NMN (β-Nicotinamide Mononucleotide), is a derivative of vitamin B3 (nicotinamide), the molecular weight is 334.221 g/mol. NMN has two isomers: α and β isomer, only β-NMN was occurred in nature, with the biological activities [1].
NMN itself is a naturally occurring species in humans and organisms. Natural NMN can be obtained from a variety of foods, and the content of NMN in some common foods are shown in the figure below. Due to the low content in food, it is difficult for humans to completely meet their needs for NMN through diet [2].
NAD+, also known as coenzyme I or nicotinamide adenine dinucleotide, is present in all living cells, and participated in thousands of biochemical reactions. Nicotinamide adenine dinucleotide (NAD+) plays an important role in various cellular metabolic reactions and is an important supporting material for cell activity.
NAD+ in human bodies decrease by half in 20 years, and the decrease of NAD during aging is considered to be a major reason of diseases and disabilities, such as hearing and vision loss, cognitive and motor dysfunction, immune deficiency, arthritis, metabolic disorders and cardiovascular diseases caused by dysregulated autoimmune inflammatory response[3]. As getting older, the decrease of NAD+ level leads to the decrease of DNA repair ability and accumulation of DNA damage, which promotes the aging process.
There are two main functions of NAD+ in human bodies. The first function is act as a coenzyme, which participates in various of biochemical reactions in cells and promotes energy metabolism (such as the oxidation of glucose, fat and amino acids). NAD+ and NADH REDOX pairs are key factors in a variety of electron-exchange-dependent biochemical reactions, especially in REDOX reactions involving redoxase-mediated hydride transfer. In these reactions, NAD+ is the electron acceptor and NADH is the electron donor. Many reactions requiring NAD(H) as a coenzyme are related to catabolism and the acquisition of metabolic energy, including alcohol metabolism, glycolysis, pyruvate oxidation decarboxylation to acetyl coenzyme A, β oxidation of fatty acid, tricarboxylic acid cycle, etc.
Apart from being a coenzyme in the reduction reactions, NAD+ can also act as a co-substrate of some non-redox reactions, that is, as a co-substrate of the sirtuins family of deacetylases, which can effectively coordinate mitochondrial function, metabolism and aging. Since then, researchers have recognized other important NAD+ consumption enzymes, such as cyclic diphosphate ribose (CADPR) synthase (including CD38 and CD157) and polydiphosphate ribose polymerase (PARP) protein families, as well as SARM1 in neurons. Researches from Dr. David Sinclair and his team have shown that the declining in NAD+ levels is caused by the protease CD38, which acts like a scissors to shred NAD+ molecules. The expression and bioactivity of CD38 gradually increased with aging, and might be several times higher in some tissues and cells [4].
The molecular size of NAD+ is too large to be directly absorbed by the human body. Directly oral ingestion of NAD+ would undergo hydrolysis in the small intestine. In theory, there is another way to supplement NAD+, by finding a method to supplement some species so that NAD+ could be synthesized in human body. There are three pathways for the synthesis of NAD+ in humans: the Preiss-Handler pathway, the de novo pathway and the salvage pathway. Although all three pathways can synthesize NAD+, they have different priorities. Among them, Preiss-Handler pathway and the de novo pathway only provide 15% of the total NAD+ in human body, and the remaining 85% of NAD+ was synthesized through salvage pathway. In other words, the salvage pathway is the key to human body to supplement NAD+ [5].
As three precursors of NAD+, nicotinamide (NAM), NMN and nicotinamide ribose (NR) could synthesize NAD+ through the salvage pathway, so these three substances have become the choices for human body to supplement NAD +.
Recently, NAM is mainly used in cosmetics in whiten purpose. Excess amount of NAM may cause skin fever, redness and pruritus. In addition, Transformation of NAM into NAD+ was also limited by the NAMPT limiting enzyme, which resulted in a poor efficiency [6].
Although NR itself has no side effects, but most of them could not converted into NMN directly. Instead, NR was firstly digested to form NAM, then participated in the synthesis of NMN, which still cannot avoid the limitation of the rate limiting enzyme. Therefore, Oral supplement of NR to increase NAD+ is also restricted [7].
