Multiple pathways for NAD+ uptake by cells – regulated by SIRT1 – ALIVE BY SCIENCE – Bioavailable NAD+ Boosters

Multiple pathways for NAD+ uptake by cells – regulated by SIRT1

SIRT1-dependent restoration of NAD+ homeostasis after increased extracellular NAD+ exposure

In this study, researchers investigated how intracellular NAD+ (iNAD) levels are regulated in several different types of healthy cells exposed to extracellular NAD+ (eNAD).

They found that iNAD+ levels reached the maximum level after 8 hours of continued exposure, after which those pathways were all downregulated.

Surprisingly, iNAD+ levels returned back to baseline levels at 24 hours, even though eNAD+ levels were still high.

In effect, when given a good supply of eNAD+, cells used all pathways to take in more NAD+.  Once iNAD+ was sufficient, pathways for importation of more NAD+ were shut down.

They found eNAD+ more than doubled the quantity of iNAD+ in the cells through 3 preferred pathways*.

  • direct import across the cell membrane of intact NAD+
  • conversion of NAD+ to NMN, then imported by the NMN transporter Slc12a8
  • further conversion of NMN to NR, then imported by NRK1

In the bloodstream, NAD+ is broken down to NMN, NR and NAM.

Once inside a cell, this process is reversed, creating NAD+, through the salvage cycle.

*NAM readily crosses the cell membrane and is metabolized to NMN, but requires NAMPT, which is the rate limiting step in the NAD+ salvage pathway and one reason why NAD+ or the higher level precursors are preferable to NAM.

SIRT1 is key regulator in maintaining iNAD+ levels

Researchers found the key regulator of this process was SIRT1.

SIRT1 senses NAD+ levels inside the cell.  As iNAD+ levels rise, so does SIRT1 activity inside the cell.  The increased SIRT1 activity signals the cell to downregulate production of enzymes used to import NAD+.  CD73, Slc12a8, and NRK1 levels decrease.

Intervention to silence SIRT1 activity resulted in 4x increase in CD73 and Slc12a8 as the cell believes it is starving for NAD+ and reaches out to grab more NAD+ and NMN from outside the cell, indicating the importance of this pathway for regulating iNAD+.

Slc12a8 utilized for NMN transport, and downregulated when not needed

Previous studies have shown the Slc12a8 enzyme is upregulated in distressed cells are low in NAD+ (r).

This study found that:

Turning off SIRT1 resulted in 4x increase in Slc12a8 as the signalling is disrupted and cells believe NAD+ levels are insufficient.

Silencing SIRT1 suffices to prompt a 4-fold increase of CD73 and Slc12a8 transcripts in resting cells.

Slc12a8 does import significant quantities of NMN

Notably, silencing NMN transporter Slc12a8 reduced the uptake of radioactivity when nicotinamide labeled [14C] NAD+ was added extracellularly indicating a contribution of this transporter on uptake of NAD+.

Slc12a8 is downregulated when cells had high iNAD+ levels.

eNAD+ exposure reduced expression of the ectonucleotidase CD73, the nicotinamide adenine mononucleotide (NMN) transporter Slc12a8, and the nicotinamide riboside kinase NRK1.

We found that transcript levels of the NMN transporter Slc12a8 were already reduced in cells after 12h eNAD+ exposure and similar to those of CD73 recovered upon washing (Fig. 3C).

Of note, we report that Slc12a8 silencing reduced [14C]-nicotinamide-labeled NAD+ uptake, confirming the role of this transporter in NAD+ uptake and its contribution to restoring NAD+ contents to control levels.

Slc12a8 is downregulated after exposure to NMN for 24 hours

We found that among NRK1, CD73, ENT1 and Slc12a8, only the expression of the latter is reduced by 24h NMN.

NAD+ crosses the cell membrane intact

This study agrees with others that show NAD+ can cross the cell membrane intact, by an as yet undiscovered transporter.

Several studies providing evidence that NAD+ crosses the plasma membrane of mammalian cells uncleaved.

The chart at left shows NAD+ increase in control group and those given AMPCP which prevents NAD+ from being metabolized to the precursors.

