Figure 1.
FK866 treatment causes a time dependent decrease in cellular NAD+/NADH and NADP+/NADPH content.
A, Western blot analysis of NAMPT and NAPRT expression in RAW 264.7 and Maf-DKO macrophages. Lysate from mouse embryonic fibroblasts (MEF) was used as positive control for NAPRT expression. Note that relative high amounts of lysate were used (intense tubulin control signal) for detection of NAPRT. B,C,E,F, Pyridine nucleotide levels in RAW 264.7 cells incubated for the indicated time periods with or without 10 nM FK866. H,I,K,L, Pyridine nucleotide levels in Maf-DKO cells incubated for the indicated time periods with or without 5 nM FK866. D,G,J,M, NAD+/NADH and NADPH/NADP+ ratios were calculated from the data in A&B, E&F, H&I, and K&L. Data in B-M represent means of three experiments performed in triplicate. ND, not detected. (N.S., not significant, *p<0.05, **p<0.01, ***p<0.001, unpaired t-test).
Figure 2.
The effect of FK866 on RAW 264.7 metabolism.
A, B, Glucose consumption and lactate production in the presence and absence of FK866. Cells were incubated in control or FK866 medium for a total of 24 hours. Glucose and lactate was measured in medium supernatants collected over the last 6 hours (18–24 h) of incubation. C, Intracellular ATP levels of RAW 264.7 macrophages treated with 5 nM FK866 for 3, 15, or 24 hours. D, Mitochondrial NAD(P)H-level. Cellular autofluorescence was measured after 24 hour incubation in control medium or medium with 5 nM FK866. E, After 24 hour pre-incubation with or without FK866, oxygen consumption was measured in suspensions of 1×106 cells in either FK866 or control medium. Control and FK866 treated cells were analyzed in parallel on the same day. The basal oxygen consumption was measured where after oligomycin, FCCP, and rotenone was added successively in order to determine the leak respiration, maximal respiration (Max), and residual oxygen consumption (Res). Data in a-c represent means ± SEM of three experiments performed in triplicate, in d means ± SEM of three experiments, and in e means ± SEM of five experiments. (*p<0.05, **p<0.01; unpaired t-test).
Figure 3.
Inhibition of NAD+ salvage synthesis with FK866 does not affect proliferation or viability of macrophages.
A, B, RAW 264.7 and Maf-KDO proliferation in the presence of different concentrations of FK866, monitored by measuring the increase in protein mass at different intervals over a 24 or 72 hour period. Data represent means of three independent experiments performed in triplicate. C, RAW 264.7 cell viability in the presence of 10 nM FK866. The appearance of the fluorescent apoptosis-specific pSIVA signal was recorded over time and the total pSIVA pixel area was determined per frame using Fiji Imaging software and plotted against time. Lines represent averages from one experiment performed in triplicate. (N.S., not significant; unpaired t-test).
Figure 4.
LPS-stimulated spreading is inhibited during NAMPT inhibition in RAW 264.7 macrophages.
A–D, RAW 264.7 cells were treated with 10 nM FK866 for 15 or 24 hours and simultaneously stimulated overnight with LPS. A representative image of each condition is shown, (Bar = 10 µm). E, To quantify spreading efficiency, RAW 264.7 macrophages expressing lifeact-EYFP were pre-treated with 5 nM FK866 for the indicated time periods, detached, resuspended, seeded in 96 well plates and allowed to adhere. Adherence and spreading of EYFP-positive cells were recorded over time. The average pixel area per cell was determined at every 10 minute interval. Lines represent means ± SEM (grey bars) of three experiments performed in duplicate.
Figure 5.
Actin reorganization is altered during NAMPT inhibition in RAW 264.7 macrophages.
Actin organization was assessed in wild type cells seeded on glass coverslips, treated with FK866 for 15 or 24 hours, and simultaneously stimulated overnight with 100/ml LPS. After fixation in 2% PFA, cellular actin was stained with phalloidin-Alexa568 and cells were imaged on a Zeiss LSM510 META confocal laser scanning microscope. (Bar = 10 µm).
Figure 6.
Phagocytosis efficiency is reduced when NAD+ salvage synthesis is inhibited.
Cells were pretreated with 10-labeled complement opsonized zymosan (COZ) particles for 30 min and analyzed by FACS. For each sample the percentage of FITC positive cells (A) and mean fluorescence of FITC positive cells (B) were measured. The phagocytic index (C) was calculated as the product of these two parameters. The internalization efficiency (D) was determined by quenching the extracellular FITC-COZ of one sample fraction with 0.05% trypan blue in potassium dihydrogen citrate/saline, pH 4.4. Unquenched fractions were used to determine the total fluorescence per cell (internalized and external particles), while quenched fractions were used to measure only the internal fluorescence per cell. Data represent means ± SEM of three assays performed in triplicate. Values were normalized to controls in b and c. (*p<0.05; paired t-test).
Figure 7.
NAD+, NMN, and NADP+ supplementation stimulate phagocytosis and give reversal of FK866 effects.
Cells were incubated for 24 hours with 100 µM NAD+, 100 µM NMN or 50 µM NADP+ in the presence or absence of 5 nM FK866 and activated overnight with LPS before they were incubated with FITC-labeled complement opsonized zymosan (COZ) particles for 30 min and analyzed by FACS. Values were normalized to the 0 nM FK866 control. Data represent normalized means ± SEM of four (0 nM FK866) or three (5 nM FK866) experiments performed in duplicate. (N.S., not significant, *p<0.05, **p<0.01; paired t-test).
Figure 8.
NAMPT-mediated salvage synthesis of NAD+ controls morphofunctional changes of macrophages: Synthesis of literature evidence.
NAD+ is required to sustain high glycolytic activity in macrophages. Metabolism of glucose yields cellular ATP which is loaded onto G-actin monomers by profilin before incorporation into an actin filament, or used to activate the Arp2/3 complex. ATP also binds to Rho GTPase Rho A, which in turn also stimulates actin polymerization (pink arrows). LPS stimulation induces the expression of the NAD+ synthesizing enzyme NAMPT (turquoise arrows). NAMPT-mediated NAD+ synthesis may regulate actin dynamics by activating Cdc42 (green arrows) or by influencing cellular metabolism and, thereby, intracellular pH and cofilin activity (purple arrows). Alternatively, NAMPT may control redox regulation of the actin cytoskeleton via mical or NADPH oxidase by regulating NADPH levels (orange arrows). GLUT, glucose transporter; NOX, NADPH oxidase; PPP, pentose phosphate pathway; ROS, reactive oxygen species; SSH-1L, slingshot-1L; TLR4, toll-like receptor 4.