The Power of NAD+ Boosting Strategies: From Metabolism to Clinical Impact


NAD+, short for nicotinamide adenine dinucleotide, is a coenzyme found within every living cell with vital roles in various fundamental biological processes, including but not limited to, energy metabolism, DNA repair, cellular communication, and enzymatic activation. Originally, NAD+ was identified for its role in enhancing fermentation in yeast, where researchers found evidence of a coenzyme whose presence was crucial for alcoholic fermentation. Though this early research predates our current understanding of NAD+, it prompted diverse biological investigations into its role in cellular redox reactions, electron transfer, glycolysis, the Krebs cycle, and fatty acid β-oxidation.

Beyond its metabolic functions, NAD+ acts as a coenzyme for vital proteins including sirtuins, polyadenosine diphosphate-ribose polymerases (PARPs), and CD38, participating in molecular deacetylation, ribosylation, glycohydrolase synthesis, and NAD+-dependent signal transduction. These actions contribute to metabolic homeostasis and cell signaling, highlighting the continuous demand for NAD+ within the body. Unsurprisingly, the mechanism of NAD+ production varies slightly across species but ultimately aims to maintain a delicate balance between production and recycling to ensure proper cellular function.

Emerging research now suggests a close link between NAD+ levels, aging, and an increased risk of age-related diseases, such as neurodegenerative disorders, cellular senescence, and cardiovascular complications. An analysis of human skin samples revealed an age-related accumulation of oxidative DNA damage, increased lipid peroxidation, and a significant (~70%) decline in NAD+ levels. Data collected on human livers indicate a 30% loss between the ages of 45–60+, and two independent MRI studies of the brain revealed a 10–25% decline in NAD+ levels from adolescence to old age. These findings align with observations in cell and rodent models, reinforcing the importance of understanding NAD+ metabolism in aging and prompting its progression to human clinical trials to evaluate whether the effects of aging can indeed be reduced or even reversed.

The mechanisms underlying the touted beneficial effects of NAD+ precursors are centered around their influence on cellular metabolism, stress response, and DNA repair through ensuring sufficient NAD+ activity. Administration of these precursors and subsequent increase in NAD+ enhances the activity of key enzymes in the citric acid cycle, sirtuin, and PARP activation. This translates to enhanced cellular resilience against oxidative stress, DNA damage, and other hallmarks of aging. However, further research is necessary to fully elucidate the specific mechanisms by which NAD+ precursors exert their effects in different tissues and cell types.

Increasing NAD+ levels is a logical first step to remediating the natural age-related decrease of the coenzyme, and this can be achieved in a facile manner through exogenous intake, a healthy diet, and exercise. B vitamins and tryptophan from dietary intake contribute to a pool of NAD+ precursors, but the majority of NAD+ is biosynthesized from internally generated and recycled precursors. In this context, nicotinamide mononucleotide (NMN) emerges as a promising candidate for NAD+-boosting interventions. As a direct precursor to NAD+, NMN readily converts within cells, potentially mitigating the age-related NAD+ decline. This potential has generated significant interest in its role as a putative anti-aging strategy, requiring rigorous scientific exploration to fully understand the safety, efficacy, and long-term consequences of NMN supplementation and the subsequent effects of NAD+ boosting in humans.

NAD+ Biosynthesis Pathways Three independent pathways, meticulously controlled by unique sets of enzymes and precursors, ensure sufficient NAD+ levels within its specific compartments: cytoplasm, mitochondria, and nucleus (Figure 1). These pathways maintain an intricate balance of NAD+ synthesis, usage, recycling, and regeneration. Serving as the predominant mechanism for maintaining cellular NAD+ homeostasis, the salvage pathway efficiently recycles NAD+ precursors, primarily utilizing nicotinamide (NAM) generated by the enzymatic breakdown of NAD+ in various cellular processes.

Dietary sources of nicotinamide riboside (NR) and NMN can also contribute to this pathway. Within this pathway, NMN serves as the key intermediate. To initiate NAD+ production, NMN is reportedly transported into cells via the recently discovered Slc12a8 transporter. Alternatively, NR enters cells through equilibrative nucleoside transporters (ENTs) and is converted into NMN by NR kinases (NRKs) in one step. NMN is then efficiently converted to NAD+ by the enzyme NMN adenyltransferase (NMNAT). The cycle continues with NAD+-consuming enzymes, such as CD38 and sirtuins, which release NAM in the reaction. NAM phorphorylribotransferase (NAMPT), the rate-limiting enzyme in this pathway, then catalyzes the conversion of NAM back to NMN, perpetuating the salvage loop. This process enables swift NAD+ production and continuous replenishment of precursors, independent of external resources.

