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

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 Km 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 Km 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

Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD+ synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry.

NAD+ is mainly synthesized from nicotinamide (Nam) by the rate-limiting enzyme Nam phosphoribosyltransferase (Nampt) and degraded to Nam by NAD+-degrading enzymes in mammals. Numerous studies report that tissue NAD+ levels decrease during aging and age-related diseases and suggest that NAD+ replenishment promotes healthy aging. Although increased expression of Nampt might be a promising intervention for healthy aging, forced expression of Nampt gene, inducing more than 10-fold increases in the enzyme protein level, has been reported to elevate NAD+ levels only 40-60% in mammalian cells. Mechanisms underlying the limited increases in NAD+ levels remain to be determined. Here we show that Nampt is inhibited in cells and that enhanced expression of Nampt activates NAD+ breakdown. Combined with the measurement of each cell's volume, we determined absolute values (μM/h) of the rates of NAD+ synthesis (RS) and breakdown (RB) using a flux assay with a 2H (D)-labeled Nam, together with the absolute NAD+ concentrations in various mammalian cells including primary cultured cardiomyocytes under the physiological conditions and investigated the relations among total cellular Nampt activity, RS, RB, and the NAD+ concentration. NAD+ concentration was maintained within a narrow range (400-700 μM) in the cells. RS was much smaller than the total Nampt activity, indicating that NAD+ synthesis from Nam in the cells is suppressed. Forced expression of Nampt leading to 6-fold increase in total Nampt activity induced only a 1.6-fold increase in cellular NAD+ concentration. Under the conditions, RS increased by 2-fold, while 2-fold increase in RB was also observed. The small increase in cellular NAD+ concentration is likely due to both inhibited increase in the NAD+ synthesis and the activation of its breakdown. Our findings suggest that cellular NAD+ concentrations do not vary dramatically by the physiological fluctuation of Nampt expression and show the tight link between the NAD+ synthesis and its breakdown

NAMPT and NAPRT activities in the absence or presence of ATP.

<p>NAMPT (15 ng) and NAPRT (20 ng) were incubated with 20 µM [¹⁴C]Nam and [¹⁴C]NA for 30 min, respectively, in the presence of 1 µM PRPP (low substrate concentration) without or with 1 mM ATP. NAMPT (96 ng) and NAPRT (100 ng) were also incubated with 50 µM [¹⁴C]Nam and [¹⁴C]NA for 7 and 6 min, respectively, in the presence of 50 µM PRPP (high substrate concentration) without or with 1 mM ATP.</p

Metabolic pathways of the salvage NAD synthesis.

<p>NaAD, NA adenine dinucleotide; <i>NMNAT</i>, NMN adenylyltransferase; <i>NaMNAT</i>, NaMN adenylyltransferase; <i>NADsyn</i>, NAD synthetase. Broken arrows indicate the possible extracellular pathway in NAD biosynthesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022781#pone.0022781-Imai1" target="_blank">[14]</a>.</p

ATP is an essential activator of NAMPT and NAPRT reactions.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022781#pone-0022781-g002" target="_blank">figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022781#pone-0022781-g003" target="_blank">3</a> are representative of at least three experiments.</p

Kinetic parameters in the NAMPT and NAPRT reactions.

<p>NAMPT (36 ng) was incubated with various Nam concentrations at a fixed PRPP concentration (300 µM) for 7 and 20 min in the presence or absence of 1 mM ATP, respectively. The enzyme (7 and 36 ng) was also incubated with various concentrations of PRPP at fixed Nam concentrations (5 and 40 µM) for 7 and 30 min in the presence or absence of 1 mM ATP, respectively. NAPRT (70 and 100 ng) was incubated with various NA concentrations at a fixed PRPP concentration (300 µM) in the presence or absence of 1 mM ATP, respectively, for 30 min. The enzyme (12 and 90 ng) was also incubated with various concentrations of PRPP at fixed NA concentrations (50 and 380 µM) for 15 and 30 min in the presence or absence of 1 mM ATP, respectively. The mutant NAPRT (H213N-NAPRT, 140 ng) was incubated with various NA concentrations at a fixed PRPP concentration (600 µM) or with various concentrations of PRPP at a fixed NA concentration (650 µM) in the presence or absence of 1 mM ATP for 60 min. <i>K_m</i> and <i>V_max</i> values represent the mean ± S.D. of at least three separate experiments.</p

PRPP, ATP, and NMN are degraded in mouse blood plasma.

<p>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 (<i>circles</i>) or 37°C (<i>triangles</i>) or in the absence of the chelators at 37°C (<i>squares</i>) 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.</p