A.N. conceived and supervised the study; V.K., M.S., A.K., and L.S. performed cell culture experiments and NMR sample preparation; K.S., K.N., and A.Y. carried out the NMR experiments and analyzed the data; M.Z. and M.K. contributed reagents, materials, and analysis tools; M.E.M. synthetized NR and NAR; A.N. and M.Z. wrote the manuscript. All authors read and approved the final manuscript.

Figure 1. Nicotinamide adenine dinucleotide (NAD+) biosynthesis in human cells. Tryptophan (Trp), pyridinic bases nicotinamide (Nam) and nicotinic acid (NA), and the nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are precursors for intracellular NAD+ synthesis. Tryptophan is converted to quinolinic acid (QA) in a series of enzymatic reactions; nicotinic acid mononucleotide (NAMN) is synthesized from QA by quinolinate phosphoribosyltransferase (QAPRT). Nicotinamide phosphoribosyltransferase (NamPRT) converts Nam into nicotinamide mononucleotide (NMN), which, in turn, is adenylated to NAD+ by nicotinamide mononucleotide adenylyltransferase (NMNAT). NA is converted to NAMN by nicotinic acid phosphoribosyltransferase (NAPRT). NAMN is adenylated by NMNAT to nicotinic acid adenine dinucleotide (NAAD), which is amidated to NAD+ by NAD synthetase (NADS). Nucleosides NR and NAR are phosphorylated to NMN and NAMN, respectively, by the nicotinamide riboside kinases (NRK). NAD+ is cleaved to Nam during NAD+-dependent protein deacylation and ADP-ribosylation. NMN might also be synthesized by an extracellular NamPRT form (eNAMPT). NAD+ can possibly be released from cells through connexin 43 hemichannels (Cx43), and can be degraded to NR by ecto-nucleotidase CD73. NR is hydrolyzed to Nam by CD157. Extracellular NAD+ can also be hydrolyzed to Nam by CD38 and CD157.

Figure 1. Nicotinamide adenine dinucleotide (NAD+) biosynthesis in human cells. Tryptophan (Trp), pyridinic bases nicotinamide (Nam) and nicotinic acid (NA), and the nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are precursors for intracellular NAD+ synthesis. Tryptophan is converted to quinolinic acid (QA) in a series of enzymatic reactions; nicotinic acid mononucleotide (NAMN) is synthesized from QA by quinolinate phosphoribosyltransferase (QAPRT). Nicotinamide phosphoribosyltransferase (NamPRT) converts Nam into nicotinamide mononucleotide (NMN), which, in turn, is adenylated to NAD+ by nicotinamide mononucleotide adenylyltransferase (NMNAT). NA is converted to NAMN by nicotinic acid phosphoribosyltransferase (NAPRT). NAMN is adenylated by NMNAT to nicotinic acid adenine dinucleotide (NAAD), which is amidated to NAD+ by NAD synthetase (NADS). Nucleosides NR and NAR are phosphorylated to NMN and NAMN, respectively, by the nicotinamide riboside kinases (NRK). NAD+ is cleaved to Nam during NAD+-dependent protein deacylation and ADP-ribosylation. NMN might also be synthesized by an extracellular NamPRT form (eNAMPT). NAD+ can possibly be released from cells through connexin 43 hemichannels (Cx43), and can be degraded to NR by ecto-nucleotidase CD73. NR is hydrolyzed to Nam by CD157. Extracellular NAD+ can also be hydrolyzed to Nam by CD38 and CD157.

Figure 2. Extracellular NAD+ intermediates support NAD+ generation in HEK293 cells. HEK293 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) containing Nam, supplemented with 10% fetal bovine serum (FBS). To inhibit NAD+ synthesis from Nam, cells were treated with FK866. Cells were also treated with NAD+ or its derivatives as indicated. Metabolic activity was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay 48 h after the treatment. Metabolic activity of untreated cells (control) was taken as 100%. Data are presented as mean ± S.D ( n = 3). Statistical analysis of differences between the groups was carried out by one-way ANOVA with post hoc comparisons using Tukey test. * indicates statistical difference at p < 0.001.

