Nicotinamide Adenine Dinucleotide in Aging Biology: Potential Applications and Many Unknowns

Abstract

Recent research has unveiled an expansive role of NAD⁺ in cellular energy generation, redox reactions, and as a substrate or cosubstrate in signaling pathways that regulate health span and aging. This review provides a critical appraisal of the clinical pharmacology and the preclinical and clinical evidence for therapeutic effects of NAD⁺ precursors for age-related conditions, with a particular focus on cardiometabolic disorders, and discusses gaps in current knowledge. NAD⁺ levels decrease throughout life; age-related decline in NAD⁺ bioavailability has been postulated to be a contributor to many age-related diseases. Raising NAD⁺ levels in model organisms by administration of NAD⁺ precursors improves glucose and lipid metabolism; attenuates diet-induced weight gain, diabetes, diabetic kidney disease, and hepatic steatosis; reduces endothelial dysfunction; protects heart from ischemic injury; improves left ventricular function in models of heart failure; attenuates cerebrovascular and neurodegenerative disorders; and increases health span. Early human studies show that NAD⁺ levels can be raised safely in blood and some tissues by oral NAD⁺ precursors and suggest benefit in preventing nonmelanotic skin cancer, modestly reducing blood pressure and improving lipid profile in older adults with obesity or overweight; preventing kidney injury in at-risk patients; and suppressing inflammation in Parkinson disease and SARS-CoV-2 infection. Clinical pharmacology, metabolism, and therapeutic mechanisms of NAD⁺ precursors remain incompletely understood. We suggest that these early findings provide the rationale for adequately powered randomized trials to evaluate the efficacy of NAD⁺ augmentation as a therapeutic strategy to prevent and treat metabolic disorders and age-related conditions.

Graphical Abstract

Graphical Abstract.

Essential Points.

• Nicotinamide adenine dinucleotide (NAD⁺) and its phosphorylated form (NADP⁺) play a fundamental role in all living organisms, carrying reducing equivalents for anabolic and catabolic processes via interconversion of their reduced forms, NADH and NADPH

• These redox pairs are essential for many biochemical reactions involved in generating cellular energy; biosynthesis of lipids, nuclei acids, and steroid hormones; protecting against oxidative stress; NAD⁺ also serves as a cosubstrate for signaling pathways, such as the sirtuin family of deacetylases, poly(ADP)-ribosyl polymerases, and cADP-ribose synthases, and Sterile Alpha and TIR Motif Containing protein 1, which are involved in metabolic regulation, DNA repair, and aging biology

• Severe NAD⁺ deficiency, as a result of eating diets deficient in vitamin B3, causes pellagra; NAD⁺ levels also decline throughout life and in many chronic age-related diseases and in response to some infections, and reduced cellular NAD⁺ bioavailability contributes to the pathophysiology of some types of metabolic disorders and age-related diseases

• NAD⁺ levels in blood and some tissues can be increased by administration of NAD⁺ precursors such as nicotinamide, nicotinamide mononucleotide, and nicotinamide riboside, or by using inhibitors of NAD⁺ degradation

• In model organisms and in preclinical models of human diseases, raising NAD⁺ levels by administration of NAD⁺ precursors or by genetic modification improves glucose and lipid metabolism; attenuates diet-induced weight-gain, diabetes, diabetic kidney disease, and hepatic steatosis; reduces endothelial dysfunction and arterial stiffness; protects heart from ischemic injury; prevents retinal degeneration; attenuates neurodegenerative disorders; and increases health span but not life span

• Early human trials of NAD⁺ precursors have shown that these medications can increase NAD⁺ levels safely in the blood and in some tissues; these early studies have reported some benefits in preventing nonmelanotic skin cancers; acute kidney injury in at-risk patients; modestly reducing blood pressure and lipid levels in older adults with obesity or overweight; and suppressing inflammation in patients with Parkinson disease, and SARS-CoV-2 infection

• Further research is needed to elucidate the clinical pharmacology, metabolism, and mechanisms of NAD⁺ precursors, and to determine their efficacy in preventing or treating metabolic disorders and age-related diseases in large, randomized trials with validated patient-important, disease-specific endpoints

The Discovery of Nicotinamide Adenine Dinucleotide and Its Essential Roles in All Forms of Life

It is now widely recognized that nicotinamide adenine dinucleotide (NAD⁺) and its phosphorylated form (NADP⁺) play a fundamental role in all forms of life, carrying reducing equivalents (H⁻) for anabolic and catabolic processes via interconversion with their respective reduced forms, NADH and NADPH, together referred to as NAD(H) and NADP(H), respectively. These redox pairs are essential for many biochemical reactions, ultimately involved in generating cellular energy in the form of adenosine triphosphate (ATP) (1, 2). NAD⁺ is converted to NADP⁺ by NAD kinase and the NADP⁺/NADPH redox couple play an important role in biosynthetic reactions in fatty acid synthesis, steroidogenesis, and nucleic acid synthesis (1, 2) and in protecting against oxidative stress by providing reducing equivalents to antioxidants such as glutathione and thioredoxin (3, 4). NAD⁺ also serves as a substrate or cosubstrate for multiple families of signaling enzymes such as the sirtuin family of deacylases, mono- and poly(ADP)-ribosyl polymerases (mARTs, and PARPs), cADP-ribose synthases (CD38 and CD157), and Sterile Alpha and TIR Motif Containing 1 (SARM1).

Generalized NAD(H) deficiency is the cause of pellagra, a condition that is fatal if not treated. Even though pellagra had been known to be associated with poverty and subsistence on corn-based marginal diets since the eighteenth century, it was not until the early years of the twentieth century that Joseph Goldberger identified pellagra as a nutritional deficiency (5, 6); some of Goldberger's experiments were conducted in prison inmates and generated substantial controversy. In 1938, Conrad Elvehjem experimentally induced pellagra (Black-Tongue Disease) in a dog model and identified nicotinic acid (NA) as the “anti-black tongue factor” or the Pellagra Preventive Factor (7). In subsequent decades, pellagra was characterized as a nutritional deficiency due to insufficient intake of niacin (the term niacin has been used at times to refer to NA specifically, but as used here refers to a group of compounds that includes NA, nicotinamide, and their derivatives) and the alternative NAD⁺ precursor tryptophan. Widespread vitamin supplementation during the World War II period essentially eliminated pellagra in the United States (8).

While searching for the yeast factors responsible for the fermentation reaction, Arthur Harden and William J. Young discovered that a heat-stable factor was necessary for the fermenting ability of the heat-sensitive fraction of yeast. Harden and Young correctly postulated that the heat-sensitive fraction contained proteins responsible for fermentation, and the heat-stable fraction contained 1 or more cofactors that were necessary for the proteins in the heat labile fraction to perform the fermentation reaction (9). Hans von Euler-Chelpin separated the components of the heat-stable fraction of the yeast cells and was awarded the Nobel Prize for identifying NAD⁺ as a dinucleotide that serves as the cofactor in the heat stable fraction of yeast cells that was necessary for the fermentation reaction. Otto Heinrich Warburg later discovered that during the fermentation reaction, the nicotinamide part of NAD⁺ accepts a hydride (H⁻) and is converted to NADH; Warburg's work uncovered the important role of NAD⁺ in certain biochemical reactions—now referred to as redox reactions (10). Arthur Kornberg purified nicotinamide mononucleotide adenylyl transferase (NMNAT), the enzyme in the NAD⁺ biosynthetic pathway that catalyzes the conversion of its direct precursor, nicotinamide mononucleotide (NMN) to NAD⁺ (11). Later, Jack Preiss and Philip Handler characterized the biochemical pathway (now referred to as the Preiss-Handler pathway) for the conversion of NA to NAD⁺ (12). In 2004, Charles Brenner discovered that nicotinamide riboside (NR) serves as a preribosylated precursor of NAD⁺ in mammalian cells via a biochemical pathway catalyzed by nicotinamide riboside kinases (NRKs) 1 and 2. These enzymes convert NR to NMN, which is subsequently converted to NAD⁺ by the catalytic action of NMNAT (13). Charles Brenner and Anthony Sauve later showed that NRKs are also able to convert nicotinic acid riboside (NAR) into NAMN, the Preiss–Handler intermediate to NAD⁺ (14, 15).

While NAD⁺ plays an essential role in redox reactions, it also serves as a co-substrate for several classes of enzymes that play important roles in signaling and metabolism. The discovery that sirtuins can extend replicative life span in yeast ushered in a new era in aging research (16) and rejuvenated interest in NAD⁺. In particular, the reports that sirtuins are NAD⁺-dependent deacylases that can extend replicative life span in yeast ushered in a new era in aging research and rejuvenated interest in NAD⁺ (16-18). Although these reports fueled extensive research on the roles of mammalian sirtuins in many areas of biology and initial reports supported a role in promoting worm and fly longevity as well (19, 20), more recent observations have challenged the view of sirtuins as conserved regulators of longevity (21). In many experiments, overexpressing sirtuins failed to influence worm or fly life span (22), and a detrimental effect has been shown for chronological life span in yeast (23). No longevity effect was observed when the sirtuin SIRT1 was overexpressed in mice (24), although a male-specific increase in longevity was reported for SIRT6 (25). Despite the controversy surrounding their role as longevity genes (21, 26), mammalian sirtuins play an important role in regulating many cellular processes in diverse tissues during aging

Other physiological roles that have been identified for NAD(H) include capping of mRNAs (27), neurotransmission (28), and calcium signaling related functions (29). These, in turn, can influence a host of downstream processes including gene expression, protein stability, calcium mobilization, DNA repair, and cell death pathways. These and other recent discoveries of the more expansive role of NAD⁺ in the biology of aging have led to a deluge of academic, pharmaceutical and public interest in augmenting NAD⁺ as a therapeutic strategy to prevent and treat metabolic disorders and other age-related diseases.

This article reviews the role of NAD⁺ in the biology of aging and metabolic disorders, the clinical pharmacology of NAD⁺ precursors as a strategy to increase NAD⁺, and offers a critical appraisal of the evidence that pharmacological interventions to restore NAD⁺ may have potential applications in preventing or treating cardiometabolic disorders and other age-related diseases.

NAD⁺ Biosynthesis and Metabolism

NAD⁺ is synthesized from nicotinamide (also known as niacinamide), NA (also known as niacin), or their intermediate forms salvaged from the diet (collectively referred to as vitamin B3, Fig. 1) (30). In addition, some NAD⁺ is produced de novo from tryptophan, primarily in the liver, and gut microbes can synthesize it from aspartate (31, 32) (Fig. 1). Notably, genetic disruption of quinolinate phosphoribosyl transferase (required for de novo synthesis) in mice does not affect NAD⁺ levels in tissues, indicating that de novo synthesis of NAD⁺ from tryptophan is not essential in mice when niacin is present in the diet (33). Conversely, mice can generate sufficient NAD⁺ exclusively from tryptophan and microbial metabolism in the absence of dietary niacin intake. However, humans are more susceptible to the development of NAD⁺ deficiency in the absence of sufficient amounts of dietary niacin. NAD⁺ biosynthesis from tryptophan is regulated by the activity of the enzyme alpha-amino-beta-carboxy-muconate-semialdehyde decarboxylase (ACMSD), which converts alpha-amino-beta-carboxy-muconate-semialdehyde to alpha-amino-beta-carboxy-muconate-semialdehyde. Higher activity of ACMSD in humans than in mice is 1 factor that likely renders humans more susceptible to the development of NAD⁺ deficiency in the absence of sufficient amounts of dietary niacin (34). Palzer et al (35) demonstrated that transgenic mice with inducible expression of human ACMSD had reduced formation of quinolinate and NAD⁺ and became niacin dependent similar to humans when ACMSD expression was high.

Figure 1.