Figure 1 – Biosynthetic pathways of NAD+ synthesis in mammalian cells – The salvage pathway is the body’s main and most efficient source of NAD+. Abbreviations: 3-HK, 3-hydroxykynurenine; 3-HAA, 3-hydroxy anthranilic acid; ACMS, 2-amino-3-carboxymuconic semialdehyde; QPRT, quinolinate phosphoribosyltransferase; NA, nicotinic acid; NAPRT, nicotinic acid phosphoribosyltransferase; NAMN, nicotinic acid mononucleotide; NMNAT, nicotinamide mononucleotide adenylyltransferase; NAAD, nicotinic acid adenine dinucleotide; NADS, NAD+ synthetase; NR, nicotinamide riboside; NMN, nicotinamide mononucleotide; NRK, nicotinamide riboside kinase; NAD+/NADH, nicotinamide adenine dinucleotide; NAM, nicotinamide; NAMPT, nicotinamide phosphoribosyltransferase; 3-HAAO, 3-Hydroxyanthranilate 3,4-Dioxygenase; NADP/NADPH, Nicotinamide adenine dinucleotide phosphate; TDO2, Tryptophan 2,3-dioxygenase; KYNU, kynureninase KFase, kynurenine formidase, KMO, Kynurenine 3-Monooxygenase.

The two remaining NAD+ biosynthetic pathways rely on dietary precursors, which can be advantageous when salvageable precursors are limited. The Preiss-Handler mechanism begins with nicotinic acid (NA)—a form of vitamin B3 or niacin found in fish, poultry, nuts, grains, and vitamin supplements. In three steps, NA is transformed into NA mononucleotide (NAMN), followed by NA adenine dinucleotide (NAAD), and finally to NAD+ by nicotinate phosphoribosyl transferase (NAPRT), NMNAT, and glutamine-dependent NAD+ synthetase (NADS) respectively.

Similar to the Preiss-Handler pathway, the de novo pathway relies on dietary tryptophan which enters cells via specific amino acid transporters, LAT-1 and hPAT4. The enzymes indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO) then convert tryptophan to N-Formylkynurenine. A series of five subsequent enzymatic reactions lead to the formation of quinolinic acid (QA). Finally, the enzyme QPRT catalyzes the conversion of QA into NAMN, which can then feed into the Preiss-Handler for NAD+ production. The liver is the primary site for NAD+ synthesis from tryptophan, as most cells lack the enzymes required for the de novo pathway. Consequently, the majority of cellular tryptophan is metabolized to NAM within the liver, released into circulation, and subsequently taken up by peripheral cells for conversion to NAD+ via the salvage pathway.

Boosting cellular NAD+ levels can be achieved through supplementation with NAD+ precursors that directly participate in NAD+ synthesis, without the need for lengthy conversion pathways. As a direct precursor, NR is one such candidate that is readily converted to NMN by NRKs, quickly integrated into the salvage pathway, and is well tolerated in moderate doses. Preclinical studies show promise of its efficacy. NR is reported to have a short elimination half-life and the effects of long-term administration are still under investigation. One step closer to NAD+ in the salvage pathway is NMN, which has been shown to increase NAD+ levels in as little as 30 min and is safe during one year of chronic dosing in mice. NMN also appears to be more stable in water, and when taken orally, is rapidly absorbed, and metabolized, with many studies showing increased NAD+ biosynthesis and the improvement of several age-related ailments. In murine models, NMN is studied extensively and has displayed several benefits such as improved mitochondrial metabolism, insulin secretion, and ischemia, to name a few. While translating pre-clinical findings to humans is always a challenge, successfully applying data from animal models could pave the way for groundbreaking solutions for age-related conditions. This data is crucial to establish NMN as a viable NAD+ boosting precursor and determine to what extent previous clinical data is applicable.

Impact of Metabolism on Supplementation Supplementation with NAD+ precursors offer a promising therapeutic approach to address declining NAD+ levels. However, a major challenge lies in the limited bioavailability of orally administered NAD+ precursors due to extensive metabolism in the gastrointestinal (GI) tract and liver.

Gastrointestinal Tract The impact of gut bacteria on NAD+ precursor metabolism and the complexities of cellular uptake mechanisms have been extensively researched in recent years. Significant progress has been made, but interesting questions remain and warrant further investigation.

