NAD+ Research

Although death is inevitable, individuals have long sought to alter the course of the ageing process. Indeed, ageing has proved to be modifiable; by intervening in biological systems, such as nutrient sensing, cellular senescence, the systemic environment and the gut microbiome, phenotypes of ageing can be slowed sufficiently to mitigate age-related functional decline.

Strategies for combating mechanisms of ageing to prevent disease, known as ‘geroprotection’, are far reaching, and currently include recommendations for exercise, diet and other aspects of lifestyle. However, these alone are not sufficient to prevent the ills of old age, and increasing efforts are directed to tackling the underlying processes of ageing. The results of these processes include damage to the genetic material and its packaging and expression, cellular senescence, and dysregulated proteostasis, mitochondrial function, nutrient sensing, intercellular communication and stem cell function. These hallmarks of ageing are causally connected, and they interact with one another to produce ageing-related decline. Currently, the most promising strategies for geroprotection include mildly lowering the activity of the nutrient-sensing network, especially the activity of mechanistic target of rapamycin protein complex 1 (mTORC1), removing senescent cells, using natural metabolites from the systemic environment that can rejuvenate stem cells, and transferring the microbiome. Increasing autophagy, probably including mitophagy, and reducing age-related inflammation are emerging as key mechanisms by which these interventions exert their effects.

An important approach is the development of small molecules, both drugs and natural products, that have geroprotective effects by combating the mechanisms of ageing. A major theme here is the prospect for indication expansion, where small molecules and agents that have good safety profiles and were previously identified for other properties are candidates for repurposing as geroprotectors.

Human ageing may be modifiable and research into ageing is entering a new and exciting phase where interventions to extend the healthspan will be tested in humans and, if validated, potentially approved for use. In addition to making the case for the clinical testing of a select set of agents, we also discuss the potential routes to testing the effects of candidates on human ageing and, if these are successful, how they could be employed to enhance the human healthspan.

Nicotinamide adenine dinucleotide (NAD+) is found in all known forms of life. As a carrier of high-energy electrons from fuel oxidation (in its reduced form, NADH), NAD+ is an essential cofactor for redox reactions in the cytosol and mitochondria.

NAD+ Homeostasis

As a substrate for major classes of enzymes such as sirtuins that cleave a carbon–nitrogen bond in NAD+ to yield nicotinamide (Nam; also known as niacinamide) and ADP- ribose (ADPR), NAD+ also regulates a variety of other cellular processes, including the cellular stress response, mitochondrial homeostasis and calcium signalling.

The different precursors to intracellular NAD metabolism—tryptophan, nicotinic acid (NA), nicotinamide, NR, and NMN—are shown, along with their extra- cellular metabolism by CD38 and CD73. The cytoplasmic and nuclear NAD+ pools probably equilibrate by diffusion through the nuclear pore. 

However, the mitochondrial membrane is impermeable to both NAD+ and NADH. Reducing equivalents generated by glycolysis are transferred to the mitochondrial matrix via the malate/aspartate shuttle and the glyceraldehyde-3-phosphate shuttle. The resulting mitochondrial NADH (malate/aspartate shuttle) is oxidized by complex I in the respiratory chain (ETC), whereas the resulting FADH2 (glyceraldehyde-3-phosphate shuttle) is oxidized by complex II. In each of the three compartments, different NAD+- consuming enzymes lead to the generation of nicotinamide, which is recycled via the NAD+ salvage pathway. Different forms of the NMNAT enzyme and sirtuins are localized in different compartments. The nature of the salvage pathway for NAD+ in mitochondria has not been fully resolved, although NMNAT3 has been found in mitochondria. NADK, NAD+ kinase.

Two key events, the activation of PARP by DNA damage and the decreased NAMPT expression associated with inflammation, lead to decreased SIRT1 and SIRT3 activity in the nucleus and mitochondria, respectively. Decreased SIRT1 activity is associated with further PARP activation and increased DNA damage. Decreased SIRT1 also leads to NF-kB activation and decreased FOXO3a activity, two factors that lead to increased inflam- mation. These contribute to the establishment of two parallel feed-forward self-reinforcing loops that further accelerate the aging process. This process is initiated earlier and faster in patients with DNA damage repair defects (such as CS, XPA, and AT). 

