NMN – nicotinamide mononucleotide improved the ability of insulin to increase glucose uptake in skeletal muscle

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A natural compound previously demonstrated to counteract aspects of aging and improve metabolic health in mice has clinically relevant effects in people, according to new research at Washington University School of Medicine in St. Louis.

A small clinical trial of postmenopausal women with prediabetes shows that the compound NMN (nicotinamide mononucleotide) improved the ability of insulin to increase glucose uptake in skeletal muscle, which often is abnormal in people with obesity, prediabetes or Type 2 diabetes.

NMN also improved expression of genes that are involved in muscle structure and remodeling. However, the treatment did not lower blood glucose or blood pressure, improve blood lipid profile, increase insulin sensitivity in the liver, reduce fat in the liver or decrease circulating markers of inflammation as seen in mice.

The study, published online April 22 in the journal Science, is the first randomized clinical trial to look at the metabolic effects of NMN administration in people.

Among the women in the study, 13 received 250 mg of NMN orally every day for 10 weeks, and 12 were given an inactive placebo every day over the same period.

“Although our study shows a beneficial effect of NMN in skeletal muscle, it is premature to make any clinical recommendations based on the results from our study,” said senior investigator Samuel Klein, MD, the William H. Danforth Professor of Medicine and Nutritional Science and director of the Center for Human Nutrition.

“Normally, when a treatment improves insulin sensitivity in skeletal muscle, as is observed with weight loss or some diabetes medications, there also are related improvements in other markers of metabolic health, which we did not detect in our study participants.”

The remarkable beneficial effects of NMN in rodents have led several companies in Japan, China and in the U.S. to market the compound as a dietary supplement or a neutraceutical. The U.S. Food and Drug Administration is not authorized to review dietary supplement products for safety and effectiveness before they are marketed, and many people in the U.S. and around the world now take NMN despite the lack of evidence to show clinical benefits in people.

The researchers studied 25 postmenopausal women who had prediabetes, meaning they had higher than normal blood sugar levels, but the levels were not high enough to be diagnosed as having diabetes. Women were enrolled in this trial because mouse studies showed NMN had the greatest effects in female mice.

NMN is involved in producing an important compound in all cells, called nicotinamide adenine dinucleotide (NAD).

NAD plays a vital role in keeping animals healthy. Levels of NAD decline with age in a broad range of animals, including humans, and the compound has been shown to contribute to a variety of aging-associated problems, including insulin resistance in studies conducted in mice. Supplementing animals with NMN slows and ameliorates age-related decline in the function of many tissues in the body.

Co-investigator Shin-ichiro Imai, MD, Ph.D., a professor of developmental biology and of medicine who has been studying NMN for almost two decades and first reported on its benefits in mice said, “This is one step toward the development of an anti-aging intervention, though more research is needed to fully understand the cellular mechanisms responsible for the effects observed in skeletal muscle in people.”

Insulin enhances glucose uptake and storage in muscle, so people who are resistant to insulin are at increased risk for developing Type 2 diabetes.

But the researchers caution that more studies are needed to determine whether NMN has beneficial effects in the prevention or management of prediabetes or diabetes in people. Klein and Imai are continuing to evaluate NMN in another trial involving men as well as women.


Nicotinamide adenine dinucleotide (NAD) is a vital metabolic redox co-enzyme found in eukaryotic cells and is necessary for over 500 enzymatic reactions. It plays a crucial role in various biological processes, including metabolism, aging, cell death, DNA repair, and gene expression (Rajman et al., 2018; Okabe et al., 2019). Thus, NAD+ is critical for human health and longevity.

The co-enzyme was first discovered by Harden and Young in 1906 as a component that enhanced the rate of alcohol fermentation in yeast extracts (Harden and Young, 1906). Over subsequent years, the chemical composition of the co-enzyme was established as an adenine, a reducing sugar group and a phosphate by Hans von Euler-Chelpin (von Euler and Myrback, 1930).

Then, in 1936, Warburg suggested that NAD+ could play a role in redox reactions (Warburg and Christian, 1936). By 1960, it was assumed that all biochemical investigations on NAD+ had been exhausted. In 1963, Chambon and Mandel reported that NAD+ is a co-substrate for the addition of poly-ADP-ribose to proteins, and this prompted a series of studies on poly-ADP ribose and poly-ADP-ribose polymerases (PARPs) (Chambon et al., 1963; Yoshino et al., 2018).