Interaction with Gut Microbiome NAD+ precursors can be categorized into two chemical groups: amidated (NR, NMN, NAM) and deamidated (NA, NAMN, NAAD). Traditionally, these groups were thought to follow distinct biosynthetic routes. Amidated precursors were known to follow the salvage pathway (NR → NMN → NAD+), while deamidated precursors fueled the Preiss-Handler pathway (NA → NAMN → NAAD → NAD+). However, this paradigm shifted with the observation of increased NAAD (a deamidated intermediate) in blood cells after NR (an amidated precursor) supplementation. This unexpected finding hinted at an interplay between the pathways, potentially involving previously unrecognized enzymatic activities in mammals for NAD+ production. Prior research identified a gut bacterial enzyme, PncC, capable of converting NMN into NAMN (a deamidated precursor) for NAD+ synthesis via the Preiss-Handler pathway. This suggests a potential role for gut bacteria in NAD+ metabolism and could explain the rise in NAAD observed after NR supplementation.

Understanding the Preiss-Handler Pathway in Simple Terms

Imagine your body needs a special kind of fuel called NAD+ to keep everything running smoothly. One way your body makes this fuel is through a process called the Preiss-Handler pathway. Let’s break it down into simple steps:

  1. Starting with Niacin (Vitamin B3):
    • Your body begins with a vitamin called niacin, which you can get from foods like fish, chicken, nuts, grains, and vitamin supplements.
  2. Making the First Ingredient (NAMN):
    • The body takes niacin and changes it into a new ingredient called NAMN (nicotinic acid mononucleotide). Think of this as mixing flour and water to make dough.
  3. Creating the Second Ingredient (NAAD):
    • Next, the body takes the NAMN and turns it into another ingredient called NAAD (nicotinic acid adenine dinucleotide). This step is like kneading the dough to get it ready for baking.
  4. Final Step to Make NAD+:
    • Finally, the body changes the NAAD into the special fuel we need, NAD+. This is like baking the dough into bread that we can eat.

So, in the Preiss-Handler pathway:

  • Niacin (Vitamin B3) → NAMNNAADNAD+

Why is this important? NAD+ is crucial for keeping our cells healthy and providing energy. The Preiss-Handler pathway is one of the ways our body makes sure we have enough NAD+ to stay healthy and energetic.

How can you help this process?

  • Eat foods rich in niacin: Include fish, chicken, nuts, grains, and niacin supplements in your diet.
  • Healthy lifestyle: Regular exercise, enough sleep, and reducing stress can support your body’s ability to make NAD+.

In simple terms, the Preiss-Handler pathway is like a cooking recipe your body follows to turn niacin (Vitamin B3) into the essential fuel (NAD+) it needs to keep you healthy and energetic.

Subsequent studies confirmed the crucial role of the gut microbiome in bridging the amidated and deamidated NAD+ pathways. In mice, orally administered NAM was deamidated by the microbial nicotinamidase, PncA, generating NA, nicotinic acid riboside (NAR), and NAAD. This microbial deamidation process was important for NAD+ synthesis in the colon, liver, and kidney. Similarly, another study showed that gut bacteria facilitated the deamidation of orally administered NMN, yielding the metabolites NAR and NAMN (in the gut and liver), and NAAD (in the liver) for incorporation into NAD+ via the de novo pathway. In germ-free mice, NMN escaped microbial deamidation and instead fueled the salvage pathway, leading to elevated levels of amidated metabolites, NR and NMN.

Surprisingly, antibiotic treatment doubled gut NAD+ metabolite levels (including NMN, NR, NAD+, and NAM) in mice, even without NMN supplementation. This finding suggests competition by gut microbiota for both dietary and endogenous NAD+ sources. Further highlighting the complexity of NAD+ metabolism, Yaku et al. revealed a two-step process in NR utilization. Initially, direct NR uptake occurred in the small intestine for up to an hour, driving NAD+ synthesis through the classic salvage pathway even in the presence of gut bacteria. Following this, the enzyme bone marrow stromal cell antigen 1 (BST1) hydrolyzed NR to NAM, which was then metabolized to NA by gut microbiota, fueling the Preiss-Handler pathway and becoming the major driver of NAD+ production. They also found that BST1 transformed NR into NAR through a base-exchange reaction using NA and NAM, providing another connection between amidated and deamidated precursors.

A key finding of these studies is that orally administered NR, NMN, and NAM predominantly undergo degradation in a process dependent on gut microbiota, resulting in the formation of NAM or NA and downstream deamidated metabolites. Only a minor portion of NAD+ precursors delivered orally integrate into tissues without significant alteration.

