASE
Alzheimer’s disease (AD) pathogenesis is widely believed to be driven by the production and deposition of the β-amyloid peptide (Aβ). Evidence now indicates that the solubility of Aβ, and the quantity of Aβ in different pools is related to disease state (r).Researchers believe that flaws in the processes governing production, accumulation or disposal of beta-amyloid are the primary cause of Alzheimer’s (r).
In studies published in 2017 and 2018 NMN decreased β-amyloid buildup, while NR did not.
Treating Heart Disease
2 separate studies to treat a form of heart disease called Friedreich’s Ataxia with NR and NMN were published in 2017. Treatment with NMN was successful, while NR did not improve cardiac function.
NMN was able to mitigate most age-associated physiological declines in mice Treatment of old mice with NMN reversed all of these biochemical aspects of aging
Raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse
Restore the mitochondrial homeostasis and key biochemical markers of muscle health in a 22-month-old mouse to levels similar to a 6-month-old mouse
This study showed supplementation with NMN was able to repair the DNA in cells damaged by radiation
The cells of old mice were indistinguishable from young mice after just one week of treatment.
NMN was immediately utilized and converted to NAD+ within 15 min, resulting in significant increases in NAD+ levels over 60 min
Administering NMN, a key NAD+ intermediate, can be an effective intervention to treat the pathophysiology of diet- and age-induced T2D
Surprisingly, just one dose of NMN normalized impaired glucose tolerance
NAD(+) levels were increased significantly both in muscle and liver by NMN
NMN-supplementation can induce similar reversal of the glucose intolerance
NMN intervention is likely to be increased catabolism of fats NMN-supplementation does mimic exercise
NMN treatment reduces brain cell death and oxidative stress These results further support the neuroprotection of NMN/NAD+
We now demonstrate that mitochondrial respiratory function was restored
NMN could restore cognition in AD model rats
NMN Treatment Rescues Cognitive impairments
NMN significantly increased the level of NAD+ in the heart
NMN protected the heart from I/R injury
NMN reduces vascular oxidative stress
NMN treatment normalizes aortic stiffness in old mice
NMN represents a novel strategy for combating arterial aging
NMN can reduce myocardial inflammation NMN thus can cut off the initial inflammatory signal, leading to reduced myocardial inflammation
Remarkably, NMN administered to FXN-KO mice restores cardiac function to near-normal levels.
Restoration of cardiac function and energy metabolism upon NMN supplementation
Remarkable decrease in whole-body EE and cardiac energy wasting
Exogenous NMN prevents photoreceptor degeneration and restores vision
NMN rescues retinal dysfunction in light-induced degeneration
Completed and pending publication
- 2016 Keio University Study – UMIN000021309
- 2017 Keio University Study – UMIN000030609 Phase 2, 8 weeks, 30 subjects
- 2017 Hiroshima University Study – UMIN000025739 24 weeks, 20 subjects, 100 & 200mg
- 2017 University of Washington Study – 8 weeks, 50 subjects
- 2017 Sinclair Metrobio study – Phase 1
- 2018 Sinclair Metrobio study – Phase 2
The Phase 1 study by Dr Sinclair has been completed, and they are ready to go forward with the Phase 2 study, so we can conclude there were positive results, and no negative side effects, else they would have to publish those immediately.
In the University of Washington study, participants are 50 healthy women between 55 and 70 years of age with slightly high blood glucose,BMI and triglyceride levels.
Using a dose of 2 capsules of 125mg NMN per day over a period of 8 weeks, researchers are testing for:
- change in beta-cell function
- works to control blood sugar
- blood vessels dilate
- effects of NMN on blood lipids
- effects of NMN on body fat
- markers of cardiovascular and metabolic health
The active supplementation portion of this study has ended, but testing of metabolic parameters will continue for 2 years after supplementation has ended. So researchers know the immediate effects and preliminary results are expected to be announced in 2018, with final results expected in 2020.
