Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site VSports app下载. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2015 Nov 6;290(45):27124-27137.
doi: 10.1074/jbc.M115.664458. Epub 2015 Sep 18.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells

Veronika Kulikova  1 , Konstantin Shabalin  2 , Kirill Nerinovski  3 , Christian Dölle  4 , Marc Niere  4 , Alexander Yakimov  2 , Philip Redpath  5 , Mikhail Khodorkovskiy  1 , Marie E Migaud  5 , Mathias Ziegler  6 , Andrey Nikiforov  7
Affiliations

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells

Veronika Kulikova et al. J Biol Chem. .

"VSports手机版" 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 VSports手机版. 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. .

Keywords: 5′-nucleotidase; NAD biosynthesis; molecular cell biology; nicotinamide; nicotinamide adenine dinucleotide (NAD); nicotinic acid; nicotinic acid riboside; nucleoside/nucleotide metabolism V体育安卓版. .

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Human cells convert nicotinic acid to nicotinic acid riboside, which is subsequently released into the culture medium. A, schematic overview of NAD biosynthetic pathways in humans. Nam is salvaged by a NamPRT to form NMN, which is adenylylated by NMN adenylyltransferase (NMNAT) to form NAD+. NA is salvaged by NAPRT to form NAMN. NAMN is then adenylylated by NMN adenylyltransferase to form nicotinic acid adenine dinucleotide (NAAD), which is converted to NAD+ by NAD synthetase (NADS). NR and NAR are salvaged by nicotinamide riboside kinase (NRK) to form NMN or NAMN. NMN and NMN are dephosphorylated to NR and NAR by cytosolic 5′-NTs. The lower panel indicates the structures of NAD+ and its metabolites. B, 700-MHz 1H NMR spectra of major NAD precursors: Nam, NA, NR, NAR, and a mixture of them. Arrows indicate peaks that were chosen for the identification and quantification of corresponding metabolites in the mixture. C, HEK293 cells were transiently transfected with vectors encoding FLAG-tagged NamPRT or NAPRT in the presence of Nam and NA in the culture medium. 3 days after transfection, culture media from control and transfected cells were analyzed by NMR spectroscopy. A 700-MHz 1H NMR spectrum of cell culture medium is shown in the uppermost panel. The region of the NMR spectra that contains peaks corresponding to NR or NAR (indicated by arrows) is highlighted by a dashed line. Neither NR nor NAR were detected in the conditioned medium from cells expressing NamPRT or from untransfected cells. The expression of NAPRT leads to release of nucleoside NAR from cells to the culture medium. D, the expression of FLAG-tagged NamPRT and NAPRT (indicated by asterisks) in HEK293 cells was confirmed by immunoblotting using antibody to FLAG peptide. Coomassie Blue staining served as a loading control. E, relative NAD levels in HEK293 cells transiently expressing NamPRT or NAPRT. The NAD level in untransfected cells (control) was taken as 100%. Data are presented as mean ± S.D. (error bars) (n = 3). The expression of NAPRT, but not NamPRT, significantly increases cellular NAD level.
FIGURE 2.
FIGURE 2.
Cytosolic 5′-nucleotidases CN-IA, CN-II, and CN-III generate nicotinic acid riboside in human cells. A and B, HEK293 (A) and HeLa S3 (B) cells were transiently transfected with vectors encoding FLAG-tagged 5′-NTs CN-IA, CN-II, CN-III, and Sdt1. The expression of FLAG-tagged 5′-NT was confirmed by immunoblotting using antibody to FLAG peptide. SOD2 served as a loading control (A). Subcellular distribution of FLAG-tagged proteins is shown in B. Cell nuclei were stained with DAPI. Scale bars, 10 μm. C, schematic representation of the experimental approach. 5′-NTs were expressed or co-expressed with NamPRT or NAPRT in human cells in the presence of Nam and NA. NR and NAR release from transfected cells to culture medium was analyzed by NMR spectroscopy. D, HEK293 (left panel) and HepG2 (right panel) cells were co-transfected with plasmid encoding NAPRT and vectors encoding the indicated 5′-NT or empty vector. 3 (for HEK293 cells) and 7 days (for HepG2 cells) after transfection, culture media from control and transfected cells were analyzed by NMR spectroscopy. 1H NMR spectra show NAR release from cells transiently expressing NAPRT. Co-expression with 5′-NT significantly increased the extracellular level of NAR.