Uptake Mechanisms Two primary pathways are currently recognized for NMN entry into enterocytes: an indirect route involving conversion to NR, and a direct route mediated by a specific NMN transporter. However, the relative importance and specific contributions of each pathway have not been determined. Initially, the prevailing model suggested NMN relies solely on an NR-mediated pathway. Here, the extracellular enzyme CD73 converts NMN to NR, which is then imported by equilibrative nucleoside transporters (ENTs) and phosphorylated back into NMN by NRK1/2 enzymes within the cell. Studies support this pathway, demonstrating the requirement of NRKs for NMN to boost NAD+ levels in muscle and liver cells. However, the slow processing time of this pathway, exceeding several hours in some studies, cannot account for rapid NMN absorption within the gut (2–3 min) and tissue uptake (10–30 min) observed in mice. Additionally, robust NMN uptake in cells despite inhibition of CD73/ENT or NRK1 further suggests an alternative route exists. The discovery of Slc12a8, a highly specific NMN transporter abundantly expressed in the gut, pancreas, liver, and white adipose tissues, provides a compelling mechanism for rapid NMN absorption and distribution. Studies demonstrate that Slc12a8 deletion in these organs significantly reduces NMN uptake and NAD+ levels, supporting its role in direct NMN transport.

A published response to the initial study challenged the validity of the Slc12a8 findings, indicating that inappropriate methods were used in the study. Agreement on accurate NMN quantification methods continues to be a challenge in NAD+ metabolism research today, due to the lack of standardized protocols. Grozio et al. provided evidence supporting the chosen analytical technique in a subsequent publication. Data interpretation was also contested, as the researchers adapted the protocols to address the inherent instability of the NMN molecule. The critique further argues that previous data showed sufficient evidence to conclude NMN was transported into the cell via conversion to NR, not via the Slc12a8 transporter. However, a key difference in the time of sampling (minutes vs. hours) indicated both routes of uptake are feasible. Tracer studies on NMN metabolism also yield inconsistent results, as some suggest significant NMN conversion to NR in the gut, with limited direct absorption. In contrast, a study detected NMN in the intestine within 10 min of oral intake, supporting the role of Slc12a8 in direct transport. Other studies have detected NMN in the intestine within 10 min of oral intake and minimal NMN-to-NR conversion in the gut when measured within minutes.

The relative contributions of NRK1/2 and Slc12a8 to NMN uptake mechanisms likely vary depending on time, cell type, tissue, and physiological conditions. This dynamic interplay was illustrated in septic mice, where NRK1/2 enzyme levels decreased significantly, while Slc12a8 expression remained stable. Notably, NMN supplementation still effectively increased NAD+ levels despite NRK1/2 pathway suppression, underscoring the critical role of alternative, NR-independent uptake mechanisms such as Slc12a8. Tissue-specific expression patterns further contribute to this complexity. Organs with inherently low NRK1 activity, such as the heart and white adipose tissue, might predominantly rely on Slc12a8-mediated uptake for NAD+ production. This could be due to differences in metabolic demands, regulatory factors, or unique cellular processes within these tissues, warranting further investigation.

Portal Delivery After absorption in the intestine, NAD+ precursors flow to the liver via the portal vein. Analysis of portal blood at three hours after oral NAM administration in mice revealed the presence of NAMN, NAR, NA, NMN, and NAM. While the major deamidated NMN metabolites, NAMN and NA, were detected, their concentrations were significantly lower (100–400-fold) compared to NAM, the predominant circulating precursor. Four hours after NR gavage, elevated levels of NA and NAR appeared in portal blood, indicating bacterial contribution to NR metabolism. These metabolites were absent in germ-free mice, highlighting the essential role of gut microbiota. Interestingly, germ-free mice also exhibited a diminished increase in circulating NAM, further emphasizing the microbiota’s influence on NAD+ precursor processing.

Hepatic First-Pass Metabolism NMN undergoes extensive first-pass metabolism in the liver when administered orally. Studies across various dosages (50 mg/kg to 500 mg/kg) consistently show that nearly all ingested NMN is converted into NAM within the liver. This minimizes the amount of intact NMN available for direct NAD+ synthesis in the liver and prevents it from reaching peripheral tissues, significantly impacting overall bioavailability.

Bypassing gut metabolism through intraperitoneal (IP) injection offers a different approach. In mice injected with radiolabeled NMN (500 mg/kg), the NMN reaches the liver first via the portal vein. While some newly generated NAD+ in the kidneys and small intestines showed incorporation of labeled NR (suggesting localized NMN conversion to NR before NAD+ synthesis), most tissues relied on NMN-derived NAM for NAD+ production. Minimal intact NMN utilization was observed in the kidney and white adipose tissue. It’s important to note that this study only assessed NMN incorporation at two and four hours, potentially missing early metabolic events. For example, a separate study using IP injection of NMN (500 mg/kg) observed that mice displayed a rapid uptake, with liver NMN levels surging 15-fold within just 15 min after injection, followed by a return to baseline by 30 min. This rapid rise and subsequent decline suggest swift NMN metabolism. Notably, NAD+ levels continued to rise steadily for 60 min, highlighting the ongoing utilization of NMN or its metabolites for NAD+ synthesis.

