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ORIGINAL RESEARCH Short-Term Cigarette Smoke Exposure Leads to Metabolic Alterations in Lung Alveolar Cells Amit R. Agarwal, Fei Yin, and Enrique Cadenas Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California

Abstract Cigarette smoke (CS)-induced alveolar destruction and energy metabolism changes are known contributors to the pathophysiology of chronic obstructive pulmonary disease (COPD). This study examines the effect of CS exposure on metabolism in alveolar type II cells. Male A/J mice (8 wk old) were exposed to CS generated from a smoking machine for 4 or 8 weeks, and a recovery group was exposed to CS for 8 weeks and allowed to recover for 2 weeks. Alveolar type II cells were isolated from air- or CS- exposed mice. Acute CS exposure led to a reversible airspace enlargement in A/J mice as measured by the increase in mean linear intercept, indicative of alveolar destruction. The effect of CS exposure on cellular respiration was studied using the XF Extracellular Flux Analyzer. A decrease in respiration while metabolizing glucose was observed in the CS-exposed group, indicating altered glycolysis that was compensated by an increase in palmitate utilization; palmitate utilization was accompanied by an increase in the expression of CD36 and carnitine-palmitoyl transferase 1 in type II alveolar cells for the transport of palmitate

Glucose is an important substrate utilized by lung alveolar type II cells for the generation of energy and for biosynthesis of pulmonary surfactant (1). The biosynthesis and secretion of pulmonary surfactant is a function of type II alveolar cells to help reduce the surface tension and airway resistance at the air–liquid interface and to promote efficient gas exchange (2). Alterations in the processing or secretion of pulmonary surfactant have been shown to cause adult respiratory distress syndrome

into the cells and into mitochondria, respectively. The increase in palmitate use for energy production likely affects the surfactant biosynthesis pathway, as evidenced by the decrease in phosphatidylcholine levels and the increase in phospholipase A2 activity after CS exposure. These findings help our understanding of the mechanism underlying the surfactant deficiency observed in smokers and provide a target to delay the onset of COPD. Keywords: cigarette smoke; alveolar cells; palmitate;

mitochondria; pulmonary surfactant

Clinical Relevance This is the first study measuring mitochondrial respiration in alveolar cells isolated from mice exposed to cigarette smoke. Cigarette smoke decreases the levels of phosphatidylcholine, the major surfactant phospholipid and up-regulates b-oxidation in alveolar cell mitochondria.

(3, 4), chronic obstructive pulmonary disease (COPD) (5), pulmonary edema (6), and infectious diseases (e.g., cystic fibrosis [7] and pneumonia [8]). Pulmonary surfactant forms a protective barrier over the alveolar epithelium to prevent infection caused by inhaled particles and microorganisms (9). Alveolar type II cells are considered stem cells in the adult lung (10) and are actively involved in alveolar repair in response to injury (11); these cells cover 60% of the alveolar epithelium (12)

and differentiate into type I cells after acute injury (13). Cigarette smoke (CS) contains more than 4,000 chemicals (14), some of which include acrolein, nicotine, nitric oxide, nitrogen dioxide, polycyclic aromatic hydrocarbons, and nitrosamines, along with additives such glycerin and sugars (15). Exposure to CS leads to an oxidant/ antioxidant imbalance in the lungs that forms the basis for the development of COPD (16). Mitochondrial metabolic

( Received in original form December 10, 2013; accepted in final form March 10, 2014 ) This work was supported by Tobacco-Related Disease Research Program grant 17RT-0171. Correspondence and requests for reprints should be addressed to Enrique Cadenas, M.D., Ph.D., Pharmacology & Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089-9121. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 51, Iss 2, pp 284–293, Aug 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0523OC on March 13, 2014 Internet address: www.atsjournals.org

