Who Wants to Live Forever? Saga of NAD+

Recent years have witnessed a resurgence of interest in nicotinamide adenine dinucleotide (NAD+) biology. This has been driven in part by the discoveries that two intermediates of NAD+ biosynthesis, nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), effectively increase NAD+ concentration in a variety of tissues, in many cases with beneficial or therapeutic effects.

NAD+ Intermediates

The scientific timeline of discovery for nicotinamide adenine dinucleotide (NAD+) is pictured below. The mystery dates back to the 1730s when the deadly disease pellagra was first described and studied. It wasn’t until the early 1900s that the nature of the disease as a nutritional deficiency was discovered, and the nutritional cures were discovered — members of the niacin family.

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It was only in the late 1990s and early 2000s that a decline in NAD was found to be associated with the processes of degenerative aging. Multiple body systems appear to suffer and age when the levels of NAD decline in the tissues of various organs.

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For many of us, we are primarily concerned with the brain and the heart, since those two organs represent a significant part of the decline in function as well as mortality, as a person ages.

So, what is NAD+ and what is it needed for? The pioneering work by Otto Warburg and co-workers in the 1930s discovered a key role for NAD+, and its phosphorylated counterpart NADP+, in hydrogen transfer biochemical reactions. The following decades unveiled how NAD+ and NADP+ are vital cofactors for most cellular oxidation/reduction reactions, where they can be reduced to NADH and NADPH, respectively, or vice versa.

The NAD+/NADH couple primarily drives oxidation reactions, while the NADP+/NADPH couple drives reductive reactions (for extensive reviews on the topic, see [10, 11]). The redox potential and relative amount of the phosphorylated and non-phosphorylated nicotinamide adenine dinucleotides is very different. For instance, NAD(H) levels in liver are twofold those of NADP(H), while in muscle they are 12-fold [11]. In addition to differences between tissues, NAD(H) and NADP(H) content is highly compartmentalized in the cell, with the mitochondria harboring the higher amounts [11]. Therefore, tissues with high mitochondrial content, such as heart or kidney, display the higher NAD(H) and NADP(H) contents. Even if cellular membranes are generally impermeable to NAM-based (di)nucleotides, the mitochondrial and cytosolic pools of NAD+ related nucleotides and their redox states are not fully independent. Instead, they are interconnected by an intricate net of molecular redox shuttles and the recently identified mitochondrial NAD+ transporter [10, 1215].

As redox cofactors, NAD(H) and NADP(H) participate in the most critical paths in cellular metabolism and mitochondrial oxidative phosphorylation. Most notably, during glycolysis, glucose can generate two molecules of glyceraldehyde-3-phosphate (G3P). This is followed by the reduction of NAD+ to NADH in the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction. Thus, glycolysis will finally render two NADH and two pyruvate molecules that can either be transformed into to NAD+ and lactate by the lactate dehydrogenase (LDH) reaction or transferred into the mitochondria. In the second case, the reducing equivalent of NADH is transported into the mitochondria via either the malate-aspartate shuttle or the glycerol-3-phosphate shuttle, while pyruvate has a dedicated transporter [10]. Once in the mitochondria, the pyruvate dehydrogenase complex will reoxidize NADH into NAD+. The mitochondrial tricarboxylic acid cycle (TCA) is a major location for the reduction of NAD+ into NADH molecules. Mitochondrial NADH can be re-oxidized to NAD+ by Complex I of the mitochondrial electron transport chain. The subsequent two electrons gained by Complex I will then be an initial step to generate a proton gradient that provides the chemiosmotic force to drive the oxidative phosphorylation of ADP to ATP, catalyzed by the F0F1-ATP synthase enzyme [10]. These processes highlight the intimate link between NAD+ and cellular ATP synthesis.

