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Bacteria in Gastrointestional Tract Metabolize NMN and Impact NAD+ Metabolome

This recently published research by Drs. Lindsay Wu, David Sinclair and others examined the pathways that oral NMN takes to becoming NAD+ in mice. It confirmed some aspects of earlier research, and also made some surprising new findings.

https://assets.researchsquare.com/files/rs-87047/v1_stamped.pdf

Several experiments were completed that all measured NAD+ metabolites in the Gastrointestinal Tract (GIT) and Liver, 4 hours after a single dose of either 50mg/kg or 500 mg/kg by oral gavage.

In addition to the control and NMN group, they also performed the same measurements in mice given antibiotics to remove bacteria from the Gastrointestinal Tract.  Some important findings were:

  • NMN is metabolized (deamidated) to NaMN and NaR by bacteria in the GIT
  • Treatment with antibiotics greatly INCREASED levels of NAD+ metabolites in GIT
  • Treatment with antibiotics DECREASED levels of NAD+ metabolites in the Liver.
  • Little NMN is metabolized directly to NAD+. 
  • Uptake of NMN by SLC12A8 enzyme may be limited. 
  • Confirms earlier work that little NMN makes it to bloodstream intact

Caveats:

  • Limited by single time point collection at 4 hours.
  • Experiments with labelled NMN used 50 mg/kg body weight and a very small number of animals, with significant discrepancies between experiments.

The cost for multiple labeling of the NMN molecules precluded the use of multiple time points.

Limited availability of isotope labelled material meant that this study used a single time point, rather than a time course which also encompassed very early time points, possibly missing the minute-order kinetics of direct NMN transport that were previously reported. (1,2)

For these reasons as well as the complexity of the isotope labeling experiments, we focus on the first experiment (figure 1) that was able to use 500 mg/kg dosage, but was also limited by a single collection time of 4 hours.

NMN is metabolized (deamidated) to NaMN and NaR by bacteria in the GIT

 

Bacteria in GIT metabolize NMN to deamidated forms of NMN (NaMN) and NR (NaR).

This chart found levels of NaR and NaMN in animals treated with NMN (blue) greatly increased vs the control group. (grey)

Those treated with antibiotics alone (yellow) or NMN + antibiotics (red) did not show an increase in NaMN, and showed a large decrease in NaR.

Takeaway: Antibiotics eliminated the bacteria in GIT which metabolize the NMN to deamidated forms of NMN and NR.

 

 

Treatment with antibiotics greatly INCREASED levels of NAD+ metabolites in GIT

 

 

As shown above, treatment with antibiotics eliminates the conversion of NMN to NaMN and NR, preserving the amidated forms in the GIT.

Levels of NR with treatment of NMN + antibiotics (red) are particularly striking.

However, it seems these massive increases in NR (and NMN) are not fully utilized to increase NAD+  in the GIT.  Levels of NAD+ are doubled (red), but are not commensurate with the increase in NR or NMN after treatment with antibiotics.

Takeaway: NMN + antibiotics result in doubling of NAD+, and massive increase of NR and NMN in GIT.

Treatment with antibiotics DECREASED levels of NAD+ metabolites in the Liver.

 

 

 

Another surprising finding is the result of antibiotics treatment on levels of NAD+ metabolites in the liver shown here.

Although NR levels in the chart above were massively increased, they did not result in an increase of any NAD+ metabolites in the liver. In fact, NR, NMN, and NAD+ levels were significantly LOWER in those animals treated with antibiotics alone (yellow), or, antibiotics + NMN (red).

This chart does show NAD+ levels in the liver approximately doubled after one dosage of NMN alone (blue), which matches up with previous studies.

Takeaway:  Elimination of bacteria appears to result in LOWER levels of NAD+ in the liver.

Little NMN is metabolized directly to NAD+.

Rather than direct incorporation via the NAD+ salvage pathway, they found a large percentage of NMN is broken down (deamidated) to the NAD+ metabolites NaMN and NaR, which created NAD+ through the de novo pathway.

Here, we show that NMN can undergo direct deamidation and incorporation via the de novo pathway, which is in part mediated by the gut microbiome.

This suggests that exogenous NMN impacts the NAD metabolome through indirect means, rather than through its direct incorporation.

Uptake of NMN by SLC12A8 enzyme may be limited.

NMN can be transported directly to NAD+ by SLC12A8, or dephosphorylation of NMN into NR by the CD73 enzyme that resides on the surface of cell membranes.  This study used 6 or 7 radioactive isotopes to trace the pathways taken to NAD+ inside cells.

For intact NMN that was not already metabolized by bacteria in the GIT, they found only 5% utilized the SLC12A8 enzyme, with the rest being dephosphorylated to NR at the cell membrane.

Only around 5% of the NMN pool was M+7 labelled.

An important caveat is the single collection time at 4 hours was long past when SLC12A8 transport has been found in other studies. (1,2)

Confirms earlier work that little exogenous NMN or NR makes it intact to bloodstream

The abundance of partially labelled NAD+ with labelling at the Nam position only is consistent with previous findings that orally delivered NMN and NR undergo cleavage at the glycosidic bond to release free Nam, with only a small proportion of orally delivered material being incorporated into tissues intact (3)

Conclusion

NAD+ metabolism is quite complex and there is a lot going on that is difficult to separate out to single pathways.  This study actually raises more questions than it answers, but we must leave many of those out to keep this review easily understood.  Researchers have a lot more to uncover.

It does confirm that little exogenous NMN (and NR) make their way to the bloodstream intact, and that bacteria play a role in regulating the uptake of NMN to NAD+, at least in mice.

It is not clear if entirely removing or bypassing the bacteria is beneficial or not. The massive increase in NAD+ metabolites in the GIT are not reflected in the Liver.