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ORIGINAL RESEARCH events are associated with redox changes (driven by oxidative stress) in the lungs after CS exposure. A number of glycolytic enzymes are sensitive to CS-induced oxidative stress along with inhibition of the mitochondrial respiratory chain complexes and consequent impairment of energy homeostasis and cell death (17). Acrolein, one of the major constituents of CS, has been shown to inhibit Complexes I and II in hepatocytes (18) and in brain mitochondria (19). Nicotine is a competitive inhibitor for Complex I (20), and nitric oxide is known to react with superoxide anion in mitochondria to yield peroxynitrite, a species known to modify irreversibly mitochondrial proteins by nitration and/or oxidation (21). The susceptibility of mitochondrial proteins to oxidative damage and subsequent dysfunction after exposure to free radicals from CS is well documented (22, 23). The ability of lung mitochondria to utilize palmitate under conditions of starvation indicates its adaptability to utilize alternate substrates for energy production (24, 25). It was previously observed that short-term CS exposure led to a substantial increase in ATP synthase activity even after metabolic alterations in glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent glycolysis (17). Alveolar type II cells increase the utilization of fatty acids after the inhibition of glycolysis in response to acrolein exposure (26). This study is aimed at establishing the metabolic alterations in alveolar type II cells after CS exposure using a novel experimental approach in which alveolar type II cells were isolated from mice exposed to air or CS under controlled conditions for 4 and 8 weeks to characterize their oxidative mitochondrial metabolism of different substrates. The recovery group was exposed to CS for 8 weeks and allowed to recover for 2 weeks before isolating alveolar type II cells for further analyses.

Materials and Methods CS Exposure

Male A/J mice (8 wk old) (Jackson Laboratories, Bar Harbor, ME) were exposed to filtered air (n = 16) or CS for 4 weeks (n = 16) or 8 weeks (n = 16) or exposed to CS for 8 weeks and allowed to recover for 2 weeks (n = 16). CS was generated from Kentucky 3R4F cigarettes

(Tobacco Research Institute, University of Kentucky, Lexington, KY) using a smoking machine (model TE-10; Teague Enterprises, Davis, CA) (27) as described elsewhere (17). All animal protocols were approved by the Department of Animal Resources at the University of Southern California.

Isolation of Primary Alveolar Type II Cells

Primary alveolar type II cells were isolated from mice exposed to CS and filtered air using the dispase (BD Biosciences, Bedford, MA) digestion–agar instillation method. Mice were killed after pentobarbital overdose, and the abdominal cavity was opened. The renal artery was severed to allow blood to flow through. The tissue around the trachea was cleared, and a suture was placed below the trachea. A small incision was made at the top of the trachea to allow a needle to pass through. The needle was held in place by tying a knot with the suture thread. PBS (20 ml) was injected through the left ventricle to perfuse the lungs, and 0.5 ml of 1% lowmelting agarose (Sigma, St. Louis, MO) was injected into the lungs through the trachea. Dispase (5 ml) was then injected into the lungs for digestion, and the lungs were excised and incubated in another 3 ml of dispase for 45 minutes. The lungs were then chopped with fine-tipped forceps in wash medium containing a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 (DMEM/F-12) (Sigma) containing 0.01% DNAase, 1 mM L-glutamine, 100 U/ml sodium penicillin G, and 100 mg/ml streptomycin. The resulting cell mixture was passed through cell strainers of pore size 100, 40, 20, 15, and 10 mM. The cells were incubated with biotinylated antimacrophage antibodies (anti-CD45, anti-CD45.1, anti-CD45.2, anti-Ter 119, and anti-CD16/32) (BD Biosciences) for 30 minutes to separate the other cell types. The purified cells were seeded onto plates coated with Laminin-I (Trevigen, Gaithersburg, MD) in medium containing DMEM/F-12, 1 mM L-glutamine, 0.25% BSA (BD Biosciences), 10 mM HEPES, 0.1 mM nonessential amino acids, 0.05% insulin-transferrinsodium selenite (Roche, Basel, Switzerland), and 100 mg/ml Primocin (Invitrogen, Carlsbad, CA) supplemented with 10% newborn bovine serum (Omega Scientific, Tarzana, CA). The contaminating

fibroblasts were removed by changing to serumless medium after 3 days of seeding. The above procedure consistently yielded 3 million cells per mouse. The cells were plated at a uniform density, and their phenotype was confirmed by staining the cells for Pro-SP C and CD45 (see Figure E1 in the online supplement). Of the cells examined, 96.12% were found to be Pro-SP C positive and CD45 negative. Lung Morphometry