The structures and some important uses of NAD⁺, NADH, NADP⁺, and NADPH in the cells. The redox couples—NAD⁺ and NADH, and NADP⁺ and NADPH—play an important role in redox reactions in which NAD and NADP serve as electron carriers during exchange of reducing equivalents in molecular reactions inside the cell. NAD⁺ also serves as a cofactor/cosubstrate for sirtuins, PARPs, CD38 and other proteins in many signaling pathways and is the only nicotinamide derived cofactor that is directly consumed with the concomitant release of nicotinamide and signaling molecules during these reactions. NADH is stable to processes responsible for the cleavage of the glycosidic bond and, therefore, serves as a protected pool of NAD⁺ in the cell. Among other processes, NADH is oxidized to NAD⁺ by complex I of the ETC and LDH. Mitochondrial NAD⁺ can be oxidized by NNT with the concomitant reduction of NADP to NADPH. NADP⁺ is also reduced to NADPH in the cytosol by the PPP enzymes, G6PDH and 6PGDH, and by reductases of folate metabolism. Crucially, NADPH is central to ROS homeostasis and is employed in anabolic processes such as the synthesis of fatty acids, nucleic acids, and steroid hormones. SIRTs, sirtuins; ARTs, ADP ribosyltransferases; SARM1, Sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1), CD38 and CD73, cluster of differentiation factor 38 and cluster of differentiation factor 73. TCA, tricarboxylic acid pathway; LDH, lactate dehydrogenase; ETC, electron transport chain; G6PDH, glucose-6-phosphate dehydrogenase; DHFR, dihydrofolate reductase; MTHFR, methylene tetrahydrofolate reductase; DPYD, dihydropyrimidine dehydrogenase; TRXR, thioredoxin reductase; NOX, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; CAT, catalase; GPX, glutathione peroxidase; FASN, fatty acid synthase; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; POR, Cytochrome P450 oxidoreductase.

Nicotinamide is converted to NMN via nicotinamide phosphoribosyltransferase (NAMPT) and subsequently converted to NAD⁺ by the catalytic action of 1 of 3 isoforms of NMN adenylyl transferase (NMNAT1-3) (36). NAD⁺ itself serves as the precursor to NADP⁺ via NAD⁺ kinase, and this process was recently shown to be reversible via nocturnin (37) or mesh1 (38). Interconversion between the oxidized and reduced forms of nicotinamide-containing species is rapid relative to synthesis and conserves the total pool size. Enzymes consuming NAD⁺ as a substrate break down a nucleosidic bond to release nicotinamide. Such enzymes include sirtuins, ARTs (PARPs and MARTs), and glycohydrolases including CD38, and SARM1 (39). Finally, enzymes of the NUDIX family, NUDT12 and NUDT13, and CD73 catalyze the hydrolysis of the pyrophosphate bond in NAD(H) to generate AMP, NMN and its reduced form, NMNH (40).

While nicotinamide can be used to regenerate NAD⁺ via the salvage pathway (41), loss of nicotinamide (and thereby NAD⁺ equivalents) occurs mainly via methylation by nicotinamide N-methyl transferase (Fig. 2). Methylated nicotinamide is fated for excretion in the urine and is typically oxidized to N-methylated 6- or 4-pyridone 3-carboxamide (Me-6-PY or Me-4-PY) by aldehyde oxidase prior to elimination (Fig. 2) (42, 43). Alternatively, unmethylated nicotinamide is oxidized by cytochrome P450 2E1 to generate nicotinamide-N-oxide (Fig. 2) (44). In addition, the nicotinamide moiety can be overoxidized while incorporated into NAD⁺ or NADP⁺, creating species that include the pyridone carboxamide adenine dinucleotides, and ribonucleotides, and ribonucleosides (Fig. 2) (45, 46). Finally, excess NA is also readily disposed of by conjugation to glycine and formation of nicotinuric acid (Fig. 2) (44). The structures of known metabolites of NAD⁺ that are thought to be unsalvageable and fated for elimination are shown in Fig. 2 (47). These metabolites of NAD⁺ and its intermediates and some additional unstable transient degradation products of NADH and NADPH (Figs. 3 and 4) have the potential to interfere with normal metabolism, as they lack redox activity but may compete with their undamaged counterparts for binding to enzymes (Fig. 4) (48, 49).

Figure 2.

Biochemical pathways for the synthesis of NAD⁺ and related metabolites. Biosynthetic intermediates and the salvage (recycling) pathway for NAD⁺ synthesis are shown in blue. The salvage pathway (recycling pathway) for NAD⁺ generation uses direct metabolites of NAD⁺ degradation. Nicotinamide (NAM) is generated from NAD⁺-consuming reactions, while nicotinamide riboside (NR) is produced either from NRH (the reduced form of NR) oxidation by N-ribosyl dihydronicotinamide : quinone reductase 2 (NQO2) or by nucleoside diphosphate linked to moiety-X (NUDIX) phosphatase-catalyzed NAD⁺ hydrolysis. The biosynthetic intermediate common to both biosynthetic precursors, nicotinamide mononucleotide (NMN), is a substrate for nicotinamide mononucleotide adenylyl transferase (NMNAT). Levels of NMN are usually low compared with NAM or NAD⁺. NAD⁺ is the only precursor to the 3′-phospho-NAD⁺, NADP. De novo biosynthetic pathway for NAD synthesis and its intermediates are shown in green. Dietary tryptophan (Trp), nicotinic acid (NA; generated by bacterial NAM deamination), aspartate (Asp), and nicotinic acid riboside (NAR) are all converted to the same biosynthetic intermediate, nicotinic acid mononucleotide (NAMN). NAMN is a substrate for NMNAT, just like NMN, and is converted to NAAD. Unlike NAD⁺, NAAD is neither a substrate for NAD⁺-consuming enzymes nor a redox cofactor. Instead, it is a direct biosynthetic precursor to NAD⁺ via NAD⁺ synthase. The expression of NAD⁺ synthase regulates this pathway. Redox-coupled biosynthetic intermediates (NR and NRH, NMN and NMNH, NAD⁺ and NADH, NADP and NADPH) are shown in black. Reduction of NAD(P) generates the NAD(P)H pools. NRH (reduced form of NR), a precursor to NMNH (reduced form of NMN) and NADH, uses adenosine kinase (AK) to enter the NAD(P)(H) biosynthetic pool. There is no known feedback inhibition of AK by either of the NAD(P)/NADPH pools; as such, once NRH enters the cell and is phosphorylated, it is retained by the cell and supplies a reliable source of NADH precursor, NMNH. As NADH is not a substrate for NAD⁺-consuming enzymes, redox biology shields the NAD(H) pools from being overly depleted. Transient metabolites of NAD⁺ are shown in purple. Nicotinic acid adenine dinucleotide phosphate (NAADP) and its reduced form NAADPH are transient species that remain under investigation, including the endogenous origin of NAADP. However, some ubiquitous redox enzymes that use NADP as a cofactor, such as G6PDH, can generate NAADPH from NAADP and abolish the signaling properties of NAADP. Several additional important byproducts of NAD⁺ consuming enzymatic reactions such as adenosine diphosphate ribose (ADPR) and cyclic adenosine diphosphate ribose (cADPR), and poly adenosine diphosphate riboside (PAR) have not been shown in this figure as they do not incorporate a nicotinoyl moiety. Nicotinic acid adenine dinucleotide phosphate exists only transiently as a signaling intermediate and is shown in magenta. As described in the text, these by-products of NAD(P)-consuming processes are major carriers of ribose and adenosine units and are shown in Fig. 2.

Figure 3.

Metabolism of NAD⁺ and its related intermediates and precursors. (A) Most readily detected metabolites of NAD⁺ and its intermediates. Dietary nicotinamide (Nam) and Nam generated from supplemented nicotinamide riboside (NR), and maybe NMN, are substrates for bacterial nicotinamidase in the gut and converted to nicotinic acid (NA). Once absorbed and in circulation, NA is rapidly converted to NAD⁺ by the Preiss-Handler Pathway unless it is in excess. Excess NA is conjugated to glycine to form nicotinuric acid (NUA) in phase II metabolism. NUA is excreted in the urine and is the only catabolite of NA. Trigonelline, N-Me-NA, is not considered an endogenous metabolite of NAD⁺ and is thought to be acquired from dietary sources. Nam that is not recycled to NAD⁺ by the salvage pathway can be methylated by nicotinamide N-methyltransferase (NNMT). This methylation process requires the methyl-donor S-adenosyl methionine (SAM) as a cosubstrate. In cells, excessive methylation of Nam promotes SAM depletion and genomic hypomethylation. Nam and methyl-Nam are readily oxidized to N-oxy-Nam and the N-methylated pyridone series, respectively. The relative abundance and product distribution of these catabolites are specific for each animal species and differ in model organisms from that in humans. Nicotinuric acid can only be generated from NA. Trigonelline (N-Me-NA), found in torrefied coffee, is detected in biospecimens like serum and urine, but its origin is unclear, and its relationship with the rest of the B3 vitaminome remains unknown. Overall, the biological properties of these circulating catabolites remain incompletely understood. 1-Methylnicotinamide, N-methyl-2-pyridone-3-carboxamide (Me-2PY), N-methyl-4-pyridone-3-carboxamide (Me-4PY), and N-methyl-6-pyridone-3-carboxamide (Me-6PY) are the major circulating and urinary metabolites of NAD⁺. Once generated, none of these catabolites can be recycled to NAD⁺. (B) Products of hyperoxidation of ribosylated nicotinamide derivatives. NAD⁺ and NADP⁺ are acceptors of electrons, in the form of hydrides, hydroxide radicals, and superoxide. While the former leads to NAD(P)H, the latter leads to the formation of hyperoxidized NAD(P) species. The hydroxide radicals and superoxide can add on 3 different positions of the nicotinamide ring and generate 3 different chemical series. Each can be metabolized into simpler units. The phosphorylated species are detected in blood and tissues, while the nucleosides and the nucleobases are found in serum and urine. While the 2, 4 and 6-isomers of 3-carboxamide pyridone ribosides have been detected, the 4-isomer of the triphosphate and dinucleotide species is the isomer most often reported. Some biochemical conversions remain uncharacterized and are indicated with dashed arrows. 2PY, 2-pyridone-3-carboxamide (2-hydroxynicotinamide); 4PY, 4-pyridone-3-carboxamide; 6PY, 6-pyridone-3-carboxamide (2-pyridone-5-carboxamide; 6-hydroxynicotinamide); 2-PYR, 2-pyridone-3-carboxamide riboside; 4-PYR, 4-pyridone-3-carboxamide riboside; 6-PYR, 6-pyridone-3-carboxamide riboside; 2-ox-NAD, 6-pyridone-3-carboxamide adenine dinucleotide; 4-ox-NAD, 4-pyridone-3-carboxamide adenine dinucleotide; 6-ox-NAD, 6-pyridone-3-carboxamide adenine dinucleotide phosphate.

Figure 4.

Unstable transient degradation products of NADH and NADPH. Isomerization of NAD(P)(H) generates isomers that are potent inhibitors of redox enzymes. Renalase catalyzes the oxidation of all NAD(P)H isomers to NAD(P); however, renalase is mainly found in kidneys and extracellularly, but it is not known whether other dehydrogenases can perform this conversion since renalase is not an obligate enzyme. Hydration of NAD(P)(H) also occurs chemically and is favored by acid conditions. Once generated, it can fully degrade to glycating species unless it is converted back to NAD(P)H by a specific epimerase-dehydratase. The absence of this enzyme is embryonically lethal.

NAD(H) and NADP(H) are compartmentalized within the cell (50), and within each compartment, the oxidized and reduced pools can be free or reversibly protein bound (51), making it challenging to understand the free concentrations available to a given enzyme. There is evidence for discrete NAD⁺ pools in the mitochondria, peroxisomes, endoplasmic reticulum, and Golgi complex (52). Transporters have been identified on peroxisomes (53) and mitochondria (54-56), but how NAD⁺ enters other compartments and how many compartments exist remains uncertain. Within each compartment, the redox ratios of NAD(H) and NADP(H) pools are independently regulated based on the metabolic reactions that employ each cofactor (1). An exception is that in the mitochondria, nicotinamide nucleotide transhydrogenase facilitates the transfer of hydride from NADH to generate NADPH (57). Our understanding of the free NAD⁺ and NADP⁺ concentrations available to enzymes, and even whether it is the total oxidized nucleotide or the redox ratio that matters for a given reaction, remains incomplete (58).