Figure 2. Extracellular NAD+ intermediates support NAD+ generation in HEK293 cells. HEK293 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) containing Nam, supplemented with 10% fetal bovine serum (FBS). To inhibit NAD+ synthesis from Nam, cells were treated with FK866. Cells were also treated with NAD+ or its derivatives as indicated. Metabolic activity was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay 48 h after the treatment. Metabolic activity of untreated cells (control) was taken as 100%. Data are presented as mean ± S.D ( n = 3). Statistical analysis of differences between the groups was carried out by one-way ANOVA with post hoc comparisons using Tukey test. * indicates statistical difference at p < 0.001.

Figure 3. Degradation of extracellular NAD+ intermediates in cultures of HEK293 cells. (A) NAD+, NMN, or NR were added to the culture medium DMEM (containing Nam), supplemented with 10% FBS as indicated. Medium was then frozen (control, 0 h) or incubated with HEK293 cells for 24 h. 1H NMR spectra of control (0 h) and conditioned medium (24 h) are presented. NAD+ (B), NMN (C), NR (D), NAAD (E), NAMN (F), or NAR (G) were added to the culture medium DMEM (containing Nam), supplemented with 10% FBS. Medium was then frozen (control, 0 h) or incubated in the presence (white circles) or absence (black circles) of HEK293 cells for 12, 24 or 48 h and analyzed by NMR spectroscopy. (B–G) represent the relative levels of NAD intermediates added to the culture media (left panels) and their degradation products (other panels). Concentrations of metabolites added to culture media (0 h) were taken as 100. Concentrations of other metabolites were proportionally recalculated. Nam values are presented as estimated Nam amounts subtracted by Nam amount in a control (0 h). Data are presented as mean ± S.D ( n = 3). Statistical analysis of differences between the groups was carried out by two-way ANOVA with post hoc comparisons using Tukey test. * indicates statistical difference at p < 0.001.

Figure 3. Degradation of extracellular NAD+ intermediates in cultures of HEK293 cells. (A) NAD+, NMN, or NR were added to the culture medium DMEM (containing Nam), supplemented with 10% FBS as indicated. Medium was then frozen (control, 0 h) or incubated with HEK293 cells for 24 h. 1H NMR spectra of control (0 h) and conditioned medium (24 h) are presented. NAD+ (B), NMN (C), NR (D), NAAD (E), NAMN (F), or NAR (G) were added to the culture medium DMEM (containing Nam), supplemented with 10% FBS. Medium was then frozen (control, 0 h) or incubated in the presence (white circles) or absence (black circles) of HEK293 cells for 12, 24 or 48 h and analyzed by NMR spectroscopy. (B–G) represent the relative levels of NAD intermediates added to the culture media (left panels) and their degradation products (other panels). Concentrations of metabolites added to culture media (0 h) were taken as 100. Concentrations of other metabolites were proportionally recalculated. Nam values are presented as estimated Nam amounts subtracted by Nam amount in a control (0 h). Data are presented as mean ± S.D ( n = 3). Statistical analysis of differences between the groups was carried out by two-way ANOVA with post hoc comparisons using Tukey test. * indicates statistical difference at p < 0.001.

Figure 4. Degradation of NAD+, NMN and NR by FBS. NAD+, NMN, or NR were added to the culture medium DMEM (without Nam), supplemented with 10% FBS or H 2 O. FBS 1 was obtained from Gibco, FBS 2 was obtained from Biochrom, and FBS 3 was obtained from HyClone. Medium was then frozen (control) or incubated for 24 h at 37 °C. Relative levels of NAD+ intermediates in the samples were then estimated using quantitative NMR spectroscopy. Amounts of metabolites added to the control culture media were taken as 100%. Data are presented as mean ± S.D ( n = 3). Statistical analysis of differences between the groups was carried out by one-way ANOVA with post hoc comparisons using Tukey test. * indicates statistical difference at p < 0.01, ** indicates statistical difference at p < 0.001.