Mice were killed by pentobarbital overdose, and the lungs were inflated using 1% low-melting agarose at a constant pressure of 25 cm H2O and fixed in 10% buffered formalin overnight. The lungs were then paraffin-embedded and cut into 5-mm sections, and the slides were stained with hematoxylin and eosin (H&E). The images were acquired using a Axioskop microscope 53 lens (Zeiss, Oberkochen, Germany). The mean linear intercept (MLI) was determined using computer-assisted morphometry with ImageJ software. The MLI was measured by converting the image to an 8-bit binary image and placing a grid of eight horizontal lines on the image equidistant from each other using ImageJ software (National Institutes of Health, Bethesda, MD). The number of times the grid line intersected the alveolar wall was counted, and an average of the eight counts was made for each figure. Four images were analyzed in this manner for each animal, and lungs from four animals per group were used to calculate the final MLI. The MLI values were obtained by dividing the length of the horizontal line with the number of intersections. No other adjustments were made to the MLI calculations to avoid bias. XF Extracellular Metabolic Flux Analysis

Mitochondrial respiration was measured using the XF Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA) according to manufacturer’s protocol. The type II cells (20,000/well) were seeded directly into XF96 plates after isolation from the CS- and air-exposed mice, and the mitochondrial respiration was measured in Krebs-Henseleit buffered medium (pH 7.4) containing 2.5 mM glucose and 0.5 mM carnitine to facilitate palmitate-BSA uptake. Substrates (glucose, 25 mM; pyruvate, 2 mM; and palmitate-BSA, 200 mM) were added through the first port to obtain basal respiration. Oligomycin (4 mM),

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ORIGINAL RESEARCH cyanide-p-trifluoromethoxyphenylhydrazone (1 mM), and rotenone (1 mM) were added one by one to measure ATP production, maximal respiration rate, and nonmitochondrial respiration (complex I driven), respectively. All values were normalized to control well by measuring the protein concentration using Bradford assay after the metabolic flux analysis.

Total Phosphatidylcholine Assay

The levels of phosphatidylcholine were measured in total cell lysates after centrifuging the lysate at 14,000 3 g to remove cell membrane contamination using kits from Abnova (Taipei, Taiwan) as described elsewhere (26). The OxiRed probe generated after phosphatidylcholine hydrolysis and its subsequent oxidation was measured colorimetrically at 570 nm.

Immunofluorescence

Immunofluorescence experiments were performed on cells fixed with 3.7% formaldehyde at room temperature for 10 minutes followed by permeabilization with 90% methanol for 5 minutes. The cells were then blocked using 3% FBS for 30 minutes followed by incubation with anti-CD36 (1:50) (Novus Biologicals, Littleton, CO) or anti-CPT1a (1:50) (Abcam, Cambridge, MA) antibody for 1 hour. The FITCconjugated secondary antibodies (1:500) were incubated along with DAPI (1:1,000) for 1 hour at room temperature in the dark. The cells were washed with PBS three times, and the images were procured using a BD Pathway 435 High-Content Bioimager (BD Biosciences).

Phospholipase A2 Activity Assay

Phospholipase (PL)A2 activity was measured in cell lysates after CS exposure using kits available from Cayman Chemicals (Ann Arbor, MI) according to the manufacturer’s protocol (26). The free thiol released after hydrolysis of arachidonoyl thio-PC at the sn-2 position by PLA2 was detected at 414 nm using 5,59-dithio-bis(2-nitrobenzoic acid). Statistical Analyses

Student’s t test assuming unequal variances and ANOVA were performed as indicated in the figure legends. Results are means 6 SD from a minimum of three experiments.

Results CS Exposure Leads to Air Space Enlargement in A/J Mice

H&E staining of lungs sections from air- or CS-exposed mice revealed increases in the MLI of 37% (from 38.3 to 52.6 mm) and 43% (55.07 mm) after 4 and 8 weeks of CS exposure, respectively. This increase in airspace enlargement was reduced by 15% (46.8 mm) in the recovery group, indicating the ability of lungs to repair alveolar damage (Figures 1A and 1B). CS Exposure Alters Mitochondrial Oxygen Consumption Rates in Type II Alveolar Cells

The effect of CS on mitochondrial respiration was examined on confluent monolayers of cells isolated from mice exposed to air or CS. Cells were supplemented with glucose 1 pyruvate or glucose or with pyruvate or palmitate-BSA in Krebs-Henseleit–buffered medium, and the increase or decrease in mitochondrial respiration after addition of substrates was noted. The absolute oxygen consumption rate (OCR) values were baselined to the

Figure 1. Cigarette smoke (CS) exposure leads to airspace enlargement in A/J mice. (A) Hematoxylin and eosin–stained images of lung sections from air- or CS-exposed mice obtained using a Zeiss Axioskop microscope (original magnification: 35). The mean linear intercept was determined using ImageJ (B) as described in MATERIALS AND METHODS. **P , 0.01 compared with control (t test). ANOVA was also performed, and statistical significance at P , 0.001 was found.