NMN is stable in the bloodstream
Sublingual NMN does bypass the liver to send the NMN direct to the bloodstream where it can be used by cells that have their own salvage pathway to increase intercellular NAD+.
In vivo, NMN is found in blood plasma. When added to blood in vitro, it is stable. (Canto,Brenner 2016)
Our results further demonstrate that while NR is spontaneously converted to NAM in cell-free plasma, NMN is more resistant to this process.
On the contrary, NMN is stable in plasma and there is no NAM increase in NMN samples up to 1 h incubation.
NR is NOT STABLE and not found in blood plasma
In both mice and humans, studies repeatedly failed to find any NR in the blood plasma at any time, even after very high dosages of NR (97, 98, 99) NR has been found at trace levels in inside blood cells.
The following quote from this Dr Brenner study also did not find NR in bloodstream after oral supplements, but was found in trace amounts after Injection.
NR varied and displayed no response to NR administration… but was detected after IP of double labeled NR.
When added to blood plasma in the lab, NR is unstable and quickly deteriorates to NAM (Canto,Brenner 2016).
∼10% of NR degraded after 10 min and ∼66% degraded after 1 h (Fig. 8e), which is further illustrated by gradual increase in NAM abundance in the samples (Fig. 8g).
NR is quickly taken up by cells and elevates NAD+ in the liver, but is not found outside the liver in blood plasma. This implies much of the overlapping benefits of NR with NMN and NAD+ are due to the increased NAD+ created in the liver.
NR is effective in liver short term
The Trammell research shows that in the liver, NMN and NAD+ must be degraded to NR before crossing the cell membrane before converting back to NMN and then NAD+. This may be why a single dose of NR increases NAD+ levels in the liver more than NMN, NAM, NA and other NAD+ metabolites as shown here.
This short term advantage for NR in the liver does not apply to all tissues, as both NMN and NAD+ have been shown to cross the cellular membrane in heart, brain, and other tissues.
NR increase in NAD+ limited by HOMEOSTASIS
The Liver is the “engine” that supplies the great majority of NAD+ to the rest of the body (Liu,2018).
In the Trammel thesis, Dr Brenner consumed 1000 mg of NR. At day 1, his NAD+ was increased by 270%.
The Elysium study used 500 mg of NR per day (plus pterostilbene). NAD+ was increased in blood plasma by 90% at 30 days, and dropped to 55% at 60 days.
The authors of the Elysium study believe that homeostasis limits the maximum increase in NAD+ that can be sustained over the long term.
High levels of NAD+ can induce homeostatic mechanisms to restrain further increases.
This may explain why those taking NR capsules often report increased energy, which seems to fade after some time. Homeostasis has brought their NAD+ levels back down and the hypothalamus isn’t getting the message to increase metabolism as it did back on day 1.
While 50% increase is helpful, keep in mind that as we age, our NAD+ levels drop in half. So the average person would need to DOUBLE their NAD+ levels – a 100% increase – to reach the levels they have in youth.
homeostasis effect on NAD+ from NMN unclear
The chart at right shows NAD+ increase measured in the liver (and soleus muscle) after 60 days of supplementation with NMN (Sinclair, 2018).
This is the best indication we have to date, but was with mice. With humans, there has been a Japanese clinical study completed, and one by Dr Sinclair, but neither has yet published the results.
We doubt they will show anywhere near this 500% increase, as NMN and NR are so closely related. But this does provide some hope that NMN is not subject to the same limits on the long-term increase of NAD+ levels as have been found with NR (above).
Sublingual NAD+ not limited by liver homeostasis
Supplying NAD+ direct to the bloodstream bypasses the liver, temporarily enabling a greater increase in NAD+ levels.
Any NAD+ (or NMN) in the bloodstream will get filtered out by the liver in 30-60 minutes.
So after an initial spike in NAD+, the same limits imposed by homeostasis in the liver will likely take effect.
This is why NAD+ clinics use slow IV drips to constantly supply NAD+ to the bloodstream rather than a single large daily injection of NAD+.
