NAD⁺ centric mechanisms and molecular determinants of skeletal muscle disease and aging

NAD⁺-dependent Sirtuins, PARPs and CD38 function

The NAD-dependent deacetylase, sirtuins play an important role in maintaining cellular processes in skeletal muscle health. The skeletal muscle stem cells change the substrate use from oxidative to glycolytic state during myogenesis, and in the muscle stem cells that undergo differentiation the NAD⁺ levels were decreased with reduced SIRT1 activity and increased H4K16 acetylation, leading to an increase in myogenic gene expression [43]. Similarly, the muscle-specific inactivation of SIRT1 in mice increased H4K16 acetylation and activation of muscle genes during myogenesis. These muscle-specific SIRT1-null mice also displayed a decrease in myofiber size, and impaired muscle regeneration and gene expression [43]. While the NAD⁺/NADH ratio is decreased during myogenic differentiation, an increase in the NAD⁺/NADH ratio has been shown to inhibit muscle gene expression through Sir2 [44].

The NAD⁺ supply also controls sirtuin’s ability to regulate various processes such as mitochondrial function, metabolism and cell cycle [45]. SIRT1 deacetylates the transcriptional regulators such as FOXO (Forkhead Box O) and PGC-1α (Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha), which increases mitochondrial biogenesis and reduces the oxidative stress [46]. SIRT1 is also important in responding to muscle injury through the regulation of NF-κB (Nuclear Factor-κB), FOXOs, MYOD1 (Myogenic Determination Protein1), and MEF2 (Myocyte Enhancer Factor2) [47]. Further, SIRT1 has been demonstrated to deacetylate the circadian core clock genes, CLOCK and BMAL1, promoting gene expression in mouse embryonic fibroblasts [47].

SIRT1 and SIRT6 both downregulate IGF-1 (Insulin-like Growth Factor 1) and alter its target AKT-mTOR pathway. This pathway is associated with skeletal muscle hypertrophy during periods of upregulation and atrophy during downregulation [48]. SIRT6 also helps to maintain muscle mass by regulating the expression of myostatin, a dominant-negative regulator of muscle mass and its activin receptor type 2b, blocking myostatin signaling [49]. SIRT6 knockout mice results in increased skeletal muscle fibrosis and degeneration associated with higher myostatin levels [49]. SIRT6 is also involved in DNA repair efficiency, and SIRT6 knockout mice demonstrated increased cellular senescence [50, 51]. In an another study involving SIRT6 knockout mice, AMP-activated protein kinase (AMPK) activity was downregulated, resulting in a reduction in fatty acid oxidation, mitochondrial oxidative phosphorylation, and glucose and lipid uptake in muscle cells suggesting SIRT6 also plays a role in energy metabolism [52].

SIRT1, SIRT3 change the mitochondrial function in a similar manner. SIRT3, being localized within the mitochondria, regulates several mitochondrial proteins such as succinate-dehydrogenase (SDH), FOXO3, manganese-dependent superoxide dismutase (Mn-SOD), and acetyl-CoA synthase [53]. During periods of fasting, mitochondrial oxidative phosphorylation is increased in part due to the expression of Sirt3, where SIRT3 is also suggested to regulate oxidative muscle fiber formation [53, 54]. Absence of SIRT3 in mice is linked with direct downregulatory effects on PGC-1α and effect mitochondrial biogenesis [55]. SIRT4 is detected in lower levels within skeletal muscle as compared to SIRT3 but thought to play a role in mitochondrial oxidative metabolism, where it acts as a negative regulator [56]. Overall, the sirtuins functioning is regulated by NAD⁺ and changes in the levels of NAD⁺ can influence SIRT activity in skeletal muscle tissue [57].

Along with sirtuins, NAD⁺ is also necessary for the proper functioning of PARPs, and CD38 [58, 59]. Poly (ADP)-ribosylation is a post-translational modification catalyzed by PARPs that involves the consecutive addition of ADP-ribose moieties from NAD⁺ to amino acids [60, 61]. PARPs are a family of proteins that partake in an array of cellular processes such as DNA repair and programmed cell death [61]. The role of PARPs in DNA repair helps to explain why PARP levels are elevated during aging, that is accompanied by increased rates of DNA damage. CD38 is a glycoprotein detected on the surface of many white blood cells or immune cells. CD38 has been described to play many roles such as regulating metabolism, immune response, inflammation, and cancer [62, 63].