NAD(H) and NADP(H) are under circadian and metabolic regulation in at least some tissues and organs (59-61). Thus, animal and human studies that seek to quantify changes in NAD⁺, NADH, NADP⁺, and NADPH must consider the time point in the circadian rhythm in addition to the feeding and physical activity status at the time of the sample collection. Although it has been widely recognized that NAD⁺ levels are wired into the circadian clock through cycling of NAMPT expression, the pentose phosphate pathway that supplies phosphoribosyl pyrophosphate for the NAMPT reaction is also under circadian control (62, 63). Moreover, nocturnin, which removes the phosphate from NADP⁺ to regenerate NAD⁺, has a unique circadian rhythm (64). Thus, multiple facets of NAD⁺ metabolism are cycling throughout the day.

As discussed earlier, there are several important interspecies differences in the biosynthesis and metabolism of NAD⁺ between mice and humans that should be considered in interpreting the mouse data in the context of human physiology and disease.

Reduced NAD⁺ Bioavailability in Aging and Disease

Severe NAD⁺ deficiency, typically as a result of prolonged exposure to a diet that is deficient in vitamin B3 and protein or drug-induced deficiency, causes pellagra (65), which is characterized by cognitive impairment, diarrhea, and dermatitis that usually is alleviated by improving diet quality or supplementing vitamin B3 (66). Recently, it has been appreciated that more subtle decreases in NAD⁺ concentration occur in some tissues due to diseases or aging (67-69). The effect of aging varies by tissue (70) and the magnitude of the effect within a given tissue varies between studies. In many cases, the data reported were generated using acidic extraction conditions, which are suitable for NAD⁺ but not NADH, leaving open the question of redox changes. Indeed, among the studies that do measure NADH with age, most report a more reduced redox state, such that NAD⁺ is decreased due to both a decrease in the total [NAD⁺ + NADH] pool size and a shift in the ratio (36, 68). It remains an open question whether falling NAD⁺ levels in aged tissues are inherently part of the aging process itself or the consequence of age-related diseases or degenerative processes (39, 71); it was recently shown that while older adults on average have lower skeletal muscle NAD⁺ concentrations than young people, the levels in exercise-trained older adults were indistinguishable from those of young controls (72). Regardless of the cause, the observation that NAD⁺ levels fall has sparked great interest in the therapeutic value of restoring them.

Pharmacologic Strategies for Increasing NAD⁺ Bioavailability

Although this review is focused on the potential of pharmacologic interventions to increase NAD⁺ levels, lifestyle interventions that improve health span, such as physical exercise, healthy diet, and alignment of activity and food intake with circadian rhythms, are known to modulate NAD⁺ levels through mediating mechanisms that remain incompletely understood (73-75). The effects of exercise interventions on NAD⁺ levels are complex and depend on the type, intensity, and duration of exercise (76). NAD⁺ levels increase after moderate but not intense chronic aerobic exercise (77) and both endurance and resistance types of exercise training upregulate the expression of NAMPT in young and older adults (78). NAD⁺ consumption is increased after an acute bout of exercise (76). Caloric restriction in mice also increases the NAD⁺ content of some tissues (79, 80) and is associated with reduced expression of PARP1 (81); feeding ketogenic diet to rats increases their NAD⁺ levels, upregulates Sirt1 mRNA levels, and reduces PARP1 levels in the hypothalamus (82).

Several pharmacologic strategies have been used to increase NAD⁺ levels: administration of NAD⁺ precursors; enhancing the activity of NAD⁺ biosynthetic enzymes; inhibition of NAD⁺ consuming enzymes (eg, CD38 (83) or PARP-1) (84); and altering the NAD⁺/NADH redox ratio. Among these strategies, NAD⁺ precursors, inhibitors of NAD⁺ consuming enzymes CD38 and PARP-1, and 1 potential redox regulator have advanced into human trials.

NAD⁺ Precursors

For the purposes of this review, we consider an NAD⁺ “precursor” to mean any molecule that supplies the chemical backbone for the nicotinamide moiety. Supplementing NAD⁺ precursors is by far the commonest strategy used to increase NAD⁺ levels (85, 86). The oral use of nicotinamide and NA as NAD⁺ precursors started in the 1950s following the elucidation that poor nutrition was the cause of pellagra. In addition, NA has lipid-lowering properties when used in pharmacologic doses and has been used clinically for this purpose (87). However, NA administration is associated with adverse effects such as flushing due to the stimulation of a G-protein coupled receptor, GPR109A (88), along with pruritis, hyperglycemia, elevation of liver enzymes, and hyperuricemia (89).

More recently, ribosylated precursors of NAD⁺, NR (13), and NMN (36), have been used in preclinical and clinical studies (90). In cultured cells and some tissues, extracellular NMN has generally been found to break down to NR prior to uptake (15, 91, 92), consistent with the view that phosphorylated nucleotides do not cross the plasma membrane. However, SLC12A8 has been described as an NMN transporter active in the intestines and certain other cell populations, raising the possibility that direct NMN uptake could occur in some settings (93). The evidence for NMN transport by SLC12A8 has in turn been questioned, renewing the controversy over whether NMN is ever taken up directly (94). In any case, both NR and NMN offer several advantages over niacin in that they avoid GPR109A-induced flushing and require less ATP to complete the synthesis of NAD⁺. Crucially, generation of NAD⁺ from each of its nonribosylated precursors requires reactions that are dependent on vitamins B1, B2, B6, and/or B9 for generating intermediates. Thus, NR and NMN, as NAD⁺ precursors, may also be more effective in the treatment of multiple vitamin deficiencies. However, the relative efficacy of NR and NMN compared with each other, niacin, or nicotinamide in vivo remains to be established.

Modes of NAD⁺ precursor administration include oral (the most common), topical (95), intravenous (96) and even intranasal (97). Intravenous administration delivers substantially more of the intact ribosylated precursors to target tissues than does oral delivery (32). The efficacy of NAD⁺ boosting after oral delivery of nicotinamide or NR depends on the gut microbiome (98-99). Although the precise metabolic fates of NAD⁺ and NADH in the human gut are unknown, pyrophosphate hydrolysis (100) and phosphorylases (101) are effective NAD⁺ degraders that likely release dietary nicotinamide, which can be used by the microbiota to generate NA (98). Topical use of NAD⁺ precursors has been successfully implemented when the hydrophilicity of the water-soluble vitamin-like species is masked with lipidic appendages (102). Here the vitamin behaves as a prodrug that requires lipase for the release of NA systemically once applied topically. Intranasal delivery of NAD⁺ precursors remains incompletely studied and although intranasal delivery of intact NAD⁺ was reported to have positive effects, there is not yet sufficient evidence to conclude that it reached target tissues intact (97). The bolus intravenous delivery of NR, NMN, NAD⁺, and NADH is being explored for increasing NAD⁺ levels systemically (103). However, the safety of intravenous administration needs further evaluation since concentrations of these species are very low in circulation under normal conditions.

NAD(H) and NADP(H) themselves are sources of vitamin B3 in foods and have also been used as supplements (104), with limited evidence supporting their value in certain circumstances (97, 105). Another approach to increasing NAD⁺ is supplementation with the reduced form of NR, NRH, that generates reduced nicotinamide mononucleotide (NMNH) via adenosine kinase (106). Unlike some NAD⁺-biosynthetic enzymes, adenosine kinase does not appear to be feedback-inhibited by NAD⁺, and NRH treatment increases NAD⁺ and NADH levels in cell cultures and mice beyond the effects reported for other approaches (106-108). NMNH also effectively increases NAD⁺ levels in vivo (109), although it remains unclear if this is due to direct uptake or liberation of NRH. Both NRH and NMNH, like NADH, are readily oxidized, making them somewhat unstable if not handled carefully, and have not been tested in humans (110).

Manipulation of NAD⁺ Metabolism

Boosting synthesis

NAD⁺ can also be increased by enhancing the activity of NAD⁺ biosynthetic enzymes. Several activators of NAMPT have been described (111-114), which could drive enhanced salvage of nicotinamide in most cell types. Alternatively, driving tryptophan catabolites into the NAD⁺ synthesis pathway in the liver and kidney by blocking their alternative fate can enhance NAD⁺ concentration in those tissues (115).

Metabolic fate of orally administered ribosylated NAD⁺ precursors

Trammell et al (116) were the first to characterize the NAD⁺ metabolome and pharmacokinetics of oral NR in humans. However, how administration of ribosylated NAD⁺ precursors, NR and NMN increases NAD⁺ concentrations in the blood and target tissues remains incompletely understood. The concept that the orally administered ribosylated precursors are primarily absorbed and converted intact into NAD⁺ in the target tissue is not supported by the mouse tracer studies (32) or the early pharmacokinetic studies (117, 118). Instead, the tracer studies in mice provide evidence of substantial first pass presystemic metabolism of NMN and NR such that only a small proportion of each of these compounds enters the systemic circulation unaltered after their oral administration (32). These mouse findings are also consistent with human studies which reveal proportionately small increases in blood NMN levels after repeated daily dosing for 28 days (117). Additional studies show that the host NAD⁺ pool can cycle between tissues and gut microbiome via secretion of nicotinamide that is converted by the gut microbiome into NA, which is then absorbed and converted to NAD⁺ (99). Studies in mice further show that oral NMN can undergo deamidation by gut microbiome and this may affect its bioavailability after its oral administration (119). How much of the oral NMN and NR in humans is absorbed intact and how much is metabolized presystemically to nicotinamide and NA and then converted to NAD⁺ in tissues remains unknown (120). The gut bacteria possess the metabolic machinery to process NAD⁺ and its precursors (98, 121) but the extent to which human digestion and the microbiome play a role in the bioavailability of NAD⁺ precursors has not been characterized. Elegant studies by the Gardell Laboratory (122) have provided information on nicotinamide metabolism in humans and demonstrated that extracellular nicotinamide is metabolized differently than nicotinamide generated intracellularly through NAD⁺ consumption. An understanding of the oral bioavailability of ribosylated NAD⁺ precursors NMN and their utilization and metabolism in different tissues is necessary for further development as potential pharmaceutical products. This information also is needed to determine the most optimum way of dosing NAD⁺ precursors.

Oral administration of NMN or NR is associated with substantial increase in blood NAD⁺ concentrations but variable increases in NAD⁺ concentrations in tissues. For instance, mouse studies have reported substantial increases in NAD⁺ levels in the peripheral blood mononuclear cells (PBMCs), brain, liver, and kidney but inconsistent changes in the skeletal muscle (123-126). In humans, it is unclear whether the poor efficacy of oral NR and NMN for inducing sustained increases in NAD⁺ levels in the muscle is due to low biodistribution of these precursors in the skeletal muscle or to increased NAD⁺ turnover in this tissue (117, 127, 128). The increase in the intramuscular concentrations of the methylated metabolites of NAD⁺—1-methyl nicotinamide, N-methyl-2-pyridone-5-carboxamide (2PY), and N-methyl-4-pyridone-3-carboxamide (4PY)—after oral NMN suggests that nicotinamide turnover in the skeletal muscle may be increased after administration of oral NAD⁺ precursors and that muscle may have a relatively fixed NAD⁺ set point (127, 128). The biosynthetic flux and metabolic fate of ribosylated precursors of NAD⁺ after repeated oral dosing have not been investigated even in mice.

Targeting NAD⁺ consuming enzymes

The decrease in NAD⁺ levels with aging and in inflammatory conditions appears to be driven primarily by increased activity of NAD⁺ consuming enzymes rather than reduced production (70). Although there are multiple enzymes that consume NAD⁺, specifically inhibiting cluster of differentiation 38 (CD38) (83) or PARP1 (129, 130) is reported to raise the steady-state NAD⁺ concentration in tissues. CD38 is a multifunctional enzyme that regulates NAD⁺ levels by degrading NAD⁺ by cleaving the N-glycosyl bond in NAD⁺ and exerts additional effects on cell signaling and carcinogenesis (131). Targeting these enzymes may be particularly appropriate when they are overactivated by inflammation (CD38) or DNA damage (PARP1), both of which increase with normal aging (132, 133). Monoclonal antibodies that block CD38 activity have been approved for the treatment of multiple myeloma (134). Similarly, an enhanced understanding of the role of PARP1 in DNA repair and cell death has led to the approval of PARP1 inhibitors for several types of cancers (135). SARM1, a multifunctional glycohydrolase, also serves as an NADase and drives high consumption when activated (136). An important safety concern is that disruption of the activity of NAD⁺ consumers may have unintended consequences. For instance, PARP1 inhibition may compromise DNA repair and CD38 loss may affect immune function and calcium signaling (137).