When administered intravenously (50 mg/kg), NMN bypasses enteric and hepatic metabolism, enabling a small portion of intact NMN to directly participate in NAD+ synthesis within the liver and kidneys. While incorporation of intact NMN was still limited, the observed differences highlight the crucial role of administration methods on NMN bioavailability.

Bloodstream Dynamics and Tissue Distribution Accurately measuring NMN in the bloodstream remains a significant hurdle, mirroring the challenges faced with defining cellular transport of NMN. Limitations in current methods and the lack of a gold standard, leading to conflicting reports. Studies show NMN detection ranging from undetectable levels to 90 µM after oral intake, hindering our understanding of NMN’s fate within the bloodstream. In general, studies using HPLC detect higher levels of NMN, whereas those using LC-MS/MS report much lower or undetectable levels of NMN.

While NMN did not directly cross the blood-brain barrier in mice after IV administration, its impact on brain NAD+ levels has proven to be substantial. NMN administration (500 mg/kg) via IP injection significantly increased hippocampal NAD+ levels by 34–39% within just 15 min, with similar effects observed in the hypothalamus. Even a lower dose, NMN (62.5 mg/kg) sustained hippocampal mitochondrial NAD+ levels for 24 h. Intravenous NMN also showed minimal muscle uptake. However, a clinical trial providing oral NMN to prediabetic women revealed elevated levels of proteins associated with insulin sensitivity in muscle tissues. Although muscle NAD+ levels remained unchanged, elevated NAD+ metabolites suggested a potential increase in muscle NAD+ turnover. A critical knowledge gap remains, however: how long do tissue NAD+ levels remain elevated after supplementation ceases in humans? Addressing this question is crucial for understanding the long-term effects of NMN.

The chosen administration route significantly impacts NMN’s fate. NMN (500 mg/kg) administered via IP injection in mice effectively boosted NAD+ levels in the liver, kidney, white adipose tissue, pancreas, and heart, with the liver showing the most prominent increase. Oral gavage (500 mg/kg) yielded similar effects in the liver but showed less efficacy in all other tissues. Clinical trials show oral supplementation increases plasma NAD+ for at least 24 h, but levels return to baseline within a month of discontinuation. In a pilot study, the administration of 300 mg IV NMN in adults not only safely elevated NAD+ levels but also uniquely reduced triglycerides. A triglyceride-lowering effect has not been observed with oral NMN, indicating route-specific metabolic variations.

Functional Diversity of NAD+ Precursors Despite sharing interconnected pathways, NAD+ precursors display diverse fates and functions, suggesting they are not simply interchangeable building blocks for NAD+ synthesis. Ongoing discoveries reveal their complexity, with each finding sparking new questions. Nevertheless, significant differences in precursor metabolism and bioavailability have been identified, highlighting their potential for tailored therapeutic interventions.

Efficacy and Therapeutic Applications of NAD+ Boosting Strategies Although NAM is the principal contributor to basal NAD+ levels in mammals, its therapeutic efficacy is limited. Unlike NMN and NR, NAM can inhibit sirtuins, a class of proteins linked to NAD+ benefits. Additionally, NAM conversion to NAD+ has inherent limitations. The initial step requires NAMPT, an energy-dependent enzyme subject to feedback inhibition, limiting its ability to substantially elevate NAD+. Elevating dietary NAM also results in a corresponding increase in NAM methylation and excretion, thereby diminishing its NAD+-boosting efficacy.

Nicotinic acid (NA) stands apart from other NAD+ precursors due to its distinctive flushing effect, even at low doses of 50 mg. This side effect arises from NA’s direct activation of the GPR109A receptor, which is independent of processes associated with NAD+ biosynthesis. Furthermore, NA exhibits lower efficacy in elevating NAD+ levels compared to NMN, NR, and NAM, rendering NA less enticing as an NAD+ booster.

NMN and NR are widely acknowledged as promising NAD+ boosters, raising NAD+ blood levels by 1.5–2.5-fold in clinical trials. However, metabolic tracing studies reveal that NMN and NR are mostly metabolized to NA or NAM in the intestine. While common logic suggests they would have similar effects and limitations as NA or NAM, they exhibit unique advantages despite their conversion. Notably, they lack the flushing side effects associated with nicotinic acid (NA) and do not appear to inhibit sirtuins, as seen with NAM. Additionally, comparative studies showed that oral NR administration was more effective than NAM or NA at raising NAD+ levels in the liver of mice, and NMN was retained in the body longer than NAM. Direct NMN transport to tissues could theoretically yield a more substantial increase in NAD+ production compared to NAM, as it bypasses the NAMPT-catalyzed reaction and is not subject to feedback inhibition.

