Impairment of Fatty Acid Oxidation in Alveolar Epithelial Cells Mediates Acute Lung Injury

Abstract

Profound impairment in cellular oxygen consumption, referred to as cytopathic dysoxia, is one of the pathological hallmarks in the lungs of patients with pathogen-induced acute lung injury (ALI). However, the underlying mechanism for this functional defect remains largely unexplored. In this study, we found that primary mouse alveolar epithelial cells (AECs) conducted robust fatty acid oxidation (FAO). More importantly, FAO was strikingly impaired in AECs of mice with LPS-induced ALI. The metabolic deficiency in these cells was likely due to decreased expression of key mediators involved in FAO and mitochondrial bioenergenesis, such as peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, carnitine palmitoyltransferase 1A, and medium-chain acyl-CoA dehydrogenase (CAD). We found that treatment of alveolar epithelial line MLE-12 cells with BAL fluids from mice with ALI decreased FAO, and this effect was largely replicated in MLE-12 cells treated with the proinflammatory cytokine TNF-α, which was consistent with downregulations of PGC-1α, carnitine palmitoyltransferase 1A, long-chain CAD, and medium-chain CAD in the same treated cells. Furthermore, we found that the BAL fluids from ALI mice and TNF-α inhibited MLE-12 bioenergenesis and promoted cell apoptosis. In delineation of the role of FAO in ALI in vivo, we found that conditional ablation of AEC PGC-1α aggravated LPS-induced ALI. In contrast, fenofibrate, an activator of the PPAR-α/PGC-1α cascade, protected mice from this pathology. In summary, these data suggest that FAO is essential to AEC bioenergenesis and functional homeostasis. This study also indicates that FAO impairment-induced AEC dysfunction is an important contributing factor to the pathogenesis of ALI.

Keywords: acute lung injury; alveolar epithelial cell; fatty acid oxidation; peroxisome proliferator–activated receptor γ coactivator-1α.

Figure 1.

Key regulatory mediators involved in fatty acid oxidation (FAO) and mitochondrial bioenergenesis are downregulated in alveolar epithelial cells (AECs) of acute lung injury (ALI) mice. (A) Primary AECs were purified from mice that were instilled intratracheally with saline or LPS (2 mg/kg) for 24 hours. Total RNAs were isolated and RNA sequencing analysis performed. Heat map of gene expression of regulators and mediators related to FAO and mitochondrial biogenesis and energenesis are presented. (B and C) The expression of the indicated genes in AECs was determined by real-time PCR (B) and Western blotting (C). (A–C) n = 3 for the saline and LPS groups, respectively; mean ± SEM; *P < 0.05, ***P < 0.001. (D) The expression of the indicated genes in total lungs was determined by real-time PCR. n = 5 for the saline and LPS groups, respectively; mean ± SEM; **P < 0.01, ***P < 0.001. PGC-1α = peroxisome proliferator–activated receptor γ coactivator-1α.

Figure 2.

AECs use both glucose and fatty acids, and FAO is impaired in AECs of mice with LPS-induced ALI. (A) Primary AECs were seeded in Seahorse XF-24 microplates and incubated with substrate-limited media for 3 hours. Basal oxygen consumption rate (OCR) was then recorded before BSA or palmitate-BSA (final concentration: 150 μM) was injected into the wells. After injection, OCR was continuously monitored until it reached the maximum value. n = 4 per group; mean ± SEM. (B) Primary AECs were seeded in Seahorse XF-24 microplates and incubated with extracellular acidification rate (ECAR) media for 1 hour. Basal OCR and ECAR were then recorded before glucose (final concentration: 10 mM) was injected into the wells. After injection, OCR and ECAR were continuously monitored until they reached the maximum values. n = 3 per group; mean ± SEM. (C) Primary AECs purified from saline or intratracheal LPS–treated mice were seeded in Seahorse XF-24 microplates and incubated with substrate-limited media for 3 hours. Basal OCR was then recorded before palmitate-BSA was injected into the wells. After injection, OCR was continuously monitored until it reached the maximum value. OCR values were normalized to total protein content of the cellular extract from each well. n = 4 mice per group; mean ± SEM.

Figure 3.

