Bioenergetic Differences in the Airway Epithelium of Lean Versus Obese Asthmatics Are Driven by Nitric Oxide and Reflected in Circulating Platelets

Abstract

Aims: Asthma, characterized by airway obstruction and hyper-responsiveness, is more severe and less responsive to treatment in obese subjects. While alterations in mitochondrial function and redox signaling have been implicated in asthma pathogenesis, it is unclear whether these mechanisms differ in lean versus obese asthmatics. In addition, we previously demonstrated that circulating platelets from asthmatic individuals have altered bioenergetics; however, it is unknown whether platelet mitochondrial changes reflect those observed in airway epithelial cells. Herein we hypothesized that lean and obese asthmatics show differential bioenergetics and redox signaling in airway cells and that these alterations could be measured in platelets from the same individual. Results: Using freshly isolated bronchial airway epithelial cells and platelets from lean and obese asthmatics and healthy individuals, we show that both cell types from obese asthmatics have significantly increased glycolysis, basal and maximal respiration, and oxidative stress compared with lean asthmatics and healthy controls. This increased respiration was associated with enhanced arginine metabolism by arginase, which has previously been shown to drive respiration. Inducible nitric oxide synthase (iNOS) was also upregulated in cells from all asthmatics. However, due to nitric oxide synthase uncoupling in obese asthmatics, overall nitric oxide (NO) bioavailability was decreased, preventing NO-dependent inhibition in obese asthmatic cells that was observed in lean asthmatics. Innovation and Conclusion: These data demonstrate bioenergetic differences between lean and obese asthmatics that are, in part, due to differences in NO signaling. They also suggest that the platelet may serve as a useful surrogate to understand redox, oxidative stress and bioenergetic changes in the asthmatic airway.

Keywords: asthma; glycolysis; metabolism; mitochondria; obesity.

FIG. 1.

Platelet and airway epithelial cell bioenergetics differs in lean and obese asthmatics. (A, B) Measured basal ECAR of (A) platelets and (B) airway epithelial cells isolated from healthy lean, asthmatic lean, healthy obese, and asthmatic obese individuals. (C, D) Representative oxygen consumption traces of (C) platelets and (D) airway epithelial cells from all four experimental groups. Basal respiration rate was measured followed by proton leak after the addition of oligomycin (O), maximal respiration after the addition of FCCP (F), and nonmitochondrial respiration after the addition of rotenone (R). (E, F) Quantification of several traces in (E) platelets and (F) epithelial cells such as those shown in (C, D), respectively. For platelet studies: healthy lean (n = 10), lean asthmatic (n = 8), healthy obese (n = 11), and asthmatic obese (n = 8). For airway epithelial cells: all groups n = 5. All data are mean ± SEM. Statistical significance was tested by one-way ANOVA. ^#p < 0.05; *p < 0.01. ECAR, extracellular acidification rate; FCCP, carbonyl cyanide-ρ-trifluoromethoxyphenylhydrazone; SEM, standard error of the mean. Color images are available online.

FIG. 2.

Platelet ECAR and maximal OCR correlate with airway epithelial ECAR and maximal OCR. The correlation between (A) basal ECAR and (B) maximal OCR in platelets and airway epithelial cells in the same individuals. Pearson R and p-value are reported for each graph. n = 5 for each experimental group (20 total). OCR, oxygen consumption rate.

FIG. 3.

Mitochondrial number and enzymatic activity are increased in cells from lean and obese asthmatic subjects. (A) Mitochondrial DNA copy number (normalized to platelet number) in platelets and from healthy lean, asthmatic lean, healthy obese, and asthmatic obese individuals. (B) Mitochondrial DNA copy number normalized to corresponding nuclear DNA copy number in airway epithelial cells from the same groups. Bars in (A, B) represent mean ± SEM. (C) Citrate synthase activity (represented as a fold change of the respective healthy lean group) in airway epithelial cells (black bars) and platelets (white bars). (D) Enzymatic activity of complexes I, II, and IV measured in airway epithelial cells and represented as a fold change of the healthy lean group. n = 10 for platelets and n = 5 for airway epithelial cells. Statistical significance determined by one-way ANOVA. ^#p < 0.05, *p < 0.01 versus respective healthy control. Color images are available online.

FIG. 4.

Platelets and airway epithelial cells show increased mitochondrial and cellular oxidant production. (A, B) Mitochondrial superoxide production in (A) platelets and (B) airway epithelial cells as well as (C, D) total cellular H₂O₂ production measured in (C) platelets and (D) airway epithelial cells from healthy lean, asthmatic lean, healthy obese, and asthmatic obese individuals. All data are individual values of each subject with bars representing the mean and standard deviation. Statistical significance determined by one-way ANOVA. ^#p < 0.05 and *p < 0.01 versus respective healthy control if not otherwise noted. n = 10 for platelets and n = 5 for airway epithelial cells. H₂O₂, hydrogen peroxide.

FIG. 5.

Arginine metabolism and NO production are altered in cells from asthmatics. (A, B) Arginase activity in (A) airway epithelial cells and (B) platelets from all four experimental groups. (C) Arginine and (D) ornithine levels in the plasma of all four experimental groups (n = X for each group). (E) Activity of NOS (expressed as fold change of healthy lean group) in airway epithelial cells (black bars) and platelets (white bars). (F) Nitrite and nitrate levels combined (expressed as fold change of healthy lean group) in airway epithelial cells (black bars) and platelets (white bars). All data expressed from healthy lean, asthmatic lean, healthy obese, and asthmatic obese groups as mean ± SEM. Statistical significance was determined by one-way ANOVA. For cell data, n = 5 for all groups. ^#p < 0.05 and *p < 0.01. NOS, nitric oxide synthase. Color images are available online.

FIG. 6.

NOS contributes to oxidant production in cells from obese asthmatic subjects. (A, B) Total cellular H₂O₂ production measured in the absence (black bars) and presence (white bars) of L-NAME (100 μM) in (A) airway epithelial cells and (B) platelets. (C) The 3-nitrotyrosine concentration measured in epithelial cells (black bars) and platelets (white bars) from healthy lean, asthmatic lean, healthy obese, and asthmatic obese individuals. n = 5 in each group. Data are mean ± SEM. Statistical significance determined by one-way ANOVA. ^#p < 0.05 and *p < 0.01. L-NAME, L-N^G-nitroarginine methyl ester.

FIG. 7.

NO inhibits respiration and complex IV but not glycolysis in cells from lean asthmatic subjects. (A, B) Basal and maximal respiratory rates in (A) airway epithelial cells and (B) platelets isolated from lean and obese asthmatic individuals and treated with (white bars) or without (black bars) c-PTIO (100 μM). (C) Basal ECAR in airway epithelial cells and platelets isolated from lean and obese asthmatic individuals and treated with or without c-PTIO. (D) Representative trace of platelets permeabilized with digitonin (20 μg/mL) and treated with TMPD (0.5 mM) and ascorbate (2 mM) to measure oxygen consumption by complex IV in the presence and absence of c-PTIO. (E) Quantification of eight traces similar to those shown in (D). Statistical significance determined by one-way ANOVA. n = 4; Data are mean ± SEM ^#p < 0.05 and *p < 0.01. c-PTIO, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide; TMPD, tetramethyl-p-phenylenediamine. Color images are available online.