Metabolism

NAD⁺ plays a central role in metabolism and acts as a substrate for signaling enzymes. The redox couple of NAD⁺/NADH is required by enzymes within the cytosol and mitochondria, serving as a regulator for the processes of glycolysis and mitochondrial oxidative phosphorylation (Fig. 2A). The process of glycolysis is performed within the cytosol during which, glucose is converted into glyceraldehyde 3-phosphate (G3P), and NAD⁺ is reduced to NADH by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in order to convert G3P into 1,3-bisphosphate [64, 65], and this process continues to generate pyruvate. In anaerobic conditions, pyruvate can be reduced to lactate by the enzyme lactate dehydrogenase (LDH), which oxidizes NADH to NAD⁺ [65]. Pyruvate that is not converted into lactate can be shuttled into the mitochondria. Once in the mitochondria, pyruvate dehydrogenase (PDH) utilizes NAD⁺ to convert pyruvate into acetyl-CoA, which is then oxidized to form adenosine triphosphate (ATP) within the tricarboxylic acid (TCA) cycle [64].

Within the TCA cycle, NAD⁺ is required in the conversion of D-isocitrate to α-ketoglutorate, α-ketoglutorate to succinyl-CoA, and malate to oxaloacetate by the enzymes isocitrate dehydrogenase, α-ketoglutorate dehydrogenase and malate dehydrogenase, respectively [66]. Malate, a precursor in this cycle, can be converted back to pyruvate by the malic enzyme (ME2) [67], which is NAD-dependent; however, it is expressed in low levels in skeletal muscle and may be involved in metabolism. Glutamate dehydrogenase 1 is a NAD⁺-dependent mitochondrial enzyme that converts glutamate to α-ketoglutarate and ammonia [68]. Similar to ME2, this enzyme is also expressed at low levels in skeletal muscle.

The malate aspartate shuttle and glyceraldehyde 3-phosphate shuttle both utilize NAD⁺/NADH to shuttle necessary metabolic substrates (Fig. 2B). In the malate aspartate shuttle, cytosolic oxaloacetate or oxalacetic acid (OAA) is reduced to malate by malate dehydrogenase (MDH1), which utilizes and oxidizes NADH to NAD⁺ [69]. Malate is shuttled into the mitochondria and converted back into OAA by malate dehydrogenase 2 (MDH2), and in this shuttling process NAD⁺ is utilized and reduced back into NADH. In the G3P shuttle, the glycolytic intermediate dihydroxyacetone phosphate (DHAP) is reduced to G3P by glycerol-3-phosphate dehydrogenase (GPDH) [70] and this conversion oxidizes NADH into NAD⁺. G3P can then be shuttled into the mitochondria and partake in other metabolic conversions, such as the conversion back to DHAP.

The processes of glycolysis and oxidative phosphorylation are necessary to generate ATP in the mitochondria, which is required for the healthy contraction and functioning of skeletal muscle. Thus proper functioning of skeletal muscle requires a high turnover of ATP and is dependent on the abundance of NAD⁺, due to its utilization in the processes of glycolysis and oxidative phosphorylation [71]. Without a sufficient supply of NAD⁺ that is needed in the processes of glycolysis and oxidative phosphorylation, these pathways that are necessary to produce cellular energy are disrupted. Nowhere is this more relevant than in skeletal muscle, which accounts for more than 70% usage of circulating insulin and glucose [72]. Research has demonstrated a hallmark of aging to be a progressive loss in skeletal muscle mass and function, potentially developing into sarcopenia. Investigation into the ageing population, including sarcopenia patients demonstrated a significant decrease in skeletal muscle biopsy NAD⁺ levels with a negative correlation with age [36, 73]. NAD⁺ levels strongly correlated with functional changes, including appendicular lean mass (ALM) index, grip strength and gait speed in sarcopenia patients [73]. Further investigation demonstrated a significant decrease in many mitochondrial genes, including those forming the electron transport chain, Complex I-V in the sarcopenia muscle biopsies [73]. Figure 3 demonstrates decreased Nampt activity and thereby decreased NAD⁺ biosynthesis during aging and in muscle diseases, including inflammation, which results in increased DNA damage and altered mitochondrial biogenesis. In addition, sex differences exist with relation to NAD⁺ levels in animal models both with baseline and during injury induced conditions such as stroke. The NAD⁺ levels were found to be lower in females compared with males or ovariectomized mice. Cerebral ischemia decreased the NAD⁺ levels, and were linked with PARP-1 and metabolic changes, which were altered in a sex-specific manner [74]. However, the NAD⁺ levels in blood plasma were not affected with age or sex in humans [75] suggesting the tissue-specific NAD⁺ biosynthetic activities.