Alveolar epithelial line MLE-12 cells use fatty acids, and FAO is impaired in MLE-12 treated with BAL fluid (BALF) from ALI mice. (A) Alveolar epithelial line MLE-12 cells were seeded in Seahorse XF-24 microplates and incubated with substrate-limited media for 12 hours. Basal OCR was then recorded before BSA or palmitate-BSA was injected into the wells. After injection, OCR was continuously monitored until it reached the maximum value. n = 5 or 6 for the palmitate-BSA or BSA group; mean ± SEM. (B) MLE-12 cells were seeded in Seahorse XF-24 microplates and treated with saline or 100 μM etomoxir (ETO) for 1 hour. The media were then replaced with FAO assay media supplemented with 150 μM palmitate-BSA and cultured for 1 hour, followed by sequential treatments with 3 μg/ml oligomycin (Oligo), 6 μM FCCP, and 1 μM rotenone (Rot) and 0.5 μM antimycin A (Ant). Real-time OCR was recorded. n = 6 or 5 for the saline or ETO group; mean ± SEM. (C) MLE-12 cells were seeded in Seahorse XF-24 microplates and incubated with ECAR media for 1 hour. Basal OCR and ECAR were then recorded before glucose (final concentration: 10 mM) was injected into the wells. After injection, OCR and ECAR were continuously monitored until they reached the maximum values. n = 7; mean ± SEM. (D) MLE-12 cells were seeded in Seahorse XF-24 microplates and treated for 16 hours with BALF pooled from five mice instilled intratracheally with saline or LPS. The media were then replaced with FAO assay media supplemented with 150 μM palmitate-BSA and cultured for 1 hour, followed by sequential treatments with 3 μg/ml oligomycin, 6 μM FCCP, and 1 μM Rot and 0.5 μM Ant. Real-time OCR was recorded. n = 6 per group; mean ± SEM. (E–F) MLE-12 cells were incubated for 16 hours with BALFs from mice instilled intratracheally with saline or LPS for 24 hours. The mRNA (E) and/or protein (F) levels of indicated genes were determined by real-time PCR and Western blotting. (E) n = 3; mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 compared with saline group. FCCP = Carbonyl cyanide 4-(trifluoro)methoxyphenylhydrazone.

Figure 4.

Proinflammatory cytokine TNF-α inhibits FAO in AECs. (A and B) MLE-12 cells were treated for 16 hours with 2 ng/ml recombinant mouse TNF-α. The protein (A) and/or mRNA (B) levels of indicated genes were determined by Western blotting and real-time PCR. **P < 0.01 compared with control group. (C) MLE-12 cells were seeded in Seahorse XF-24 microplates and treated with 2 ng/ml or 5 ng/ml mouse TNF-α in Opti-MEM overnight. The media were then replaced with FAO assay media supplemented with 150 μM palmitate-BSA and cultured for 1 hour, followed by sequential treatments with 3 μg/ml oligomycin, 6 μM FCCP, and 1 μM rotenone and 0.5 μM antimycin A. Real-time oxygen consumption rate was recorded. n = 6 per group; mean ± SEM. con = control; MEM = Eagle’s minimum essential medium.

Figure 5.

Blocking FAO decreases intracellular ATP levels and promotes AEC apoptosis. (A–C) MLE-12 cells were treated for 16 hours with 100 μM ETO (A), BALFs from mice instilled intratracheally with saline or LPS (B), or 2 ng/ml mouse TNF-α (C). Intracellular ATP levels were determined using Luminescent ATP Detection Assay Kit. n = 4; mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 compared with respective control group. (D and E) MLE-12 cells were treated for 16 hours with BALFs from mice instilled intratracheally with saline or LPS. The cells were harvested and incubated with annexin V–FITC and propidium iodide (PI). Cell apoptosis was analyzed by flow cytometry (D). Percentages of apoptotic cells were plotted (E). (F and G) MLE-12 cells were pretreated for 1 hour with 100 μM ETO, followed by incubation with 2 ng/ml mouse TNF-α for 16 hours. Cell apoptosis was analyzed by flow cytometry (F). Percentages of apoptotic cells were plotted (G). (D–G) n = 3; mean ± SD; **P < 0.01, ***P < 0.001. (H) MLE-12 cells were transduced with control lentivirus or lentivirus that expressed mouse PGC-1α. Levels of PGC-1α were determined 1 day after transduction. (I) MLE-12 cells were transduced with control lentivirus or lentivirus that expressed mouse PGC-1α. The cells were then plated in Seahorse XF-24 microplates overnight. The media were then replaced with FAO assay media supplemented with 150 μM palmitate-BSA and cultured for 1 hour, followed by sequential treatments with 3 μg/ml oligomycin, 6 μM FCCP, and 1 μM Rot and 0.5 μM Ant. Real-time oxygen consumption rate was recorded. n = 6 per group; mean ± SEM. (J) MLE-12 cells were transduced with control lentivirus or lentivirus that expressed mouse PGC-1α. The cells were treated with TNF-α overnight. The expression of the indicated genes were determined by real-time PCR. n = 3; mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001. (K) The cell transduction and treatment were conducted as in J, and annexin V–FITC and PI assay performed. Percentages of apoptotic cells were plotted. n = 4; mean ± SD; **P < 0.01, ***P < 0.001.