Modulating the redox state

Separately from changing the [NAD⁺ + NADH] pool size, free NAD⁺ can be substantially affected by the NAD⁺/NADH redox ratio. Providing oxidized metabolites such as pyruvate or limiting flux through glycolysis can shift the cytosolic NAD⁺/NADH redox ratio in favor of NAD⁺ (via lactate dehydrogenase in the case of pyruvate) facilitating NAD⁺-dependent reactions (1). Another strategy to manipulate the NAD⁺/NADH redox ratio is activating a futile cycle for the NAD(P)H:quinone oxidoreductase 1 (NQO1) (138) that shifts the NAD⁺/NADH ratio and thereby increases NAD⁺ levels. This approach is being considered in applications such as preventing cisplatin-mediated acute kidney injury and promoting ROS-induced apoptosis in cancer cells (139, 140); a molecule that employs this strategy, KL1333, is entering human trials (141). KL1333 is an orally available, small organic molecule that reacts with NQO1 as a substrate and increases the intracellular NAD⁺ levels via NADH oxidation. A water-forming NADH oxidase from Lactobacillus brevis (LbNOX) has also been used as a genetic tool to increase the NAD⁺/NADH ratio in human cells (142, 143). Although such genetic tools are unlikely to be used in humans, they create new opportunities for mechanistic exploration in cells and model organisms.

Effects of NAD⁺ Augmentation by Pharmacolgic Approaches in Preclinical Models

For a more in-depth discussion of the extensive work in this area, the reader is directed to recent reviews (36, 86). Extensive preclinical data on the use of NA to lower lipids are not discussed because this effect is generally not observed with other precursors and is therefore likely to be independent of NAD⁺. Overall, preclinical studies suggest many potential benefits of increasing NAD⁺ (Table 1), but the optimal intervention strategy and underlying mechanisms remain to be fully elucidated. Here, we focus primarily on the effects of pharmacologic approaches to increase NAD⁺ levels. The strongest rationale for the use of NAD⁺ precursors is the evidence that NAD⁺ is depleted or suspected to be depleted in many of the conditions described below (123, 124, 144-148).

Table 1.

Preclinical studies

Physiologic effect/benefit	Model(s)	Intervention(s)	Selected references
Effect on health span
Longevity effects: improved health span but not life span in wild-type mice	20-month-old mice (modest increase)	400 mg/kg/day NR in diet	(149)
	Mice on chow or HFD (no effect)	0.5-1 g/kg (of diet) NAM in diet	(150)
	HET3 mice on chow (no effect)	1000 ppm NR in diet	(151)
Physiologic effects on metabolism and metabolic disorders
Improved insulin sensitivity/glucose tolerance	Nampt+/− mice	500 mg/kg NMN IP once	(152)
	Mice on HFD	500 mg/kg/day NMN	(125)
	Mice on HFD	400 mg/kg/day NR in diet	(123)
	Mice on HFD	0.5-1 g/kg (of diet) NAM in diet	(150)
Improved insulin secretion	Nampt+/− mice	500 mg/kg NMN IP once	(152)
	Mice on HFD	500 mg/kg/day NMN	(125)
	Mice on fructose diet	500 mg/kg NMN IP once	(153)
Decreased body weight gain	Mice on HFD	400 mg/kg/day NR in diet	(123)
	Mice on HFD ± streptozotocin	3 g/kg (of diet) NR in diet	(145)
	Aged mice	100-300 mg/kg/day NMN in diet	(124)
Decreased liver fat	Mice on HFD	500 mg/kg/day NMN IP	(154)
	Mice on Western diet	400 mg/kg/day NR in diet	(84)
	Mice on chow or HFD	0.5-1 g/kg (of diet) NAM in diet	(150)
	Mouse model of NAFLD
Effects on muscle performance, endurance, and physical function
Increased mitochondrial biogenesis/improved function	Mice on HFD	400 mg/kg/day NR in diet	(123)
	Aged mice	500 mg/kg/day NMN IP	(155)
Increased endurance	Mice on HFD	400 mg/kg/day NR in diet	(123)
	Aged mice	400 mg/kg/day NMN in drinking water	(156)
Improved blood flow to the muscle and vascular function	Aged mice	400 mg/kg/day NMN in drinking water	(156)
	Aged mice	500 mg/kg/day NMN IP	(96)
Cardiovascular Disease
Prevention/improvement of heart failure with reduced and preserved ejection fraction	Mice with transverse aortic constriction	500 mg/kg/day NMN IP	(157)
	Heart-specific SRF KO mice	400 mg/kg/day NR in diet	(158)
	Mice on HFD + L-NAME (HFpEF)	400 mg/kg/day NR in diet	(159)
	Mouse and rat HFpEF	40 mM NAM in drinking water	(160)
Reduces aortic atherogenesis progression and improves plaque stability	Apoe −/− mice Double Ldlr−/− and Apoe−/− mice	0.25% or 1% NAM in drinking water	(161, 162)
Prevents or attenuates ischemia and reperfusion myocardial injury	wild type adult rat	10-20 mg NAD+ intravenously	(163)
	Aged male rats	100 mg NMN intraperitoneally	(164)
Kidney disease
Acute kidney injury	Cisplatin-induced kidney injury	400 mg/kg NAM IP twice	(165)
	Cisplatin-induced kidney injury	500 mg/kg/day NMN IP	(166)
	Ischemia-reperfusion and cisplatin acute injury (ineffective in chronic models)	400-800 mg/kg/day NR in diet	(167)
Diabetic kidney disease	Diabetic db/db mice with nephropathy	500 mg/kg/day NMN in saline daily	(168)
Degenerative neurological and aging conditions
Slowed progression in Alzheimer's disease models	Tg2576 mice	250 mg/kg/day NR in diet	(169)
	APP(swe)/PS1(ΔE9) double transgenic mice	100 mg/kg/2 days NMN SC	(170)
	Aβ1-42 oligomer infused Wistar rats	500 mg/kg/day NMN IP	(171)
Slowed progression of congenital muscle disorders	Mice with mitochondrial myopathy	400 mg/kg/day NR in diet	(172)
	Mice with mitochondrial myopathy	400 mg/kg/day NR in diet	(173)
	Mdx mice (DMD model)	400 mg/kg/day NR in diet	(174)
Slowed disease progression in mice with premature aging syndromes	Atm−/− mice (life span extended)	12 mM NR in drinking water	(175)
	Csbm/m mice	500 mg/kg/day NR IP	(176)
Other physiologic effects
Protected from hemorrhagic shock	Long-Evans rats with severe blood loss	400 mg/kg/day NMN in drinking water	(177)
Improved early development of offspring	Mice and rats, supplementation of lactating mothers	3 g/kg (of diet) NR in chow	(178)
Improved liver regeneration	Mice with 2/3 partial hepatectomy	400 mg/kg/day NR in drinking water	(179)
	CCl4-treated wild-type and telomerase knockout mice	5 mM NMN in drinking water	(180)
	Mice with 2/3 partial hepatectomy	250 mg/kg/day NAM IP	(181)
	Mice with 2/3 partial hepatectomy	400 mg/kg/day NR in drinking water	(182)

Abbreviations: APP, amyloid precursor protein; HFD, high-fat diet; HFpEF, heart failure with preserved ejection fraction; IP, intraperitoneally; NAM, nicotinamide; NAFLD, nonalcoholic fatty liver disease; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside; SC, subcutaneously.

NAD⁺ Augmentation in Metabolic Disorders

Obesity

Obesity induced by feeding high-fat diet to mice is associated with reduced expression levels of intracellular NAMPT as well as NAD⁺ in the liver and white adipose tissue (123-125). Paradoxically, the plasma levels of an extracellular secreted form of NAMPT (eNAMPT, also known as visfatin) are increased in obesity. While the functional role of eNAMPT remains to be fully elucidated, it has been suggested to act both as a hormone and as an NAD⁺ biosynthetic enzyme (183). Recently, eNAMPT from adipose tissue was shown to be contained in vesicles and to correlate with hypothalamic NAD⁺ concentration, potentially indicating a new NAD⁺ mediated signaling axis (184). Oral administration of NAD⁺ precursors, NMN or NR, restores NAD(H) levels in mice with diet-induced obesity. NR administration was reported to enhance energy expenditure and attenuate weight gain induced by feeding high-fat diet to mice (123). Although the effect on weight gain has been reproduced (84), only modest (185) or no (186) benefits were observed in other studies. Long-term administration of NMN also has been reported to be associated with increased energy expenditure and physical activity and reduced weight gain with aging (124).

Diabetes mellitus

Diabetes induced by feeding high-fat diet in mice is associated with reduced NAD⁺ levels in many tissues including white adipose tissue and liver (125). In mice, NAD⁺ augmentation by oral administration of NMN as well as NR protects against diet-induced glucose intolerance and diabetes and improves whole body and liver insulin sensitivity (125, 145). In some studies, these effects have been associated with upregulation of SIRT1 and SIRT3, deacetylation of FOXO1, and induction of SOD2 and NDUFA9 (an oxidoreductase enzyme complex in the electron transport chain), improvements in mitochondrial function, running ability, and cold tolerance (123). NMN reduces oxidative stress and the inflammatory response to feeding of high-fat diet (125). Long-term NMN administration in mice prevents the age-related decline in NAD⁺ and the development of insulin resistance with aging (124). Similarly, administration of nicotinamide improves glucose intolerance associated with diet-induced obesity in mice (150). Nicotinamide administration has been reported to increase β cell mass (187) without increasing NAD⁺ levels in the pancreas in rodent models of type 1 diabetes and rescues streptozotocin-induced β cell damage (188).

Diabetic kidney disease

In addition to the classical Kimmelsteil Wilson lesions in the glomeruli (189), diabetic kidney disease (DKD) is characterized by pathologic changes in nearly all kidney compartments, including the vasculature, tubules, and interstitium (190-192). Indeed, tubulointerstitial disease is the best indicator of progressive DKD (193, 194). Treatments targeting glomerular hemodynamics through blockade of the renin-angiotensin-aldosterone system have been the main approach for slowing the progression of DKD, but DKD often progresses despite such therapy (195, 196). One of the most exciting advances in the management of type 2 diabetes mellitus has been the introduction of a tubule-directed therapy: interfering with the tubular reabsorption of glucose through inhibition of sodium-glucose cotransporter 2 (197, 198). Pioneering studies by Hasegawa et al (199) have unveiled the important role of tubule-podocyte interactions in the pathogenesis of DKD and the role of NAD⁺ as a key mediator of the tubular-podocyte crosstalk (126). Podocyte-specific Sirt1 hyperexpression prevented the development of glomerular abnormalities in streptozotocin-induced diabetes, while podocyte-specific genetic disruption of SIRT1 worsened glomerular abnormalities (126). SIRT1 expression in the tubule epigenetically regulates Claudin-1 expression in podocytes through DNMT1-mediated gene methylation (199). Thus, SIRT1-NAD⁺ pathway mediates the paracrine interaction between the tubule and the adjacent podocyte.

As further evidence of the important role of NAD⁺ in the pathogenesis of DKD, the Hasegawa Laboratory (168) treated db/db mice with NMN daily for 2 weeks. NMN treatment increased SIRT1 expression in the kidneys of db/db mice, attenuated the increase in urinary albumin to creatinine ratio, and provided histopathologic protection with less mesangial expansion and foot process effacement without a significant change in hemoglobin A1c (HbA1c). NMN treatment also provided a survival benefit in db/db mice (168). Remarkably, the benefits of 2-weeks of NMN treatment persisted 14 weeks post-treatment, potentially through epigenetic gene modulation (legacy effect) by NAD⁺-dependent sirtuin deacetylase (168, 200). Thus, several lines of preclinical evidence support the premise that NAD⁺ augmentation could be an attractive strategy for the treatment of DKD.