NAD and Its Precursors

The puzzle of NAD goes to the heart of how our bodies work. We cannot simply say “the heart does this,” “the liver does this,” “the kidneys do this,” “the brain does this,” etc. Beneath such glib layers of explanation lie the deeper biochemical and physiological functions and processes which enable these organs to fulfill their roles. Everything requires energy. If our body tissues lose their grasp on the energy they need, they begin to deteriorate — to age. That is not the only cause of aging, but it sits at the center of the cascade of causes.

An aging person (anyone over the age of 30) can mitigate part of this “tissue energy crisis” by supplying the body with precursors of NAD. Some of the precursors of NAD are pictured below:

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NMN (nicotinamide mononucleotide) and NR (nicotinamide riboside) are two of the most common precursors of NAD used by the public in the form of nutritional supplements. There is a bit of a controversy over which of the two supplements are more effective. There has even been a suggestion by one scientist that NR may inadvertently contribute to some forms of cancer metastasis. Time and more research will help to sort out these questions.

These NAD precursors are not a magic bullet. But careful research is helping us to discover some details about how they may be of benefit to us.

Human patients with stage D heart failure orally supplemented with NR for 5–9 days (0.5–1 g/day) displayed increased whole blood NAD+ levels, mitochondrial function in peripheral blood mononuclear cells (PBMCs) and a large reduction in pro-inflammatory cytokine (IL-1b, IL-6 and IL-18) gene expression [221]. This suggests that NR could help reducing the pro-inflammatory systemic state in in cardiovascular complications. Along the same lines, when healthy elderly individuals where supplemented with NR (1 g/day) for 21 days no major changes were observed in body composition or cardiovascular parameters, yet a decrease in circulating levels of inflammatory cytokines (IL-2, IL-5, IL-6) was also observed [222]. Therefore, two independent studies point towards the concept that NR supplementation could reduce systemic inflammatory signals.

NAD Precursors

It can be difficult to discover the subtleties at work when we try to untangle the complex interactions of genes and the natural components of our bodies, with the environment. Part of that is due to the clumsiness of the tools that we are using in our research. It takes time to build the tools that we need. But as we stumble awkwardly ahead using our clumsy tools, we discover clues, little by little.

Recently, NR showed some promising outcomes in treating patients suffering from ataxia–telangiectasia (AT), a rare neurodegenerative disease, causing severe disability. AT is caused by mutation of the Ataxia-Telangiectasia Mutated (ATM) gene, encoding the ATM kinase, a master regulator of DNA damage resolution [247]. Initial work in mice suggested that dietary NR supplementation (12 mM in the drinking water) increased the lifespan of the short-lived Atm−/− mice, correlating with the normalization of mitochondrial architecture due to improved mitophagy and a better resolution of DNA repair [225]. In an open-label, proof-of-concept clinical study, 24 patients with AT were treated with NR (25 mg/kg/day) during four consecutive months. NR supplementation led to improvements in diverse kinetic and speech parameters [227]. Interestingly, these benefits disappeared 2 months after NR withdrawal [227], further strengthening that the benefits could be genuinely due to NR treatment.

NAD Precursors

A newer precursor to NAD has recently been discovered which may prove significantly better in many respects than either NMN or NR. It is a reduced form of NR called dihydronicotinamide riboside (NRH). More on this discovery:

Parallel work by the Sauve lab and our group identified a reduced version of NR, dihydronicotinamide riboside (NRH), as a new NAD+ precursor in mammalian cells and tissues [128, 263] (Fig. 2). Despite the structural similarity with NR, the biological properties of NRH turned out to be particularly surprising. NRH can sharply increase NAD+ levels in cultured cells, being far more potent than any other NAD+ precursor described to date [128, 263]. After entering the cells, predominantly through ENTs [128], NRH uses a unique path to drive NAD+ synthesis. Surprisingly, the ability of NRH to act as a NAD+ precursor does not rely on NRK activity. Using chemical inhibitors, Giroud-Gerbetant et al. identified adenosine kinase (AK) as the enzyme that initiated the conversion of NRH to NAD+ [128]. This finding was later confirmed by the Sauve lab using cellular fractionation methods [264]. The phosphorylation of NRH by AK renders dihydronicotinamide mononucleotide (NMNH), which is then adenylated by NMNAT enzymes to generate NADH, which is then oxidized to NAD+ [128, 264]. Thus, NRH defines a new path towards NAD+ synthesis relying on the activity of AK. The use of AK for the initial catalysis step and that it acts by increasing NADH might be critical in understanding why NRH action in cultured cells is so vastly superior to all other precursors. Interestingly, as with NMN, NMNH can be used as an effective extracellular NAD+ precursor, but also require dephosphorylation to NRH prior to cellular uptake [265].

