What is NADH

NADH is short for reduced nicotinamide adenine dinucleotide. NADH is a coenzyme composed of ribosylnicotinamide 5′-diphosphate coupled to adenosine 5′-phosphate by pyrophosphate linkage. It is found widely in nature and is involved in numerous enzymatic reactions in which it serves as an electron carrier by being alternately oxidized (NAD+) and reduced (NADH). NADH is the reduced form of NAD+ and NAD+ is the oxidized form of NADH ¹. NAD+ and NADH participate in reactions such as glycolysis, the tricarboxylic acid cycle (citric acid cycle), and oxidative phosphorylation,  participating in multiple redox reactions in cells ². In addition, it serves as a substrate for several enzymes involved in DNA damage repair, such as the sirtuins (silent information regulator 2 or Sir2) and poly (ADP-ribose) polymerases (PARPs) ³. The human serum NADH concentration was found to be in very small amount (50 nM to 1.2 μM) ⁴.

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme found in all living cells. NAD+ (nicotinamide adenine dinucleotide) serves both as a critical coenzyme for enzymes that fuel reduction-oxidation reactions, carrying electrons from one reaction to another, and as a cosubstrate for other enzymes such as the sirtuins and poly(adenosine diphosphate–ribose) polymerases. Cellular NAD+ concentrations change during aging, and modulation of NAD+ usage or production can prolong both health span and life span.

The NAD⁺/NADH ratio also regulates the activity of various metabolic pathway enzymes such as those involved in glycolysis, Kreb’s cycle (also known as tricarboxylic acid cycle or citric acid cycle), and fatty acid oxidation ⁵. Intracellular NAD⁺ is synthesized de novo from L-tryptophan, although its main source of synthesis is through salvage pathways from dietary vitamin B3 (Niacin) as precursors. NAD⁺ is utilized by various proteins including sirtuins (silent information regulator 2), poly ADP-ribose polymerases (PARPs) and cyclic ADP-ribose synthases. The NAD⁺ pool is thus set by a critical balance between NAD⁺ biosynthetic and NAD⁺ consuming pathways. Raising cellular NAD⁺ content by inducing its biosynthesis or inhibiting the activity of poly ADP-ribose polymerases (PARPs) and cyclic ADP-ribose synthases via genetic or pharmacological means lead to sirtuins activation. Sirtuins (silent information regulator 2) modulate distinct metabolic, energetic and stress response pathways, and through their activation, NAD⁺ directly links the cellular redox state with signaling and transcriptional events. NAD⁺ levels decline with mitochondrial dysfunction and reduced NAD⁺/NADH ratio is implicated in mitochondrial disorders, various age-related pathologies as well as aging.

Sirtuins (silent information regulator 2 or Sir2) proteins are a family of evolutionarily conserved nicotinamide adenine dinucleotide (NAD⁺)-dependent protein deacylases harboring lysine deacetylase, desuccinylase, demalonylase, demyristoylase and depalmitoylase activity ⁶ or an ADP-ribosyltransferase activity ⁷. Mammals contain seven sirtuins (SIRT1–7) that are locacted in different subcellular compartments i.e. nucleus (SIRT1, SIRT6 and SIRT7), cytosol (SIRT2), and mitochondria (SIRT3, SIRT4 and SIRT5) ⁸ and are implicated in a wide variety of biological functions including control of cellular metabolism and energy homeostasis, aging and longevity, transcriptional silencing, cell survival, proliferation, differentiation, DNA damage response, stress resistance, and apoptosis ⁹. Since sirtuins are NAD⁺-dependent enzymes, the availability of NAD⁺ is one of the key mechanisms that regulate their activity. Sirtuins therefore serve as “metabolic sensors” of the cells as their activity is coupled to changes in the cellular NAD⁺/NADH redox state, which is largely influenced by the availability and breakdown of nutrients ¹⁰. Thus, NAD⁺ is not only a vital cofactor/coenzyme but also a signaling messenger that can modulate cell metabolic and transcriptional responses. Changes in cellular NAD⁺ levels can occur due to modulation of pathways involved in NAD⁺ biosynthesis and consumption. Reduced NAD⁺ levels have been reported in mitochondrial and age-related disorders, and NAD⁺ levels also decline with age ¹¹. Boosting cellular NAD⁺ levels serves as a powerful means to activate sirtuins, and as a potential therapy for mitochondrial as well as age-related disorders.