Nonalcoholic Fatty Liver Disease

In a mouse model of diet-induced nonalcoholic fatty liver disease, hepatic NAD⁺ levels as well as the activities of SIRT1 and SIRT3 are reduced (201). Disruption of NAMPT expression either by genetic knockout of Nampt or by using a pharmacologic inhibitor increases lipid accumulation and chronic inflammation in the liver and impairs hepatic insulin sensitivity (202). NR administration protects mice against aging-induced nonalcoholic fatty liver disease–like phenotype (203). In another study, NR was reported to attenuate liver damage and the development of hepatic steatosis in prediabetic mice fed high-fat diet (145). Sirt1 overexpression also is associated with reduced fat accumulation in response to high-fat diet in mice (204). Both NR and nicotinamide promote regeneration of the liver after partial hepatectomy (179, 181), and NMN is protective against the hepatotoxin carbon tetrachloride (180).

Skeletal Muscle Mass and Performance, and Physical Function

NAD⁺ reduction to NADH occurs during glycolysis, pyruvate dehydrogenase, lipid β-oxidation, and TCA cycle. Most of the free energy released during oxidation is retained in the reduced coenzymes NADH and FADH₂ generated during these processes (205). During respiration, electrons generated from NADH and FADH₂ are eventually transferred to O₂ (205). The oxidation of NADH to NAD⁺ in mitochondria by action of Complex I and in cytoplasm by lactate dehydrogenase is essential for ATP production (205). A substantial fraction of mitochondrial ATP production during aerobic exercise is linked to Complex I activity.

NAD⁺ precursors improve the aerobic capacity and physical activity of older mice, and protect against age-related physiological decline, mitochondrial dysfunction, and glucose intolerance (123, 124, 206). Aging is associated with reduced muscle microvasculature, blood flow, and oxygen delivery and reduced endurance (207, 208). Administration of an NAD⁺ precursor, NMN, to aged mice is associated with increased muscle capillarity, improved blood flow to the muscle, and longer running time to exhaustion than young mice and a lower postexercise lactate level, consistent with improved aerobic capacity (208). Mice with endothelial cell-specific knock down of SIRT1 exhibited reduced exercise capacity and a reduced number of capillaries (208). In contrast, increased expression of endothelial SIRT1 was associated with increased muscle capillary density and exercise capacity (208). The effects of NMN on physical endurance in aging mice were augmented by exercise (208).

Increasing NAD⁺ facilitates SIRT1 activity, which in turn activates PGC-1α and the FOXO family of proteins that govern mitochondrial biogenesis and function. Increased SIRT1 activity prevents muscle atrophy and promotes muscle growth in mice (209-211). Exogenous NAD⁺ precursors can increase mitochondrial oxygen consumption and ATP production in mammalian cells (209, 210, 212, 213). Genetic depletion of NAMPT in adult skeletal muscles leads to fiber degeneration and progressive loss of both muscle strength and treadmill endurance (209). Thus, NMN administration would be expected to enhance mitochondrial biogenesis and efficiency resulting in improved muscle bioenergetics and fitness (208, 212, 213). The administration of NMN in old mice also has been reported to reverse age-related mitochondrial dysfunction (124, 149). NR is protective in the mdx model of Duchenne muscular dystrophy (174) and maintains muscle mass in a model of cancer cachexia (214). Beltra et al (215) reported downregulation of Nrk2, an enzyme involved in catalyzing the conversion of NR to NMN in the NAD biosynthetic pathway and NAD⁺ depletion in a preclinical model of cancer cachexia. Administration of nicotinamide increased NAD⁺ levels in the muscle and prevented the loss of body weight and muscle mass. In line with these findings, the expression levels of Nrk2 also were reduced in patients with cancer and associated with impaired energy metabolism and a catabolic state (215). Taken together, these preclinical data have led to the hypothesis that NAD⁺ augmentation by administration of NAD⁺ precursors will improve aerobic capacity in young men and women and the effects of NAD⁺ augmentation on aerobic capacity and other measures of physical performance will be augmented by high intensity physical exercise.

However, there is a notable lack of evidence and even some counter evidence to the idea that NAD⁺ boosting could be beneficial in young, healthy mice. While NR increases the endurance of obese mice, it causes only a trend toward improvement in lean animals (123); a study in young rats showed a trend toward decreased, rather than increased swimming ability (216). Recently, intravenous NR was used to more effectively increase NAD⁺ levels in the muscles of young mice (compared with oral dosing) but was found to have no effect on respiratory capacity or insulin sensitivity, despite modestly reducing weight gain on Western diet (217). Moreover, young mice fed a mildly obesogenic diet with very high dose NR developed glucose intolerance (218).

Acute Kidney Injury

The highly metabolically active cells of the kidney tubule have high energy needs that are linked with mitochondrial quality control, inflammatory responses, and autophagy (219). NAD⁺ is an essential cofactor for these processes that are critical for cellular health (36, 124, 126, 200). Acute kidney injury is characterized by substantial decreases in NAD⁺ levels (165, 199, 200). De novo NAD⁺ biosynthesis is impaired in patients undergoing cardiac surgery and in critically ill patients at risk of acute kidney injury (165). NMN, NR, NRH, and nicotinamide have all been reported to protect against acute kidney injury caused by cisplatin or ischemia in mice (107, 165-167). Moreover, a decrease in NAD⁺ and related metabolites is 1 of the most prominent metabolic markers for kidney disease in humans, suggesting that these lessons from rodents may have translational potential (220).

Effects of NAD Augmentation on Life Span and Health Span

The data generally do not support a substantial benefit of NAD⁺ precursors on life span in wild type mice. Nicotinamide at several doses failed to improve survival in C57BL/6 mice, despite metabolic benefits (150). A small trial of NR initiated late in life produced a modest, but significant increase in remaining life span in C57BL/6 mice (149). However, a larger study that started the supplementation earlier in HET3 mice (albeit at a lower dose) failed to demonstrate any effect on life span (151). NR has been shown to be protective in models of progeroid syndromes caused by DNA repair defects including ataxia telangiectasia and Cockayne syndrome through mechanisms that include improving mitophagy and mitochondrial quality control (175, 176). The survival in ataxic mice is dramatically extended. NR further prevents hearing loss induced by noise or Cockayne syndrome (144, 221). Overall, the existing preclinical literature support a benefit of NAD⁺ boosting on health span and mitigation of disease, but not on life span in otherwise healthy rodent models.

NAD⁺ Augmentation in Cardiovascular Disease

NMN and NR have been reported to reverse age-related endothelial dysfunction and reduce oxidative stress (150). In C57BL/6 mice, oral supplementation of NMN reversed vascular aging, including endothelial dysfunction and aortic stiffening by reducing oxidative stress, increasing nitric oxide bioavailability, and normalizing intrinsic arterial wall stiffness by reducing fibrosis (207).

Oral nicotinamide supplementation in Apoe null mice reduces the oxidation of Apo B containing lipoproteins, upregulated aortic interleukin-10 expression, downregulated tumor necrosis factor α expression and prevented aortic atherogenesis (161). The anti-atherogenic effect of nicotinamide are associated with a shift towards an anti-inflammatory cytokine and macrophage profile (161). Administration of 1-methylnicotinamide, a metabolite of nicotinamide, also prevents the progression of atherosclerotic lesions and improved endothelial function and inflammation in Ldlr^−/−/Apoe^−/− double knockout mice (222). Pharmacologic inhibition as well as genetic disruption of PARP1, an NAD⁺ consuming enzyme, reduces atherosclerotic plaque size and improved plaque stability without affecting the lipoprotein profile in Apoe^−/− mice (223). PARP1 inhibition has also been reported to improve endothelial function and attenuate atherogenesis in rabbits (224).

NAD⁺ supplementation protects the heart from ischemia/reperfusion injury in several preclinical models (163-228). Ischemia-induced myocardial injury in dogs is associated with increased activity of NAD⁺-consuming enzymes and depletion of myocardial NAD⁺, NADH, NADP⁺, and NADPH. Furthermore, other studies have shown that NMN administration increases myocardial NAD⁺ levels and attenuates cardiac ischemia/reperfusion injury in older rats (164) and mice (225). In mouse models of preeclampsia, administration of nicotinamide reduced blood pressure in the mother and prevents fetal growth restriction and premature birth (226, 227).

Heart failure

Both NMN and NR have shown promising results across multiple rodent models of heart failure (157-230). Heart failure is characterized by alterations in NAD⁺ metabolism and impaired myocardial energetics. These include a modest decrease in the expression of Nampt and a strong induction of Nrk2, which might suggest a shift to NR-dependent NAD⁺ synthesis (158). In a mouse model of dilated cardiomyopathy and heart failure, NR administration was reported to improve myocardial function (158). Another study reported increase in cellular NAD⁺ content and improvements in left ventricular structure and function in cardiomyopathy caused by laminin A/C gene mutations (231). In a mouse model of heart failure with preserved ejection fraction (HFpEF), alterations in myocardial mitochondrial function have been associated with impairment of fatty acid oxidation, SIRT3 downregulation and NAD⁺ deficiency. NR supplementation in this mouse model of HFpEF improved mitochondrial function, cardiac remodeling, and improved diastolic function and exercise tolerance (159). In Dahl salt-sensitive rats, a preclinical model of HFpEF, nicotinamide supplementation reduced blood pressure and diastolic dysfunction by improving myocardial bioenergetics, cardiomyocyte stiffness, calcium-dependent relaxation (160).

The downstream mechanisms by which NAD⁺ precursors exert their diverse beneficial effects remain incompletely understood and for almost all of these benefits, a head-to-head comparison of various NAD⁺ precursors has not been performed. Many of the effects of NAD⁺ boosting or restoration likely depend on redox reactions that are vital to cellular function (eg, glycolysis). In these cases, inactivating the pathways to test dependence is not an option, making it difficult to prove a definitive mechanism. It can also be misleading to infer a common mechanism simply because 2 interventions affect the same phenotype—for instance, NR accelerates liver regeneration and loss of the NAD⁺-dependent sirtuins SIRT1 or SIRT3 slows it, but the benefit of NR is preserved in the absence of SIRT1 or SIRT3 (182).

Neurodegenerative Diseases

In normally aged mice, NAD⁺ boosting with NMN or NR has been associated with reducing a number of age-associated phenotypes such as cataracts, impaired neurovascular coupling, and reduced numbers and renewal potential of neural stem cells (124, 149, 232). NAD⁺ augmentation exerts neuroprotective effects in preclinical models of several neurodegenerative diseases such as Parkinson disease (233), Alzheimer disease (234), amyotrophic lateral sclerosis (148), and Cockayne syndrome (221). In models of neurodegenerative disease, especially Alzheimer disease, both NMN and NR have resulted in improved performance in cognitive tasks (169, 235). NR administration has been reported to prevent noise-induced hearing loss and spiral ganglia neurite degeneration in mice (144).

Clinical Trials of NAD⁺-Boosting Compounds

Several clinical trials have assessed the effects of orally consumed NAD⁺-boosting compounds (116, 118, 127, 236-249). Many of these studies have been performed using NR or NMN individually (116, 118, 127, 236-249) (Tables 2 and 3) or NR combined with the polyphenol pterostilbene or other compounds (250-254) (Table 4) or with nicotinamide alone or in combination with other compounds (165, 255, 256). These trials have been conducted mostly in middle-aged and older healthy adults or people with chronic disorders, although younger adults were included in some studies. The outcomes assessed in these studies have included the circulating or tissue levels of NAD⁺ and its metabolites, safety, physiological functions, and/or clinical markers. Although very few studies have been large enough or long enough to meaningfully evaluate disease outcomes, phase 1 and phase 2 trials have provided important data on physiologic effects and early evidence of potential benefits.

Table 2.