Figure 4. Degradation of NAD+, NMN and NR by FBS. NAD+, NMN, or NR were added to the culture medium DMEM (without Nam), supplemented with 10% FBS or H 2 O. FBS 1 was obtained from Gibco, FBS 2 was obtained from Biochrom, and FBS 3 was obtained from HyClone. Medium was then frozen (control) or incubated for 24 h at 37 °C. Relative levels of NAD+ intermediates in the samples were then estimated using quantitative NMR spectroscopy. Amounts of metabolites added to the control culture media were taken as 100%. Data are presented as mean ± S.D ( n = 3). Statistical analysis of differences between the groups was carried out by one-way ANOVA with post hoc comparisons using Tukey test. * indicates statistical difference at p < 0.01, ** indicates statistical difference at p < 0.001.

Figure 5. Degradation of extracellular NAD+ intermediates in cultures of HEK293 cells grown in serum-free medium. The indicated NAD+ intermediates were added to the serum-free medium (SFM) Pro293A-CDM. Medium was incubated in the presence or absence of HEK293 cells for 12, 24, or 48 h, as indicated. Relative levels of NAD+ intermediates in culture medium were then estimated using quantitative NMR spectroscopy. Amounts of metabolites added to culture media (control, 0 h) were taken as 100%. Nam values are presented as estimated Nam amounts subtracted by Nam amount in a control (0 h) medium. Data are presented as mean ± S.D ( n = 3). Statistical analysis of differences between the groups was carried out by one-way ANOVA with post hoc comparisons using Tukey test. * indicates statistical difference at p < 0.001, ** indicates statistical difference at p < 0.05.

Figure 5. Degradation of extracellular NAD+ intermediates in cultures of HEK293 cells grown in serum-free medium. The indicated NAD+ intermediates were added to the serum-free medium (SFM) Pro293A-CDM. Medium was incubated in the presence or absence of HEK293 cells for 12, 24, or 48 h, as indicated. Relative levels of NAD+ intermediates in culture medium were then estimated using quantitative NMR spectroscopy. Amounts of metabolites added to culture media (control, 0 h) were taken as 100%. Nam values are presented as estimated Nam amounts subtracted by Nam amount in a control (0 h) medium. Data are presented as mean ± S.D ( n = 3). Statistical analysis of differences between the groups was carried out by one-way ANOVA with post hoc comparisons using Tukey test. * indicates statistical difference at p < 0.001, ** indicates statistical difference at p < 0.05.

Figure 6. The effect of equilibrative nucleoside transporter (ENT) inhibition on NR, NMN, and NAD+ utilization by HEK293 cells. HEK293 cells were cultivated in the serum-free medium Pro293A-CDM in the presence of NA, NR, NMN, or NAD+ at a concentration of 1 µM, 10 µM, or 100 µM, as indicated (A) or in the presence of NAD+ (B), NMN (C) or NR (D) at a concentration of 100 µM. Cells were also treated with inhibitors of equilibrative nucleoside transporters S-(4-nitrobenzyl)-6-thioinosine (NBTI) or dipyridamole (Dip). Nam utilization was inhibited by FK866 addition. (A) Metabolic activity was measured by MTT assay 72 h after the treatment. Metabolic activity of untreated cells was taken as 100. (B) Relative levels of NAD metabolites in culture media were estimated using quantitative NMR spectroscopy 48 h after the treatment. Amounts of NAD+, NMN, NR, and Nam in control culture media incubated without cells were taken as 100. Data are presented as mean ± S.D ( n = 3). Statistical analysis of differences between the groups was carried out by two-way ANOVA with post hoc comparisons using Tukey test. * indicates statistical difference at p < 0.001 vs. the FK866-treated cells, # indicates statistical difference at p < 0.001 vs. treatment with the 10 µM NR, + indicates statistical difference at p < 0.01 vs. the treatment with corresponding NAD precursor only. (B–D) Relative levels of NAD metabolites in culture media were estimated using quantitative NMR spectroscopy 48 h after the treatment. Concentrations of NAD+, NMN, NR, and Nam in control culture media incubated without cells ( w / o cells) were taken as 100. Data are presented as mean ± S.D ( n = 3). Statistical analysis of differences between the groups was carried out by one-way ANOVA with post hoc comparisons using Tukey test. * indicates statistical difference at p < 0.001, ** indicates statistical difference at p < 0.01.