Figure 1. NADH redox reaction

Figure 2. NADH and NAD+ redox metabolism

It is known, as aging progresses, nicotinamide adenine dinucleotide (NAD⁺) levels decrease and are involved in age-related metabolic decline and mitochondrial dysfunction ¹². Elevated NADH to NAD+ ratio further suggests that older individuals of both sexes are unable to utilize NADH as effectively as the younger adults. This observation has direct bearing on the mitochondrial oxidation. NADH is perhaps not oxidized efficiently in the older and female adults than the younger individuals, i.e. less of the energy pool (ATP) in the older adults. Recent studies have shown that a reduction in NAD⁺ is a key factor for the development of age-associated metabolic decline. Increased NAD⁺ levels in vivo results in activation of pro-longevity and health span-related factors. Also, it improves several physiological and metabolic parameters of aging, including muscle function, exercise capacity, glucose tolerance, and cardiac function in mouse models of natural and accelerated aging.

It has been shown that the cellular NAD⁺ pool is determined by a balance between the activity of NAD-synthesizing and NAD-consuming enzymes ¹³. In previous publications, it was demonstrated that expression and activity of the NADase CD38 increases with age and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity (see Figure 3 below) ¹⁴. It was also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo. That indicates that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases ¹⁴. CD38 was originally identified as a cell-surface enzyme that plays a key role in several physiological processes such as immune response, inflammation, cancer, and metabolic disease ¹⁵.

Type 2 diabetes has become an epidemic due to calorie-rich diets overwhelming the adaptive metabolic pathways. One such pathway is mediated by nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD⁺ biosynthesis and the NAD⁺-dependent protein deacetylase SIRT1. The results presented in this study in mice demonstrated that nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD⁺ biosynthesis is severely compromised by high fat diet and aging, contributing to the pathogenesis of type 2 diabetes ¹⁶. Strikingly, nicotinamide mononucleotide (NMN), a product of the nicotinamide phosphoribosyltransferase (NAMPT) reaction and a key NAD⁺ intermediate, ameliorates glucose intolerance by restoring NAD⁺ levels in high fat diet-induced type 2 diabetes mice. Nicotinamide mononucleotide (NMN) also enhances hepatic insulin sensitivity and restores gene expression related to oxidative stress, inflammatory response, and circadian rhythm, partly through SIRT1 activation. Furthermore, NAD⁺ and NAMPT levels show significant decreases in multiple organs during aging, and nicotinamide mononucleotide (NMN) improves glucose intolerance and lipid profiles in age-induced type 2 diabetes mice ¹⁶.

The recent development of potent and specific CD38 inhibitors ¹⁷, together with the novel findings highlighting the role of NAD⁺ replacement therapy and CD38 in age-related diseases such as hearing loss and Alzheimer’s ¹⁸, indicate that CD38 inhibition combined with NAD precursors may serve as a potential therapy for metabolic dysfunction and age-related diseases.

Figure 3. NAD decline due to increases in CD38/NADase during aging

[Source ¹²]

NAD+ biosynthesis, consumption and compartmentalization

The mammalian NAD⁺ biosynthesis occurs via de novo and salvage pathways, and involves four major substrates including the essential amino acid l-tryptophan (Trp), nicotinic acid (NA), nicotinamide (NAM), and nicotinamide riboside (NR) ¹⁹. De novo biosynthesis of NAD⁺ starts from dietary L-tryptophan (Trp) which is catalytically converted to N-formylkynurenine by either indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO) and is the first rate limiting step. N-formylkynurenine is then converted by a series of four enzymatic reactions to α-amino-β-carboxymuconate-ε-semialdehyde (ACMS) which is unstable and hence undergoes either complete enzymatic oxidation or non-enzymatic cyclization to quinolinic acid (see Figure 4). The second rate limiting step involves the catalytic conversion of quinolinic acid to nicotinic acid mononucleotide (NAMN) by quinolinate phosphoribosyl transferase (QPRT). Next, NAMN is converted to nicotinic acid adenine dinucleotide (NAAD) by one of the three isoforms of nicotinamide mononucleotide adenylyltransferase (NMNAT) enzyme. The human NMNAT1 is localized in the nucleus, NMNAT2 is found in the Golgi and cytosol, whereas NMNAT3 is localized in both mitochondria and cytosol ²⁰. The final step of de novo biosynthesis is the amidation of NAAD by NAD synthase (NADS) enzyme (see Figure 4) ¹⁹. The de novo pathway contributes only a minor fraction to the total NAD⁺ pool, however, its importance is stressed by the human disease pellagra which is caused by dietary deficiency of Trp and NAM intermediate, leading to diarrhea, dermatitis, dementia and ultimately death ²¹. However, pellagra is easily treated by dietary supplementation of L-tryptophan (Trp) or niacin (vitamin B3) (i.e. nicotinic acid, nicotinamide and nicotinamide riboside). The primary source of NAD⁺ biosynthesis is the salvage or Preiss-Handler pathway which utilizes dietary niacin as precursors (Figure 4). The salvage pathway involves catalytic conversion of nicotinic acid to nicotinic acid mononucleotide by nicotinic acid phosphoribosyltransferase (NAPT), which is subsequently converted to NAD⁺ by the action of nicotinamide mononucleotide adenylyltransferase (NMNAT) and NAD synthase (NADS) enzymes. The nicotinamide and nicotinamide riboside are converted to nicotinamide mononucleotide (NMN) by the action of nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide riboside kinase (NRK) enzymes respectively. Finally, nicotinamide mononucleotide (NMN) is enzymatically converted to NAD⁺ by nicotinamide mononucleotide adenylyltransferase (NMNAT).