Summary of clinical trials with NR supplementation

Author, year	Population	Intervention	Main outcomes and findings
Trammell et al, 2016	A healthy 52-year-old male (n = 12)	7 days of NR (1000 mg/day)	Increased blood cellular NAD+ bioavailability
Airhart et al, 2017	Healthy adults (21-50 years; n = 8)	8 days of NR (daily dose escalating from 250-2000 mg over the trial)	Treatment was safe and well-tolerated. Blood NAD+ and NR levels increased
Dollerup et al, 2018, 2019, 2020	Obese middle-aged/older men (40-70 years; n = 40)	12 weeks of placebo or NR (1000 mg 2×/day)	Treatment was well-tolerated but had no effect on skeletal muscle NAD+ or insulin sensitivity
Martens et al, 2018	Healthy middle-aged/older adults (55-79 years; n = 24)	6-week crossover design: placebo vs NR (500 mg 2×/day)	Treatment was well-tolerated and blood cellular NAD+ metabolism increased
Elhassan et al, 2019	Healthy older men (median: 75 years; n = 12)	21-day crossover design: placebo vs NR (500 mg 2×/day)	Treatment was well-tolerated and skeletal muscle NAD+ metabolism increased
Conze et al, 2019	Middle-aged adults (40-60 years; n = 133)	8 weeks of placebo, 100 mg NR, 300 mg NR, or 1000 mg NR	Treatment was well-tolerated at all doses and caused dose-dependent increase in urine, plasma, and cellular NAD+ metabolites
Remie et al, 2020; Nascimento et al, 2021	Overweight/obese adults (45-65 years; n = 13)	6-week crossover design: placebo vs NR (1000 mg/day)	Treatment had no effect on insulin sensitivity, mitochondrial function or brown adipose tissue activity
Zhou et al, 2020	Patients with stage D heart failure (27-64 years; n = 4)	5-9 days of NR (2000 mg/day)	Whole blood NAD+ levels were increased. Expression of genes for proinflammatory cytokines were reduced in PBMCs
Stocks et al, 2021	Active young men (mean 23 years; n = 8)	7-day crossover design: placebo vs NR (500 mg 2×/day)	Increased NAD+ metabolites in skeletal muscle with treatment. Treatment did not alter substrate metabolites or mitochondrial respiration
Brakedal et al, 2022	Patients with Parkinson's Disease (n = 30)	30 days of placebo or NR (1000 mg/day)	Treatment was safe, increased cerebral NAD+, and altered cerebral metabolism
Wu et al, 2020	Healthy adults (mean: 24 years; n = 35)	7 days of placebo or NR (1000 mg/day)	Whole blood NAD+ metabolite levels increased with treatment vs placebo

Abbreviations: NAD⁺, nicotinamide adenine dinucleotide; NR, nicotinamide riboside; PBMC, peripheral blood mononuclear cell.

Table 3.

Summary of trials with NMN supplementation

Author, Year	Population	Intervention	Main outcomes and findings
Yoshino et al, 2021	Overweight/obese postmenopausal women with prediabetes (n = 25)	10 weeks of placebo or NMN (250 mg/day)	Treatment increased blood cellular NAD+, increased muscle insulin sensitivity and had no effect on body composition
Liao et al, 2021	Young/middle-aged recreationally trained runners (n = 48)	6 weeks of placebo, low dosage NMN (300 mg/day), medium dosage NMN (600 mg/day), or high dosage NMN (1200 mg/day). Groups followed same aerobic exercise program.	Select improvements in submaximal cardiorespiratory parameters were observed in the medium and high dosage groups
Kim et al, 2022	Healthy older adults (65+ years; n = 108)	12 weeks of placebo (Am), placebo (Pm), NMN (250 mg Am), or NMN (250 mg Pm)	Treatment had no effect on sleep quality or fatigue
Okabe et al, 2022	Healthy adults (20-65 years; n = 29)	12 weeks of placebo or NMN (250 mg/day)	Treatment was safe and increased whole blood NAD+ metabolome
Huang, 2022	Healthy middle-aged adults (40-65 years; n = 62)	60 days of placebo or NMN (300 mg/day)	Treatment increased serum NAD+ metabolome
Igarashi et al, 2022	Healthy older men (65+ years; n = 42)	12 weeks of placebo or NMN (250 mg/day)	Treatment was safe and increased whole blood NAD+ metabolome. Gait speed and grip strength were increased
Pencina et al, 2022 (MIB-626)	Overweight/obese adults (55-80 years; BMI 28-40 kg/m2; n = 32)	14 days of 1×/day MIB-626 (1000 mg/day), 2×/day MIB-626 (2000 mg/day), or placebo	Treatment was safe. MIB-626 dose dependently increased whole blood NAD+ metabolome
Askasaka et al, 2022	Frail older men (65+years; n = 15)	24 weeks of placebo or NMN (250 mg/day)	Treatment was safe. No change in gait speed or grip strength
Pencina et al, 2022	Healthy overweight or obese adults, 45 years or older (n = 30)	Placebo or NMN (1000 mg twice daily) for 28 days	NMN treatment significantly increased blood NAD+, body weight; diastolic blood pressure, total cholesterol, low-density lipoprotein cholesterol, and nonhigh-density lipoprotein cholesterol decreased significantly more in the NMN group than placebo

Abbreviations: BMI, body mass index; NAD⁺, nicotinamide adenine dinucleotide; NMN, nicotinamide mononucleotide.

Table 4.

Summary of trials with NR co-administered with additional compounds

Author, year	Population	Intervention	Main outcomes and findings
Dellinger et al, 2017	Healthy adults (60-80 years; n = 115)	8 weeks of placebo, NRPT 1 × (250 mg NR + 50 mg PT), or NRPT 2 × (500 mg NR + 100 mg PT)	Treatment was well-tolerated and caused dose-dependent increase in blood NAD+
de la Rubia et al, 2019	People with ALS (n = 20)	16 weeks of placebo or EH301 (1200 mg)	Treatment improved ALS functional rating score
Altay et al, 2021	Adults positive for COVID-19 (18-66 years; n = 397)	14 days of placebo or “metabolic activators” including NR. All patients received standard care	Plasma markers of NAD+ bioavailability increased. Shorter recovery time with active treatment vs placebo. Blood markers of inflammation were improved with treatment vs placebo
Jensen et al, 2022	Older adults, 55-80 years (n = 32)	14 days of placebo or 1000 mg NR plus 200 mg pterostilbene	Compared with placebo, NR plus pterostilbene did not improve recruitment of muscle stem cells or other markers of muscle regeneration in response to an experimentally induced muscle injury

Abbreviations: ALS, amyotrophic lateral sclerosis; NAD⁺, nicotinamide adenine dinucleotide; NR, Nicotinamide Riboside; NRPT, nicotinamide riboside plus pterostilbene; PT, pterostilbene.

Because the primary focus of this review is on the potential benefits of chronic treatment with NAD⁺-boosting precursors, we only discuss multiple dose studies. The duration of the included trials ranged from 5 days to 16 weeks. We also excluded the literature on the lipid-lowering effects of niacin (see recent reviews on niacin (257-261). Although niacin may increase NAD⁺ bioavailability, its salutary effects on lipids are likely independent of NAD⁺ per se and it is not viewed as an attractive candidate in health span–promoting settings because of its side effects, such as flushing, pruritis, hyperglycemia, hyperuricemia, and liver enzyme elevations (259). Finally, we excluded trials of NAD⁺ augmentation in some congenital conditions (eg, ataxia telangiectasia) that included mostly children (262) as our central focus is healthy aging. The use of NRH and NMNH has been so far limited to only a few preclinical studies in mice (107-109). At present, the lack of human safety data is an obstacle for these compounds, despite very effective NAD⁺ boosting.

The Effect of NAD⁺ Precursors on NAD⁺ Bioavailability

NR supplementation

Multiple dose studies of up to 2000 mg NR daily have reported an increase in NAD⁺ levels in the whole blood, PBMCs, and possibly brain in healthy adults and in patients with some clinical disorders (116, 127, 236, 237, 241, 243, 249). NR administration is also associated with increases in the levels of NAD⁺-related metabolites in plasma, skeletal muscle, and urine, including a notable increase in nicotinic acid adenine dinucleotide (NAAD) in PBMCs (31). However, oral NR administration is associated with only a minimal increase in NR concentrations in plasma and only a modest or no increase in NAD⁺ levels in the skeletal muscle (240, 243). Marked interindividual variability has been observed in the magnitude of the increases in NAD⁺ concentrations in response to NR supplementation (Table 2).

NMN supplementation

Chronic oral supplementation of NMN in doses ranging from 250 mg/day to 1000 mg twice daily from 2 to 12 weeks consistently increases blood NAD⁺ levels in healthy older men and women, middle-aged and older men and women with overweight or obesity, and postmenopausal women with prediabetes (118, 128, 246, 263, 264). The increases in blood NAD⁺ levels with oral NMN administration are strongly associated with the administered dose (118). NMN administration is associated with a substantial increase in NAD⁺ metabolites—nicotinamide, N-methyl nicotinamide, and N-methyl-2-pyridone-5-carboxamide (2PY) in the plasma and urine (118) (Table 3).

Supplementation with other regimens and compounds

A trial in which NR was administered with pterostilbene for 8 weeks found dose-dependent increases in NAD⁺ in whole blood in healthy men and women, 60 to 80 years of age (251). Administration of NR along with L-serine, N-acetyl-L-cysteine, and L-carnitine tartrate to patients with COVID-19 was reported to increase biomarkers of NAD⁺ metabolism in plasma (250) (Tables 4 and 5).

Table 5.

Summary of trials with nicotinamide alone or with other compounds

Author, year	Population	Intervention	Main outcomes and findings
Poyan Mehr et al, 2018	Patients undergoing cardiac surgery	0, 1, or 3 g nicotinamide daily for 3 days	Smaller increase in serum creatinine and troponin levels in nicotinamide-treated groups relative to placebo
Gustavo De Morales et al, 2022	42 patients, 40-80 years, with treated open-angle glaucoma and moderate visual field loss in at least 1 eye	Ascending doses of nicotinamide (1000-3000 mg daily) plus pyruvate (1500-3000 mg) vs placebo daily	Short-term improvement in visual field relative to placebo
Chen et al, 2023	Adults with 2 or more nonmelanoma skin cancers resected in the past 5 years (n = 386)	Placebo or nicotinamide 500 mg twice daily for 12 months	nicotinamide treatment was associated with 23% lower incidence of new nonmelanoma skin cancer than placebo

A few trials that used enteric-coated capsules containing NAD⁺ or NADH in patients with schizophrenia (265, 266), Alzheimer disease (267-269), and chronic fatigue syndrome (104, 270-273) found an increase in blood NAD⁺ levels. One trial of NADH supplementation found increases in blood NADH, but reductions in NAD⁺ concentrations (272). A study assessing the combination of nicotinamide and D-ribose daily for 8 days in healthy men and women also reported increases in blood NAD⁺ levels (274).

Safety of NAD⁺ Augmentation Strategies

At the doses (up to 2000 mg daily) and treatment durations (up to 24 weeks) studied to date, NR and NMN have been safe and well-tolerated, with few adverse events and no drug-related serious adverse events (116-118, 127, 236-249, 263). Nicotinamide has been safely administered at daily doses of up to 3000 mg for up to 1 year (165, 255, 256). The side effects commonly associated with niacin such as flushing, itching, hyperuricemia, hyperglycemia, and elevation of transaminases (260, 261) have not been reported with NMN, NR, or nicotinamide. NMN, NR, and nicotinamide do not bind GPR109A that mediates the flushing response to niacin (88). The data is more limited with other compounds but early phase trials have not revealed any safety concerns.

NAD⁺ augmentation and cancer risk

The complex and context-specific relation between NAD⁺ augmentation and cancer risk has been the subject of some debate recently. NAD⁺ plays an important role in DNA repair and the age-related decrease in NAD⁺ levels may increase the risk of DNA damage and cancer (275, 276). Therefore, increasing NAD⁺ level has been postulated as a strategy for reducing cancer risk (277). Oral nicotinamide treatment was shown to reduce the incidence of new nonmelanotic skin cancers and actinic keratoses in high-risk patients (255). Senescent cells that accumulate with aging contribute to age-related decline in NAD⁺ levels (132, 278) and have previously been shown to promote tumorigenesis in mice (279).

However, there is also a concern that high NAD⁺ levels might support the metabolism of cancer cells, such as their preferential use of the glycolytic pathway even in the presence of sufficient oxygen and normal mitochondrial function (the Warburg Effects), enabling tumor progression and survival (280). There has been concern that increased levels of NAD⁺ may potentially promote cancer cell growth, metastases, and resistance to chemotherapy (281). Increasing NAD⁺ by administration of precursors may also upregulate the proinflammatory SASP (Senescence-Associated Secretory Phenotype) which in some contexts may be tumorigenic (282). Suppressing the activity of NAMPT to reduce NAD⁺ levels by the use of small molecule inhibitors and induce cell death has been suggested as an anticancer strategy although the efficacy of NAMPT inhibitors in clinical trials has been limited (283, 284).

