Welcome to part two of a two-part series exploring NAD+ biology; you can find part one here. There are already a myriad of excellent reviews on NAD+ biology in general [1-3], so, in this article, we will be mainly focusing on the NAD+ precursor nicotinamide mononucleotide (NMN).

NMN and synthesis of NAD+

NMN can be readily found in low amounts in a wide variety of foods, such as fruit, vegetables, and meat, but it is only recently that its potential has been investigated in animal models.

NMN is produced from nicotinamide (NAM), a form of water-soluble vitamin B3, and 50-phosphoribosyl-1-pyrophosphate (PRPP) by nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting NAD+ biosynthetic enzyme found in mammals. NMN is also created from nicotinamide riboside (NR) via a phosphorylation reaction mediated by nicotinamide riboside kinase (NRK).







The conversion of NMN into NAD+ is facilitated by nicotinamide mononucleotide adenylyltransfereases (NMNATs), rate-limiting enzymes that are present in all organisms. Data from rodent studies has shown that NMN can increase NAD+ biosynthesis in multiple tissues, including the pancreas, liver, adipose tissue, heart, skeletal muscle, kidneys, eyes, and blood vessels [4-15].

It is still not totally clear if NMN can cross the blood-brain barrier (BBB), as it may be too large to pass through the membrane from the bloodstream into the brain. However, studies show that intraperitoneal injection increases NAD+ in multiple brain regions, including the hippocampus and hypothalamus, within 15 minutes of administration [16-17]. This strongly suggests that NMN can pass through the BBB and increase NAD+ synthesis in the brain.

NAD+ and the implications for therapies against age-related diseases

A year-long study of wild-type C57BL/6 mice showed that NMN is tolerated well [18]. The mice were given up to 300 mg/kg during the study and suffered no adverse reactions or toxic effects. This suggests that NMN has therapeutic potential, and there is a growing amount of research that shows it has beneficial effects on a varied range of physiological functions, meaning that it may have broad implications as a therapy for treating age-related diseases.

So far, there have been several observed benefits of NMN administration. Some studies suggest that beta cells in the pancreas are sensitive to changes in NAD+ levels and to NMN treatment. A single injection of NMN at a dose of 500 mg/kg in mice increases glucose-stimulated insulin production, thereby improving glucose tolerance in age- and diet-induced diabetic mice [19-20]. This also improved the situation in NAMPT knockout mice and in aged wild-type and beta cell-specific SIRT1-overexpressing mice [21-23].







Data also suggests that NMN improves the activity of insulin along with its production. Mouse studies show that treatment with NMN improves hepatic insulin resistance induced by a high-fat diet by restoring NAD+ synthesis, increasing the activity of SIRT1, a critical signaling molecule that interacts with NAD, and reducing gene expressions associated with oxidative stress, inflammation and circadian rhythms [24].

Other studies show that long-term NMN consumption suppresses age-related inflammation in adipose tissue and improves whole-body insulin sensitivity in normally aging wild-type C57BL/6 mice [25]. NAD+ synthesis is impaired in obese and aged mice, so this study suggests that adipose tissue NAD+ could be a suitable target for insulin resistance, key risk factors for type 2 diabetes, and cardiovascular disease.

The administration of NMN has also been shown to improve the function of mitochondria in multiple organs and tissues. Mice treated with NMN have been found to have increased mitochondrial oxidative phosphorylation in skeletal muscle tissue; this likely helps with weight control by increasing whole-body energy expenditure during normal day-to-day function and movement [26]. This also leads to the improvement of skeletal muscle mitochondrial oxidative metabolism and endothelial function along with reversal of vascular aging in mice [27-29]. NMN also appears to address retinal degeneration via interaction with the mitochondrial sirtuins SIRT3 and SIRT5, at least in NAMPT knockout mice [30].

In terms of the brain, NMN appears to improve various neuronal functions, with administration improving both cognition and memory in mouse and rat models of Alzheimer’s disease [31-33]. NMN also appears to have neuroprotective properties and protects neurons from death following ischemia or intracerebral hemorrhage [34-35]. NMN also reduces the age-related loss of neural stem cells in the dentate gyrus of wild-type C57BL/6 mice [36].

Moving to the kidneys, NMN appears to inhibit acute renal injury via a SIRT1-mediated response [37]. NMN has also been shown to improve DNA damage repair from radiation [38].







More research is needed

While it is clear that NMN has beneficial effects in multiple tissues and organs in rodents; indeed, there are various conditions that show significant loss of NAD+ levels, there are also a number of unknown things about NAD+ that should be resolved.

For example, it is still unclear what downstream mechanisms are mediating the beneficial effects of improved NAD+ synthesis. NAD+ is involved in the activity of poly ADP ribose polymerases (PARPs), sirtuins, ADPribosyl cyclases, and mono-ADP ribosyltransferases while serving as a cofactor in redox reactions for a myriad of enzymes.

On one hand, we know that the inhibition or deletion of sirtuins blocks the positive benefits of NAD+ repletion, which spotlights the key role these enzymes have in working in unison with NAD+ [39]. On the other hand, the inhibition of NAD+ consuming enzymes, such as PARP1/2 and CD38, give similar benefits to increasing NAD+ via therapeutically increasing it [40-43].

Given the complex interactions at play here, it will take a considerable research effort to discern what exactly is going on, how these various benefits are conveyed, and the exact downstream mechanisms at play here. As part of that process, it is also critical to carefully assess any potential negative side effects of NAD+ therapies, particularly their intermediates.







While no evidence to date suggests that increasing NAD+ promotes cancer development, there are concerns that boosting NAD+ may help already established tumors to grow [44], especially given the recent finding that NAD+ boosting increases the development of the vascular system by facilitating SIRT1-mediated crosstalk between endothelial cells and muscle tissue [45]. SIRT1 has been shown to have both pro- and anticarcinogenic effects in a context-dependent manner [46]. So far, there is no observed increased cancer incidence in mice, but it is something to consider for the future development of therapies that increase cellular NAD+.

Conclusion

The results of mouse studies suggest that NAD+ repletion approaches hold great potential, but as always in science, we should be cautious. Thus, further preclinical and clinical studies are needed to establish the long-term safety of NMN as a human therapeutic.

Fortunately, there are currently ongoing human trials for NMN being conducted with a view to establishing toxicity and safety profiles over the long term, so we should have data in due course that will inform us of where to go next.

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