Turning Back the Clock in Aging Cells

Today, we take a look at NAD+ biology, its history, how it relates to aging, and the potential for therapies against age-related diseases via the NAD+ precursor molecule Nicotinamide mononucleotide (NMN).

What is NAD+?

Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells. It is a dinucleotide, which means that it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base, and the other contains nicotinamide.

In metabolism, NAD facilitates redox reactions, carrying electrons from one reaction to another. This means that NAD is found in two forms in the cell; NAD+ is an oxidizing agent that takes electrons from other molecules in order to become its reduced form, NADH. NADH can then become a reducing agent that donates the electrons it carries. The transfer of electrons is one of the main functions of NAD, though it also performs other cellular processes, including acting as a substrate for enzymes that add or remove chemical groups from proteins in post-translational modifications.

NAD is created from simple building blocks, such as the amino acid tryptophan, and it is created in a more complex way via the intake of food that contains nicotinic acid (niacin) or other NAD+ precursors. These different pathways ultimately feed into a salvage pathway, which recycles them back into the active NAD+ form.

Nicotinamide adenine dinucleotide (NAD+) biology has seen a great deal of interest in the last few years, partially due to the discovery of two precursors of NAD+ biosynthesis, nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), which both increase NAD+ in multiple tissues.

Accumulating evidence suggests that NAD+ systemically declines with age in a variety of organisms, including rodents and humans, which contributes to the development of many age-related diseases. For this reason, there is a great deal of interest in creating potential interventions that increase NAD+ levels via precursors, thus delaying or even preventing certain aspects of age-related functional decline and diseases.

History of NAD+ research

Before we talk more about NAD+ precursors, let’s take a look at the history of NAD+ biology in order to get a better idea of the background and research that has taken place.

We have known about NAD+ for over a hundred years. Harden and Young originally discovered NAD+ back in 1906, when they discovered that it increased the rate of fermentation in yeast [1]. In the years following this, Euler-Chelpin, a German-born Swedish biochemist, identified that NAD+ was a nucleotide and, in 1929, received the Nobel Prize in Chemistry for his research on the fermentation of sugar and the enzymes involved in the resulting complex reactions.

In 1937, the American biochemist Conrad Elvehjem identified two vitamins, nicotinic acid and nicotinamide, which were deficient in people suffering from pellagra [2]. In 1949, Elvehjem showed that nicotinamide was able to prevent pellagra in dogs by increasing the production of NAD+ [3]. This discovery ended up with nicotinic acid and nicotinamide being classified as vitamin B3, and they are considered to be the original NAD+ precursors.

The first NAD+ gold rush

In 1963, there was a surge of interest in NAD+, when Chambon, Weill, and Mandel showed that NMN activated a previously unknown DNA-dependent polyadenylic acid-synthesizing nuclear enzyme [4]. This research subsequently led to the discovery of poly-ADP-ribose and poly-ADP-ribose polymerases (PARPs), which are part of a family of proteins involved in a number of cellular processes, including DNA repair, maintaining genomic stability, and programmed cell death [5].

In 1965, Hayaishi and his team started to reveal the NAD+ pathway, how it was created via tryptophan, and the NAD+ intermediate nicotinic acid mononucleotide (NMN) [6]. In 1966, Gholson suggested that there was an active turnover cycle of NAD+, which, at the time, performed “an important but as yet unknown function” relating to cellular metabolism [7]. A decade later, in 1976, this was confirmed by Rechsteiner and his team when they published evidence for the rapid turnover of NAD+; this further supported the suggestion that NAD+ had another major function in eukaryotic cells in addition to its established role in oxidation and reduction [8].

In 1989, Lee and his research team incubated NAD+ with extracts from sea urchin eggs, discovering a new NAD+ metabolite, cyclic ADP-ribose [9]. The enzyme ADP-ribosyl cyclase was initially identified in 1991 by Lee and his team in Aplysia, a type of large sea slug [10]. It was later found to exist in mammals as CD38, which was confirmed by Lee, Walseth, and Lee in 1992 [11].

The second NAD+ gold rush

There was another surge of interest in NAD+ biology that happened in the year 2000 and has lasted until the present. The study that started the renewed interest was published by Guarente in 2000, which showed that yeast SIR2 (silent information regulator 2) and the related mouse ortholog, SIRT1, both had NAD+ dependent protein deacetylase activity [12]. Adding further fuel to the fire, in 2001, human nicotinamide/nicotinic acid mononucleotide adenylyltransferase (NMNAT) was successfully isolated [13]. NMNAT is an important NAD+ biosynthetic enzyme, so its isolation and characterization was an important step.

Next, nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme responsible for converting nicotinamide into NAD+, was isolated and characterized between 2002–2004 [14–15].

In 2004, nicotinamide riboside was shown to lead to the creation of NAD+ via nicotinamide riboside kinases (NRKs). This study demonstrated that nicotinic acid mononucleotide was not the only path to NAD+ biosynthesis [16].

That brings us to the end of our potted history of NAD+; almost half a century after the first surge of interest, we are now enjoying a new era of research and discovery in NAD+ biology.

Nicotinamide mononucleotide and NAD+

There are already some excellent reviews on NAD+ biology in general [17–19], so, in this article, we will be mainly focusing on the NAD+ precursor nicotinamide mononucleotide (NMN).

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 [20–31].

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 [32–33]. 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 [34]. 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 [35–36]. This also improved the situation in NAMPT knockout mice and in aged wild-type and beta cell-specific SIRT1-overexpressing mice [37–39].

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 [40].

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 [41]. 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, which is a key risk factor 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 [42]. This also leads to the improvement of skeletal muscle mitochondrial oxidative metabolism and endothelial function along with reversal of vascular aging in mice [43–45]. NMN also appears to address retinal degeneration via interaction with the mitochondrial sirtuins SIRT3 and SIRT5, at least in NAMPT knockout mice [46].

In 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 [47–49]. NMN also appears to have neuroprotective properties and protects neurons from death following ischemia or intracerebral hemorrhage [50–51]. NMN also reduces the age-related loss of neural stem cells in the dentate gyri of wild-type C57BL/6 mice [52].

In the kidneys, NMN appears to inhibit acute renal injury via a SIRT1-mediated response [53]. NMN has also been shown to improve DNA damage repair from radiation [54].

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 losses 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+ [55]. 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 [56–59].

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. 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 [60], 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 [61]. SIRT1 has been shown to have both pro- and anticarcinogenic effects in a context-dependent manner [62]. 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 in 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 therapy.

Fortunately, there are currently ongoing human trials for NMN being conducted at Brigham and Women’s Hospital in order to establish 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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