NMN and NR: the classical NAD+ precursors
Nicotinamide adenine dinucleotide, or NAD+, is an extremely versatile molecule that participates in hundreds of enzymatic reactions in the cell. This versatility makes NAD+ essential for the cell’s well-being, and disturbances in NAD+ metabolism have been linked to a number of prevalent diseases in society, such as diabetes, neurodegenerative diseases, kidney injury, and others.
Since declined NAD+ has been identified in all these diseases, one can easily foresee that NAD+ replenishment could have some kind of therapeutic effect. In fact, several years ago, it was demonstrated that administration of NAD+ precursors (also called NAD+ boosters) such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), led to NAD+ repletion and protected against several diseases in animal models. For example, diabetic mice supplemented with NAD+ precursors show improved insulin sensitivity and glucose tolerance, while mouse models of Alzheimer’s have improved cognitive function.
“Our study establishes reduced nicotinamide mononucleotide (NMNH) as the second member of the new family of reduced NAD+ precursors”
Although these preclinical studies were really promising, the first clinical trials recently showed that the effects of NMN and NR are very mild in humans. NR supplementation, for instance, was able to increase NAD+ levels in blood, but failed to do so in other tissues, such as the muscle. This inefficacy in raising NAD+ might explain why NR has no apparent effect on total energy expenditure, blood glucose or insulin sensitivity in humans.
Many questions arise from these studies. For example, are these NAD+ precursors effective when there is no basal NAD+ deficiency? It does not seem to be the case. Do we maybe need to administrate them for longer periods? Or is it that NAD+ metabolism is so tightly regulated in humans that these molecules are not powerful enough to produce a shift? One thing is clear, we need to look for new molecules to add to the current repertoire of NAD+ boosters if we want to overcome these limitations.
Reduced nicotinamide mononucleotide (NMNH): a new NAD+ booster
In our new study, we identify a reduced form of NMN, termed NMNH, as a new and very effective NAD+ enhancer. First, we develop a method for its synthesis and purification and demonstrate that NMNH is more potent and faster than NMN in several cell lines, especially in brown adipocytes. Moreover, by using several inhibitors of key enzymes of the NAD+ synthesis pathway, we also identify NMNH metabolic route to NAD+, which is different to that of NMN and NR.
NMNH protects against ischemic damage in a cell model of kidney disease
The great efficiency of NMNH to induce NAD+ levels made us think about its possible therapeutic uses. To test this, we used a cellular model for acute kidney injury. These cells, called TECs (Tubular Epithelial Cells), are subjected to low oxygen concentrations, generating a hypoxic status that resembles ischemic (lack of oxygen) damage.
NMNH was also able to potently increase NAD+ in these cells, especially under hypoxic conditions and, more importantly, to reduce the expression of one of the main markers of kidney damage, Kim-1 (kidney injury molecule 1). Interestingly, NMNH significantly increased the expression of the Mitochondrial Transcription Factor A (Tfam), which is known to play a crucial role in mitochondrial activity in TECs, and the antioxidant enzymes peroxiredoxin 1 (Prdx1) and glutathione peroxidase 1 (Gpx1), which are crucially involved in resistance to renal ischemic damage. Finally, we demonstrate that NMNH induces the flux through the pentose phosphate pathway and, at the same time, leads to an enrichment in the number of cells in the S phase of the cell cycle, in which they prepare for the proliferative stage (see article for more details).
Together, our results in kidney cells demonstrate that NMNH supplementation reduces cellular damage and enhances repair by targeting NAD+ regeneration, mitochondrial activity and nucleotide metabolism
NMNH is well tolerated and effectively increases NAD+ levels in mice
At this point, we were already really happy with our results in cells, but we needed to know if NMNH would also work in a whole organism (the idea is to use it in humans in the future, right?). To prove this, we injected C57BL/6N mice with vehicle (PBS), or 250 mg/kg of NMN or NMNH and compared NAD+ levels in different tissues after 24 hours. Strikingly, NMNH administration was able to sustain a 2-fold NAD+ increase in blood for at least 20 hours following the first injection, while NAD+ levels declined back to basal levels just four hours after NMN administration. Other tissues also showed nice increases in NAD+ levels after NMNH administration, as you can see in the figure below.
Altogether, our results in mice demonstrate that NMNH is also an efficient NAD+ booster in vivo, and is able to induce a robust increase in NAD+ levels in a variety of tissues.
The new era of reduced NAD+ precursors
Our study with NMNH in The FASEB Journal and the previous one with a reduced form of nicotinamide riboside (NRH) in Molecular Metabolism, together with reports by others demonstrating that NRH is a superb NAD+ enhancer, open doors for a new generation of highly efficient NAD+-enhancing molecules that could succeed where the classical NAD+ precursors NMN and NR have failed, e.g. boosting NAD+ levels in humans.
New studies will need to shed light on the effectivity of these new compounds to prevent or cure disease in animal models and, what is more important, to bring these compounds to the clinic. The new era of reduced NAD+ precursors is here.
Enhancing NAD 