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. 2018 May 1;27(5):1067-1080.e5.
doi: 10.1016/j.cmet.2018.03.018.

Quantitative Analysis of NAD Synthesis-Breakdown Fluxes

Ling Liu  1 , Xiaoyang Su  2 , William J Quinn 3rd  3 , Sheng Hui  4 , Kristin Krukenberg  5 , David W Frederick  3 , Philip Redpath  6 , Le Zhan  7 , Karthikeyani Chellappa  3 , Eileen White  7 , Marie Migaud  8 , Timothy J Mitchison  9 , Joseph A Baur  10 , Joshua D Rabinowitz  11
Affiliations

Quantitative Analysis of NAD Synthesis-Breakdown Fluxes

Ling Liu et al. Cell Metab. .

Abstract

The redox cofactor nicotinamide adenine dinucleotide (NAD) plays a central role in metabolism and is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. NAD concentration falls during aging, which has triggered intense interest in strategies to boost NAD levels. A limitation in understanding NAD metabolism has been reliance on concentration measurements. Here, we present isotope-tracer methods for NAD flux quantitation. In cell lines, NAD was made from nicotinamide and consumed largely by PARPs and sirtuins. In vivo, NAD was made from tryptophan selectively in the liver, which then excreted nicotinamide. NAD fluxes varied widely across tissues, with high flux in the small intestine and spleen and low flux in the skeletal muscle. Intravenous administration of nicotinamide riboside or mononucleotide delivered intact molecules to multiple tissues, but the same agents given orally were metabolized to nicotinamide in the liver. Thus, flux analysis can reveal tissue-specific NAD metabolism.

Keywords: NAD; NADH; flux quantification; isotope tracers; mass spectrometry; mononucleotide; niacin; nicotinamide; redox cofactor; riboside.