The cellular abundance of NAD⁺ is also regulated by its breakdown since NAD⁺ serves as a degradation substrate for multiple enzymes including sirtuins, poly ADP-ribose polymerases (PARPs) and cyclic ADP (cADP) ribose synthases which cleave NAD⁺ to produce nicotinamide and an ADP-ribosyl product ²². For instance, the deacetylase activity of mammalian sirtuins uses NAD⁺ to cleave the acetyl group from ε–acetyl lysine residues of target proteins to generate nicotinamide and 2′O-acetyl-ADP-ribose. Sirtuins are activated in response to nutrient deprivation or energy deficit which triggers cellular adaptations to improve metabolic efficiency. Poly ADP-ribose polymerases’s are activated in response to DNA damage (e.g. DNA strand breaks) and genotoxic stress, and use NAD⁺ to catalyze a reaction in which the ADP ribose moiety is transferred to a substrate protein. The cADP-ribose synthases (e.g. CD38 and CD157) use NAD⁺ to generate cADP-ribose which serves as an intracellular second messenger. The members of poly ADP-ribose polymerases and cADP-ribose synthase family show increased affinity and lower K_m for NAD⁺ compared to sirtuins, indicating that their activation critically impacts intracellular NAD⁺ levels and determines if it reaches a permissive threshold for sirtuin activation ²³. Multiple studies also suggested that PARP activity constitutes the main NAD⁺ catabolic activity, which drives cells to synthesize NAD⁺ from de novo or salvage pathways ²⁴.

The intracellular NAD⁺ levels are typically between 0.2 and 0.5 mM in mammalian cells, and change during a number of physiological processes ²³. Since the nucleus, cytosol and mitochondria are equipped with NAD⁺ salvage enzymes, the compartment-specific NAD⁺ production activates distinct sirtuins to trigger the appropriate physiological response. The NAD⁺/NADH levels also vary with the availability of dietary energy and nutrients. For instance, tissue NAD⁺ levels decrease with energy overload such as high-fat diet ²⁵ and display circadian oscillations with a 24 hour rhythm in the liver, which is regulated by feeding ²⁶. During energetic stress such as exercise, calorie restriction and fasting in mammals, the NAD⁺ levels increase leading to sirtuin activation, which is associated with metabolic and age-related health benefits (Figure 5) ²⁷. Decreased sirtuins (e.g. SIRT1 and SIRT3) expression is associated with various age-related pathologies ²⁸ and their overexpression has been reported to enhance overall mitochondrial and metabolic health in age-related disorders as well as mitochondrial diseases ²⁹.