Muscular Dystrophies

Inherited muscle diseases show effects of decreased muscle performance, mass, and function not attributed to aging [76]. Muscular dystrophies are defined as genetic alterations that result in progressive skeletal muscle weakness and deterioration. Duchenne’s muscular dystrophy (DMD) is the most common type of muscular dystrophy, and as such has the most strides being made in terms of its treatment. DMD is characterized by the degeneration of skeletal muscle due to the absence of the protein dystrophin [77], has a co-occurrence of low muscle NAD⁺ abundance [78]. Major consumers of NAD⁺ such as PARPs and N-methyltransferase (NNMT) are elevated in DMD patients and other muscular diseases. Further, in the muscles of mdx mouse model of human DMD, the levels of NAD⁺ were significantly reduced along with a reduction in NAMPT activity and increase in PARP activity [79]. In mdx mice, treatment with resveratrol, a SIRT1 activator, helped to relieve the pathophysiology of this muscular dystrophy [80]. Given the role of NAD⁺ in skeletal muscle functioning and aging, it is hopeful to think that the effects of NAD⁺ biosynthesis in DMD animal models could be extended to other muscular dystrophies and muscle diseases as a therapy.

Metabolic diseases

Skeletal muscle is a major site of energy expenditure and metabolic functioning. Metabolic disorder and disease such as obesity and type 2 diabetes mellitus (T2DM) affect skeletal muscle, with far reaching inflammatory effects that lead to reductions in the amount of available NAD⁺ [81]. A major cause of skeletal muscle metabolic disorder is due to high-fat and high-sugar diet, leading to obesity. Skeletal muscle samples from mice fed a high-fat diet (HFD) displayed high levels of PARP-2, a NADase enzyme in the PARP protein family. These mice also showed decreased activation of the SIRT1/PGC-1α pathway, a pathway that is important in mitochondrial biogenesis and function [82]. Further delineation is observed in diabetes-induced muscle injury with type 1 diabetes demonstrating a decline in NAD⁺ levels likely the result of increased activation of PARP consequence of low NAD⁺ levels remain unknown, however, ATP levels were significantly decreased [83].

Given the roles of NAD⁺ in metabolic regulation and sirtuin activity, it is reasonable to hypothesize if metabolic health could be improved by NAD⁺ supplementation, and if NAD-dependent proteins like PARP could be inhibited to elevate the NAD⁺ levels. Surprisingly, in a study involving the overexpression of NAMPT, which resulted in a 50% increase in mouse skeletal muscle NAD⁺ levels did not alleviate the negative effects of a HFD or induce mitochondrial biogenesis [84]. This raises questions as to the tissues in which NAD⁺ levels to be increased, and contribute to positive effects within skeletal muscle.

Inflammation

Insulin resistance is known to co-occur with the aging process in humans [85] including inflammation. The relationship between inflammation and insulin resistance is cyclic, in which inflammation can cause insulin resistance and insulin resistance leads to inflammation [86] (Fig. 4). Skeletal muscle inflammation has emerged as a player in the development of T2DM [87, 88]. Skeletal muscle inflammation is the result of multiple causes, such as obesity. With obesity levels increasing with age, chronic low grade inflammation is also on the rise [89]. Adipose tissue and skeletal muscle both secrete inflammatory cytokines, such as IL-1β, IL-6, and TNF- α [90].