Mitochondrial function is diminished as a result of decreased SIRT3 activity, leading to mitochondrial protein hyperacetylation, whereas decreased SIRT1 is associated with decreased TFAM (necessary for mitochondrial DNA replication and transcription) and decreased PGC-1a (necessary for mitochondrial biogenesis). Possible therapeutic interventions to restore NAD+ levels are illustrated for each of the key enzymes (red arrows).

Regulation of SIRT1. SIRT1 is a NAD+ dependent histone/protein deacetylase that acts by removing the acetyl group from lysine residues of protein substrates or histones in the presence of the cofactor NAD+(nicotinamide adenine dinucleotide). The nicotinamide (NAM), the substrate deacetylated on lysine residues, and the molecule 20-O-acetyl-ADP- ribose are the products of this reaction. Therefore, is implicit that two important factors for its regulation are NAD+, the essential cofactor for the enzymatic reaction, and NAM, a feedback inhibitor. Also the activation of the enzymes involved in NAD+ salvage pathway plays an important role in this context. 

NAM phoshoribosyltransferase (Nampt) catalyzes the conversion of NAM to NAD+, and therefore, it activates SIRT1 by both increasing cellular NAD+ and diminishing NAM. NAMPT, in turn, is positively regulated by exercise and nutritional depletion; both conditions can stimulate the synthesis of SIRT1 and the activation of AMPK. Indeed, exercise, physical efforts, or stress conditions can cause a lowering of energy levels, an ATP decrement, and an AMP/ATP ratio increase. In this metabolic condition, AMPK, once activated, is able to increase ATP levels, stimulate the NAD+ increase by boosting transcription of the NAD+ biosynthetic enzyme NAMPT, and thus activate SIRT1.Whereas there is a relationship of reciprocal activation between SIRT1 and AMPK, there is a relationship of reciprocal inhibition between SIRT1 and mTOR. Indeed, SIRT1 is able to directly activate TSC2, an inhibitor of mTOR, and to inhibit AKT, a key factor for the activation of mTOR. On the other hand, NAM,a SIRT1 inhibitor, isable to activate AKT and mTOR.

Sirtuins are involved in many reactions that regulate cellular metabolism through the NAD+- dependent deacetylation or deacylation of proteins including histones, such as H3, H4 and H1, transcription factors and co-activators, including p53, NF-κB, PPARγ co-activator 1α
(PGC1α) and FOXO1, and signal- ling regulators of metabolism, including protein kinase A, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR)13. Sirtuins also utilize NAD+ to remove other kinds of post-translational protein modifications, including malonylation and succinylation. In all of these reactions, the ADPR generated from NAD+ becomes an acceptor for the post-translational group removed from the target protein.

Nicotinamide adenine dinucleotide (NAD+) is required for distinct reactions in glycolysis, fatty acid oxidation (FAO) and the tricarboxylic acid (TCA) cycle that lead to the generation of ATP via the electron transport chain (ETC) in mitochondria. In healthy renal tubular epithelial cells, this ATP is utilized to provide the energy required for key functions, such as solute transport and maintenance of membrane integrity. Efficient FAO also prevents the potentially toxic accumulation of storage fats. NAD+ might also exert renoprotective effects via its interactions with sirtuins and PPARγ co-activator 1α (PGC1α). Sirtuin 1 (SIRT1) activates PGC1α via NAD+-dependent deacetylation and PGC1α in turn promotes NAD+ biosynthesis via the de novo pathway by co-ordinately upregulating the expression of the genes encoding enzymes in this pathway.