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ORIGINAL RESEARCH time point just before the addition of substrates. The decrease in basal respiration after addition of oligomycin served as an indicator for ATP production (Table 1). The basal respiration on glucose 1 pyruvate increased significantly by 10% after 8 weeks of CS exposure (Figure 2B), along with a nonsignificant increase in ATP production (Table 1). Cyanide-ptrifluoromethoxyphenylhydrazone was added to uncouple mitochondrial respiration from oxidative phosphorylation, and maximal respiration was recorded. The difference between maximal respiration rate and basal respiration is indicated as the spare respiratory capacity in Table 2. The spare respiratory capacity did not change significantly in alveolar type II cells isolated from mice exposed to air or CS. The observed H1 leak of 20% (Figure 2A) may seem high when compared with basal respiration but falls to 12.5% when comparing with the maximal respiration. A part of the 40% respiration observed after addition of rotenone may be ascribed to fatty acid metabolism donating reducing equivalents at the coenzyme Q site of the respiratory chain. The change in respiration on glucose or pyruvate when supplied individually was more substantial. OCR (basal respiration) from alveolar type II cells isolated from mice exposed to CS decreased significantly by 13 and 10% on glucose after 4 and 8 weeks of CS exposure, respectively, indicating a decrease in pyruvate formation from glycolysis (Figures 2B and 2C). This decrease was reversed when supplied with pyruvate directly, and a 5 and 10% increase in mitochondrial respiration was found after 4 and 8 weeks of CS exposure, respectively (Figures 2B and 2C). Accordingly, ATP production decreased by 38% (40.68–25.21%) and 47% (40.76–21.57%) while metabolizing glucose after 4 and 8 weeks of CS exposure,

respectively (Table 1), and increased by 22% (43.27–53.04%) and 34% (43.72–58.85%) while using pyruvate after 4 and 8 weeks of CS exposure, respectively (Table 1). The ability of alveolar type II cells mitochondria to utilize pyruvate after CS exposure at a higher rate when supplied directly suggests an increasing need of substrates other than glucose for ATP production and an impairment of glycolysis. Accordingly, the basal respiration and ATP production on palmitate-BSA increased by 8 and 13% after 4 weeks of CS exposure and by 13 and 8% after 8 weeks of CS exposure, respectively (Figure 2B; Table 1). Mitochondrial respiration on pyruvate was still higher in the recovery group but was reversed when supplied with palmitate-BSA. The spare respiratory capacity was found to increase on pyruvate and palmitate-BSA, although not significantly, after 8 weeks of CS exposure (Table 2). CS Exposure Alters Extracellular Acidification Rates in Type II Alveolar Cells

The extracellular acidification rates, which serve as an indicator for glycolysis, did not change significantly in alveolar type II cells respiring on glucose or glucose 1 pyruvate in cells isolated from mice exposed to CS for 4 weeks (Figure 3A) but decreased significantly in cells respiring on glucose after 8 weeks of CS exposure (Figure 3B). This alteration in glycolysis was reversed in the recovery group, and an increase was observed while metabolizing glucose and pyruvate or glucose alone (Figure 3C).

antibody using the immunofluorescence technique. The expression of CD36 receptor increased by 38 and 83% in type II alveolar cells isolated from mice exposed to CS for 4 and 8 weeks, respectively, thus suggesting an increase in fatty acid uptake in these cells (Figures 4A and 4B). This increase was reversed in the recovery group. Translocation of fatty acids into mitochondria takes place by a two-step process mediated by the carnitine-palmitoyl transferase system: the expression of carnitine-palmitoyl transferase-1 increased significantly by 69% after 8 weeks of CS exposure, and this increase was reversed in the recovery group (Figures 4C and 4D). CS Exposure Leads to Increased Phosphatidylcholine Breakdown in Type II Alveolar Cells