The inflammatory cytokines and oxidative stress decreases NAD⁺ biosynthesis by the intracellular nicotinamide phosphoribosyl transferase (iNAMPT) [81]. The skeletal muscle-specific NAMPT deficient mice resulted in a dramatic reduction of NAD⁺ levels along with fiber degeneration and muscle weakness [91]. Whereas, overexpression of NAMPT in aging mice helped to preserve NAD⁺ levels, as well as muscle mass and function [91]. In an another study, involving the reduction of NAMPT in mice resulted in a metabolic shift from oxidative to glycolytic, which was associated with acetylation of the SIRT6 targets within the SIRT6/HIF1α axis [38].

A metabolic shift away from glucose oxidation is positively correlated with insulin resistance [92, 93]. It is thought that mitochondrial dysfunction could contribute to age-related insulin resistance, however, the mechanisms by which mitochondria effect insulin resistance are not fully understood [87, 92, 94]. In mice that had genetic ablation of SIRT2, the insulin-induced glucose uptake occurring in skeletal muscle was decreased compared to control groups [95]. This result implicates SIRT2, as a cytosolic and mitochondrial deacetylase, with a possible role in the mitochondrial dysfunction resulting in insulin sensitivity.

Changes in metabolism

SIRT1 is a predominant sirtuin isoform that has been studied well for its effects on mitochondrial functioning. SIRT1 directly affects the activity of an important regulator of mitochondrial biogenesis, PGC-1α [46]. PGC-1α is a coactivator of multiple transcription factors that work together to increase mitochondrial biogenesis. The activation of SIRT1 by resveratrol deacetylates PGC-1α, enhancing its transcriptional activity with increased mitochondrial biogenesis in skeletal muscle [96].

The activation of sirtuin is negatively correlated to the activity of NADases. CD38 is a NADase that actively increases with aging and is thought to lead to mitochondrial dysfunction in part due to changes in SIRT3 activity [97]. Mice that are deficient in CD38 demonstrate higher metabolic rates with reduced risk of obesity, which is attributed to the enhanced mitochondrial function through the upregulation of sirtuin activity [98, 99]. While the observed decline in skeletal muscle-specific SIRT3 with age is due to higher acetylation levels and decreased mitochondrial functioning [100, 101]. Within the mitochondria are enzymes and proteins that can be acetylated and regulate mitochondrial biogenesis through their activation or inhibition.

Mitochondrial dysfunction is thought to be involved in the muscle aging process [102, 103]. There are multiple factors playing into the deterioration of mitochondrial function, implicating NAD⁺-dependent protein acetylation/deacetylation as a cause [100, 104]. In mice and humans, higher acetylation levels within skeletal muscle mitochondria were associated with lower exercise capacity and increased acetylation of fatty acid β-oxidation enzymes [105]. Acetylation of these enzymes are related to reductions in the capacity of the mitochondria to carry out processes of fatty-acid oxidation within skeletal muscle [105, 106]. The results from this study implicated SIRT3 in the regulation of acetylation levels, however, the abundance of NAD⁺ and NAD⁺/NADH were not shown to differ significantly between the experimental and control groups [105].

Muscle damage and repair

The genetic ablation of SIRT1 in mouse muscle stem cells impaired muscle function [107]. However, the study was not able to directly link SIRT1 levels to muscle structure, function, and recovery following injury. Whereas, the skeletal-muscle specific SIRT1-null mice showed muscular fragility attributed to impaired membrane resealing and repair [108]. Increased DNA damage occurs with aging and results in either cell death or cellular senescence through mechanisms in healthy cells that prevent unchecked cell growth [109]. A senescence-associated secretory phenotype is observed in senescent cells, which activates PARP-1 and CD38 [58]. While aging is reported to upregulate SIRTs, there is a decline in NAD⁺ abundance seen partly due to competition from other NAD⁺-dependent enzymes, including CD38 and PARP-1 [58, 59]. A major cause for the decline in NAD⁺ levels during aging is CD38 [110]. Aging is linked to elevated levels of CD38, but not PARP1 or SIRT1, indicating CD38 as a major culprit contributing to NAD⁺ deficiencies [97]. However, SIRT1 regulates cell survival through the interaction of DNA damage proteins, including Ku70, FOXL2, NBS1 (Nibrin), WRN (Werner Syndrome ATP-dependent Helicase), and XPC (Xeroderma Pigmentosa Complementation Group C) [111]. SIRT1 also helps to modulate apoptosis through the interaction of FOXO proteins, demonstrated specifically in the activation of FOXO3 that can potentiate its ability to resist oxidative stress [112].