Figure 4. NAD⁺ biosynthesis

Footnotes: Schematic representation of de novo and salvage pathways for NAD⁺ biosynthesis. In mammals, the de novo biosynthesis starts from l-tryptophan (Trp) which is enzymatically converted in a series of reactions to quinolinic acid (QA). Through quinolinate phosphoribosyltransferase (QPRT) enzyme activity, QA is converted to nicotinic acid mononucleotide (NAMN), which is then converted to nicotinic acid adenine dinucleotide (NAAD) by nicotinamide mononucleotide adenylyltransferase (NMNAT) enzyme. The final step in de novo biosynthesis is the amidation of NAAD by NAD synthase (NADS) which generates NAD⁺. The salvage pathway involves NAD⁺ synthesis from its precursors, i.e. Nicotinic acid (NA), nicotinamide (NAM) or nicotinamide riboside (NR). NA is catalytically converted to NAMN by the action of nicotinic acid phosphoribosyltransferase (NAPT). NAM is converted by nicotinamide phosphoribosyltransferase (NAMPT) to nicotinamide mononucleotide (NMN), which is also the product of phosphorylation of NR by nicotinamide riboside kinase (NRK) enzyme. Finally, NAMN is converted to NAD by the action of NMNAT and NADS enzymes, whereas NMN is converted to NAD by the NMNAT enzyme. Multiple enzymes break-down NAD⁺ to produce NAM and ADP-ribosyl moiety, however only sirtuins are depicted in this figure

[Source ³⁰]

Figure 5. Boosting NAD+ levels is beneficial for health and lifespan

Footnotes: NAD⁺ is a rate-limiting cofactor for the enzymatic activity of sirtuins. Boosting intracellular NAD⁺ levels by physiological (e.g. exercise, calorie restriction, fasting) or pharmacological [e.g. resveratrol, sirtuin activating compounds (STACs)] interventions, and inducing NAD⁺ biosynthesis through supplementation with precursors (e.g. NA, NAM, NR) or inhibition of NAD⁺ consuming enzymes (e.g. PARP-1, CD38) leads to activation of sirtuins (e.g. SIRT1, SIRT3). SIRT1 deacetylates and activates transcriptional regulators (e.g. PGC-1α, FOXO1), whereas SIRT3 deacetylates and activates multiple metabolic gene targets (e.g. succinate dehydrogenase, superoxide dismutase 2), which in turn regulate mitochondrial biogenesis and function. Supplementation with NR or PARP inhibitors extends lifespan in worms by inducing the UPR^mt stress signaling response via Sir-2.1 activation, which then triggers an adaptive mitohormetic response to stimulate mitochondrial function and biogenesis. Improved mitochondrial function associated with mitohormesis or metabolic adaptation can attenuate the impact of mitochondrial diseases, aging as well as age-related metabolic and neurodegenerative disorders. The physiological and pharmacological interventions that boost NAD⁺ levels are highlighted in yellow and pink respectively whereas the pathways that produce and consume/decrease NAD⁺ levels are highlighted in green and red respectively

[Source ³⁰]

What does NADH do?

NAD⁺ and its phosphorylated and reduced forms including NADP⁺, NADH, and NADPH (reduced nicotinamide adenine dinucleotide phosphate) are vital in regulating cellular metabolism and energy production. NAD⁺ functions as an oxidoreductase cofactor in a wide range of metabolic reactions and modulates the activity of compartment-specific pathways such as glycolysis in the cytosol, and tri-carboxylic acid (TCA) cycle, OXPHOS, fatty acid and amino acid oxidation in the mitochondria. For instance, NAD⁺ is converted to NADH at the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step of glycolysis, a pathway that generates pyruvate from glucose ³¹. In the mitochondrial compartment, NAD⁺ is converted to NADH at multiple steps in the tricarboxylic acid cycle (citric acid cycle) in which acetyl-coenzyme A is oxidized to carbon dioxide. Mitochondrial NADH is then oxidized by furnishing reducing equivalents to complex I in the ETC through a series of redox reactions that generate ATP from ADP by OXPHOS. The NAD⁺/NADH ratio thus regulates multiple metabolic pathway enzymes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase. In contrast to NAD⁺/NADH, the NADPH/NADP⁺ ratios are maintained high in both cytosol and mitochondrial compartments, to maintain a reducing environment ³². NADPH plays a key role in reductive biosynthesis and cellular defense against oxidative damage ³³. For instance, NADPH serves as a cofactor for P450 enzymes that detoxify xenobiotics, acts as a terminal reductant for glutathione reductase which maintains reduced glutathione levels during oxidative defense, and also serves as a substrate for NADPH oxidase that generates peroxides for release during oxidative burst processes in the immune system ³³.

Increased NAD+ levels protects against mitochondrial and age-related disorders

Mitochondrial disorders represent one of the most common forms of heritable metabolic disease in children ³⁴. Reduced NAD⁺/NADH ratio is strongly implicated in mitochondrial disorders and, age-related disorders including diabetes, obesity, neurodegeneration and cancer ³⁵. NAD⁺ levels also decline during aging in multiple models including worms, rodents and human tissue ³⁶. Increasing evidence suggests that boosting NAD⁺ levels could be clinically beneficial, as it activates the NAD⁺/sirtuin pathway which yields beneficial effects on multiple metabolic pathways.