FIGURE 3.
FIGURE 3.
CN-II and CN-III generate NR and NAR in vitro by dephosphorylation of NMN and NAMN, respectively. A, His-tagged proteins CN-IA, CN-II, CN-III, and Sdt1 were purified after overexpression in E. coli and analyzed by SDS-PAGE. B, schematic representation of the experimental approach. NMN/NAMN 5′-nucleotidase activities of purified proteins were measured using a colorimetric method to detect Pi released during the reaction. The formation of NR and NAR in these reactions was detected by HPLC. C, pH optima for the indicated 5′-nucleotidases were estimated using corresponding preferred substrates. The 5′-nucleotidase activity is presented as μmol of released Pi/min/mg of purified protein. Data are presented as mean ± S.D. (error bars) (n = 3). D, relative 5′-nucleotidase activities of the indicated 5′-NTs with various mononucleotides that were used as substrate at 5 mm. The specific activities (see Table 1) with corresponding preferred substrates were taken as 100%. Data are presented as mean ± S.D. (error bars) (n = 3). E, the generation of NR and NAR from mononucleotides NMN and NAMN by CN-II, CN-III, and Sdt1 proteins was confirmed by HPLC. AU, absorbance units.
FIGURE 4.
FIGURE 4.
CN-II and CN-III require millimolar concentrations of NMN or NAMN for efficient catalysis in vitro. Michaelis-Menten kinetics of CN-II (A), CN-III (B), and Sdt1 (C) for the indicated substrates are shown. The Km and Vmax values were determined by nonlinear regression using SigmaPlot. Data are presented as mean ± S.D. (error bars) (n = 3).
FIGURE 5.
FIGURE 5.
HepG2 cells expressing NAPRT release NAD precursors, which thereby support neighboring cells that are unable to metabolize Nam or NA. HepG2 cells, which lack NAPRT activity, were cultivated in the presence of NA. Nam utilization was inhibited by FK866 addition as indicated. A, cells die in the presence of FK866. Overexpression of NAPRT restores the ability to use NA as an NAD precursor and recovers cell survival. Cell viability was measured by MTT assay 7 days after the transfection of cells with vector encoding FLAG-tagged NAPRT. Viability of untreated untransfected cells (control) was taken as 100%. Data are presented as mean ± S.D. (error bars) (n = 3). B, the expression of FLAG-tagged NAPRT (indicated by an asterisk) was confirmed by FLAG immunoblotting analysis. Coomassie Blue staining served as a loading control. C, cells transiently transfected with vectors encoding the indicated FLAG-tagged proteins or with empty vector. Cell nuclei were stained with DAPI. Scale bars, 100 μm. D, relative NAD levels in cells transiently expressing FLAG-tagged NAPRT. The NAD level in untreated untransfected cells (control) was taken as 100%. Data are presented as mean ± S.D. (error bars) (n = 3). The expression of NAPRT-FLAG partially recovered the NAD level in FK866-treated cells. E, two populations of cells were separated by growing in a co-culture system (upper panel). Overexpression of NAPRT-FLAG in cells growing in a 24-well plate considerably increased the viability of untransfected cells growing in cell culture inserts (CCI) in the presence of FK866 and NA (lower panel). Cell viability was measured by MTT assay 7 days after the transfection. Viability of untreated untransfected cells (control) growing in cell culture inserts was taken as 100%. Data are presented as mean ± S.D. (error bars) (n = 3). The p value was calculated using Student's t test. PET, polyethylene terephthalate; NMNAT2, NMN adenylyltransferase 2.