The levels of phosphatidylcholine—the major surfactant phospholipid—decreased significantly (by 61%) in type II alveolar cells isolated from mice exposed to CS for 8 weeks; the decrease is expected to reflect the decrease in surfactant biosynthesis (Figure 5A) and was not reversed in the recovery group. The release of palmitate from the sn-2 position of phosphatidylcholine by the activity of PLA2 could explain the decrease in the levels of phosphatidylcholine and the increased mitochondrial respiration on palmitateBSA. Accordingly, a significant increase in PLA2 activity after 8 weeks of CS exposure was found (Figure 5B).

Discussion CS Exposure Leads to Increased Transport of Palmitate into Type II Alveolar Cell Mitochondria

The uptake of palmitate by type II alveolar cells is mediated by the CD36 receptor. The expression of the receptor was measured by staining the cells with anti-CD36

The increase in MLI after CS exposure indicates the destruction of alveoli and an enlargement of air spaces, which are indicators for an emphysematous lung. This increase was reversible after smoking cessation, and alveolar repair was almost

Table 1. Effect of Cigarette Smoke Exposure on ATP Production in Alveolar Type II Cells Substrate Glucose 1 pyruvate Glucose Pyruvate Palmitate

Control 44.76 40.68 43.27 41.26

6 6 6 6

3.2* 9.6 4.2 7.0

4 wk CS Exposure 43.21 25.21 53.04 54.15

6 6 6 6

2.4 4.0 5.8† 9.2†

Control 43.76 40.76 43.72 47.71

6 6 6 6

7.3 3.1 0.6 3.7

8 wk CS Exposure 52.42 21.57 58.85 54.93

6 6 6 6

15.3 5.2† 5.7† 2.8†

Control 44.44 42.36 42.76 47.82

6 6 6 6

7.1 3.3 4.6 2.0

Recovery 47.73 45.50 53.73 45.99

6 6 6 6

12.2 10.8 8.6 10.0

Definition of abbreviation: CS, cigarette smoke. *Values are % change in basal respiration after addition of oligomycin indicated as ATP production. † P , 0.05 compared with control (t test).

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Figure 2. CS exposure alters mitochondrial oxygen consumption rates (OCRs) in type II alveolar cells. (A) Time course of OCR of type II alveolar cells respiring on either glucose or palmitate-BSA. Type II alveolar cells were isolated from mice exposed to air or CS for 8 weeks. Changes in OCR on glucose (25 mM) 1 pyruvate (2 mM), glucose (25 mM), pyruvate (2 mM), or palmitate-BSA (200 mM) measured using the XF Extracellular Flux Analyzer as described in MATERIALS AND METHODS after 4 weeks of CS exposure (B), 8 weeks of CS exposure (C), and 8 weeks CS exposure followed by 2 weeks of recovery (D). *P , 0.05 and **P , 0.01 compared with control (t test). FCCP, cyanide-p-trifluoromethoxyphenylhydrazone.

complete after 2 weeks of smoking cessation (Figures 1A and 1B). The decrease in MLI in the recovery group suggested the ability of the alveolar epithelium to repair the damage caused by CS insult. A longer period of recovery may be expected to

further decrease the MLI. However, because no compliance measurements were made we cannot be certain of the extent of the recovery. We believe the actual destruction may not be reversible but may be repairable by the stem cell potential of alveolar type

II cells, which could utilize the metabolic shift to increase fatty acid metabolism as a survival mechanism and differentiate into type I cells. The inability of the alveolar epithelium to completely recover after withdrawal of CS exposure may also be

Table 2. Effect of CS Exposure on Spare Respiratory Capacity in Alveolar Type II Cells Substrate Glucose 1 Pyruvate Glucose Pyruvate Palmitate

Control 40.78 15.32 27.92 23.45

6 6 6 6

3.2* 3.3 9.0 2.2

4 wk CS Exposure 42.51 16.4 45.75 23.69

6 6 6 6

9.5 5.2 9.2 3.9

Control 38.05 11.76 37.54 19.7

6 6 6 6

11.06 5.8 6.4 4.4

8 wk CS Exposure 44.66 10.48 46.01 27.56

6 6 6 6

17.0 1.0 19.3 4.4

Control 34.12 15.89 38.65 20.27

6 6 6 6

13.3 5.7 14.6 9.5

Recovery 31.3 16.45 29.29 22.72

6 6 6 6

11.7 3.6 17.7 9.4

Definition of abbreviation: CS, cigarette smoke. *Values are Dmaximal – basal (%), which represents the difference between maximal respiration and basal respiration and indicates the spare respiratory capacity.