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Figures

Figure 1
Figure 1
Quantitation of NAD turnover in cell culture. (a) Switching the media from unlabeled to [2,4,5,6-2H] nicotinamide (2H-NAM) results in NAD labeling without otherwise perturbing cellular pool sizes or fluxes. Fast labeling implies high fluxes relative to pool-size. (b) Isotopic fractions of intracellular NAM and NAD after switching to 2H-NAM in T47D cells; U, unlabeled fraction; L, labeled fraction. (c) Labeling schematic. (d) NAD labeling dynamics after switching to 2H-NAM in T47D cells. Symbols, experimental data (mean ± s.d., n=3); lines are to guide the eye. See also Figure S1 and Table S1.
Figure 2
Figure 2
NAD kinase flux. (a) Approach to calculate NAD consumption by NAD kinase (f1, forward flux). (b) Labeling dynamics; symbols, experimental data (n=3); lines, fit to differential equations in (a). **p<0.01, paired t-test; dots, experimental data, n=3. See also Figure S1.
Figure 3
Figure 3
NAD utilization in cell lines. (a) NAD concentration and labeling in T47D cells treated with olaparib (10μM, PARP1/2 inhibitor). Olaparib was added simultaneously with switching cells into 2H-NAM. Symbols, experimental data (mean ± s.d., n=3); lines, fit to equations corresponding to model in (b) (see STAR Methods). (b) Approach to calculate NAD consumption by different enzymes, based on assumption of fixed NAD production flux and decreased consumption flux upon adding inhibitor. (c) Fitted NAD efflux based on NAD concentration, cell growth rate, and isotope labeling in the presence or absence of 10 μM olaparib as shown in panel (a). Horizontal line within box, best fit; box, interquartile range; whisker, 95% confidence intervals. (d) PARylation activity, and PARP-mediated NAD consumption as measured by isotope tracing in the presence and absence of 10 μM olaparib are not correlated across five breast cell lines. Lysate and MagPlex beads were incubated together overnight to measure PARylation activity (Krukenberg et al., 2014). Data are mean ± s.d., n = 3. (e) Total NAD consumption fluxes in XPA-deficient or XPA-restored cells treated with DMSO or olaparib, calculated from 2H-NAM labeling in the first 8 h of drug treatment. Results are normalized to untransfected XPA-deficient cells; data are mean ± s.d., n = 3; * p<0.05, paired t-test. (f) NAD concentration and labeling in T47D cells incubated simultaneously with 2H-NAM and zeocin (250μg/ml, to induce DNA double strand break), with or without olaparib, for 8 h. Data are mean ± s.d., n = 3. (g) Increase in total NAD consumption flux based on data in (f) (mean with 95% confidence interval). (h) NAD concentration and labeling in T47D cells treated with sirtinol (25 μM, Sirtuin 1/2 inhibitor). Sirtinol was added simultaneously with switching cells into 2H-NAM. Symbols, experimental data (n=3); line, fit to equations, (i) Same as (h) but with dual PARP and SIRT1/2 inhibition. (j) Decrease in NAD consumption, calculated based on first 4 hours after drug exposure in T47D cells, for olaparib (10 μM, PARP1/2 inhibitor), sirtinol (25 μM, Sirtuin 1/2 inhibitor), EX527(10 μM, Sirtuin 1/2 inhibitor), and co-treatment of olaparib (10μM) and sirtinol (25μM) (mean with 95% confidence interval). (k) Fraction of NAD directed toward supporting growth in different cell lines cell lines, as determined by experimental measurements of growth rate relative to NAD isotope labeling rate (mean with 95% confidence interval). (I) Pie graphs indicating NAD fates in differentiated myocytes (C2C12 cells) and proliferating T47D and MCF7 cells. Consumption routes in C2C12 cells and MCF7 cells were determined as for T47D cells (see Figure S2 for data in C2C12 cells and MCF7 cells). See also Figure S2 and Table S2.
Figure 4
Figure 4
Relationship between NAD concentration and flux in cell lines. (a) NAD concentration and labeling in T47D cells treated with FK866 (100nM, NAMPT inhibitor). FK866 was added simultaneously with switching cells into 2H-NAM. Symbols, experimental data (mean ± s.d., n=3); line, fit to equations corresponding to the illustrated kinetic scheme, which assumes that NAMPT fully blocks NAD synthesis and NAD consumption is proportional to its concentration (“first-order kinetics”). (b) NAD concentration before and after labeling for 5 h. T47D cells were pre-treated with 1× NAM (standard DMEM condition), 0.1× NAM, or 0.01× NAM for 1 week and labeled with the same concentration of 2H-NAM, or were pre-treated with 5× NR for 4 days and labeled with the same concentration of 2H,13C-NR. Data are mean ± s.d., n = 4. (c) Correlation between NAD concentration and consumption flux based on data in (b). (d) Correlation between t1/2 for NAD labeling by 2H-NAM and t1/2 for NAD depletion upon adding FK866 (100 nM) across 12 cell lines. Each dot represents one cell line. For data by cell line, see Table S3. (e) Across the same 12 cell lines, NAD flux correlates poorly with labeling t1/2. (f) NAD flux correlates more strongly with intracellular NAD concentration. See also Table S3.
Figure 5
Figure 5
Contributors to NAD biosynthesis in vivo in mice. (a) Concentration of NAD contributors (log scale, mean ± s.d., n=4). (b) Schematic of tryptophan (Trp) and NAM tracer metabolism. 13C-Trp was infused via jugular vein at 5nmol/g/min and 2H-NAM at 0.3nmol/g/min; Tryptophan to NAD flux (f1), NA to NAD flux (f2), NAM uptake from circulation (f3), and NAMPT flux (f4). (c) Serum and tissue isotope labeling of NAM from 5 h [U-13C]Trp infusion (left), or from 5 h 13C-NA infusion (right) (mean ± s.d., n=3). (d) Serum isotope labeling of NAM from 2H-NAM infusion. Symbols, experimental data (mean ± s.d., n=3); lines are to guide the eye. (e) NAM labeling from 2H-NAM infusion. (f) Labeled NADP(H) relative to labeled NAD(H) in tissues after 5 h 2H-NAM infusion. (g) NAD(H) concentration across tissues. (h) Labeled fractions of NAM, NAD and NADPH in tissues after 1 h, 2 h, 5 h of 2H-NAM infusion. For (e) -(h), data are mean ± s.d., n=3. For related cell culture experiments, see also Figure S3, S4 and S5, Table S1 and Table S4.
Figure 6
Figure 6
NAD turnover in tissues. (a) Quantitative NAD fluxes in tissues, based on metabolic flux analysis informed by LC-MS measurement of metabolite labeling in serum and tissues after separate infusions of 13C-Trp, 13C-NA and 2H-NAM. Values shown are fluxes (unit: μM per hour) from the best fit flux sets for network in (6b). For complete flux sets, see Table S5. Fluxes shown for tryptophan and NA reflect net assimilation into NAD. For NAM, there is significant net export from liver and kidney. For these 2 tissues, we show separately the uptake and excretion fluxes of NAM, as determined by modeling of the tissue labeling data. For all other tissues, NAM uptake and excretion are balanced, and we show only a single value corresponding to the exchange rate between the tissue and circulation. (b) Total NAD production flux (f1 +f2 +f4) across tissues and relevant NAD enzyme protein expression levels based on antibody staining from http://www.proteinatlas.org/. (c, d) Across tissues, NAD production flux (panel b) correlates with inverse labeling half-time but not NAD concentration. (e) NAD labeling half-time across cell lines and corresponding mouse tissues. (f) NAD labeling half-time and Trp fractional contribution in HepG2 cells, primary hepatocytes, and in vivo liver. Bars are mean with 95% confidence intervals. See also Figure S6, Table S5, S6 and S7.
Figure 7
Figure 7
NR and NMN are effectively delivered to tissues by IV, but not oral administration. (a) Schematic of 2H,13C-NR and 2H,13C-NMN metabolism in vivo. NAD made directly from NR or NMN is M+2 labelled. NAD made from NAM derived NR or NMN is M+1 labeled. Previously made NAD, or NAD made unlabeled NAM is unlabeled (M+0). (b) Stability of NR and NMN standards in PBS, DMEM with 10% DFBS, mouse serum, or mouse blood. Symbols are experimental data (mean ± s.d., n=3); lines are single exponential fits. (c) Circulating NAM from tail bleeds at the indicated times after a 50 mg/kg bolus of 2H,13C-NR or 2H,13C-NMN by oral gavage or by IV injection. (d) Corresponding circulating NR and NMN. (e) Corresponding tissue NAD labeling. Data are mean ± s.d., n = 3. See also Figure S4 and Figure S7.
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