NMNH Potently Enhances NAD+ and Suppresses Glycolysis, the TCA Cycle, and Cell Growth

NAD+ is essential for biological processes such as metabolism and DNA repair, and its decline is associated with age-related diseases. Supplementation with NAD+ precursors, such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), has been shown to increase cellular NAD+ levels and confer health benefits. Recent studies suggest that the reduced form of NR, NRH, is a better NAD+ booster than NR or NMN, and can increase resistance to cell death caused by genotoxins. Additionally, NRH is orally available and has been shown to prevent acute kidney injury in mice. So researchers explored whether the reduced form of NMN(NMNH) can increase cellular NAD+ levels and regulate biological processes.

NMNH Increased Cellular NAD+ Levels Both In Vitro and In Vivo

The researchers conducted NMNH (100 μM) treatment and found that increased the level of cellular NAD+ by 5-fold in HepG2 cells, whereas 100 μM NMN only slightly increased the level of NAD+, as determined by mass spectrometry. (As shown on figure 1-A)
Author treated C57BL/6J male mice with 340 mg/kg NMN or NMNH via intraperitoneal injection and measured liver NAD+ concentrations by mass spectrometry. It featured that found that the liver NAD+ level was 4-fold higher in NMNH-treated mice than in PBS-treated mice (As shown on figure 1-C). Additionally, the liver NAD+ level in NMNH-treated mice was 1.5-fold higher than in NMN-treated mice.

Figure 1

The experiment results indicated that NMNH is an efficient NAD+ enhancer, and is a more potent in vivo NAD+ enhancer than NMN.
But while treated C57BL/6J male mice with 50, 100, 500, or 1000 mg/kg NMNH via intraperitoneal injection every other day for a week and found that the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were not elevated (As shown on figure 2), which suggested that NMNH was not liver-toxic at high dosage.
It demonstrated that NMNH treatment increased mouse liver NAD+ contents in a dosage-dependent manner, and 1000 mg/kg NMNH treatment increased the mouse liver NAD+ level.

Figure 2

NMNH Inhibited Glycolysis and the TCA Cycle

The author explores the NMNH transform routine to NAD+ as shown on figure 3.

Figure 3

And in terms of the experiment result, NADH levels were 2.5-fold higher in NMNH-treated HepG2 cells than in untreated and NMN-treated cells. Figure 4 also uncovered that 340 mg/kg NMNH treatment increased the NADH level by 3-fold in the mouse liver, while 340 mg/kg NMN treatment increased the NADH level by 1.7-fold.

Figure 4

However, NMN treatment revealed distinct regulation of glycolysis and the TCA cycle. NMN only induced a mild decrease of 2PG/3PG and PEP in glycolysis (Figure 3E) and almost no observable reduction of the TCA cycle in cells (Figure 3F). These results suggested that NMNH causes very significant suppression of glycolysis and the TCA cycle, while NMN also hindered the glycolysis but to a much milder extent.

Figure 5

NMNH Repressed Cell Growth

Data-dependent quantitative proteomic analysis was performed to identify differentially expressed proteins (DEPs) of HepG2 under 1 mM NMNH treatment for 12 h.  And to further examine the effects of NMNH on cell growth, researchers treated 786-O cells with different concentrations of NMNH. 786-O cells, derived from a clear cell renal cell carcinoma patient, exhibit a typical Warburg phenotype and their growth is dependent on glycolysis.

Figure 6

It revealed that NMNH inhibited cell growth in 786-O cells at 50 μM, whereas 250 μM NMNH was needed to effectively inhibit cell growth in HK-2 cells (as shown in figure 6), which was an immortalized proximal tubule epithelial cell line from a normal adult human kidney.

Conclusion

NAD+ precursors in reduced form (NMNH and NRH) are better NAD+ enhancers than the corresponding ones in oxidized form both in vitro and in vivo. Meanwhile, we confirmed that long-term administration of NMNH is safe to mice. NMNH effectively increased cellular NAD+ content and this process was mainly dependent on NMNAT. NMNH is a potent NAD+ enhancer in vitro and in vivo. NMNH also significantly increased cellular NADH levels, repressed glycometabolism, and inhibited cell growth.

Reference: Liu, Y., Luo C., etal, Reduced Nicotinamide Mononucleotide (NMNH) Potently Enhances NAD+ and Suppresses Glycolysis, the TCA Cycle, and Cell Growth, Promote research, 2021