As a precursor of NAD+, NMN could avoid the restriction of the rate-limiting enzyme. The absorption of NMN in human body was rapidly, and it could convert to NAD+ directly. Therefore, NMN can be used as the most direct and rapid method to supplement NAD+.
At present, there are three main methods to produce NMN: chemical synthesis method, fermentation method and enzymatic catalyzed synthesis method.
Chemical synthesis method: Chemical synthesis method is the relatively simple way to produce NMN, and the cost of this method is much lower than the other two methods. Chemical synthesis method usually requires several steps, and the overall yield is not high. The purity of the product could be improved through further separation and purification. NMN has two types of diastereomers: α-NMN and β-NMN. The pure β-NMN cannot be selectively synthesized via chemical synthesis method, and the chiral purity of NMN synthesized through this method is always low. The byproducts produced in this method are not naturally exists in human body, most of them are chemical impurities (mainly chemical solvents etc.). Although the impurities are trace amount, they still might be accumulated in the body, causing the harmful to human body for a long period.
Fermentation method: The product of this method is mainly represented by Shinkowa from Japan. The high technical barriers and the low product yield leads to an expensive price of the product.
Enzyme catalyzed synthesis method: this is what we usually called enzymatic method. This method simulates the metabolic pathway of NMN in human body, express the enzyme in vitro, and realize the enzyme catalysis reaction in vitro to produce NMN. The production process of enzymatic method is the same as in vivo, giving no extra byproducts (purity above 99%). Enzymatic methods can be further divided into one-step enzymatic method and full enzymatic method. One-step enzymatic method is to synthesize NMN through one-step enzymatic catalyzed reaction using chemical synthesized NR as raw material. Full enzymatic method using the core starting material from natural source, which further synthesize NMN by simulating the multi-step enzymatic reaction in vivo. Full enzymatic method does not involve chemical materials, it is safe and has high technical barriers.
Full enzymatic method is a common production method of NMN at present, with high technical barriers. Several key catalytic enzymes are expensive, accounting for about 80% of the cost in the whole production process, but this method is the safest and most efficient way. NMN produced by the full enzymatic method is superior to other production methods in terms of product purity, safety and stability, and it is the first choice of the major manufacturers in the market.
In 2013, Professor David Sinclair’s research group in Harvard Medical School reported that, one week after oral ingestion of NMN by 22-month-old old mice, the NAD+ level of mice was increased, and the key biochemical criterions levels related to mitochondrial homeostasis and muscle function was recovered to the equivalent to 6-month-old young mice. This research was published on Cell [8].
In 2012, Dr. Yoshino J et al. from School of Medicine, University of Washington, found that intraperitoneal injection of NMN can promote the biosynthesis NAD+ in mice, and enhance insulin sensitivity or insulin secretion to improve impaired glucose tolerance. This method is an effective intervention for diet and age-induced T2D (type 2 diabetes) [9].
Sirtuins are a family of NAD+ dependent deacetylase proteins, which play a major regulatory role in almost all cellular functions, affecting physiological processes such as inflammation, cell growth, circadian rhythm, energy metabolism, neuronal function and stress resistance. In 2019, Professor Kane E et al. from the Department of Genetics, Harvard Medical School, founded that NMN, as an important precursor of NAD+ synthesis in vivo, plays many beneficial roles (such as improving metabolism and protecting cardiovascular system, etc.) by activating Sirtuins after it enhances the level of intracellular NAD+ [10].
PARPs (Poly ADP ribose polymerase) are DNA repair enzymes localized in the cell nucleus, which catalyze DNA repairment. NMN is an important reagent for the synthesis of PARPs in biological cells. In 2017, Professor David Sinclair’s research group in Harvard Medical School discovered how NAD+ repairs DNA damage. As NAD+ levels gradually decline during aging, the DNA repairment enzyme PARP1 is increasingly bound by DBC1 (breast cancer deletion factor 1) to form the PARP1 DBC1 complex, which hinders PARP1 from repairing damaged DNA. Increasing the level of NAD+ interferes the formation of PARP1 DBC1 complex, thus restoring the DNA repairment activity of PARP1. A reduction of DNA damage was found after one week of oral NMN in old mice exposed to radiation. The research results are published on Science [11].