In the last decade, new interests in NAD+ emerged because of its association with sirtuins, a family of NAD-dependent protein deacylases (SIRT1–7) (Rajman et al., 2018). Roy Frye showed that mammalian sirtuins could metabolize NAD+ and that NAD+ had a protein ADP-ribosyltransferase activity (Frye, 1999). Guarente and Imai made a phenomenal discovery that yeast SIR2 (silent information regulator 2) and the mouse ortholog SIRT1 have NAD+-dependent protein deacetylase activity (Imai et al., 2000).

Previously, several studies had shown that sirtuins play a critical role in regulating multiple cellular functions, such as cell growth, energy metabolism, stress resistance, inflammation, and circadian rhythm neuronal function, among others (Imai and Yoshino, 2013; Rajman et al., 2018).

The deficiency of NAD+ is closely associated with diverse pathophysiologies, including type 2 diabetes (T2D), obesity, heart failure, Alzheimer’s disease (AD), and cerebral ischemia. The NAD+ levels decline in multiple organs with age, and this contributes to the development of various age-related diseases (Yoshino et al., 2011; Gomes et al., 2013; Mouchiroud et al., 2013; Mills et al., 2016). Therefore, NAD+ supplementation could be an effective therapy for the treatment of the conditions mentioned above.

Nicotinamide mononucleotide (NMN) is one of the intermediates in NAD+ biosynthesis and is a bioactive nucleotide formed by the reaction between a phosphate group and a nucleoside containing ribose and nicotinamide (NAM) (Poddar et al., 2019). NAM is directly converted to NMN by nicotinamide phosphoribosyltransferase (NAMPT).

The molecular weight of NMN is 334.221 g/mol (Poddar et al., 2019). There are two anomeric forms of NMN named alpha and beta, and the latter is the active form (Poddar et al., 2019). NMN is found in various types of natural foods, such as vegetables, fruits, and meat. Edamame and broccoli contain 0.47–1.88 and 0.25–1.12 mg NMN/100 g, respectively, whereas avocado and tomato contain 0.36–1.60 and 0.26–0.30 mg NMN/100 g, respectively. However, raw beef only contains 0.06–0.42 mg NMN/100 g (Mills et al., 2016). Recent preclinical studies have demonstrated that the administration of NMN could compensate for the deficiency of NAD+, and NMN supplementation was able to effect diverse pharmacological activities in various diseases.

In this review, NAD+ biosynthesis pathways and the possible reason for its age-related decline are described. Also, a summary of studies on the role of NAD+ deprivation in causing human diseases and how the application of NMN could have positive effects on those diseases is provided.

NAD+ Biosynthesis Pathways

Three different NAD+ biosynthesis pathways have been described in mammalian cells (Figure 1): (1) Preiss–Handler, in which NAD is synthesized from nicotinic acid (NA); (2) de novo synthesis, which starts from tryptophan; and (3) salvage pathway, which is most predominant in mammalian cells.

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FIGURE 1
Biosynthetic pathways of NAD+ in mammalian cells includes de novo, Preiss–Handler, and salvage pathways, and the salvage pathway is the main source of NAD+. NAD, nicotinamide adenine dinucleotide; NA, nicotinic acid; NAPRT, nicotinic acid phosphoribosyltransferase; NAMN, nicotinic acid mononucleotide; NAAD, nicotinic acid adenine dinucleotide; NADS, NAD+ synthetase; NMNAT, nicotinamide/nicotinic acid mononucleotide adenylyltransferase; ACMS, 2-amino-3-carboxymuconate semialdehyde; QA, quinolinic acid; QPRT, quinolinate phosphoribosyltransferase; NAM, nicotinamide; NAMPT, nicotinamide phophoribosyltransferase; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside; NRK, nicotinamide riboside kinase.

he Preiss–Handler Pathway
This pathway starts with conversion of NA to the nicotinic acid mononucleotide (NAMN) by the enzyme nicotinic acid phosphoribosyltransferase (NAPRT) (Preiss and Handler, 1958). Afterward, NAMN is used for nicotinic acid adenine dinucleotide (NAAD+) biosynthesis by nicotinamide/nicotinic acid mononucleotide adenylyltransferase (NMNAT1/2/3). Finally, NAD+ synthetase (NADS) transforms NAAD+ to NAD+ with ammonia and ATP action as extra ingredients (Yang and Sauve, 2016).