FIGURE 6.
FIGURE 6.
Nicotinic acid riboside released from HepG2 cells is taken up and used as NAD precursor by neighboring cells. HepG2 cells were cultivated in the presence of NA, NR, or NAR as indicated. Nam utilization was inhibited by FK866 addition as indicated. A, proposed scheme of nucleoside NR and/or NAR release from intrinsically NAPRT-deficient HepG2 cells overexpressing NAPRT-FLAG. B, extracellular nucleosides NR and NAR support NAD synthesis and cell viability in the presence of FK866. Cell viability was measured by MTT assay 7 days after the treatment. Viability of untreated cells (control) was taken as 100%. Data are presented as mean ± S.D. (error bars) (n = 3). C, 700-MHz 1H NMR spectra of cell culture medium obtained from control (transfected with empty vector) cells or from cells transiently transfected with vector encoding NAPRT. Cell culture medium was analyzed by NMR 7 days after transfection. D, low micromolar concentrations of nucleosides NR and NAR were sufficient to maintain viability of FK866-treated cells. Cell viability was measured by MTT assay 7 days after the treatment. Viability of untreated cells (control) was taken as 100%. Data are presented as mean ± S.D. (error bars) (n = 3). NAAD, nicotinic acid adenine dinucleotide.
FIGURE 7.
FIGURE 7.
Generation and release of NAR and NR by human cells. A, HEK293 cells with or without transient overexpression of NAPRT (as indicated) were cultured for 4 days in the presence of Nam and NA. The medium was then mixed 2:1 with fresh medium and used to culture HepG2 in the presence or absence of FK866 as indicated. The preconditioned medium was renewed every 24 h. Viability of the HepG2 cells was assessed after 7 days of culture. B, the experiment was conducted as in A except untransfected HEK293 or HeLa cells were used to generate the preconditioned medium. As a control, NAR was added to the medium as indicated. The data in A and B are represented as mean ± S.D. (error bars) (n = 3). C, NMR analyses of media from untransfected HEK293 and HeLa cells indicate release of NAR and likely NR (asterisk) from HeLa, but not HEK293, cells. X designates an unidentified peak. D, proposed mechanism of riboside generation from NA and Nam and their release from human cells. NAR can be generated from NAMN by human cytosolic 5′-nucleotidases and released from cells to the culture medium. A similar mechanism is proposed for the generation and release of NR. NUDT, Nudix hydrolase; NMNAT, NMN adenylyltransferase; NAAD, nicotinic acid adenine dinucleotide; NADS, NAD synthetase.
See this image and copyright information in PMC

References (VSports app下载)

    1. Nikiforov A., Kulikova V., and Ziegler M. (2015) The human NAD metabolome: functions, metabolism and compartmentalization. Crit. Rev. Biochem. Mol. Biol. 50, 284–297 - PMC - PubMed
    1. Houtkooper R. H., Cantó C., Wanders R. J., and Auwerx J. (2010) The secret life of NAD+: an old metabolite controlling new metabolic signaling pathways. Endocr. Rev. 31, 194–223 - PMC - PubMed
    1. Haigis M. C., and Sinclair D. A. (2010) Mammalian sirtuins: biological insights and disease relevance. Annu. Rev. Pathol. 5, 253–295 - PMC - PubMed
    1. Hassa P. O., and Hottiger M. O. (2008) The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases. Front. Biosci. 13, 3046–3082 - PubMed
    1. Koch-Nolte F., Kernstock S., Mueller-Dieckmann C., Weiss M. S., and Haag F. (2008) Mammalian ADP-ribosyltransferases and ADP-ribosylhydrolases. Front. Biosci. 13, 6716–6729 - PubMed

Publication types

MeSH terms

LinkOut - more resources