Another interesting aspect of NRH is that, unlike NR, it is not degraded in mouse plasma [128]. Accordingly, the intraperitoneal injection of NRH led to larger increases in NAD+ than those observed with NR [128]. Unlike NR, NRH could be detected in circulation after oral administration. More precisely, it was detected in the low micromolar range when gavaged at 250 mg/kg, which is consistent with promoting significant effects on NAD+ levels in cultured cells [128]. To date, two studies in mice support the case that NRH could have therapeutic applications.

NAD Precursors

More on NRH from a bulk manufacturer

You may be able to obtain NRH by shopping around, but it may be awhile before it becomes commonly available on your supermarket shelves.

More on the common precursors NR (nicotinamide riboside) and NMN (nicotinamide mononucleotide):

NAD Intermediates

As outlined above, it is clear that both NMN and NR have beneficial effects in multiple conditions in rodents. Indeed, there are a number of pathophysiological conditions that show significant decreases in tissue NAD+ levels (Table 3). Although both compounds have been tested in some models, no side-by-side comparisons have been conducted between NMN and NR. Therefore, even though both compounds are capable of enhancing NAD+ biosynthesis, there might be certain interesting differences in their effects on these pathophysiological conditions. Additionally, in the vast majority of cases in which NMN and NR are effective, it still remains unclear what downstream mechanisms mediate their beneficial effects. NAD+ is required as a cosubstrate for PARPs, sirtuins, ADP-ribosyl cyclases, and mono-ADP ribosyltransferases, but also serves as a redox cofactor for countless enzymes (Figure 1B). In several cases, deletion of sirtuins has been shown to block key benefits of NAD+ supplementation, supporting a role for these enzymes (Brown et al., 2014; Gomes et al., 2013; Guan et al., 2017; Martin et al., 2017).

NAD Intermediates

Most of the studies on these NAD precursors have been done on animal models, specifically mice of various derivations. Human studies have had mixed results. Since our ignorance is far greater than our knowledge when it comes to this complex topic — as with most complex topics — there is only a small basis for recommending the use of any of the above supplements.

Nevertheless, given the current state of knowledge and ignorance, the potential benefits of supplementation with NAD+ precursors (combined with SIRT boosters such as resveratrol) seem to outweigh the risks — at least in healthy persons past the age of 50.

The least expensive approach to supplementation with NAD+ precursors, to my knowledge, is to take nicotinamide and ribose as separate supplements, but at the same time. The two supplements are absorbed separately and recombined as NR, and then converted to NMN (see picture above). On the other hand, genetic differences between people — and between their microbiomes in the gut — probably cause differences in individual abilities to boost NAD+ levels using the above approach. Everything the gut absorbs goes to the liver, where it is subject to “first pass” metabolic modification and dismantling into component parts.

Keep in mind that some new cancer treatments in development are based upon blocking NAD+ in cancer cells. Too much NAD+ boosting in patients with certain cancers may interfere with the effect of their chemotherapy. In other words, keep up to date with your cancer screening. If you are being treated for cancer with chemotherapy, ask your doctor whether NAD+ boosters could interfere with the treatment. This caution is important for a relative few persons considering supplementation with NAD+ boosters.

As I always say, supplementation with most of the things in vitamin stores will accomplish little more than creating some very expensive urine. Certain supplements, however, may make a big difference in the quality of life for persons with particular morbidities — including the decline in function associated with aging.

Bonus video: One man’s opinion on which is the better precursor of NAD+

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