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NMNH acts as a potent NAD+ enhancer in vitro

 

Once we had established a reliable synthesis and purification method and correctly identified the compound obtained as NMNH, our next aim was to test if this reduced precursor was able to act as an NAD+ enhancer in cells. To do so, we supplemented AML12 mouse hepatocytes with different concentrations of either NMN or NMNH. At every concentration tested, NMNH caused a much more significant increase in cellular NAD+ levels than NMN (Figure 2A). In fact, NMNH was able to significantly increase NAD+ at a 10 times lower concentration (5 µM) than that needed for NMN, and reached a plateau at supplementation concentrations of 500 µM. At this concentration, NMNH achieved an almost 10-fold increase in the NAD+ concentration, while NMN was only able to double NAD+ content in these cells, even at 1 mM concentration. NMNH also proved to be a faster precursor, resulting in a significant increase in NAD+ levels within 15 minutes (Figure 2B). Interestingly, upon NMNH supplementation, NAD+ steadily increased for up to 6 hours and remained stable for 24 hours, while NMN reached its plateau after only 1 hour, most likely because the NMN recycling pathways to NAD+ had already become saturated.

 

Supplementation with NAD+ enhancers has emerged as a valid strategy to treat or alleviate features of metabolic and age-related diseases in several preclinical models. To assess the in vivo effects of NMNH in comparison with NMN, we injected C57BL/6N mice with vehicle (PBS) or 250 mg/kg of NMN or NMNH, which was followed by sequential blood sampling (at 1, 4, 20 hours) and, after 24 hours, sacrifice for tissue collection.

 

NMNH showed a potent NAD+-boosting effect in blood, efficiently increasing NAD+ levels after 1 hour and to a much higher extent than NMN (Figure 6A). Strikingly, while blood NAD+ content after NMN supplementation declined to basal levels after just 4 hours, NMNH administration sustained a 2-fold NAD+ increase for at least 20 hours following the first injection. This was also reflected in the blood samples taken 4 hours after the second IP injection, as an increase in NAD+ was only detected in NMNH-treated animals (Figure 6A).

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  • To further prove that NMNH can act as a potent NAD+ booster in vitro, we supplemented different murine (AML12 and T37i) and human (HepG2, skin fibroblasts, SY5Y, and HeLa) cell lines with the optimal dose of 500 µM for 24 hours (Figure 2D). NMNH supplementation increased NAD+ content in all these cell types and was more potent than NMN at the same concentration. While NMN increased NAD+ levels 1.3-2.4-fold, NMNH outperformed NMN and was able to increase NAD+ from 2.5-fold in HepG2 hepatocytes, the least responsive cell line, up to 19-fold in the brown adipocytes cell line T37i (Figure 2D). NMNH was also a remarkably potent NAD+ booster in AML12 hepatocytes (9.3-fold) and fibroblasts (8-fold).

    To analyze the effect of NMNH administration on other NAD+-related metabolites, we performed targeted semiquantitative metabolomics in AML12 hepatocytes. After 24 hours, NMNH not only led to an increase in NAD+ levels, but also an enhancement in NADH, although the increase in NADH was less pronounced (~2.8-fold) than that of NAD+ (~9-fold) (Figure 2E). This result confirms that, as previously described for NRH,45 NMNH increases NAD+ content over NADH, leading to a higher NAD+/NADH balance. In accordance, we could not detect any changes in cellular α-hydroxybutyrate (α-HB) content (Figure 2E), a recently described marker for increased reductive stress.47

    NMNH supplementation was also accompanied by an increase in NADP+ and NADPH levels, most likely due to the activity of NAD+ kinase over elevated NAD+.48

    Strikingly, NMNH itself could only be detected when it was administered to cells, suggesting that under physiological conditions, this molecule is absent or not detectable with our experimental setup. This was also the case for NRH, which increased only upon NMNH supplementation, suggesting a common metabolism for these two molecules.

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