Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

Abstract

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

Welcome to the Family: Identification of the NAD+ Transporter of Animal Mitochondria as Member of the Solute Carrier Family SLC25

“…With regard to the pyridine base, NAD + can be synthesized from several different precursors in animals: nicotinamide (Nam) and nicotinic acid (together known as vitamin B3), tryptophan, and nicotinamide riboside (NR). They are obtained from diet and imported into cells by various SLC transporters (SLC5A8, SLC22A13, and members of the SLC29 family for vitamin B3; e.g., SLC7A5 and SLC36A4 for tryptophan) [4,5]. The majority of NAD + is synthesized from nicotinamide, which is also released by NAD + consuming signalling reactions (Figure 1).…”

NAD+ and NAFLD – caution, causality and careful optimism

“…Future studies may reveal whether mechanisms for NMN uptake are tissue-or cell-type specific. In contrast to NMN, NR uptake seems to be mediated by equilibrative nucleoside transporter (ENT) 1, 2 and 4 (Kropotov et al 2021). Once in the cytosol, NAD + is transported into the mitochondria by SLC25A51 (also known as MCART1) and loss of this transporter was found to impair mitochondrial respiration and block NAD + uptake into the mitochondria (Girardi et al 2020;Kory et al 2020;Luongo et al 2020).…”

The role of NAD and NAD precursors on longevity and lifespan modulation in the budding yeast, Saccharomyces cerevisiae

“…On hydrolyses of the amide group of NAM with Pcn1, the NA are produced (Hong and Huh 2021 ; Ghugari et al 2020 ), thereby suggesting that at mutant of PNC1, NAM concentration could be tremendously increase while also inhibiting sirtuin functions (Mei and Brenner 2014 ; Jiang et al 2016 ). At this convergence point, nicotinamide mononucleotide adenylyltransferase 1 & 2 (Nma1 and Nma2) participates in the conversion of NaMN to nicotinic acid adenine dinucleotide (NaAD) by adenosine monophosphate (AMP moiety) addition (Pinson et al 2019 ), which undergoes amidation by glutamine (Q)-dependent NAD synthetase (Qns1) to yield NAD molecules (Kropotov et al 2021 ; Chi and Sauve 2013 ). Aside from Qns1 which carries out amidation of NaAD to NAD, there exist also other de novo route which uses molecular oxygen as a substrate (Bna2, Bna4 and Bna1), thus suggesting utilization of anaerobic cellular growth conditions on the salvage pathways for NAD synthesis (Croft et al 2020 ).…”