The Effects of NAD⁺ Precursors on Physiologic and Health Outcomes

Effects on body composition, metabolism and diabetes mellitus

Although body composition and nighttime metabolic rate were reported to be improved following 6 weeks of treatment with NR in 1 study of men and women, aged 45-65 years, with overweight or obesity (240), most other studies of up to 12 weeks have found no effects on body composition, blood glucose or insulin levels, energy expenditure, or brown adipose tissue activity (118, 237).

Yoshino et al (128) determined the effects of NMN supplementation on body composition, and skeletal muscle insulin sensitivity and insulin signaling in a randomized controlled trial in 25 postmenopausal women with prediabetes who were obese or overweight (body mass index 25.3-39.1 kg/m²). The participants were randomized to receive either placebo or NMN 250 mg orally daily for 10 weeks. NMN treatment for 10 weeks was associated with increased levels of NAD⁺ in the PBMCs and its circulating metabolites but did not increase the intramuscular NAD⁺ concentrations (128). Peripheral (mainly reflective of muscle) insulin sensitivity, assessed as the rate of insulin-stimulated glucose disposal during the hyperinsulinemic euglycemic clamp, improved after 10 weeks of NMN treatment but did not change in placebo-treated participants (128). Markers of muscle insulin signaling (phosphorylation of muscle AKT and mTOR) and muscle remodeling (eg, platelet-derived growth factor) were upregulated by NMN treatment. However, NMN treatment had no significant effect on plasma glucose, insulin, free fatty acid, lipids, fat mass, fat-free mass, intra-abdominal adipose tissue, and intrahepatic triglyceride content, blood pressure. The other trials also have not shown significant improvements in blood glucose or insulin levels in middle-aged and older adults (118, 237, 238).

As discussed earlier, nicotinamide administration increases β cell proliferation and attenuates hyperglycemia in rodent models of diabetes (187, 188). In a relatively small, randomized trial, the first-degree relatives of patients with type 1 diabetes, who had anti-islet cell antibodies, were randomized to receive 1200 mg/m² oral nicotinamide or placebo daily (285). A greater proportion of participants randomized to nicotinamide remained diabetes-free than those randomized to placebo (285). A subsequent larger trial of nicotinamide vs placebo in a first-degree relatives of patients with type 1 diabetes who had positive anti-islet cell antibodies did not confirm the findings of the first trial and found similar frequencies of incident type 1 diabetes, presence of anti-islet cell antibodies, and no significant differences in the first phase insulin release, and insulin sensitivity between the nicotinamide and placebo arms of the trial (286).

Effects on blood pressure

In a randomized trial, 6 weeks of treatment with NR in healthy middle-aged and older adults with elevated systolic blood pressure at baseline was associated with a significant reduction in systolic blood pressure and in arterial stiffness (238). In this trial, 6 weeks of NR treatment did not significantly change vascular endothelial function (flow-mediated dilation), resting metabolic rate, body mass, body mass index, percent body fat, plasma glucose-insulin regulation, motor function, maximal aerobic exercise capacity, or submaximal exercise performance (238).

In another trial of healthy older adults, treatment with the combination of NR and pterostilbene for 8 weeks was associated with improvements in diastolic BP and liver function (reduced plasma alanine transaminase), at the low (250 mg/day) dose, and performance in 30-second chair stand and 6-minute walk test at the 500 mg/day dose (251). Some other trials of up to 12-week duration that did not specifically focus on blood pressure as an outcome measure have reported no significant changes in systolic or diastolic blood pressure compared with placebo (eg (237)).

Effects on muscle performance and physical function

Exercise training increases the levels of NAD⁺, and the expression NAMPT (78, 287), the rate-limiting enzyme in the NAD⁺ salvage pathway and increases the activity of sirtuins (287). Therefore, the effects of exercise training and NAD⁺ precursors could be complementary or even additive. In a study of Chinese trained runners (245), the participants were given daily doses of 300 mg, 600 mg, or 1200 mg NMN for 6 weeks in addition to running for 50 to 60 minutes 5 or 6 times each week. The administration of the 1200 mg NMN daily plus training was associated with greater increases in the percentage of maximum oxygen uptake power at first ventilatory threshold, and power at second ventilatory threshold compared with the control group (245). NAD⁺ augmentation has been reported to increase muscle acetyl carnitine in healthy adults (240). In another placebo-controlled, randomized trial (246), administration of 250 mg NMN daily to healthy older men for 6 or 12 weeks was associated with greater improvements in walking speed and grip strength compared with placebo but did not affect fat-free or fat mass. However, no change in grip strength was detected in older men following 3 weeks of supplementation with 1000 mg/day NR (127). In the longest trial reported to date, patients with mitochondrial myopathy were treated with NA for 4 to 10 months, resulting in statistically significant improvements in functional capacity, increased mitochondrial biogenesis, and elevated blood NAD⁺ levels (288). Other short-term single or multiple dose studies have reported inconsistent effects of NAD⁺ augmentation on measures of muscle performance and aerobic capacity in humans (263, 289). Overall, although the preclinical data of the effects of NAD⁺ augmentation on aerobic performance are promising and some early studies have reported modest improvements in function, substantially larger trials of longer duration are needed to determine whether NAD⁺ augmentation in older adults with functional limitations can induce meaningful improvements in performance-based and self-reported measures of physical function.

To determine whether treatment with NR and pterostilbene increases skeletal muscle stem cells and improves muscle regeneration after injury induced by electrically induced eccentric muscle in older adults, Jensen et al (254) randomized healthy older adults to receive either 1000 mg NR plus 200 mg pterostilbene or placebo for 2 weeks. Treatment with NR plus pterostilbene did not have any significant effect on skeletal muscle stem cell number, recruitment, or proliferation, muscle fiber area, central nuclei, and embryonic myosin heavy chain expression (254). In another trial (290), treatment of with NR was associated with increase in muscle mitochondrial DNA and expression levels of genes associated with mitochondrial biogenesis but no consistent changes in satellite cell number and body composition.

Acute kidney injury

The energetic needs of the highly metabolically active cells of the kidney tubule are linked with energy generation and mitochondrial quality control, inflammatory responses, and autophagy (219). NAD⁺ is an essential cofactor for these processes that are critical for cellular health (36, 124, 126, 200). Acute kidney injury is characterized by decreases in NAD⁺ levels (165, 199, 200, 291), as is chronic kidney disease in humans (220). The de novo NAD⁺ biosynthesis is impaired in patients undergoing cardiac surgery and in critically ill patients at risk of acute kidney injury (165). In a phase 1 placebo-controlled study, administration of nicotinamide was associated with a dose-related increase in circulating NAD⁺ metabolome and a smaller increase in serum creatinine than placebo in patients undergoing cardiac surgery who were at risk for acute kidney injury (165).

Clinical trials for other conditions

Skin cancer and actinic keratosis

nicotinamide has been reported to protect the skin against damage and actinic keratosis caused by ultraviolet light (275). A randomized placebo-controlled trial treated 382 participants who had 2 or more nonmelanoma skin cancers resected in the preceding 5 years with either nicotinamide or placebo for 1 year (255). The participants randomized to nicotinamide had significantly lower incidence of new nonmelanoma skin cancers and actinin keratosis than placebo.

Effects on systemic inflammation

NAD⁺ precursors have been shown to reduce the circulating and tissue levels of inflammatory markers in patients with a number of disease states. Short-term (5-9 days) treatment with NR reduced the expression of proinflammatory cytokine genes in PBMCs of patients with heart failure (242). In another study of young healthy adults and patients with systemic lupus erythematosus, NR inhibited lipopolysaccharide-stimulated interferon activation in myeloid cells (244). Moreover, NR supplementation reduced plasma markers of inflammation in overweight older men (127).

SARS-CoV-2 infection

A growing body of evidence suggests that NAD⁺ depletion due to its increased consumption as a part of the innate immune response contributes to the dysregulated and exaggerated inflammatory response to SARS-CoV-2 (146), acute respiratory distress syndrome, and multi-organ damage including acute kidney injury. A placebo-controlled trial of a combined regimen of NR, L-serine, N-acetyl-L-cysteine, and L-carnitine tartrate in patients infected with SARS-CoV-2 reported shorter time to symptom-free recovery (5.7 vs 9.2 days) and a greater reduction in inflammatory markers compared with placebo group (250). In another nonrandomized trial, nicotinamide treatment was associated with lower rates of a composite endpoint of death and progression to renal replacement therapy, and significantly less increase in serum creatinine among patients hospitalized with SARS-CoV-2 and acute kidney injury than no treatment (292). These findings need confirmation in prospective, adequately powered, randomized trials.

Neurological, neurocognitive, and psychiatric disorders

In a phase 1 trial (249), NR treatment of patients with Parkinson disease was associated with increased NAD⁺ concentrations in the brain, changes in cerebral metabolism, a modest improvements in motor function, and a trend toward improved ratings of disease severity (249). The changes in motor function or disease severity, and inflammatory markers did not differ significantly between NR- vs placebo-treated participants (249). Even though the trial was not powered for clinically meaningful outcomes, the trial's findings are important in demonstrating that orally administered NAD⁺ precursor, NR, was able to (1) increase intra-cerebral NAD⁺ levels and the CSF levels of NAD⁺ metabolite (or N-methyl-6-pyridone-3-carboxamide: Me-6-PY); and (2) alter brain metabolic activity. These findings provide the rationale for larger and longer-term studies of NAD⁺ precursors in neurodegenerative disease such as Parkinson disease and Alzheimer disease.

Increasing intracellular NAD⁺ through administration of NAD⁺ precursors, such as NR and NMN, has been shown to prevent or attenuate Alzheimer disease neuropathology, and improve cognitive function in preclinical models (169, 234, 293, 294). In contrast to many Alzheimer disease drugs in development that target 1 mechanism, NAD⁺ precursors may prevent Alzheimer disease neuropathology by multiple mechanisms: by inducing a switch to nonamyloidogenic processing of amyloid precursor protein due to increased α-secretase activity; reducing the synthesis of oligomerized Aβ peptides; preventing microglia-dependent Aβ toxicity; attenuating neuroinflammation; improving mitochondrial energetics; promoting neuronal regeneration; and improving insulin action (169, 234, 293, 294). No human studies have been conducted to date to determine whether these precursors cross the blood-brain barrier or engage the target mechanisms in humans. A few small trials using various precursors in patients with Alzheimer disease have reported inconsistent results (267-269). Studies assessing the effects of enteric-coated capsules containing NADH also have shown inconsistent improvements in cognitive rating scales in patients with Alzheimer disease (267).

A clinical trial of NR plus pterostilbene in patients with amyotrophic lateral sclerosis (ALS) reported improved ALS functional rating score, pulmonary function, and muscle strength vs placebo. Some benefits persisted in patients who continued treatment for up to 1 year in the open-label treatment arm (252). Although these data are encouraging, they need confirmation in larger trials because of the trial's small size and high dropout rate (252).

Administration of enteric-coated capsules containing NAD⁺ to patients with schizophrenia did not improve symptoms after 3 weeks of treatment but these clinical trials were not sufficiently long or large to observe clinically meaningful treatment effects (265, 266).

Research Gaps and Opportunities for the Development of Pharmacologic Approaches for NAD⁺ Augmentation

Opportunities for Advancing Our Understanding of the Clinical Pharmacology and the Mechanisms of Therapeutic Efficacy

In spite of the promise of the potential applications of NAD⁺ precursors, our understanding of the absorption, presystemic and systemic metabolism of NAD⁺ precursors, the site/s and metabolic pathways of their conversion to NAD⁺, and their tissue bioavailability remains limited. It remains unclear how orally administered NAD⁺ precursors increase NAD⁺ levels in the blood and in some tissues (eg, blood and brain) but not in others (eg, muscle). An improved understanding of the metabolism and clinical pharmacology of NAD⁺-boosting compounds is necessary to guide the selection of dose, optimum route of administration, and development of optimized strategies to enhance NAD⁺ bioavailability in the target tissues (eg, muscle and brain. The role of the gut microbiome in the presystemic metabolism of orally administered NR and NMN remains poorly understood, but appears important in rodent models (98, 99).