Pharmacological activation of NAD⁺ production has recently been used to treat mouse models of mitochondrial diseases. For instance, treatment of cytochrome c oxidase (COX) deficiency caused by SURF1, SCO2 or COX15 genetic mutations in mice, with AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), partially rescued mitochondrial dysfunction and improved motor performance ³⁷. These findings could be explained by the fact that AMPK stimulates NAD⁺ production, consequently activating SIRT1 which promotes energy production and homeostasis ²⁸. Oral administration of NAD⁺ precursor, NR in mitochondrial myopathy mice harboring a pathogenic mutation in the mtDNA helicase—Twinkle, effectively delayed myopathy progression, by increasing mitochondrial biogenesis, preventing mitochondrial ultrastructural abnormalities, mtDNA deletion formation and activating the mitochondrial unfolded protein (UPR^mt) response ³⁸. In addition, NR supplementation and reduction of NAD⁺ consumption by a specific PARP inhibitor significantly improved mitochondrial respiratory chain defect and exercise intolerance, in a mouse model of COX deficiency caused by SCO2 mutation ²⁹.

Besides improving mitochondrial function, boosting NAD⁺ levels with resveratrol, nicotinamide riboside or nicotinamide mononucleotide (NMN) also corrects metabolic disturbances in mice caused by high fat diet ²⁸. Nicotinamide mononucleotide (NMN) administration ameliorates glucose intolerance and insulin resistance in diet- and age-induced type 2 diabetic mice ³⁹ and rectifies glucose-stimulated insulin secretion and glucose intolerance in NAMPT-deficient animals, by restoring NAD⁺ levels ⁴⁰. Interventions using NAD⁺ precursors or poly ADP-ribose polymerase inhibitors were also shown to be neuroprotective. For instance treatment with nicotinamide mononucleotide (NMN) or nicotinamide riboside precursors, protected against axonal degeneration and hearing loss in mice ⁴¹. Raised NAD⁺ levels after calorie restriction, nicotinamide or nicotinamide riboside treatment attenuated increase in β-amyloid content and oxidative damage, preventing cognitive decline and neurodegeneration in rodent models of Alzheimer’s disease ⁴². PARP-1 (poly ADP-ribose polymerase 1) activation also occurs in neurodegenerative DNA repair disorders including xeroderma pigmentosum group A (XPA) and Cockayne syndrome group B, and treatment with specific PARP inhibitors rescues defective phenotypes in XPA mutant worms and Cockayne syndrome group B mutant mice respectively ⁴³. However, PARP-2 (poly ADP-ribose polymerase 2) deleted mice were glucose intolerant and exhibited pancreatic dysfunction, implying that these results may interfere with other beneficial consequences of PARP inhibition, and hence warrant further investigation on the safe clinical use of these inhibitors ⁴⁴. Because poly ADP-ribose polymerase inhibitors enhance oxidative metabolism and improve metabolic flexibility, these compounds are being tested in phase III trials as anti-cancer agents ⁴⁵.

Increasing NAD⁺ levels by treatment with nicotinic acid and nicotinamide precursors has been shown to inhibit metastasis and breast cancer progression in response to mitochondrial complex I defect in mice ⁴⁶. However, reducing NAD⁺ bioavailability is reported to have an antineoplastic effect in various tumor cell types, as cancer cells rely on increased central carbon metabolism and biomass production to sustain an unrestricted growth ⁴⁷. The exact role of sirtuins in cancer remains controversial with dichotomous functions being reported, for example multiple studies have shown that SIRT1, SIRT3 and SIRT5 can act as tumor promoters or tumor suppressors under different cellular conditions, tumor stage and tissue of origin ⁴⁸. However, SIRT4 is only shown to have a tumor suppressor function ⁴⁹. Further research is needed to understand why and how certain sirtuins have both oncogenic or tumor-suppressive roles, and how this dual action may be best exploited for cancer management.