Isotope labeling studies revealed an unexpected mechanism for NAD+ elevation. While NMN and NR are thought to directly elevate NAD+ as precursors, oral administration of labeled versions surprisingly led to an increase in unlabeled NAD+ metabolites. This suggests indirect effects on endogenous NAD+ biosynthesis, potentially through activation of an unknown signaling pathway. Further investigation is needed to elucidate this novel mechanism and its contribution to the unique effects of NMN and NR. NMN offers a potential advantage over NR due to its direct cellular uptake, independent of NRK1/2 enzymes. This tissue-specific requirement for NRK1/2 conversion could limit NR’s efficacy in certain cell types, highlighting the potential therapeutic benefit of NMN in scenarios where NR might be less effective.

More recently, researchers have explored the effects of enzymes related to NAD+ synthesis, namely ACMSD, as possible therapeutic targets for boosting NAD+ levels. The enzyme α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) governs the rate at which ACMS is cyclized to form QA and continue the de novo synthesis cycle. In both C. elegans and murine models, the inhibition of ACMSD was shown to increase NAD+ synthesis in a dose-dependent manner and subsequently increase SIRT1 and improve mitochondrial function. This mechanism appears to be conserved evolutionarily between these models suggesting that the same may apply to humans, but further research is necessary to determine the extent of this link.

Physiological Context and Precursor Efficacy The therapeutic efficacy of different precursors can diverge even within the same physiological context. For example, NR and NMN effectively promote hematopoiesis, but NA and NAM do not. Oral supplementation with NR, but not NAM, restored NAD+ levels in the heart and improved cardiac function in a mouse model of cardiomyopathy. While NMN supplementation has been shown to improve cardiac function in a model of Friedreich’s ataxia cardiomyopathy, similar effects were not observed for NR.

Aging disrupts NAD+ balance, prompting the intestine to upregulate the NMN transporter Slc12a8. This regulatory mechanism forms a feedback loop, enabling NMN supplementation to effectively restore NAD+ levels. Conversely, with age and disease, there can be a decline in NAMPT activity and expression, which plays a crucial role in the salvage pathway for NAD+ production by converting NAM to NMN. This downstream bottleneck impedes the efficacy of NAM supplementation for NAD+ repletion under such conditions. In contrast, NMN bypasses the NAMPT step in the salvage pathway, demonstrating superior capability to elevate NAD+ levels and alleviate dysfunction associated with NAMPT deficiency.

NRK2, a key enzyme in the salvage pathway for NAD+ biosynthesis, exhibits stress-induced upregulation. This is observed in injured neurons, muscle subjected to injury or high-fat diets, and stressed cardiac tissue. This suggests a protective mechanism, as NR or NMN supplementation effectively reduces disease severity in most conditions that exhibit stress-induced elevations in NRK2.

Human Studies Discussion Human trials exploring NMN supplementation have yielded promising results. However, the outcomes exhibit notable inconsistencies, underscoring areas for improvement and guiding future research endeavors towards resolving these discrepancies. Understanding the nuanced factors influencing NMN’s efficacy—such as timing, dosage, and target populations—is essential to characterize its clinical significance.

Modulation of NAD+ Levels Human trials measuring NMN’s impact on serum NAD+ levels show mixed results, highlighting significant challenges in measuring this metabolite. While Yi et al. reported consistent dose-dependent increases in serum NAD+ and NMN, Katayoshi et al. found undetectable levels. Further complicating the picture, Huang et al. observed a non-significant increase in serum NAD+/NADH, contradicting prior studies demonstrating efficacy at similar or lower doses.

Investigations into the impact of NMN supplementation on NAD+ concentrations reveal distinct NAD+ metabolic profiles within various blood cell fractions. Yamane et al. observed a significant increase in plasma NAD+ levels following daily NMN supplementation (250 mg) for 3 months in healthy volunteers, peaking within the first month and remaining elevated throughout supplementation. Similarly, Okabe et al. noted a doubling of NAD+ levels after one month of NMN supplementation, with sustained elevation until supplementation cessation. Notably, Okabe et al. assessed NAD+ in whole blood, capturing both circulating and cellular NAD+, while Yamane et al. focused on plasma, crucial for systemic distribution. This discrepancy elucidates markedly lower NAD+ concentrations in plasma reported by Yamane et al., approximately 100 times lower than in whole blood. Intriguingly, NMN concentration was tenfold higher when measured in isolated plasma than when measured in whole blood by Okabe et al., highlighting the importance of sample type in NAD+ metabolism data interpretation.

Supplementation appears to influence NAD+ metabolism beyond simple conversion to NAD+. Okabe et al. observed increases in NAMN, a deamidated metabolite of NMN, alongside elevated NAD+. Igarashi et al. also reported increased levels of NAD+ precursors (NR, NAR) after NMN supplementation, mirroring findings in animal studies.

