Strategies to restore levels of the enzyme cofactor nicotinamide adenine dinclueotide (NAD) late in life to maintain health by treatment with NAD precursors, such as nicotinamide mononucleotide (NMN), represent an exciting area of research in aging and age-related diseases. A study in Nature Metabolism provides an answer to the hotly debated yet fundamental question: how NMN actually gets into cells.
The enzyme cofactor NAD is essential for life. Originally discovered in 1906 as an accelerator of yeast fermentation, NAD is once again at the forefront of biology, thanks largely to research, again in yeast, that has identified the oxidized form of NAD (NAD+) as a signalling molecule that dictates the health and lifespan of eukaryotes1,2,3. In yeast, increasing NAD levels through treatment with metabolic precursors extends lifespan, and in aged mice, this extends lifespan and improves motor coordination, eye function, bone density, insulin sensitivity, liver and kidney function, physical endurance, muscle strength, and the function of stem cells and mitochondria2,3.
These findings have raised an important question: how do cells take up these precursors to make NAD?4,5 In this issue of Nature Metabolism, Grozio et al.6 identify the transporter for NMN, the immediate precursor to NAD, helping to explain how mammals absorb and manufacture NAD.
There are three main approaches to raising NAD+ levels in mammals: inhibiting NAD destruction by CD38 (ref. 7) or SARM1 (ref. 8); inhibiting the enzyme ACMSD, which siphons off NAD precursors in the de novo NAD synthesis pathway9; and providing NAD+ precursors such as nicotinamide riboside (NR)10 and NMN3.
Once taken up by cells via equilibrative nucleoside transporters, NR is phosphorylated by nicotinamide riboside kinase (NMRK1 and NMRK2 (NRK1/2)) to generate NMN, which is then immediately converted to NAD+ by nicotinamide mononucleotide adenylyltransferases (NMNATs; Fig. 1)11. NR and NMN have been used in preclinical animal studies5, and NR raises NAD+ levels in blood lymphocytes of human subjects10.
A question that has been debated for years is whether NMN is simply a pro-drug of NR. For NMN to enter cells, it was thought to require a dephosphorylation step on the extracellular surface of cells to convert it to NR before being taken up by the equilibrative nucleotide transporters (ENTs) and then re-phosphorylated by NRK back into NMN11. This view is consistent with the metabolism of nucleoside-based drugs, such as HIV inhibitors, which are usually first dephosphorylated in the gut. It remains unclear whether the rapid kinetics of NMN uptake exhibited by certain cell types can be explained by a similar mechanism3.
This new study from Grozio et al.6 identifies a previously characterized amino acid and polyamine transporter called Slc12a8 (ref. 12) as an NMN transporter. Slc12a8 has a number of surprising attributes. It requires sodium and not chloride for NMN co-transport and is highly selective for NMN, excluding even nicotinic acid mononucleotide (NaMN), which differs from NMN by only one atom.
It is important to note that the discovery of an NMN transporter by no means diminishes the importance of uptake via dephosphorylation11. It does, however, enrich our knowledge by providing a new mechanism through which the absorption and distribution of NAD precursors might be differentially regulated. For example, the authors find that Slc12a8 is highly enriched in the small intestine of mice, with expression that is at least 100-fold higher in this tissue than in fat or brain tissue. Interestingly, the expression of Slc12a8 increases in the intestines of old mice as NAD levels decline4,13, suggesting that upregulation during aging is a compensatory mechanism.
The authors speculate that Slc12a8 in the gut endothelium serves to take up NMN from naturally occurring dietary sources, such as fruits, vegetables, and milk, or from the breakdown of NAD+. But the amount of NMN needed to raise NAD in a mouse or human is far beyond what is available naturally. Both NMN and NR are present in food3,14 but at concentrations of less than 1 mg per kg of food, whereas hundreds of milligrams per dose are needed to raise NAD+ in humans3. And given that cells make their own NAD from tryptophan, it is unclear whether dietary uptake of NMN can meaningfully influence NAD+ levels or whether Slc12a8 affects NAD synthesis beyond the liver, where NMN is primarily metabolised15. One intriguing possibility is that NMN could be produced by the microbiome. If so, NMN transport may prove more important than the low NMN levels in food imply.
There is no doubt that this new work will spark a debate about the relative contributions of NMN transport and the previously described mechanism11, which could be addressed by head-to-head comparisons of NAD precursors and by following the in vivo metabolism of isotopically labelled NAD precursors in knockout animals. Given that NAD precursors are sold as supplements and are under development as pharmaceuticals, this new understanding of NAD physiology is an important addition to the field, but also raises new questions about where NAD precursors are taken up and processed, and where they naturally come from.