Today, we are going to take a look at the topic of NAD+, its precursor, nicotinamide mononucleotide, and the debate surrounding the ability of these molecules to pass through the cell membrane.
What is NAD+?
Nicotinamide adenine dinucleotide (NAD) is a coenzyme encountered in all living cells. It is a dinucleotide, which means that it consists of two nucleotides joined via their phosphate groups. One nucleotide contains an adenine base, and the other contains nicotinamide, and it is this structure that gives NAD its name.
In metabolism, NAD helps to facilitate redox reactions, carrying electrons from one reaction to another. This means that NAD can be found in two forms within the cell; NAD+ is an oxidizing agent that takes electrons from other molecules in order to become its reduced form NADH, which can then become a reducing agent that donates the electrons it is carrying. This movement from one place to another of electrons is one of the key functions of NAD, though it also has many other cellular functions.
Not only is NAD+ a redox cofactor, but it is also a critical signaling molecule that controls cell function and survival in response to environmental changes such as nutrient intake and cellular damage. Age-related changes to the level of NAD+ in the cell influences mitochondrial function, nutrient sensing and metabolism (chemically processing and using food), redox reactions, circadian rhythm, immune and inflammatory responses, DNA repair, cell division, protein-to-protein signaling, chromatin, and epigenetics (changes in genes expression).
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 including nicotinamide mononucleotide (NMN) and Nicotinamide riboside (NR). These different pathways ultimately feed into a salvage pathway (bottom right of the diagram below), which recycles them back into the active NAD+ form.
Can NAD+ or NMN pass through the plasma membrane?
Recently, there has been some debate in academia regarding the ability of nicotinamide adenine dinucleotide (NAD+) and one of its precursors, nicotinamide mononucleotide (NMN), to pass through the plasma membrane. It has been suggested that NMN cannot pass through the plasma membrane. However, there are some issues with this, given that there is research suggesting that this is not always the case and that NMN and/or NAD+ can cross the membranes of at least some cell types.
As far back as 2009, there is data that throws doubt on this assumption in the publication: Detection and pharmacological modulation of nicotinamide mononucleotide (NMN) in vitro and in vivo .
Also, evidence that intracellular NMN contents promptly increase when the nucleotide is added to the culture media indicates that plasma membrane is permeable to this nucleotide. These findings suggest that the pharmacologic effects of exogenous NMN in cultured cells and mice are due to cellular uptake and changes in NAD contents.
The matter is far from settled, as we can see from what Dr. David Sinclair says in his recent review Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence .
Whether or not NMN is taken up by a transporter is currently the subject of debate [3-4]. Brenner, Cantó and colleagues argue that NMN is not taken up quickly enough to invoke the presence of a transporter and that both NAD+ and NMN undergo extracellular degradation to generate permeable precursors that can be taken up by cells .
However, things are not that clear, and it is plausible that there could be specificity of cell types and NAD+ precursors. It could be the case that some cell types readily take up NAD+ or NMN and some do not; thus, finding out which do and do not should be the priority here. It is not at all unusual for such specificity to be encountered in biological systems, so this is something that needs to be determined during human clinical trials.
On the other hand, Imai argues that this is likely a cell-type-specific phenomenon and that some cell types can rapidly take up NMN . If so, the identification of the putative transporter will help resolve the debate and help identify which cell types and tissues are able to transport NMN across the plasma membrane. Additional studies with isotopically labeled NAD+ precursors to trace the uptake and metabolism of these molecules should help answer these questions.
Dr. Sinclair also mentions here the possibility that there are as yet undiscovered transporters that facilitate the passage of NAD+ and NMN into the cell via the plasma membrane. Transporters (or membrane transport proteins) are proteins involved in the transport of ions, molecules, and macromolecules and they exist within and span the cell membrane allowing them to transport ions and molecules through them.
Transporters play an important role in cell survival, as these proteins make the interaction between the external and internal environment of a cell possible, importing and exporting substances in and out of the cell. In this case, Dr. Sinclair is suggesting that there are likely additional undocumented transporters that can facilitate the movement of NAD+ and NMN into the cell waiting to be discovered.
Earlier research also supports the idea that NAD+ can cross at least some cell membranes in certain types of cells, in this case, cardiomyocytes likely via an undocumented transporter . In this 2009 paper, we note the following section about extracellular NAD and its ability to enter the cell:
Exogenous addition of NAD was capable of maintaining intracellular levels of NAD and blocking the agonist-induced cardiac hypertrophic response in vitro as well as in vivo.
