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

Andrey Kropotov,Maria Svetlova,Andrey Nikiforov,Mikhail Khodorkovskiy,Mathias Ziegler,Julia Sudnitsyna,Marie E Migaud,Veronika Kulikova,Ljudmila Solovjeva,Kirill Nerinovski,Alexander Yakimov

doi:10.3390/ijms22031391

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.

Highlights

It has been shown that the reduced form of nicotinamide riboside (NR), NRH, can enter NAD+ biosynthesis via phosphorylation by adenosine kinase [6,7]
We confirmed that pharmacological inhibition of equilibrative nucleoside transporter (ENT) suppresses NAD+ synthesis from NR in cultured HEK293 cells
Thereafter, the culture medium was collected and analyzed by (Figure 2C). These results suggest that members of the ENT family can mediate NR uptake

Summary

Introduction

Alternative NAD+ precursors have been studied that have the potential to efficiently replenish cellular pools of the coenzyme. It has been shown that the reduced form of NR, NRH, can enter NAD+ biosynthesis via phosphorylation by adenosine kinase [6,7]. The deamidated forms of Nam and NR, nicotinic acid (NA) and its ribosylated form (NAR) are utilized as NAD+ precursors. While NA has been well known as another form of vitamin B3 that is converted to NAD+ in the Preiss-Handler pathway, the role of NAR as NAD+ precursor still awaits comprehensive characterization. Studying the potential of alternatives to Nam for NAD+ biosynthesis is important, because imbalances in NAD+ homeostasis are associated with a wide range of pathologies. A significant drop in the NAD+ level is observed with the development of metabolic [8,9]

Methods

Results

Conclusion