NAD⁺-dependent acetylation and deacetylation

Although, there are several NAD⁺-dependent enzymes in the cellular milieu, sirtuins represent a unique and important class of histone modifiers/erasers known as HDACs (Histone Deacetylases) contributing to the epigenetic control of gene expression. Sirtuins erase or remove acetyl groups from the specific amino acids of histone tails. The acetylation and deacetylation of lysine residues at the N-terminal tail of histone proteins has gene regulatory effects. Acetylation neutralizes the positive charge of lysine, reducing the electrostatic interactions and results in a less densely packed form of chromatin. This form of chromatin is referred to as euchromatin and is often associated with active transcription. Whereas, the tightly packed DNA, known as heterochromatin is associated with deacetylation and lower levels of transcription or gene silencing. Histone proteins are acetylated and deacetylated typically by HATs (Histone Acetyl Transferases) and HDACs, respectively.

Aging is known to coincide with a decrease in histone acetylation and there are four different groups of HDACs classified thus far, playing various roles in skeletal muscle development. Class I enzymes contain HDACs 1-3, and 8, Class II contain HDACs 4-7, 9 and 10, Class III HDACs contain Sirtuins (SIRT) 1-7 in mammals and Sir2 in yeast, and Class IV contain HDAC 11 [22]. Class I and II HDACs utilize Zn as a cofactor to catalyze deacetylation of histone/non-histone substrates. Whereas, Class III HDACs or sirtuins utilize NAD⁺ as a cofactor for deacetylation of their substrates. Although, there are several class III HDACs, their activity is regulated or restricted to their subcellular localization. SIRT 1, 2, 6 and 7 are predominantly nuclear, and because NAD⁺ is relatively a small molecule, it can shuttle in and out of the nucleus. This shuttling integrates the availability of NAD⁺ through metabolic processes and deacetylation of histone, which will turn off gene expression [113, 114].

NAD⁺ is an important cofactor playing a key role as a cellular rheostat in enzyme catalysis and whose levels dictate the global acetylation or deacetylation patterns brought about by sirtuins, relating metabolic flux and gene expression. Sirtuins interact with not only histones, but also with several other important transcriptional factors and proteins involved in cellular signaling networks. They can also remove several other acyl groups from histones (Lysine-K) like benzoyl, propionyl, butyryl, etc. [115, 116]. The roles of these covalent modifications and the consequence of acyl groups removal by sirtuins remains to be unexplored from a normal cellular physiology or pathology point of view.

Sirtuins also regulate the activity of several transcriptional factors such as NF-kB, FOXO1 and other important proteins involved in transcriptional, developmental and signaling cascades [117-120]. The protein type and subcellular localization are factors that affect whether the removal of acetyl groups from specific amino acids of proteins, which will lead to either loss-of-function or gain-of-function. Table 2 shows the list of various histone and non-histone substrates of sirtuins, and their corresponding cellular outcomes.

Association between NAD⁺ and DNA methylation

DNA methylation occurs at the 5’-C position in a CpG (5’-Cytosine-phosphate-Guanine-3’) dinucleotide and is generated and maintained by DNMTs (DNA methyl transferases). There are four DNMTs found in eukaryotes, including DNMT1 and DMNT3s (DNMT3A, DNMT3B, and DNMT3L). DNMT1 is primarily active during cell division, maintaining DNA methylation, while DNMT3s are responsible for de novo cytosine methylation and maintenance [121]. Promoter hypermethylation typically occurs at the CpG islands and causes gene repression. Within the skeletal muscle tissue, hypermethylation and consequent gene repression are reported to increase with age [122].