Bruzzone et al. have shown that connexin 43 (Cx43) channels are permeable to extracellular NAD . Because cardiomyocytes express a large amount of Cx43 channels, we tested the effect of exogenous NAD in the presence of a Cx43 channel blocker, carbenoxolone. The results showed that in the presence carbenoxolone NAD treatment failed to block agonist-induced ANF release from nuclei, thus demonstrating that exogenous NAD is likely to enter into cardiomyocytes via the Cx43 channels.
There is also this from the 2011 paper Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis which again suggests extracellular NAD crosses the plasma membrane directly under as yet unknown conditions :
Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.
In the present study we report that exposure to eNAD substantially increases the dinucleotide cellular pool, suggesting plasma membrane permeability.
In conclusion, the present study furthers our understanding of the biochemistry of NAD, underscoring its pharmacotherapeutic potential. Development of strategies able to increase the intracellular pool of NAD in specific cellular compartments may represent a pharmacological challenge for the next future.
We see additional evidence that there may be a currently unknown transporter in the 2018 publication Nicotinamide adenine dinucleotide is transported into mammalian mitochondria :
Here we present evidence that mitochondria directly import NAD.
Taken together, our experiments confirm that despite the lack of any recognized transporter, mammalian mitochondria, like their yeast and plant counterparts, are capable of importing NAD.
At least two studies have previously reported evidence for uptake of NAD, leading the authors to propose that intact NAD crosses the plasma membrane and subsequently enters the mitochondria directly.
This observation suggests that a mitochondrial transporter for NMN may also await discovery.
In summary, we show that mammalian mitochondria are capable of directly importing NAD (or NADH). This finding strongly suggests the existence of an undiscovered transporter in mammalian mitochondria.
Once again this is further support that there are transporters awaiting discovery that do facilitate the movement of NAD+ and NMN directly into the cell in at least some types of cells.
Taken together, this data suggests that the uptake of NAD+, NMN, and other precursors may be cell-type dependent and reliant on as yet unknown transporters; therefore, understanding these cell-specific interactions relies on further human studies. It may even turn out to be the case that for NAD+ repletion, a combination of precursors may be the solution rather than simply just one. Like many things concerning the biology of aging, things are rarely black and white, and this is why more research is important.
While the currently published studies on NMN have been only conducted in mice, the Sinclair lab is currently conducting human clinical trials at Brigham and Women’s Hospital, so, hopefully, we should have some published data available soon.
You may also be interested to learn that we are hosting the NAD+ Mouse Project by the David Sinclair Lab over at Lifespan.io, our research crowdfunding platform. Launched on September 18th, this project will attempt to discover more about NAD+ biology and its effects on lifespan. If we can raise the funds to help the lab start running this experiment, we are a step closer to answering some of the questions raised here.
 Formentini, L., Moroni, F., & Chiarugi, A. (2009). Detection and pharmacological modulation of nicotinamide mononucleotide (NMN) in vitro and in vivo. Biochemical pharmacology, 77(10), 1612-1620.
 Rajman, L., Chwalek, K., & Sinclair, D. A. (2018). Therapeutic potential of NAD-boosting molecules: the in vivo evidence. Cell metabolism, 27(3), 529-547.
 Mills, K.F., Yoshida, S., Stein, L.R., Grozio, A., Kubota, S., Sasaki, Y., Redpath, P., Migaud, M.E., Apte, R.S., Uchida, K., et al. (2016). Long-term administration
of nicotinamide mononucleotide mitigates age-associated physiological decline in mice. Cell Metab. 24, 795–806.
 Ratajczak, J., Joffraud, M., Trammell, S.A., Ras, R., Canela, N., Boutant, M., Kulkarni, S.S., Rodrigues, M., Redpath, P., Migaud, M.E., et al. (2016). NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells. Nat. Commun. 7, 13103.
 Pillai, V. B., Sundaresan, N. R., Kim, G., Gupta, M., Rajamohan, S. B., Pillai, J. B., … & Gupta, M. P. (2010). Exogenous NAD blocks cardiac hypertrophic response via activation of the SIRT3-LKB1-AMP-activated kinase pathway. Journal of Biological Chemistry, 285(5), 3133-3144.
 Bruzzone, S., Guida, L., Zocchi, E., Franco, L., & De Flora, A. (2001). Connexin 43 hemi channels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells. The FASEB Journal, 15(1), 10-12.
 Pittelli, M., Felici, R., Pitozzi, V., Giovannelli, L., Bigagli, E., Cialdai, F., … & Chiarugi, A. (2011). Pharmacological Effects of Exogenous NAD on Mitochondrial Bioenergetics, DNA Repair and Apoptosis. Molecular pharmacology, mol-111.
 Davila, A., Liu, L., Chellappa, K., Redpath, P., Nakamaru-Ogiso, E., Paolella, L. M., … & Baur, J. A. (2018). Nicotinamide adenine dinucleotide is transported into mammalian mitochondria. Elife, 7, e33246.