Our data also suggest that eNAD+ can cross the plasma membrane uncleaved. Indeed, in the presence of an extracellular concentration of AMPCP leading to an almost complete CD73 inhibition (see Fig. 2H), eNAD+ can still prompt significant iNAD+ increases.

iNAD+ increase is not dependent on NAM and NR

Interestingly, these cells do not totally depend on importation of NAM and NR to increase iNAD+.

NR is imported into cells by NRK1, and NAM depends on NAMPT.

The chart at left shows the increase in iNAD+ is lower when NRK1 and NAMPT are silenced, but not eliminated.

This shows again that  other pathways of iNAD+ increase are also in effect.

We found that NAMPT or NRK1 silencing did not prevent iNAD+ increase after eNAD+ exposure (Fig. S2 D and E), supporting the hypothesis that, at least in part, NAD+ was transported intact.

The CD73-independent uptake of radioactivity in cells exposed to labeled NAD+ might be ascribed to intact NAD+ transportation.

 

Different cell types

The majority of this research focused on human liver cells, however, they found the same effect in several different cell types.

We also evaluated the effects of eNAD+ over time in SHSY-5Y (neuroblastoma), HT29 (colorectal adenocarcinoma) and RPTEC (renal proximal tubule epithelial) cells, and found similar kinetics of iNAD+ contents upon NAD+ exposure. (Fig. 1A)

We investigated the kinetics of iNAD+ levels in different cell types challenged with prolonged exposure to extracellular NAD+ (eNAD+). Surprisingly, we found that after the initial increase, iNAD+ contents decreased back to control levels (iNAD+ resetting).

 

When iNAD+ levels are high, all NAD+ import pathways are downregulated

According to the authors:

Surprisingly, we found that after the initial increase, iNAD+ contents decreased back to control levels (iNAD+ resetting).

Indeed, eNAD+ exposure reduced expression of the ectonucleotidase CD73, the nicotinamide adenine mononucleotide (NMN) transporter Slc12a8, and the nicotinamide riboside kinase NRK1.

Several studies providing evidence that NAD+ crosses the plasma membrane of mammalian cells uncleaved.

 

eNAD+ is beneficial to health

The authors of this research point out that numerous studies in mice have shown a benefit to cellular health from supplying supplemental NAD+.

We previously reported that iNAD+ contents increase upon a brief exposure of HeLa cells to eNAD+ and that this increase confers significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide or N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) [32]. These findings are in line with numerous reports also showing cytoprotective properties of eNAD+

although a putative plasma membrane NAD+ transporter is yet to be identified, several membrane carriers and enzymes have been involved in the uptake of NAD+-cleaved products [42, 43, 44]. Specifically, eNAD+ is cleaved into NMN and AMP by CD73 [38,39] and by CD203a (also known as PC-1) [58], or converted into adenosine diphosphate ribose (ADPR) and NAM by CD38 and its paralog CD157 [40,59,60].

Regardless of the mechanisms underpinning NAD+ uptake, various studies report that the increased availability of eNAD+, both in vitro and in vivo, prompts metabolic and signaling responses that, as a whole, improve bioenergetics and resistance to stress.

 

Conclusion

This study found that eNAD+ is efficiently imported by several pathways, and regulated by a robust system that prevents excessive NAD+ buildup.

The SIRT1 regulation of iNAD+ levels demonstrated in this research should be comforting to those worried about potential negative side effects from supplementation with NAD+ and its immediate precursors, as the cells studied here have an efficient means of limiting importation of excess NAD+ and precursors.

While the NR pathway is important, it is one that both NAD+ and NMN can utilize.  NR does not utilize the other 2 pathways as it is quickly metabolized to NAM outside of cells.

Numerous studies have clearly demonstrated that NR is unstable in the bloodstream . While it is useful as an additional pathway for the last step, it is not the best option for supplementation vs NMN and NAD+ which are far more stable in the bloodstream and have multiple pathways to increase intracellular NAD+.

Since NAD+ is by far the most stable of these metabolites in the bloodstream and is able to utilize these pathways to increase intracellular NAD+, this study lends significant support to the use of NAD+ supplementation (vs NMN or NR) as a means of restoring NAD+ inside of cells.