Methylated Cytosines in CpG islands are recognized by methyl CpG binding proteins (CBP), and these further recruit histone deacetylases (HDACs/Sirtuins), histone methyl transferases (HMTs), and chromatin remodeling complexes to co-repress transcription, ultimately leading to gene repression (Fig. 5A). This process occurs in an opposite direction where promotor hypomethylation cause gene activation. 5-methylcytosine and 5-hydroxymethylcytosine is called the 5^th and 6^th base, respectively apart from the normal Adenine, Thymine, Guanine and Cytosine nucleotide bases, since they play a role in gene regulation mediated by the action of DNMTs and TET (ten-eleven-translocase) enzymes. The 5-methylcytosine and 5-hydroxymethylcytosine play an important role in connecting epigenetic control of gene expression and metabolism.

Aging is a complex process and involves gradual but substantial changes in gene expression and metabolic pathways mainly dependent on environmental factors like food (folate intake), physical exercise, etc. Dietary folate, choline, and vitamin B12 are important methyl donors and regulate DNA and histone methylation. In addition, α-ketoglutarate is essential for the action of TET enzymes and links the TCA cycle to DNA methylation and gene expression. As shown in Figure 2B, the reduction of NAD⁺ is required to generate α-ketoglutarate from isocitrate. Without adequate supply of NAD⁺, the TCA cycle will not be able to run efficiently, limiting the amounts of α-ketoglutarate formed from this pathway and hindering TET activity [123].

The genome-wide association studies (GWAS) performed in humans surveying the DNA methylation status in skeletal muscle DNA display the role of epigenetics during normal aging. In a study performed on post-mitotic skeletal muscle tissue of 48 healthy males, half of the individuals aged between 18-27 years and the other half aged between 68-89 years showed differences in the differentially methylated CpG nucleotides[124]. This study identified over 2,114 genes within the aged skeletal muscle having at least one dmCpG site located intragenically. Within the genes observed to be differentially methylated in aging individuals was Tubulin-folding co-factor D (TBCD), containing the highest number of intragenic dmCpG sites. TBCD is a component of microtubules that can result in the disruption of microtubule pathways, if altered, affecting motor neuron’s function. The NFATC1 (Nuclear Factor of Activated T Cells 1) gene was also identified to be of interest, having a large number of intragenic dmCpG sites and playing a role in skeletal muscle transcription regulation [124]. A similar study was performed on the whole blood of 1,550 female twins aged between 17-82 years in an effort to identify the relationship between DNA methylation levels and the skeletal muscle mass variations with age. Seven regions were identified to be of importance due to their location in or near genes previously noted in muscle-related studies. These genes being DNAH12 (Dynein Axonemal Heavy Chain 12), CAND1 (Cillin-Associated NEDD8-Dissociated Protein 1), CYP4F29P (Cytochrome P450 Family 4 Subfamily F Member 29, pseudogene), and ZFP64 (Zinc Finger Protein 64) [125].

The effect of methylation patterns within skeletal muscle stem cell on aging was also investigated in a clinical study. This study used muscle stem cells obtained from 22 adults, 7 young and 14 aged individuals, demonstrates that DNA methylation is affected by age and regulates genes controlling cell quiescence [126]. In the muscle samples from aged individuals, methylation of SPRY1 (Sprouty RTK Signaling Antagonist 1) negatively impacted the abundance of the stem cell pool, caused by a failure of re-quiescence in the activated stem cells. It was shown that the myogenic potential is preserved in these stem cells, however, the ability to return to quiescence is impaired after the activation process is triggered. Overall, the suppression of SPRY1 by DNA methylation leads to fewer reserve cells and impairs muscle stem cells self-renewal [126].