PGC1α promotes mitochondrial quality control and ATP production via interrelated process that include mitochondrial biogenesis and the induction of mitophagy via transcription factor EB (TFEB). SIRT3 also utilizes NAD+ to directly promote healthy mitochondrial function and SIRT1 may limit stress signalling through the pro-apoptotic Jun N-terminal kinase (JNK) pathway. In addition to its effects on mitochondria, PGC1α signalling can induce the production of vascular trophic molecules, such as VEGF. In tubular epithelial cells, a PGC1α-dependent product of FAO, β- hydroxybutyrate (β-OHB), may signal the production of vasodilator prostaglandins that can maintain renal blood flow during conditions such as shock that would otherwise promote renal ischaemia. Phosphorylation of NAD+ to NADP+ may potentiate defence against oxidant stress induced by inflammation, toxins, or ischaemia–reperfusion injury by promoting the reduction of glutathione (GSH) and through the vasodilator nitric oxide (NO•). Mitochondrial quality control may also limit the burden of free radicals emanating from injured mitochondria.

Ageing is a complex process and no geroprotective intervention has ameliorated all of its features, although DR has so far come the closest. Genetic studies in model organisms have indicated that combinatorial interventions targeting different pathways can be the most effective in ameliorating ageing. The same is likely to be true of pharmacological interventions and, indeed, combinatorial treatments in yeast, C. elegans and Drosophila have been more effective than administration of single agents. The evidence from animal studies and our understanding of human ageing indicate that multiple approaches to ameliorating the effects of ageing should be pursued in parallel.

Nutrient-responsive signalling pathways that maintain health and extend lifespan.

 

Calorie or dietary restriction increases the concentrations of metabolic effectors such as nicotinamide adenine dinucleotide (NAD+) and AMP while reducing the concentrations of glucose, amino acids and lipids. Exogenous administration of nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) or the nicotinamide phosphoribosyltransferase (NAMPT) activator P7C3 can increase NAD+ levels. Calorie restriction also reduces the concentrations of the hormonal effectors insulin, insulin-like growth factor 1 (IGF1) and growth hormone (GH). These effectors stimulate or inhibit the activity of metabolic sensors such as the sirtuins (SIRTs), AMP kinase (AMPK), target of rapamycin (TOR), insulin–IGF1 signalling (IIS) and forkhead box O (FOXO) transcription factors.

Sirtuin-activating compounds (STACs) such as SRT1720 and SRT2104 can directly activate SIRT1, whereas rapamycin is a direct inhibitor of TOR. Metformin indirectly activates AMPK. These metabolic sensors regulate downstream activities such as DNA repair, mitochondrial biogenesis and function, stress resistance, stem cell and telomere maintenance, autophagy, chromatin modifications, reduced inflammation,and translation fidelity. 

The net effect is to tip the scale in favour of homeostasis and compressed morbidity, resulting in a disease-free, more youthful-like state.

A final approach is to avoid drugs altogether and develop natural products as supplements to slow ageing. These compounds are less tightly regulated than are drugs, and many are already legally marketed as treatments for a wide range of conditions, often with- out clear clinical evidence to support their use. In the context of geroprotection, a combination of two com- pounds to modulate NAD+ and sirtuin activity is being marketed and has undergone limited human testing.

NAD+ metabolism is a highly dynamic process and all four NAD coenzymes — NAD+, NADH, NADP+ and NADPH — have redox and signalling functions. NAD+ serves as a cofactor for most enzymes that oxidize substrates and is itself reduced to NADH. Conversely, NADPH often functions as a cofactor for enzymes that reduce substrates. 

Thus, NAD+ homeostasis has key roles in cellular catabolism and anabolism. In addition, NAD+ can undergo cleavage to generate ADPR or signalling molecules, act as an acceptor for acyl and acetyl modifications removed from proteins via sirtuins and undergo phosphorylation to form NADP+.

NAD+ consumption is catalysed by three classes of enzymes: sirtuins, poly(ADP-ribose) polymerases (PARPs) and cyclic ADPR (cADPR) synthetases.
Ageing is a conserved phenomenon across all species and imposes an ever-increasing risk of dysfunction and death in older organisms.