The dose selection for NAD⁺ precursors in clinical trials remains empiric. The recommended dietary allowance for vitamin B3 is ∼16 mg (niacin equivalent) daily for an adult male and 14 mg (niacin equivalent) daily for an adult female; the equivalent doses of oral NR and NMN would be ∼38 mg daily and ∼44 mg daily, respectively. Yet, in early phase trials, the NMN and NR doses of 1000 mg daily or more have been required to substantially increase blood NAD⁺ levels (eg, (118)).

The downstream mechanisms by which NAD⁺ precursors exert their diverse beneficial physiological and clinically important effects in the target populations also remain incompletely understood. Some effects of augmenting NAD⁺ levels likely depend on alterations in redox biology and bioenergetic balance. More data are needed from interdisciplinary collaborations between clinical and basic scientists in clinical trials to identify mechanisms of the beneficial physiological and clinical effects, including the roles of reduced inflammation and oxidative stress, improved mitochondrial function, other hallmarks of aging-related mechanisms, and the role of circulating factors released from cells and tissues in response to NAD⁺ boosting therapies that may act as signaling molecules on remote tissues to improve physiological function.

Opportunities for Harmonizing Analytical Methods and Measurements

While techniques for measurements of NAD⁺ and its metabolome have been published (295) and have become more widely available, the acquisition and processing of human samples has yet to be standardized. The interlaboratory differences in the methods for measuring NAD⁺, NADH, NADP⁺, and NADPH have limited a meaningful comparison among studies. NAD⁺, NADP⁺, NADH, and NADPH are chemically unstable (296-298) and each is sensitive to sample collection conditions, such as freeze/thaw cycles and pH. The collection of blood samples in the presence of an acid stabilizes NAD⁺ but degrades NADH and NADPH. Therefore, standardization of sample collection procedures that can be used widely in the setting of clinical trials is necessary to ensure preanalytical stability and minimize variability among laboratories.

An in situ analysis using ultrahigh field NMR spectroscopy is currently the most accurate method for the measurement of NAD⁺, and NADH levels due to the elimination of variables related to extraction and handling (299). Analysis of NAD⁺ levels in the muscle and brain by 7 T ¹H NMR spectroscopy has provided invaluable insight on the effect of aging on the brain NAD⁺ levels (300). High sensitivity ³¹P-NMR spectroscopy (299, 301, 302) can provide simultaneous measurement of NAD⁺, NADH and NAD⁺/NADH ratio in vivo (299). However, further refinement of the MRS procedures is needed to optimize quality and intensity of NAD⁺ and NADH signals and minimize interference from each other and from other pyrophosphate containing metabolites (303). Also, standardization of ³¹P-MRS procedures with optimized modeling is needed to facilitate quantification of molar concentrations of NAD⁺ and NADH in the tissue and an accurate assessment of the redox state (299).

Accurate and harmonized measurements of NAD⁺ metabolites are necessary to inform the bioavailability of NAD⁺ precursor pools, the systemic NAD⁺ abundance, the NAD⁺ usage, and NAD⁺ disposal. For instance, an increase in nicotinuric acid is indicative of an increase abundance of circulating NA, while an increase in methylated species would indicate an increased abundance of nicotinamide and/or NA and potentially associated with a shift in the turnover of the 1-carbon pathway. Similarly, an increase in the abundance of the ribosylated hyperoxidized species is associated with increased abundance in reactive oxygen species, and loss of functional NAD(P)(H).

Opportunities for Advancing Translation of Basic Biology into Approved Therapies for Metabolic Disorders and Age-Related Conditions

The data from preclinical studies and early human trials provide strong rationale for further investigations of the clinical pharmacology and biological mechanisms, as well as the efficacy of NAD⁺ precursors in carefully selected age-related conditions and diseases that can constitute approvable indications. Notably, no safety concerns have emerged from short-term studies of oral NAD⁺-boosting compounds, NMN and NR of up to 16 weeks, but long-term safety remains to be established. Mechanisms for pooling of safety data prospectively from multiple trials in real time would offer a more robust and real-time evaluation of drug safety than has existed today.

Short-term trials of up to 16 weeks have demonstrated that NR and NMN, when administered in appropriate doses of up to 2000 mg daily in middle-aged and older healthy adults and patients with some clinical disorders, increase blood NAD⁺ levels and the levels of NAD⁺ metabolites in the blood and some human tissues and fluids, including PBMCs, brain, cerebral spinal fluid, and urine. NAD⁺ levels are not increased in the skeletal muscle after oral administration of NAD⁺ precursors, although the levels of NAD⁺ metabolites have been found to be increased.

Some previous studies also have been limited by the variable quality of over-the-counter formulations; limited knowledge of the clinical pharmacology and metabolism; empiric dosing regimens; issues with preanalytical stability of samples; interlaboratory differences in assays (see above); and trial design issues such as the framing of indications, study populations, and endpoints. Several early phase trials have provided useful information about physiologic effects but clinical trials have not been of sufficient size or duration to enable rigorous evaluation of the efficacy in improving health and disease outcomes in patients with specific disease conditions. Very few trials were conducted in people with prespecified approvable indications or included clinically meaningful disease endpoints. Studies to date have been limited to small single-site trials: multisite efficacy trials of NAD⁺ boosting compounds (NMN; NR) in larger, properly powered cohorts are needed. The previous trials have been of relatively short duration (up to 16 weeks); substantially longer intervention trials are needed to determine the effects of NAD⁺ augmentation on clinically meaningful outcomes.

The use of Good Manufacturing Practice (GMP) grade formulations that meet regulatory requirements is necessary for ensuring participant safety and enabling comparison of data across studies. Recently, the US Food and Drug Administration, citing the provisions of Section 201(ff) of the Federal, Food, Drug, and Cosmetic Act, has noted that compounds that are under investigation as a potential new drug under an IND can no longer be marketed as dietary supplements. Consequently, NMN which is under investigation as a potential new drug, can no longer be marketed as a dietary supplement. The relevant issue for the clinical trial design is that the use of GMP grade formulations that meet regulatory requirements and that have undergone rigorous preclinical toxicology studies as well as early phase pharmacokinetic and pharmacodynamic studies is necessary for ensuring participant safety, rational dosing, reproducibility, and rigor.

Conclusions

NAD⁺ plays a pivotal role in energy generation, redox reactions, cellular signaling and as co-substrate in many biochemical pathways that play important roles as regulators of health span. NAD⁺ levels can be raised in the blood and in some organs in humans by administration of oral NAD⁺ precursors and by inhibitors of its degradation. The preclinical data are promising. It remains uncertain whether aging itself could be an indication for drugs that target mechanisms of aging; therefore, careful framing of indication/s is required for these drug/s to be approved. Emerging evidence suggests benefit in preventing acute kidney injury in at-risk hospitalized patients (eg, those undergoing cardiac surgery and those with SARS-CoV-2 infection); modest improvements in blood pressure in middle-aged and older adults with obesity or overweight and increased blood pressure at baseline; and suppression of inflammation in older patients, and in those with heart failure, Parkinson disease, and SARS-CoV-2 infection. NAD⁺ augmentation in patients with SARS-CoV-2 infection has been reported to shorten time to symptomatic recovery. These promising early findings need confirmation in larger randomized trials. The measures of whole-body insulin sensitivity, circulating glucose and A1c have generally not shown consistent improvements in early phase trials. In spite of the strong preclinical evidence of improved aerobic capacity and running time in older mice, the data in healthy adults without functional limitations have been variable. Further studies are needed to determine the effects of NAD⁺ augmentation with and without physical exercise on muscle performance, aerobic capacity, and other measures of physical function in older adults with functional limitations, especially among diverse populations that are unable to engage in or adhere to exercise interventions. The data on the efficacy of NAD⁺ augmentation in animal models of DKD and Alzheimer disease, and preliminary findings in patients with Parkinson disease are promising and need further confirmation in randomized trials in older adults with these conditions.

Demonstration of efficacy in randomized trials in specific age-related diseases and conditions could provide the rationale for evaluating the efficacy of NAD⁺ augmentation to reduce the incidence of multiple age-related conditions in older adults with multimorbidity. Such trials would allow testing of the geroscience hypothesis that posits that interventions aimed at modifying aging biology can delay or prevent multiple diseases of aging (304, 305); these studies will require large sample size, long intervention durations, and substantial resource allocation. Because of the increasing prevalence of metabolic disorders around the world, powerful global trends towards the aging of human populations, growing societal burden of metabolic disorders and other age-related diseases, and the promising preclinical and early clinical trial data, accelerated efforts are needed for the clinical development of NAD⁺ augmentation as a strategy to prevent and treat age-related diseases.

Abbreviations

2PY

N-methyl-2-pyridone-5-carboxamide

4PY

4-pyridone-3-carboxamide

6PY

6-pyridone-3-carboxamide (2-pyridone-5-carboxamide; 6-hydroxynicotinamide)

ACMSD

alpha-amino-beta-carboxy-muconate-semialdehyde decarboxylase

ADPR

adenosine diphosphate ribose

AK

adenosine kinase

ATP

adenosine triphosphate

CD38

cluster of differentiation 38

CD73

cluster of differentiation 73

DKD

diabetic kidney disease

G6PDH

glucose-6-phosphate dehydrogenase

HFpEF

heart failure with preserved ejection fraction

Me-NA

N-methyl nicotinic acid trigonelline

Me-4-PY

N-methyl-4-pyridone-3-carboxamide

Me-6-PY

N-methyl-6-pyridone-3-carboxamide

NA

nicotinic acid

NAAD

nicotinic acid adenine dinucleotide

NAADP

nicotinic acid adenine dinucleotide phosphate

NAADPH

reduced form of nicotinic acid adenine dinucleotide

NAD(OH)H

6-hydroxo-NADH (R/S)

NADP(OH)H

6-hydroxo-NADPH (R/S)

NAMN

nicotinic acid mononucleotide

NAD⁺

nicotinamide adenine dinucleotide

NADH

reduced form of nicotinamide adenine dinucleotide

NADP

nicotinamide adenine dinucleotide phosphate

NADPH

reduced form of NADP

[NAD⁺ + NADH]

total NAD pool

NADS

NAD synthase

NADK

NAD kinase

NAM

nicotinamide

NAMPT

nicotinamide phosphoribosyltransferase

NAPRT

nicotinic acid phosphoribosyltransferase

NAR

nicotinic acid riboside

NR

nicotinamide riboside

NRH

reduced form of NR

NRK

nicotinamide riboside kinase

NMN

nicotinamide mononucleotide

NMNAT

nicotinamide mononucleotide adenylyl transferase

NMNH

reduced form of NMN

NNT

nicotinamide nucleotide transhydrogenase

NQO1

NAD(P)H dehydrogenase [quinone]1

NUA

nicotinuric acid

NUDIX

nucleoside diphosphate linked to moiety-X (NUDIX) hydrolases

PBMC

peripheral blood mononuclear cell

PNP

purine nucleoside phosphorylase

QPRT

quinolinate phosphoribosyl transferase

SARM1

Sterile Alpha and TIR Motif Containing 1

Disclosures

The writing of this review article was not supported by funding from any grant or other sources. Dr. Shalender Bhasin reports receiving research grants from the National Institute on Aging, National Center for Medical Rehabilitation Research of the National institute of Child Health and Human Development, the National institute of Nursing Research, AbbVie, Transition therapeutics, Metro International Biotechnology, and FPT; consulting fees from Novartis and Aditum; and equity interest in FPT and Xyone Therapeutics. These research grants are managed by the Brigham and Women's Hospital and potential conflicts are overseen by the Massachusetts General Brigham Office of Industry Interaction in accordance with institutional guidelines. Dr. Marie Migaud reports receiving research support from the National Institutes of Health, the Translational Research Institute for Space Health, and Elysium Health; she holds multiple patents on the manufacturing of NAD⁺ precursors and derivatives, and their applications. Dr. Joseph Baur reports receiving research funding and materials from the NIH, Pfizer, Elysium Health and Metro International Biotech; and consulting fees from Pfizer, Elysium Health, and Cytokinetics; he holds a patent for using NAD⁺ precursors in liver injury. Dr. Nicolas Musi reports receiving support from the National Institutes of Health. Drs. Douglas Seals reported no conflicts of interest.