Dietary Supplements and Pharmacological Treatments

The baseline requirement of NAD⁺ is mainly met by the dietary tryptophan or niacin, which serves as the precursor of NAD⁺ synthesis. The other dietary NAD⁺ precursors such as NAM and NR are also incorporated into the intracellular NAD⁺ pool [135]. Increasing the NAD⁺ biosynthesis and availability more than that met by our daily dietary intake is considered beneficial for human health, especially in metabolic and muscle diseases such as obesity, diabetes, muscular dystrophies, and other neurodegenerative diseases [135]. The pharmacological use of NR has advantages over the use of other precursors such as NA or NAM. NR appears to be more effective in increasing the intracellular NAD⁺ levels with less side effects than the NA or NAM precursors [135]. A study utilizing younger mice fed with NR demonstrated improved endurance suggesting enhanced mitochondrial functioning resulting in a higher oxidative capacity as compared with the vehicle treated group of mice [136]. These studies demonstrate that NR supplementation increased the pool of NAD⁺ and led to a significant increase in the activity of SIRT1 and SIRT3 within skeletal muscle. Furthermore, sirtuin activity increased the expression of PGC-1α, thereby increasing the mitochondrial biogenesis. In a separate study involving aged men given oral NR supplementation there was an increase in the muscle NAD⁺ metabolome, as evidenced by a two-fold increase in muscle nicotinic acid adenine dinucleotide (NaAD), a biomarker of NAD⁺ synthesis [137], however, NAD⁺ levels were not increased. Interestingly, the NR supplementation was also associated with the downregulation of genes involved in glycolysis, TCA cycle and mitochondrial energy metabolism within muscle. Further analysis of these results did not show increased mitochondrial copy number, respiration, or citrate synthase activity. NR-mediated changes were not observed in sirtuin functioning, as changes in the acetylation levels of muscle proteins were not detected. While these results did not demonstrate enhanced sirtuin activity, the study provided insights into decreased inflammatory effects that were observed with NR supplementation. In addition, NR supplementation led to a reduction in the levels of circulating inflammatory cytokine such as IL-2, IL-5, IL-6, and TNF-α [137].

Recent reports utilizing NR supplementation in the ageing population sought to identify skeletal muscle content in metabolic disease, including obese and insulin-resistant men. Similar results were observed with NR supplementation having little to no effect on skeletal muscle mitochondria. Interestingly, NR-supplementation resulted in a significant decrease in NAMPT skeletal muscle protein expression [138]. Further, NAMPT expression correlated with key mitochondrial proteins, including the electron transport complex (ETC) I, succinate dehydrogenase, and sirtuin 3.

Metabolic enzyme activity profiling of mice suggested that NAMPT and NRK1/2-mediated salvage pathways are the main biosynthetic routes within skeletal muscle to replenish NAD⁺ pools [27]. NMN and NR are the substrates and product of these two enzymes, respectively. Supplementation using these NAD⁺ precursors was seen to result in a 2-fold increase of NAD⁺ biosynthesis within the skeletal muscle myotubes. NAMPT knockout mice treated with NAD⁺ supplements showed high levels of NAD⁺, confirming that the NAD⁺ precursors of NMN and NR can be salvaged independently of NAMPT. This suggests the potential of NRK2 as a skeletal muscle-specific target to improve NAD⁺ abundance and has provided new context for the role of NAMPT as a rate-limiting step of NAD⁺ salvage. NRK2 knockout in mice had no effect on the NAD⁺ abundance, demonstrating redundancy within the NAD⁺ salvage pathway [27]. In a 12-month long NMN supplementation using mice as subjects, enhanced energy metabolism, suppressed weight gain, and increased physical activity and insulin sensitivity were observed [139]. Overtime, the NAD⁺ levels were decreased in skeletal muscle tissue, however, the mitochondrial respiration rate in skeletal muscle was enhanced. Furthermore, a 10-week treatment of NMN supplementation in obese prediabetic women enhanced skeletal muscle insulin sensitivity and signaling [135]. These results show promise in the potential use of NRK2 as a skeletal muscle-specific target in conjunction with NMN and NR supplementation to improve NAD⁺ abundance.