De novo Synthesis From Tryptophan
The eight-step de novo synthesis pathway is initiated by indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO) that convert tryptophan to N-formylkynurenine (Salter et al., 1991). Through formamidase (KFase), N-formylkynurenine is then transformed to be kynurenine, to which hydroxyl is added via kynurenine 3-hydroxylase (K3H). The product, 3-hydroxy-kynurenine, is converted to 3-hydroxyanthranilate followed by 2-amino-3-carboxymuconate semialdehyde (ACMS) via kynureninase (Kyase) and 3-hydroxyanthranilate-3,4-dioxygenase. ACMS then cyclizes to form quinolinic acid (QA) that participates in NAMN biosynthesis with quinolinate phosphoribosyltransferase (QPRT) (Yang and Sauve, 2016). The last two steps are the same as the Preiss–Handler pathway that NAD+ is synthetized sequentially from NAMN and NAAD by NMNAT 1/2/3 and NADS.

The Salvage Pathway

The salvage pathway is the primary source of NAD+ in mammalian cells. The degradation of NAD+ and subsequent generation of NAM (as a by-product) are achieved by NAD-consuming enzymes, such as sirtuins, PARPs, CD38, CD157, and sterile alpha and TIR motif-containing protein 1 (SARM1) (Okabe et al., 2019).

There are only two steps in the salvage pathway. The rate of NAD+ synthesis in this pathway is mostly determined by NAMPT that converts NAM and 5-phosphoribosyl-1-pyrophosphate (PRPP) to NMN in the first step. Then, NMN, the substrate for NAMNT, is conjugated to ATP and converted to NAD in the second step.

The NAMPT exists in two forms in mammals, that is, intracellular NAMPT (iNAMPT) in the cytoplasm and nucleus and extracellular NAMPT (eNAMPT) in the plasma or extracellular space (Revollo et al., 2007). The SIRT1-dependent deacetylation of iNAMPT predisposes the protein to secretion in adipocytes (Yoon et al., 2015). Various types of cells, including mature adipocytes, pancreatic β-cells, myocytes, epithelial cells, and hepatocytes (Revollo et al., 2007; Garten et al., 2010; Zhao et al., 2014) secrete and release eNAMPT to the plasma or extracellular space.

Also, another NAD precursor, nicotinamide riboside (NR), is incorporated into cells using equilibrative nucleoside transporters (ENTs) (Nikiforov et al., 2011) and phosphorylated to NMN by nicotinamide riboside kinase (NRK1/2) intracellularly (Ratajczak et al., 2016). Conversion of extracellular NMN to NR mediated by enzyme CD73 is required for cell uptake and intracellular synthesis of NAD+ (Grozio et al., 2013; Ratajczak et al., 2016). NAD+ biosynthesis in kidney and brown adipose tissue has been shown to decrease after administration of NMN in NRK1 knockout mice (Ratajczak et al., 2016).

However, a recent study identified Slc12a8 as a specific transporter of NMN, which is highly expressed in the small intestine (Grozio et al., 2019). In the study, Slc12a8 expression was upregulated in the small intestines of aged mouse in response to a decrease of NAD+. These findings suggested that the uptake pathway of NMN could be via a cell- or tissue-specific manner.

Given that the salvage pathway is the main and the most efficient route for NAD+ biosynthesis, NMN or NR supplementation is becoming the preferred option of improving NAD+ levels that is devoid of side effects. Currently, increasing numbers of clinical trials using NMN and NR have been approved and are geared toward the treatment of various diseases, which further demonstrate that NMN is a suitable and safe drug for use in humans.

Effect of Aging on NAD+ Levels
NAD+ Biosynthetic Pathways Decline With Age

The decline in NAD+ biosynthetic pathways in the course of aging could be a possible explanation for the reduction of NAD+ levels. NAMPT controls NAD+ levels, thereby influencing the activity of NAD-dependent enzymes, including sirtuins and PARPs.

A study demonstrated that the NAD+ levels and NAMPT protein levels declined significantly in multiple organs, including the pancreas, white adipose tissue (WAT), and skeletal muscle of old mice (Yoshino et al., 2011). However, exercise training increased NAMPT expression in the skeletal muscles (Costford et al., 2010). NAD+ levels and exercise capacity were preserved in aged transgenic mice with muscle-specific NAMPT transgene expression (Frederick et al., 2016). These results suggested that the deficiency of NAMPT result in a reduction of NAD+ levels in the aged mice, and exercise may elevate NAMPT expression, thus restoring the NAD+ levels (Figure 2).