Frequent dosages throughout the day of our NAD+ sublingual tablets provide a steady supply of NAD+ direct to the bloodstream, avoiding the limits imposed by homeostasis in the liver.
Importance of Hypothalamus for Energy Metabolism
The hypothalamus in particular has emerged as an integrating, superordinate master regulator of whole-body energy homeostasis.
In summary, the hypothalamus plays a key role in the regulation of appetite and food intake both in humans and rodents.
Hypothalamic inflammation impairs the effects of insulin and leptin, contributing not only to hyperphagia and obesity development but also to the associated dysregulation of glucose homeostasis.
Energy homeostasis is controlled mainly by neuronal circuits in the hypothalamus and brainstem.
The hypothalamus is the region of the brain that controls food intake and body weight.
Leptin and insulin signal the status of body energy stores to the hypothalamus.
These peripheral hormones influence their effects on energy homeostasis either by activating or inhibiting the activity of the orexigenic or anorexic peptides within the hypothalamus.
Hypothalamus as master aging clock
- Sublingual delivery is required for all NAD+ metabolites and precursors to avoid digestion in the stomach and liver.
- NAD+ clinics use slow drip IV of NAD+ to avoid the stomach and liver. They are exploding in popularity, but the extreme cost and time required for treatment severely limit their application for the general public.
- Sublingual NAD+ delivery solves the bioavailability problem and mimics the slow drip delivery used successfully by NAD+ IV clinics.
- Sublingual NAD+ is not subject to the homeostasis that limits NAD+ increase with NR (and perhaps NMN), as it is supplied directly to the bloodstream.
- We believe the ability of NAD+ to increase metabolism through the hypothalamus has a great impact on the entire body. This is accomplished directly from increased NAD+ circulating in blood plasma, and not from NMN or NR.
- Other organs such as heart, liver, kidney, and lungs also clearly benefit from increased circulating NAD+, but there is evidence NR an NMN may have similar effectiveness.
- Exogenous NR increases circulating NAD+ levels, but after several weeks, that increase is severely limited by homeostasis.
- Exogenous NMN elevates NAD+ levels similar to NR, but seems to be less limited by homeostasis. Publication of recently completed research should shed more light on that question.
NMN demonstrates a remarkable ability to rapidly restore vascular growth that has not been shown with use of NR or NAD+.
Sublingual NAD+ will lead to a greater increase in circulating NAD+ than NR or NMN supplements.
OTHER WAYS TO INCREASE NAD+
There are a number of lifestyle changes you can make to increase NAD+ in you body.
It’s well known that Calorie Restriction (CR) can extend longevity by 30–50% in many mammals (32)
CR has also been shown to increase NAD+ levels in the body , thru these pathways:
- Lowering blood glucose levels minimizes inflammation, which consumes NAD+.
- The ketone body BHB signals to increase AMPK to produce more NAD+
- Burning Ketones for fuel instead of glucose requires 1/2 as much NAD+
KETOSIS BOOSTS NAD+
Ketosis is a metabolic state in which fat provides most of the fuel for the body. It occurs when there is limited access to glucose (blood sugar), which is the preferred fuel source for many cells in the body.
Intermittent or Periodic Ketosis is also effective at extending lifespan and likely achieves much of the benefit (36,37).
Some research even shows more benefit from a cyclical rather than a full time Ketogenic Diet (71).
The Red bars in the chart at left show the increased levels of the Ketone Body BHB produced from a cyclical Keto diet, resulting in Increased NAD+ and greatly improved neurological function and health.
EXERCISE THAT BOOSTS NAD+
Researchers are finding that 2-3 short bouts of High Intensity Interval Training (HIIT) per week is far more effective at lowering inflammation (and increasing NAD+), especially among older adults (55)
Exercise is very effective at boosting AMPK and NAD+, especially when performed at times of low blood glucose levels ( more about HIIT ).
Short bouts of HIIT accomplishes the goal, while avoiding overtraining from endurance workouts which increases inflammation and consumes NAD+ (55).
Decline of NAD+ during Aging, 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.