As an important enzyme in the NAD⁺ salvage pathway, NAMPT has been investigated as a target to increase the abundance of viable NAD⁺ within skeletal muscle cells. In mice that had muscle-specific overexpression of NAMPT, NAD⁺ levels were consequently higher but were not seen to regulate any targets of oxidative metabolism, however, an enhanced exercise capacity was observed [84, 91]. NAMPT and NRK2 both function within the NAD⁺ salvage pathway. Whereas, the Preiss-Handler pathway is another biosynthetic pathway for the generation of NAD⁺ in cells. In both these pathways NMNAT3 is present as a common enzyme (Figs. 1A, B). In mice, the administration of the Nampt activator P7C3 (Fig. 1C), a small molecule neuroprotective compound belonging to the class of aminopropyl carbazole has also led to increased NAD⁺ biosynthesis and availability [140-143]. Recently, we demonstrated that administration of P7C3 in the diabetic db/db mice increased insulin sensitivity and improved muscle function via. Nampt activation [30].

NMNAT3 has been established as the only enzyme in the NAD⁺ synthesis pathway that is localized in the mitochondria. This brings into question whether NMNAT3 could be seen as a target to increase NAD⁺ abundance within the mitochondria, however, NMNAT3 requires the import of cytosolic NMN for NAD⁺ production [144]. Despite this limitation, NMNAT3-overexpressing mice had significantly increased mitochondrial NAD⁺ levels within skeletal muscle tissue, accompanied by increased fatty acid oxidation [145]. Inconsistent with these results is the demonstration that NMNAT3 knockout in mice does not result in a significant difference in mitochondrial NAD⁺ levels [145]. This could be due to a redundancy in the enzymatic activity. More work needs to be done on NMNAT3 expression to determine its viability as a pharmacological target in humans to increase NAD⁺ biosynthesis.

Exercise

There are many different pathways that can be affected by exercise and is considered as the gold standard to improve the aging muscle function. AMPK plays a key role in the regulation of ATP levels within the cell through the upregulation or downregulation of anabolic and catabolic pathways, respectively [146]. Because AMPK acts as an energy sensor and exercise utilizes ATP (the bodies energy currency), it is clear that exercise would result in increased AMPK activation [147]. AMPK signaling was seen to indirectly activate SIRT1 by altering intracellular NAD⁺/NADH ratio. The effects of AMPK on NAD⁺ levels help to explain the increased mitochondrial biogenesis that is observed along with increased oxidative capacity of skeletal muscle and reduced signs of skeletal muscle fragility [148, 149].

Sirtuin activity is demonstrated to be enhanced with exercise, unsurprisingly given the elevated effects exercise is reported to have on intracellular NAD⁺ levels [57, 150]. It has also been suggested that SIRT1 can be activated by an acute load of exercise, while exercise training can activate both SIRT1 and SIRT3 [57]. In human subjects of 65 years of age and older, master athletes in this age group had higher protein levels of SIRT1 and SIRT3 [151].

NAMPT decreases with age in skeletal muscle, helping to explain the decline in NAD⁺ levels that is also seen during aging. Recent studies demonstrate that distinct modes of exercise can impact the NAD⁺ salvage capacity in the skeletal muscle. Aerobic and resistance exercise training was compared in a study involving young and aged individuals to assess whether these exercise modes have varying effects on NAD⁺ salvage pathways [152]. The increase in NAMPT levels was positively correlated with VO₂ peak and lean body mass. At the end of 12 weeks, aerobic exercise training increased NAMPT levels by 28% in older individuals and just 12% in younger individuals. Resistance exercise training was seen to increase NAMPT levels by 30% in older individuals and 28% in younger individuals [152]. While these numbers demonstrate aerobic and resistance training modes have similar impacts in aged individuals, what is interesting is the difference that is seen in the effects of the younger participants. If resistance exercise can improve NAMPT levels more strikingly than aerobic exercise in the younger participants, it suggests that resistance exercise has the potential to be used in younger populations as a method to prevent or prolong the effects of NAD⁺ deficiencies later in life. It would be interesting to follow the impact of resistance exercise from young to old age to study the viability of this exercise mode in preventing NAD-deficiency-mediated aging compared to aerobic exercise groups.