Growing evidences have shown that sirtuins are essential factors those delay cellular senescence and extends the organismal lifespan through the regulation of diverse cellular processes. Therefore, we summarize the evidences and controversies regarding the roles of different sirtuins on aging and lifespan extension, and systematically elucidate the functions and pathways of sirtuins on aging and lifespan extension.

Among the mammalian sirtuins, sirt1 has been the most extensively characterized for its role in aging. Although much of the attention has gone to sirt1 and its protective effects against the onset of chronic diseases, its effect on longevity remains unconvincing. Sirtuins other than sirt1 are also reported to exert a prolongevity effect. Recently, sirt2 has been found to be a key modulator of ageing, and it extends lifespan in the BubR1 mice model. 

Additionally, sirt3 is the only sirtuin that has been shown to be associated with human aging; some (but not all) studies have linked polymorphisms in the sirt3 genomic locus to survival in elderly individuals. However, no pan- or tissue-specific transgenic animal models overexpressing sirt3 to determine whether sirt3 overexpression confers lifespan extension or protects against age-associated pathologies have been described in the literature currently, and some newer studies failed to confirm these correlations in other populations. In contrast, recent work on sirt6 suggests that this sirtuin might hold the most potential for actual life-span extension. Loss of sirt6 causes severe metabolic defects and rapid aging. In addition, global sirt7

depletion contributes to premature ageing, especially in the backbone, white adipose tissue and the heart.

Leucine amplifies the effects of NA on lipid metabolism, hyperlipidemia and atherosclerosis in mice, at least in part by activation of the AMPK/Sirt1 axis. This combination may be a potential therapeutic alternative for hyperlipidemia and atherosclerosis.

Leucine lowers NAD+ activation energy for sirtuins. Robust synergy at very low concentrations of sirtuin activators mimics the effects of caloric restriction/ high NAD+ concentration.

Leucine amplifies the effects of NA on lipid metabolism, hyperlipidemia and atherosclerosis in mice, at least in part by activation of the AMPK/Sirt1 axis. This combination may be a potential therapeutic alternative for hyperlipidemia and atherosclerosis.

Leucine lowers NAD+ activation energy for sirtuins. Robust synergy at very low concentrations of sirtuin activators mimics the effects of caloric restriction/ high NAD+ concentration.

These redox reactions are critical in enabling the most basic function of the cell, namely, the harvesting of energy in the form of ATP from fuel substrates such as glucose, amino acids and fatty acids. Without NAD+, fuel cannot be converted into energy.

Inhibition of CD38 activity by quercetin in cells (IC50 = 16.4 6 1.8 mmol/L) resembles the effect on the recombinant protein. Furthermore, It is found that quercetin promotes an increase in intracellular NAD+ in a dose-dependent manner. To further confirm this effect, researchers incubated cells in PBS and measured intracellular NAD+ levels over time.They found that in untreated cells, NAD+ levels decrease with time, probably as a result of the removal of NAD+ precursors from the culture media. However, when the cells were treated with quercetin, NAD+ levels were stable over time, suggesting that inhibition of CD38 is enough to maintain intracellular NAD+ levels in the absence of NAD+ precursors.

Curcuma longa has various therapeutic effects on different diseases. However, it has limited tissue distribution, low serum levels and apparent rapid metabolism in human. To increase Curcuma longa bioavailability, Piper nigrum, known as a natural adjuvant increases the bioavailability of Curcuma longa.

Anti-inflammatory mechanisms of curcumin including inhibition of nuclear factor kappa-B.

Although a number of challenges remain, including regulatory hurdles, clinical design questions, incompletely validated biomarkers of human ageing and commercial challenges to bringing the new interventions to market, it is likely that strong evidence will emerge in the near future for feasible strategies to delay human ageing. Administering these interventions in a safe manner based on solid and promising cellular signal pathways that is inclusive of everyone regardless of financial capacity is needed, and this approach could tilt medical treatment away from ‘sick’ care and towards broad spectrum prevention, a major advance that can revolutionize medicine, maximizing the improvement of life quality and mitigating the soaring costs of age-associated chronic diseases.

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