In 2019, Professor Nina Klimova et al. from School of Medicine, University of Maryland, found that intraperitoneal injection of NMN to experimental rat could significantly increase the level of mitochondrial NAD+ in the brain hippocampus and the level of ATP in the brain tissue, thus improving the biological energy metabolism of the body. In addition, NMN could also improve the activity of antioxidant enzymes in mitochondria and reduce the production of reactive oxygen species [12].
In 2018, Professor David Sinclair’s research group found that after 2 months of oral supplement of NMN to 18-month-old old mice, the number and density of capillaries both returned to the levels of younger mice, as well as increased resting muscle perfusion and dissolved oxygen levels. Their exercise endurance increased by 58 to 80 percent, and the blood lactate levels dropped after exercise. These results are published on Cell [13].
In 2017, Professor Margaret J. Morris’s research group from School of Medicine, University of New South Wales, conducted a series of studies on the role of NMN in weight management, and found that both NMN and exercise could reduce the body obesity, improved the glucose tolerance and mitochondrial function in genetically obese mice. However, they found that NMN seems to have a stronger effect than exercise on hepatic adipose catabolism (HADH) and anabolism (FASN), the possibly reason might be the injection of NNM increased the level of NAD+ in body, thus activated Sirtuins protein, which increased the catabolism and anabolism of liver fat [14][15].
The heart is the most important organ in body, and it is crucial to maintain the function of heart. In 2014, Dr. Yamamoto T et al. from New Jersey Medical School found that Intraperitoneal injection of NMN in mice could significantly increase the level of NAD+ in their heart and prevent the decrease of NAD+ during ischemia. NMN protects the heart from ischemia reperfusion injury [16].
Neurovascular dysfunction may cause early vascular and neurodegenerative cognitive impairment. Risk factors such as diabetes, hypertension, obesity, lack of physical activity and smoking are all associated with vascular dementia and Alzheimer's disease. Maintaining neurovascular function is important for the prevention of neurodegenerative diseases. In 2019, Dr. T Arantini S et al. from the Health Science Center, University of Oklahoma, found that Intraperitoneal injection of NMN had a significant protective effect on cerebral micrangium in aging mice. NMN could decrease the oxidative stress on cerebral microvascular endothelial, improved endothelial function and saved neurovascular coupling (NVC) response in the cortex of aging mice, then could help to improve cerebral cortical function [17].
Insulin Resistance (IR) refers to the decreased sensitivity of the target organ to the action of Insulin, that is, the normal dose of Insulin produces less than the normal biological effect of a state. Insulin sensitivity is described as the degree of insulin resistance. This could result in the low degree of carbohydrate decomposition. There are two major reasons caused of type 2 diabetes: low insulin production and low insulin sensitivity.
In 2016, Kelly L. Stromsdorfer et al. from the University of Washington, found that decreased NAD+ levels in adipose tissue in obese and elderly mice were associated with severe insulin resistance in multiple organs. Supplementation of NMN in drinking water can reverse insulin resistance and reduce plasma free fatty acid concentration in mice with insulin resistance caused by inactivation of specific enzymes [18].
Aging is the cumulative result of stress and strain, injury and infection, immune responses weakness, nutritional imbalances, metabolic disorders, etc. Aging can be delayed by reducing the accumulation of aging factors through nutritional supplements, or by developing good living habits.
In 2016, Professor Shinichiro Imai’s research group from University of Washington published their long-term experiment with oral NMN in mice. Mice treated with oral NMN for 12 months were compared with normal aging mice treated without NMN. Oral NMN quickly converted to NAD+ in tissues and effectively eliminated various physiological declines associated with aging: Prevented age-related weight gain, improved metabolism, increased physical strength, improved insulin sensitivity and plasma fat indicators, improved vision, improved immune function, prevention of bone density decline, etc. Moreover, NMN did not have any toxicity or side effects [2].
