Inhibition of nicotinamide phosphoribosyltransferase: cellular bioenergetics reveals a mitochondrial insensitive NAD pool

Abstract

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.

FIGURE 1.

Effect of FK866 on NAD and ATP contents in various cell cultures under basal and PARP-1-activating conditions. Concentration-dependent effects of FK866 on NAD (A) and ATP (B) contents in various cultured cell types. Basal contents of NAD and ATP contents were comparable among different cell types and comprised between 10.4 ± 4.4 and 23.5 ± 7.8 nmol/mg of protein, respectively. Time course of the effects of FK866 at 100 μm and 100 nm on NAD (C) and ATP (D) contents in HeLa cells. Effects of FK866 (100 μm, 30 min preincubation) on NAD (E) and ATP (F) contents of cultured cells exposed 1 h to the PARP-1 activating compound MNNG (100 μm). Each point/column represents the mean ± S.E. of at least three experiments conducted in duplicate.

FIGURE 2.

Effects of various compounds on FK866-induced NAD depletion in HeLa cells. A, effects of the PARP-1 inhibitor PHE, the MART inhibitors novobiocin (NOV) and mIBG, and the Sirt-1 inhibitor EX-257 (EX) on NAD depletion prompted by FK866 (100 μm/1 h). Each drug was preincubated 30 min at the indicated concentrations. B, additive effects of the PARP-1 inhibitor PHE (30 μm) and MART inhibitors novobiocin (NOV, 1 mm) and mIBG (100 μm) on reduction of NAD depletion prompted by FK866 (100 μm/1 h). C, effects of FK866, the PARP-1 inhibitor PHE and MART inhibitors novobiocin (NOV) and mIBG (all at 100 μm) on in vitro activity of purified PARP-1. D, effects of kynurenine (KYN, 200 μm) and the kynurenine monooxygenase inhibitor Ro-618048 (RO, 30 μm) on NAD contents in cells under control conditions or exposed to FK866 (100 μm/1 h). KYN and RO have been preincubated 60 min. Each column represents the mean ± S.E. of three experiments conducted in duplicate. A, *, p < 0.05; **, p < 0.01 versus FK866. B, *, p < 0.05; **, p < 0.01 versus control. D, *, p < 0.05; **, p < 0.01 versus control; §, p < 0.05, versus FK866. Analysis of variance and Tukey's post hoc test were used.

FIGURE 3.

Effects of FK866 on subcellular NAD contents, poly(ADP-ribose) formation, and ATP production. Quantitation of cytosolic and mitochondrial NAD contents in HeLa cells exposed to FK866 at 100 μm for 1 h (A) or 100 nm for 24 h (B). C, Western blotting evaluation of the effects of FK866 (100 nm/24) on poly(ADP-ribose) (PAR) formation prompted by the PARP-1 activator MNNG (100 μm). Note that poly(ADP-ribosyl)ated proteins appear as a smear due to their highly increased molecular weight. D, visualization of autofluorescence in cells under control conditions or exposed to FK866 at 100 μm for 1 h or 100 nm for 24 h. E, quantitation of cell autofluorescence of cells shown in D. F, ATP content of cells exposed or not to 100 nm FK866 for 24 h, cultured overnight in the absence of glucose, and pulsed for 1 h with 1 mm pyruvate (PYR). Values represent the % of ATP of cells cultured in the presence of glucose (control). G, ATP production by mitochondria isolated from control or FK866-challenged cells and exposed 5 min to 1 mm pyruvate (PYR) or 1 mm pyruvate plus 3 μm ADP. In A, B, F, and G, each column represent the mean ± S.E. of 7 (A), 5 (B), or 3 (F and G) experiments. In C the blot is representative of three different experiments. In D an experiment representative of 4 is shown. In E each column represents the mean ± S.E. of the fluorescence present in each microscopic field (3 fields per slide have been acquired). A and B, *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control. Analysis of variance and Tukey's post hoc test were used.

FIGURE 4.

LC-MS/MS evaluation of cytosolic and mitochondrial concentrations of FK866 in HeLa cells. A, ion transition spectrum of FK866 fragmentation. B, cytosolic and mitochondrial concentrations of FK866 in cells exposed to 100 μm for 1 h. Values are the mean of three independent experiments.

FIGURE 5.

Subcellular localization of Nampt in HeLa cells. A, Western blot analysis of Nampt in whole extract, or cytoplasmic, nuclear, and mitochondrial fractions. Apoptosis inducing factor (AIF) is shown as a mitochondrial marker. B, fluorescence of cells transfected with cytosolic GFP (Cyt-GFP), Nampt-GFP, or mit-GFP together with that of their mitochondria stained with TMRE is shown. The merging of green and red fluorescence is also shown. Note that TMRE fluorescence becomes yellow after merging with that originating from mit-GFP, whereas remains red or appears orange after merging with that of Cyt-GFP or Nampt-GFP. C, magnifications of areas delimited by dotted lines in B are shown. Note the negative image of the TMRE-positive mitochondria in the cytoplasmic area of cells expressing Nampt-GFP. In A, a representative blot of two is shown. In B and C, representative images of 3 experiments are shown. Bars = 4 μm (A) and 0.2 μm (B).

FIGURE 6.

Effects of PARP-1 inhibition of boosting of the kynurenine pathway on the effects of FK866 on NAD contents in various mouse organs. Mice were injected intraperitoneally with FK866 (100 mg/kg) and subcutaneously with PARP-1 inhibitors PJ34 (10 mg/kg) or kynurenine (100 mg/kg) and NAD contents were measured in different tissues 8 h later. Each column represents the mean ± S.E. of 5 mice. *, p < 0.05 versus vehicle. Analysis of variance and Tukey's post hoc test was used.