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Figure 3. CS exposure alters mitochondrial extracellular acidification rates in type II alveolar cells. Changes in extracellular acidification rate (ECAR) on glucose (25 mM) 1 pyruvate (2 mM) or glucose measured using the XF Extracellular Flux Analyzer as described in MATERIALS AND METHODS after 4 weeks of CS exposure (A), 8 weeks of CS exposure (B), and 8 weeks of CS exposure followed by 2 weeks of recovery (C). *P , 0.05 and **P , 0.01 compared with control (t test).

interpreted as premature/accelerated aging of lung in response to CS-induced oxidative stress. This would mean that the compliance measurements would not recover to control levels, but the rate of loss in lung function would revert to normal (28). This would account for the slight improvement in life expectancy seen in people who quit smoking. CS exposure is known to contribute to pulmonary cell senescence, which may impair the ability of

lungs to repair the alveolar damage and this may require chronic exposure (29, 30). The experimental model in this study, consisting of a maximum of 8 weeks exposure of mice to CS, may be viewed as acute exposure; 6 months of using this model of smoke exposure system in mice is required to induce disease pathology comparable to human disease (31). A number of studies have shown an increase in MLI of 30 to 40% after CS exposure (32–36), but there have also been studies reporting 10 to 15% increases in MLI after CS exposure. The reason for this discrepancy may be differences in smoke exposure paradigms, type of cigarette used, strains of mice, or techniques used to measure mean linear intercept. The percent changes in basal respiration after addition of substrates, though small, indicate the ability of alveolar cells to respond to external stress conditions. The difference between basal respiration on glucose and palmitate-BSA was exacerbated not only by altered glycolysis but also by increased b-oxidation after CS exposure. This indicates a metabolic shift in alveolar type II cells from glucose (glycolysis) to palmitate (b-oxidation; most likely from dipalmitoyl-phosphatidylcholine) for energy production. The decrease in basal respiration after 4 and 8 weeks of CS exposure while metabolizing glucose (Figures 2A–2C) can be attributed to the inactivation of GAPDH, a central glycolytic enzyme, upon S-glutathionylation (17). This was also supported in type II alveolar cells by the decrease in the extracellular acidification rate after 8 weeks of CS exposure (Figure 3B) while metabolizing glucose. ATP production decreased accordingly after 4 and 8 weeks of CS exposure (Table 1). Because glutathionylation is a reversible posttranslational modification, the decrease in basal respiration and ATP production were reversed in the recovery group. Our previous work showed that CS-induced inhibition of GAPDH in the glycolytic pathway resulted in a shift of glucose metabolism toward the pentose phosphate pathway with concomitant up-regulation of glucose-6-phosphate dehydrogenase. The latter is the rate-limiting enzyme of the pentose phosphate pathway and also supports the increase in the levels of NADPH after CS exposure (17). This shift in metabolic flux to the pentose phosphate pathway has also been observed in

Caenorhabditis elegans under oxidative stress (37) and is known to be a survival mechanism for neurons in response to nitrosative stress (38, 39). The increase in the levels of NADPH helps promote the reduction of oxidized glutathione and to maintain the redox environment in the reduced state. This suggests that the energyproducing glycolytic pathway is not entirely inhibited and that the alveolar cells are using a modified fuel mix. The spare respiratory capacity did not change significantly in type II alveolar cells isolated from mice exposed to CS; alteration in OCR was mainly substrate driven, and no damage was found to the electron transport chain complexes. The constitutive reserve capacity can be expected to be maintained after short-term CS exposure. Data in Table 2 indicate that CS exposure does not alter the spare respiratory capacity, but its values are inherent in the type of substrate being metabolized: the spare respiratory capacity while metabolizing pyruvate was the highest (because pyruvate was readily available for metabolism by the pyruvate dehydrogenase complex, supply of acetylCoA to the tricarboxylic acid cycle, and funneling reducing equivalents through the respiratory chain); conversely, glucose and palmitate require metabolism by glycolysis and b-oxidation to furnish substrates to the tricarboxylic acid cycle (Table 2). We have shown previously (17) that short-term CS exposure leads to reversible changes in energy metabolism and cellular redox status. The levels of glutathione were found to be increased in the lung homogenates and were supported by the up-regulation of the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase. The mitochondrial redox status was also found to be maintained in a reduced state by the increase in levels of NADPH and glutathione after CS exposure. A number of redox-related genes, including glutathione peroxidases (Gpx1, Gpx3, Gpx4), superoxide dismutase 1 and superoxide dismutase 2, peroxiredoxin 5, and glutathione reductase, were also significantly up-regulated after 8 weeks of CS exposure. These changes were found to be reversed in the recovery group, and we believe that 8 weeks of CS exposure may not be enough to cause irreversible damage to redox-related proteins. The levels of proinflammatory mediators (keratinocyte chemoattractant and macrophagemonocyte chemoattractant protein) were

