Abstract
NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors.
Highlights
Nicotinamide riboside (NR) and nicotinic acid riboside (NAR) can serve as precursors of Nicotinamide adenine dinucleotide (NAD) in human cells
Human Cells Convert Nicotinic Acid to Nicotinic Acid Riboside, Which Is Subsequently Released into the Culture Medium—To study the release of nicotinamide riboside (NR) and NAR from human cells, we first optimized a 1H NMR-based experimental approach to detect NAD precursors in culture medium
We explored whether human cells growing in the presence of Nam and NA as NAD precursors can produce and release NR or NAR into the culture medium
Summary
Nicotinamide riboside (NR) and nicotinic acid riboside (NAR) can serve as precursors of NAD in human cells. Recombinant human cytosolic 5-nucleotidases (5-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and catalyze NR and NAR formation in vitro. NAD is used as a substrate and is degraded by several families of regulatory proteins such as protein deacetylases (sirtuins), ADP-ribosyltransferases, poly(ADP-ribose) polymerases, and ADP-ribosyl cyclases, which govern vital processes including gene expression, progression of the cell cycle, insulin secretion, DNA repair, apoptosis, and aging [1,2,3,4,5,6] Proper control of these NAD-dependent metabolic and signaling processes depends on how efficiently cells can maintain their NAD contents.
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