Declining NAD⁺ levels during aging compromise mitochondrial function in multiple model organisms, which can be restored via NAD⁺ precursor supplementation or poly ADP-ribose polymerase inhibition. For instance, nicotinamide mononucleotide or nicotinamide riboside administration in aged mice or worms respectively, reversed mitochondrial dysfunction by restoring NAD⁺ levels ⁵⁰. Moreover, nicotinamide riboside administration or poly ADP-ribose polymerase inhibition in worms extended lifespan by activating the UPR^mt response via Sir-2.1 (worm SIRT1 ortholog) and mitonuclear protein imbalance, which in turn induced a mitohormetic response to improve mitochondrial function (Figure 5) ⁵¹. Inducing UPR^mt genes such as Hsp60 paralogs in Drosophila also prevented mitochondrial and age-dependent muscle dysfunction, thereby promoting longevity ⁵².

Modulation of NAD+ levels by pharmacological compounds

Besides physiological processes, NAD⁺ levels can be modulated pharmacologically. Resveratrol—a polyphenolic compound found in red wine has been shown to indirectly stimulate NAD⁺ production by activating the energy sensor AMP-activated protein kinase (AMPK) ⁵³. Increased NAD⁺ subsequently stimulates SIRT1 activity, which in turn activates PGC-1α and FOXO family of proteins that govern mitochondrial biogenesis and function (Figure 5) ⁵³. SIRT1 is also amenable to intervention by small molecules such as SIRT1-activating compounds (STACs) that exert beneficial effects on age-related metabolic abnormalities ²⁸. NAD⁺ levels can be directly raised by supplying NAD⁺ biosynthetic precursors/intermediates, or by inhibiting NAD⁺ consuming enzymes with specific inhibitors (Figure 5). For instance, supplementation of nicotinic acid, nicotinamide riboside or nicotinamide mononucleotide compounds increase NAD⁺ levels in both cultured cells and mouse tissues²⁸. Because nicotinamide riboside can be metabolized both in the nucleus and mitochondria, its supplementation raises the nuclear and mitochondrial NAD⁺ levels, thereby activating nuclear SIRT1 and mitochondrial SIRT3 respectively ²⁸. Pharmacological activation of NAD⁺ thus stimulates the activity of multiple sirtuin in a compartment-specific manner to exert its beneficial effects on multiple metabolic pathways which is in contrast to SIRT1 activating compounds’s that specifically stimulate the activity of SIRT1 pathway. Treatment of mice or cultured cells with poly ADP-ribose polymerase and CD38 specific inhibitors has also been shown to induce NAD⁺ levels that activate sirtuins ⁴⁵.

Summary

Based on the current evidence, both NAD⁺ precursors and poly ADP-ribose polymerase inhibitors seem as promising candidates for boosting NAD⁺ levels in cell culture and animal models. However, there are several key questions that remain unanswered ⁵⁴.

1. First, whether different pharmacological, genetic and physiological manipulations that boosts NAD⁺ production lead to enhanced activity of all sirtuin enzymes or whether only a few family members are activated, especially considering the fact that some sirtuins may have opposing actions?
2. Second, how sirtuins located in different subcellular compartments differ in their enzyme kinetics towards NAD⁺ availability?
3. Third, what may be the optimal dosages, routes of administration, efficacy and bioavailability of compound drugs that raise intracellular NAD⁺ levels for human application?

Future studies that are directed towards understanding these would be highly relevant in designing therapeutic strategies aimed at selective activation of specific sirtuins, and would also aid in translating the results for human clinical application. It is possible that some of the NAD⁺ boosting drugs show adverse side effects in humans which could preclude their use and/or may be acceptable for only those inherited conditions that are highly devastating. It is also important to determine if nicotinamide riboside could be valid substitute to avoid undesirable side effects of other NAD⁺ precursors such as nicotinic acid and nicotinamide, for instance when used as lipid lowering drugs ⁵⁵.

In addition, future studies are required to examine the UPR^mt pathway in vivo in mammalian models to identify key signaling molecules involved in mitochondrial protective mechanisms, which will further advance our understanding of the diseases associated with mitochondrial dysfunction, and will allow discovery of new targets to modulate this pathway. Finally, it remains to be determined whether or not boosting NAD⁺ levels could extend lifespan in higher organisms. Although much remains to be done, based on the steadily growing evidence, the pharmacological modulation of NAD⁺ levels via NAD⁺ precursors and poly ADP-ribose polymerase inhibitors appears to be an attractive and valid strategy to enhance oxidative metabolism and mitochondrial biogenesis, and holds a significant therapeutic potential in the clinical management of mitochondrial and age-related disorders.

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