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Figure 4. CS exposure leads to an increase in the expression of CD36 and CPT1 in type II alveolar cells. Immunofluorescence analysis for changes in the expression of CD36 (A) and CPT1 (C) in alveolar type II cells performed by staining for DAPI- (blue) and FITC-labeled CD36 or CPT1 (green). The changes in expression were quantified using Image J software for CD36 (B) and CPT1 (D). *P , 0.05 compared with control (t test). ANOVA was also performed, with values of P , 0.001 for CD36 and P , 0.01 for CPT1. AU, arbitrary units.

not altered significantly after CS exposure. Thus, a longer duration of CS exposure may be needed to observe inflammatory responses (17). Inflammation is one of the major pathophysiological alterations observed with chronic CS exposure (40, 41). The increase in proinflammatory cytokines shown after acute CS exposure at total particulate matter concentrations of 300 mg/m3 (42) is not attainable with the Teague smoke exposure system used in the current study with a maximum total particulate matter concentration of 80 to 90 mg/m3 and has been shown by others (43) not to induce neutrophil infiltration in CS-exposed lungs. Thus, the animal model used in our study may not be clinically relevant to COPD from an inflammation perspective. It is under these molecular 290

events that we observe a metabolic shift in alveolar cell mitochondria. The biosynthesis and secretion of pulmonary surfactant takes place exclusively in type II alveolar cells (2). Phosphatidylcholine is stored in lamellar bodies and serves as a structural marker for the identification of type II alveolar cells. Although glucose is the preferred substrate for energy production in type II alveolar cells under normal conditions (1, 44), palmitate is preferred under altered physiologic states, such as starvation (24, 25) or altered glycolysis (26). Thus, an increase in palmitate metabolism may support the decrease in substrate availability due to altered glycolysis. We have also previously shown a dosedependent increase in oxidation of

palmitate-BSA in primary mice alveolar type II cells after acrolein exposure (26). A number of b-oxidation–related genes (e.g., Acadl and Acadm) were found to be significantly up-regulated after 8 weeks of CS exposure in our previous study (17). Type II alveolar cells mostly depend on circulating fatty acids and express a specific receptor (i.e., CD36 or fatty acid translocase) for the uptake of palmitate (45, 46). This uptake has been shown to be saturable and energy dependent in nature (45). We found a significant, dosedependent, and reversible increase in the expression of CD36 on alveolar type II cells isolated from mice after 4 and 8 weeks of CS exposure (Figures 4A and 4B). The uptake of palmitate into the mitochondria is a two-step process facilitated by CPT1 on

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Figure 5. CS exposure leads to decreased levels of phosphatidylcholine and increased phospholipase (PL)A2 activity in type II alveolar cells. Changes in phosphatidylcholine levels (A) and PLA2 activity (B) after air and CS exposure in type II alveolar cells as described in MATERIALS AND METHODS. *P , 0.05 and **P , 0.01 compared with control (t test). ANOVA was also performed, with values of P , 0.001 for phosphatidylcholine and P , 0.05 for PLA2 activity.