NAD+ levels are dynamic, and influenced by diet, activity, and circadian rhythms. Studies suggest short-term fluctuations and a return to baseline within 2 h after daytime NMN administration. Long-term monitoring indicates stable NAD+ levels except for NMN-induced increases and exercise-related elevations. Intravenous NMN transiently elevates NAD+ and activates SIRT1/CD38 pathways.

Interestingly, NMN’s effects on NAD+ levels appear tissue specific. Studies by Yoshino et al. and Pencina et al. observed increased NAD+ in PBMCs (blood cells) but not muscle tissue following NMN supplementation. Furthermore, Qiu et al. showed reduced PBMC NAD+ in hypertensive patients, with NMN supplementation partially restoring these levels alongside lifestyle changes. These findings highlight the complexity of NMN’s influence on NAD+ metabolism and the need for further research on its tissue-specific effects and potential interactions with other interventions.

Sleep Regulation and Quality Studies evaluating the effects of NMN on sleep using single doses (100–500 mg) and short-term supplementation (250 mg for 8 weeks) showed no significant changes in sleep measures. However, a longer-term study (12 weeks) showed hints of potential benefit. Afternoon NMN intake (250 mg) in this study reduced drowsiness in older adults, suggesting timing of the dose may play a role. In a population with pre-existing sleep difficulties, 12 weeks of daily NMN supplementation (180 mg) yielded statistically significant improvements in sleep quality, latency, and daytime function. These findings suggest NMN’s effect on sleep may depend on dose, duration, and potentially time of administration, with greater benefit observed in those with existing sleep issues.

Physical Performance While some studies report improved gait speed, grip strength, and lower limb function, others show no significant effects on walking distance or in diabetic/obese populations. Interestingly, NMN can improve muscle insulin sensitivity without impacting mitochondrial function or overall performance, suggesting a more nuanced effect on muscle health. Additionally, higher NMN doses (600–1200 mg) might benefit trained athletes in terms of aerobic capacity. Timing of administration (afternoon vs morning) may also influence outcomes. Overall, the evidence for NMN’s efficacy in enhancing physical performance remains inconclusive and warrants further investigation.

Cardiometabolic Health Human trials investigating NMN’s impact on cardiovascular health parameters yield mixed results. Several studies observed encouraging results, including reductions in weight, blood pressure, and cholesterol levels. However, others have reported no significant changes in insulin sensitivity, lipid profiles, or vascular function. Notably, positive effects on blood pressure and arterial stiffness were observed in hypertensive individuals and those with higher baseline glucose/BMI, suggesting potential benefits in specific subpopulations. It is important to note that larger and longer-term clinical trials with more defined populations are needed to make comparisons to established therapeutic approaches to cardiometabolic health are essential to evaluate the relative clinical value of NMN and guide future research efforts. Larger, more comprehensive studies with diverse participant groups will allow for better comparisons with existing treatments for heart and metabolic health. These studies are essential to evaluate the relative clinical value of NMN and guide future research efforts.

Glucose Metabolism and Regulation In prediabetic women, 250 mg/day NMN significantly enhanced muscle insulin sensitivity, but these effects were not observed systemically. Additionally, a small trial in postmenopausal women receiving 300 mg/day NMN for 8 weeks reported reduced HbA1c and increased adiponectin, suggesting potential anti-inflammatory and insulin-sensitizing properties. Supplementation of NMN in healthy adults led to a significant increase in postprandial insulin levels after two months, suggesting enhanced glucose-stimulated insulin secretion. However, the significance of this finding in healthy individuals remains uncertain. Adding another layer of complexity, a large cross-sectional study involving over 1394 adults (most with existing metabolic conditions) found that those with the highest NAD+ levels had a three times greater risk of metabolic syndrome compared to those with the lowest levels. These contradictory findings highlight the need for further research, particularly long-term studies, to understand NMN’s sustained effects on various health markers. Additionally, the studies involved diverse populations with different metabolic conditions. The observational study suggests a potential link between NAD+ levels and specific metabolic profiles, emphasizing the importance of individualized approaches.

Overall Well-Being and Quality of Life A clinical trial showed significant improvements in subjective general health scores at day 60 for all NMN doses, with earlier improvements (day 30) in higher dose groups. While another study did not reach statistical significance, it observed a trend towards improvement in the NMN group. Postmenopausal women in a small NMN supplementation study (8 weeks) reported subjective improvements in allergies, joint pain, overall well-being, recovery, cognitive clarity, and hair quality.

Telomere Lengthening A small-scale study demonstrated significant telomere lengthening in PBMCs of male volunteers after 30 days of NMN (300 mg/day) supplementation. Telomeres continued to elongate at 60 days and nearly doubled from baseline by 90 days of supplementation. NMN’s impact on telomeres may be linked to stabilizing telomeres and preventing tissue damage through its effect on NAD+ and the SIRT-1 pathway.

