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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.

SUBMITTER: Kropotov A

PROVIDER: S-EPMC7866510 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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

Kropotov Andrey A Kulikova Veronika V Nerinovski Kirill K Yakimov Alexander A Svetlova Maria M Solovjeva Ljudmila L Sudnitsyna Julia J Migaud Marie E ME Khodorkovskiy Mikhail M Ziegler Mathias M Nikiforov Andrey A

International journal of molecular sciences 20210130 3

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD<sup>+</sup>) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD<sup>+</sup> 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 ...[more]

PMID: 33573263

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Project description:Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [3H]uridine uptake by NBMPR was biphasic, with IC50 values of 11.3 nM for ENT1 and 9.6 μM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1- and ENT2-mediated [3H]uridine uptake revealed no difference in Jmax (16.53 and 30.40 pmol cm-2 min-1) and an eightfold difference in Kt (13.6 and 108.9 μM). The resulting fivefold difference in intrinsic clearance (Jmax/Kt) for ENT1- and ENT2 transport accounted for observed inhibition of [3H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC50 values of ∼200 µM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [3H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENT: This study characterizes an in vitro model using S-[(4-nitrophenyl)methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.

Dataset Information

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

Publications

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

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