the outer mitochondrial membrane; CPT1 expression was up-regulated in alveolar type II cell mitochondria isolated from mice after CS exposure (Figures 4C and 4D). Our previous microarray performed on mouse lungs exposed to air or CS also indicated increased gene expression of Cpt1a and Slc25a20 (17); the latter encodes for the carnitine/acylcarnitine translocase, which is located on the inner mitochondrial membrane and helps translocate acylcarnitine to the mitochondrial matrix. Slc25a20 increased significantly after 8 weeks of CS exposure (17). The activity of CPT2 releases acyl-CoA into the matrix to undergo b-oxidation. It may be possible

that CS exposure could be associated with the death of cells that were not able to survive due to a compromised ATP generation, whereas cells with up-regulated b-oxidation would be isolated after death. This suggests that up-regulation of b-oxidation is a survival mechanism for alveolar type II cells after CS exposure. The recovery group was included to minimize this possibility; this group did show a decrease in b-oxidation after 2 weeks of withdrawal of CS exposure. Phosphatidylcholine represents approximately 80% of the surfactant phospholipid (2, 47), and the levels decreased significantly after 8 weeks of CS

exposure (Figure 5A). In addition to phosphatidylcholine, pulmonary surfactant is known to contain small amounts of other phospholipids, such as phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine (48). It may be possible that a decrease in phosphatidylcholine biosynthesis up-regulates the biosynthesis of pulmonary surfactant utilizing other phospholipids but could not be expected to recover the total function of pulmonary surfactant in the absence of phosphatidylcholine. Surfactant deficiency as a result of alterations in the levels of phospholipids or inactivation of surfactant proteins is a common observation in smokers and patients with COPD (49, 50). The biosynthesis of phosphatidylcholine is also dependent on the glycolytic pathway because its glycerol backbone originates from glycolysis (2). The inhibition of GAPDH would promote the biosynthesis of phosphatidylcholine by the glycerol-3phosphate pathway. However, this increase is not observed because of the increased activity of PLA2 after 4 and 8 weeks of CS exposure (Figure 5B), which catalyzes the release of the fatty acid at the sn-2 position (mostly palmitate) of the surfactant phospholipid (29, 30). The released palmitate can be converted to palmitoylCoA by the activity of palmitoyl-CoA synthase and then taken up into the mitochondrion by the CPT system. A decrease in the levels of phosphatidylcholine and an increase in PLA2 activity have also been observed in alveolar type II cells after acrolein exposure (26). The gene encoding for PLA2, Pla2g4a, was also found to be up-regulated after CS exposure in our microarray analysis and was one of the five genes that did not return to its original level in the recovery group (17). In the recovery group, a decrease in levels of phosphatidylcholine was observed because the up-regulation of GAPDH-mediated glycolysis would prevent an increase in the phosphatidylcholine levels. Also, the recovery period of 2 weeks may not be sufficient for the phosphatidylcholine levels to recover to the normal levels. This is supported by the failure of Pla2g4a gene expression levels to return to normal levels in the recovery group (17), further indicating that the decrease in PLA2 enzyme activity is due to the switch to glucose as the preferred substrate in alveolar cell mitochondria.

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ORIGINAL RESEARCH The decrease in ATP flux as a result of glycolytic impairment hinders the utilization of glucose as the principal substrate for energy production after CS exposure. This impairment was compensated by increasing the breakdown of the energy-rich fatty acids to maintain the ATP flux. The increase in fatty acid metabolism is supported by the increase in uptake of circulating fatty acids and by their release through the activity of PLA2. The up-regulation of mitochondrial transport of fatty acids by the CPT system helps rescue the decreasing energy production that may occur in systems with impaired glycolysis. These alterations in mitochondrial substrate utilization may have implications for efficient gas exchange at the air–liquid interface and may provide a target to delay the onset of CS-induced alveolar damage in

the form of GAPDH. These results may explain the decrease in free fatty acid levels observed in patients with COPD (51). A high-fat, low-carbohydrate diet has also been shown to cause improvement in lung function in patients with COPD (52). A decrease in body weight in mice exposed to CS was also observed in our first study (17). As with smokers, the mice in the recovery group regained weight in 2 weeks. It could be speculated that the inactivation of GAPDH and the decrease in glycolysis prevent the use of glucose as an energy source and, along with increased breakdown of fatty acids, contribute to the malnutrition observed in patients with COPD (53, 54). Thus, it may only be in metabolism-related aspects that this animal model is clinically relevant to COPD; whether the findings in this study could be

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