Accumulating evidence has demonstrated that the levels of NAD+, which is essential for vital activity, declines with age in various organs and tissues throughout the body [10]; thus, intracellular enhancement of NAD+ synthesis helps prevent aging-related diseases such as diabetes, Alzheimer's disease, and cardiopathy [21]. NAD+ is intracellularly generated using tryptophan, niacin, nicotinamide riboside (NR), and NMN [8]. However, these molecules are not effective in increasing NAD+ levels because tryptophan is also used in neurotransmission and protein synthesis [22], and niacin supplementation is associated with side effects, such as flushing and liver damage due to overconsumption [23]. Consequently, research has mainly focused on using NR and NMN as substrates for NAD+ synthesis. Recent studies have demonstrated that NR is mostly decomposed by intestinal bacteria when orally administered to humans [24]; however, NMN is rapidly released into the blood via uptake by intestinal epithelial cells via Scl12a8 [11]. We hypothesized that NMN supplementation increased NAD+ levels more efficiently than that obtained using NR, even when administered intravenously, because Scl12a8 is expressed in almost every cell as well as intestinal epithelial cells.
NMN, which is a precursor of NAD+, has been proven to increase NAD+ levels via uptake into the body in both rodents and humans [13, 25]. Although NMN has been studied using various administration methods such as oral, intraperitoneal injection, and intravenous injection to enhance NAD+ synthesis [8], the efficacy and safety of intravenous administration in humans have not yet been verified. Our study showed that a single intravenous dose of 300 mg of NMN enhanced NAD+ activity in blood cells without affecting these cells, including erythrocytes, leukocytes, and platelets, and major markers in the liver, heart, pancreas, and kidneys.
We hypothesized that NMN-mediated activation of mitochondria relieves fatigue because NAD+ plays an important role in oxidative phosphorylation involved in ATP synthesis in mitochondria [26]. In this study, intravenous NMN administration to healthy subjects showed a tendency for fatigue recovery, although the difference was not significant (Table 1). We expect that a considerable recovery from fatigue will be obtained in studies on intravenous NMN administration in relatively larger cohorts.
NAD+ is involved in the regulation of circadian rhythms, and an increase in intracellular NAD+ levels via intravenous NMN administration is expected to improve sleep quality [20]. Based on the Pittsburgh Sleep Quality Index (PSQI) results, we found that sleep quality improved significantly at one month after intravenous NMN administration (Table 1). We speculate that oral administration of NMN did not significantly improve sleep quality in a previous study [13], whereas intravenous administration improved sleep quality in the present study by increasing NAD+ levels in the suprachiasmatic nucleus of the hypothalamus and spreading throughout the body before NMN could be metabolized in the liver.
Body temperature, blood pressure, pulse, and oxygen saturation were not significantly affected via intravenous administration of NMN (Fig. 1a-d). There was no change in the levels of metabolic markers of sugars and proteins after intravenous NMN administration (Fig. 1e-i); however, there was a significant decrease in TG level even though there was no change in cholesterol level (Fig. 1j-m). Several studies have demonstrated that NAM is produced when NAD+ is used as a coenzyme by sirtuin family proteins after NAD+ is synthesized from NMN [14]. Previous studies have suggested that NAM activates the NA-specific receptor GPR109A when hydrolyzed to yield NA [27]. GPR109A is a predominantly expressed G protein symbiotic receptor in fat cells, and GPR109A is activated in adipocytes via NA to suppress the decomposition of TG and reduce the amount of free fatty acids (FFAs) released into the blood; subsequently, FFA in blood is taken up by the liver and TG is synthesized. The activation of GPR109A by NA leads to the suppression of TG synthesis in blood [28]. Therefore, we speculate that NAM produced as a by-product of intravenously administered NMN metabolism mediated the suppression of TG synthesis. No reduction in TG levels was observed in previous clinical trials on oral administration of NMN [13], suggesting that NMN may be metabolized via different pathways between oral and intravenous administration. This is thought to be caused by NMN being metabolized in cells throughout the body by avoiding the first passage through the liver via intravenous administration; the proportion of the produced GPR109A-bound NA on the membrane of adipocytes is increased. A previous study reported that the increase in TG levels in the plasma and liver of mice deficient in adipocyte-specific NAMPT1, an enzyme that synthesizes NMN from NAM, indicates that increased intracellular NMN synthesis plays a critical role in decreasing TG levels. Therefore, NMN is involved in the reduction of TG levels [29].
There was no noticeable change in levels of metabolic markers of any of the organs after intravenous NMN administration (Fig. 2–3). The amount of NAD+ was significantly increased at NMN administration to 2 h after administration compared to that at before administration, and the total NAD+ levels showed a significant increase except at 3 h after administration (Fig. 4a-b). In contrast, since the amount of NADH had a large experimental error and an accurate value could not be detected (Fig. 4c), the ratio of NAD+ to NADH could not be determined accurately (Fig. 4d). The current clinical trial on intravenous NMN administration demonstrated that NMN helped increase the amount of NAD+ in blood cells; however, the amount of NADH should be reevaluated in a relatively larger cohort. Although the experimental error was large and no significant increase was observed (Fig. 4e-f), nuclear SIRT1 tended to be activated 30 min to 1 h after NMN administration, similar to the increase in NAD+ level (Fig. 4e).
In conclusion, our clinical study demonstrated that NMN administration at 300 mg is tolerated by humans because it does not cause significant damage to blood cells, liver, pancreas, heart, and kidneys when injected intravenously, and effectively increases the amount of NAD+ in blood cells. In addition, intravenous administration of 300 mg of NMN may help prevent aging-related diseases such as diabetes, Alzheimer’s disease, and heart disease, because our findings suggest that SIRT1 may be activated. Future studies should establish activation conditions for sirtuin family proteins, including SIRT1, following intravenous administration of NMN, and verify the safety of intravenous administration of NMN several times over a long period of time.