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FIGURE 2
Hypothetic molecule mechanisms of NAD+ decreased with aging. Oxidative stress, DNA damage, and chronic inflammation are increased with aging, which results in accelerated NAD degradation via activation of CD38 and PARPs, or dysregulation of NAMPT. Finally, decreased levels of NAD+ lead to various metabolic and age-associated diseases.

Inflammation and oxidative stress caused by aging have been shown to reduce the NAMPT-mediated NAD+ biosynthesis (Yoshino et al., 2011; Figure 2). Besides, Nampt gene encoding is controlled by BMAL1/CLOCK complex, a heterodimeric complex of core circadian transcription factors, which is suppressed by inflammatory cytokines (Cavadini et al., 2007). Therefore, the development of chronic inflammation in the course of aging may contribute to the inhibition of NAMPT-mediated NAD+ biosynthesis and CLOCK/BMAL-mediated circadian machinery (Imai and Guarente, 2014).

NAD+-Consuming Enzymes Are Activated With Age
PARP1
PARPs were initially considered to be DNA damage repair agents in the 1960s (Chini et al., 2017). The accumulation of DNA damage during aging could activate PARP, among which PARP-1 acts as a major cellular NAD+-consuming enzyme (Imai and Guarente, 2014). Cockayne syndrome (CS) is an aging-related progressive neurodegeneration that occurs as a result of mutations in either Cockayne syndrome group A (CSA) or B (CSB) proteins (Gitiaux et al., 2015; Scheibye-Knudsen et al., 2014). In CS mice, PARP inhibitor or NAD+ supplementation reversed decline in SIRT1 activation and mitochondrial function caused by aberrant PARP activation (Scheibye-Knudsen et al., 2014). Consistently, another inhibitor of PARP, PJ34, or knockout boosted the levels of NAD+, SIRT1 activity, and oxidative metabolism (Bai et al., 2011).

CD38
The CD38 enzyme and its homolog CD157 were initially described as plasma membrane antigens on thymocytes and T lymphocytes. Their role in NAD+ consumption have been revealed; that is, CD157/BST-1 could hydrolyze NR (Preugschat et al., 2014) and CD38 hydrolyzes NAD+ to generate NAM, adenosine diphosphoribose (ADPR), and cyclic ADPR (cADPR). In addition, CD38 also hydrolyzes cADPR (De Flora et al., 2004) and NMN (Grozio et al., 2013).

In mammals, the level of NAD+ and mitochondrial function decreased partially through regulation of SIRT3 as the expression and activity of CD38 protein increased in various tissues during aging (Camacho-Pereira et al., 2016). Administration of CD38 inhibitors elevated intracellular NAD+ level (Escande et al., 2013; Boslett et al., 2017). Consistently, CD38 knockout mice displayed significantly higher NAD+ level in multiple organs (Young et al., 2006).

Sterile Alpha and TIR Motif-Containing 1 (SARM1) Protein
The toll/interleukin-1 receptor (TIR) domain of sterile alpha and TIR motif-containing 1 (SARM1) protein presents NADase activity (Rajman et al., 2018) that is involved in axonal degeneration after axon injury. In response to neuronal injury, the TIR domain of SARM1 cleaves NAD+ to generate ADP ribose (ADPR) and cyclic ADPR, which may contribute to axonal degeneration (Essuman et al., 2017). Paradoxically, overexpression of enzymes in NAD+ biosynthesis pathway or supplying NR could inhibit SARM1-induced axon destruction (Gerdts et al., 2015).

In summary, there are many ways of restoring NAD+ level depletion caused by aging or other diseases, including improving NAMPT expression, providing NAD+ precursors, or inhibiting NAD+, consuming enzymatic activities of PARP, CD38, and SARM1. Currently, supplementation with NMN or NR is considered a viable and highly efficient strategy of increasing NAD+ levels (Figure 3).

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FIGURE 3
Nicotinamide mononucleotide exerts pharmacological effects by increasing intracellular NAD+ levels. Extracellular NMN is cleavage by CD73, which yields NR that is incorporated into cells using equilibrative nucleoside transporters (ENTs). NMN is converted to NAD+, which produces beneficial effects on cell, including mitochondrial function, DNA repair, gene expression, anti-inflammation and cell survival.

reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198709/


More information: “Nicotinamide mononucleotide increases muscle insulin sensitivity in prediabetic women” Science (2021). science.sciencemag.org/lookup/ … 1126/science.abe9985

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