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Cited by 117Sirtuins and Resveratrol in MedicineBiochemical and Molecular ResearchFolate and B Vitamins Research

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Nicotinamide Phosphoribosyltransferase/Visfatin Does Not Catalyze Nicotinamide Mononucleotide Formation in Blood Plasma

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Hara, Nobumasa, Yamada, Kazuo, Shibata, Tomoko, Osago, Harumi, Tsuchiya, Mikako·2011·PLoS ONE
DOI 10.1371/journal.pone.0022781PMC PMC3149623
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Abstract

Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined K(m) values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases K(m) values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of "NAMPT-mediated systemic NAD biosynthesis." Our study would advance current understanding of visfatin physiology.

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Figures

Figure 1 Metabolic pathways of the salvage NAD synthesis. NaAD, NA adenine dinucleotide; NMNAT, NMN adenylyltransferase; NaMNAT, NaMN adenylyltransferase; NADsyn, NAD synthetase. Broken arrows indicate the possible extracellular pathway in NAD biosynthesis [14].
Figure 1 Metabolic pathways of the salvage NAD synthesis. NaAD, NA adenine dinucleotide; NMNAT, NMN adenylyltransferase; NaMNAT, NaMN adenylyltransferase; NADsyn, NAD synthetase. Broken arrows indicate the possible extracellular pathway in NAD biosynthesis [14].CC BY · PubMed Central
Figure 2 ATP is an essential activator of NAMPT and NAPRT reactions. NAMPT (A, 15 ng) and NAPRT (B, 20 ng) were incubated with 20 µM [14C]Nam and [14C]NA for 6 and 7 min, respectively, in the presence of 1 µM PRPP and indicated concentrations of ATP. The amounts of NMN (A) and NaMN (B) formed were determined. Data in figures 2 and 3 are representative of at least three experiments.
Figure 2 ATP is an essential activator of NAMPT and NAPRT reactions. NAMPT (A, 15 ng) and NAPRT (B, 20 ng) were incubated with 20 µM [14C]Nam and [14C]NA for 6 and 7 min, respectively, in the presence of 1 µM PRPP and indicated concentrations of ATP. The amounts of NMN (A) and NaMN (B) formed were determined. Data in figures 2 and 3 are representative of at least three experiments.CC BY · PubMed Central
Figure 3 PRPP, ATP, and NMN are degraded in mouse blood plasma. PRPP, ATP, or NMN (150, 300, or 60 pmol, respectively) was incubated with the blood plasma (30 µl) in the presence of EDTA/EGTA at 4 (circles) or 37°C (triangles) or in the absence of the chelators at 37°C (squares) for the indicated times. After the incubation, the amounts of PRPP (A), ATP (B), and NMN (C) remaining at each time point in the plasma were determined and expressed as percent of those at 0 min.
Figure 3 PRPP, ATP, and NMN are degraded in mouse blood plasma. PRPP, ATP, or NMN (150, 300, or 60 pmol, respectively) was incubated with the blood plasma (30 µl) in the presence of EDTA/EGTA at 4 (circles) or 37°C (triangles) or in the absence of the chelators at 37°C (squares) for the indicated times. After the incubation, the amounts of PRPP (A), ATP (B), and NMN (C) remaining at each time point in the plasma were determined and expressed as percent of those at 0 min.CC BY · PubMed Central

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Metadata

Authors
Hara, N., Yamada, K., Shibata, T., Osago, H., & Tsuchiya, M.
Affiliations
Shimane University (JP)
Corresponding author
Hara, Nobumasa
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-08-03
Volume
6
Issue
8
Pages
e22781
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0022781
PMC
PMC3149623
OpenAlex
W2131005104
Cited by
116
References
37
Citations by year
2025: 7 · 2024: 7 · 2023: 7 · 2022: 12 · 2021: 4 · 2020: 9 · 2019: 6 · 2018: 8 · 2017: 14 · 2016: 4 · 2015: 9 · 2014: 8 · 2013: 10 · 2012: 11
FWCI
4.57
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Institutional Repositories DataBase (IRDB) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Japan Society for the Promotion of Science (21580139)
Topics
  • Health Sciences › Medicine › Geriatrics and Gerontology › Sirtuins and Resveratrol in Medicine
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Molecular Research
  • Health Sciences › Nursing › Nutrition and Dietetics › Folate and B Vitamins Research
Keywords
visfatin, nicotinamide mononucleotide, NAD biosynthesis, blood plasma, enzyme kinetics, substrate Km, ATP stimulation, 5-phosphoribosyl-1-pyrophosphate, nicotinamide
MeSH terms
Animals, Humans, Mice, Inbred C57BL, Niacinamide, Nicotinamide Mononucleotide, Organophosphorus Compounds, Plasma, Mice, Nicotinamide Phosphoribosyltransferase

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