Figure 6.

Ablation of AEC PGC-1α aggravates LPS-induced ALI. (A) AECs were purified from the control PGC-1α^fl/fl and AEC PGC-1α^−/− mice and levels of the indicated genes determined. n = 3; mean ± SEM; *P < 0.05, **P < 0.01. (B) Mice were treated with tamoxifen as in A. AECs were purified, seeded in Seahorse microplates in FAO assay media supplemented with 150 μM palmitate-BSA and cultured for 1 hour, followed by sequential treatments with 3 μg/ml oligomycin, 6 μM FCCP, and 1 μM Rot and 0.5 μM Ant. Real-time OCR was recorded. n = 2 per group. (C and D) The control PGC-1α^fl/fl and AEC PGC-1α^−/− mice were instilled intratracheally with saline or LPS (2 mg/kg in 50 μl saline). At 24 hours after treatment, the mice were killed and BALF (0.5 ml for 3 times/mouse) and lungs collected. BALF was centrifuged to collect supernatants and cell pellets. Red blood cells in the cell pellets were lysed. The whole lungs were homogenized in 3-ml protein extraction buffer. Levels of indicated proinflammatory cytokines in BALF (C) and lung extracts (D) were determined by ELISA. (E and F) Total protein levels (E) and leukocyte numbers (F) in the BALFs. *P < 0.05. (G) Lungs of experiments similar to C were fixed with 10% neutral-buffered formalin and tissue slides prepared. Hematoxylin and eosin staining was performed. (H) Lungs of experiments similar to C were fixed and tissue slides prepared. Immunohistochemistry (IHC) assays for cleaved caspase 3 were performed. Rabbit IgG was used as IHC negative control. (G and H) Original magnification ×100; scale bars: 200 μm.

Figure 7.

The activator of PGC-1α/peroxisome proliferator–activated receptor-α cascade, fenofibrate, attenuates LPS-induced ALI in mice. C57BL/6 mice (8 wk old) were instilled intratracheally with saline or LPS (2 mg/kg in 50 μl saline). At 4 hours after the intratracheal treatment, the mice were injected intraperitoneally with vehicle or fenofibrate (100 mg/kg body weight). At 24 hours after saline or LPS instillation, the mice were killed and BALF (0.5 ml for 3 times/mouse) collected. BALF was centrifuged to collect supernatants and cell pellets. Red blood cells in the cell pellets were lysed. Levels of indicated proinflammatory cytokines (A), protein concentrations (B), and total leukocyte numbers (C) in BALF was determined. (A–C) n = 3, 5, 3, 5 mice, respectively; mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. (D) Primary AECs were purified from mice injected intraperitoneally with vehicle or fenofibrate. Levels of the indicated genes in the cells were determined. n = 3; mean ± SEM; *P < 0.05, **P < 0.01. (E) Primary AECs purified from saline- or LPS-instilled mice injected intraperitoneally with vehicle or fenofibrate were seeded in Seahorse XF-24 microplates and incubated with substrate-limited media for 3 hours. Basal OCR was then recorded before BSA or palmitate-BSA was injected into the wells. After injection, OCR was continuously recorded until stable levels were reached. Fold change in average OCR values were plotted. n = 4, 4, 2; ***P < 0.001.