Side Effects and Safety Considerations A growing body of human trials, currently encompassing 19 published studies, has investigated the safety of NMN supplementation. Early studies focused on single doses, with Irie et al.’s work demonstrating no adverse effects following ingestion of up to 500 mg NMN. Subsequent research has explored chronic administration, with studies reporting good tolerability for doses ≤500 mg over at least a month. Higher doses (600–1250 mg) administered for up to 6 weeks have also shown no significant side effects in Liao et al. and Fukamizu et al.s’ trials. Notably, Pencina et al. observed positive health outcomes alongside increased NAD+ levels in a study using 1000 mg NMN twice daily for 2 weeks.

While existing evidence is encouraging, a definitive understanding of NMN’s safety profile is still under development. Limitations in current research include short durations, small sample sizes, and a lack of diverse participants. Additionally, the potential for long-term effects, interactions with medications, and safety in individuals with pre-existing conditions require further exploration. To definitively establish a Tolerable Upper Intake Level (UL), long-term studies with age-specific considerations are crucial. Overall, NMN appears safe at moderate doses in healthy individuals, but ongoing, long-term research is essential to ensure its safe application in a wider population. Animal studies offer a glimpse into the potential hazard of increasing NMN as more comprehensive trials have been conducted using mice. It is reported that there is no carcinogenicity or tumorigenicity associated with prolonged NMN exposure for the purposes of increasing NAD levels. It seems, however, that during cancer progression, NAD boosting has shown deleterious effects by promoting cell survival, growth and propagation resulting in resistance to typical treatments and increased levels of inflammation. This possible discrepancy between humans and mouse models can only be remedied with comprehensive long-term studies.

APPENDIX 1- Understanding NAD+ and Its Benefits: A Simple Guide

What is NAD+?

NAD+ (nicotinamide adenine dinucleotide) is a molecule found in all living cells. Think of it as a helper molecule that plays a vital role in keeping our cells healthy and functioning properly. It helps convert food into energy, repair damaged DNA, and supports overall cell health.

Why is NAD+ Important?

As we age, the levels of NAD+ in our bodies decrease. This decline is linked to various signs of aging and age-related diseases, such as memory loss, muscle weakness, and heart problems. By maintaining or boosting NAD+ levels, we might slow down aging and improve overall health.

Benefits of Boosting NAD+ Levels

  • More Energy: NAD+ helps our cells produce energy, making us feel more energetic.
  • Better Brain Health: Higher NAD+ levels support brain function, potentially improving memory and focus.
  • Cell Repair: NAD+ helps repair damaged DNA, which can prevent diseases and improve longevity.
  • Healthy Aging: Maintaining NAD+ levels can reduce the signs of aging, such as wrinkles and muscle weakness.
  • Improved Metabolism: NAD+ supports metabolic processes, which can help with weight management and overall health.

How to Boost NAD+ Levels

There are several ways to increase NAD+ levels in your body:

Diet: Eating foods rich in NAD+ precursors (substances that help create NAD+) can help. These include:

  • Dairy milk (contains Nicotinamide Riboside or NR)
  • Fish
  • Chicken
  • Green vegetables
  • Whole grains

Supplements: Taking NAD+ precursors as supplements can effectively boost NAD+ levels. The most common supplements include:

  • Nicotinamide Riboside (NR): Converts to NAD+ in the body.
  • Nicotinamide Mononucleotide (NMN): Directly converts to NAD+ and is quickly absorbed by the body.

Exercise: Regular physical activity can naturally increase NAD+ levels.

Healthy Lifestyle: Reducing stress, getting enough sleep, and maintaining a balanced diet all support higher NAD+ levels.

    Taking NAD+ Supplements

    • Nicotinamide Riboside (NR) Supplements: These are easy to find and take. Studies suggest they can raise NAD+ levels by 1.5 to 2.5 times.
    • Nicotinamide Mononucleotide (NMN) Supplements: These are also effective and may be even quicker at increasing NAD+ levels. They are absorbed rapidly when taken orally.

    Safety and Side Effects

    NAD+ supplements are generally safe for most people. However, it’s always best to talk to a healthcare provider before starting any new supplement, especially if you have underlying health conditions or are taking other medications.

    In Summary

    NAD+ is crucial for energy production, DNA repair, and overall cell health. Boosting NAD+ levels can help improve energy, brain function, and healthy aging. You can increase NAD+ by eating certain foods, taking supplements like NR and NMN, exercising, and leading a healthy lifestyle. Always consult with a healthcare professional before starting new supplements.

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