Department of Pediatrics, National Jewish Medical and Research Center, and University of Colorado Health Sciences Center, Denver, Colorado 80206
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ABSTRACT |
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Impairment of lung aconitase activity, citric acid cycle, and mitochondrial respiration by hyperoxia necessitates the elevation of glycolysis for energy production and of pentose shunt activity for reducing equivalents. The molecular mechanisms that allow increased glucose utilization are unknown. Adult male and female rats were adapted to sublethal hyperoxia, equivalent to 83% oxygen at sea level, or air for 7 days. Lung RNA and protein increased in hyperoxia (197 and 57%, respectively), whereas total DNA was unchanged. In hyperoxia, lung total hexokinase (HK) activity increased threefold, and mRNAs for HK-II and -III were specifically upregulated. HK-I mRNA was unchanged. mRNAs for HK-II and -III gradually increased during the first 72 h in hyperoxia. HK-II mRNA was significantly elevated at 72 h, preceding changes in lung cell populations. Although virtually absent in air, HK-II activity was highly expressed in hyperoxia. Among lung glucose transporters, specific expression of mRNAs for GLUT-4 (insulin dependent) and sodium-glucose cotransporter-1 was decreased, whereas that for GLUT-1 was minimally changed. Adaptation to hyperoxia involves coordinated changes in gene expression for the proteins regulating pulmonary glucose transport.
messenger ribonucleic acid; lung; glycolysis; monosaccharide transport proteins; energy metabolism
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INTRODUCTION |
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EXPOSURE TO HIGH CONCENTRATIONS of oxygen is often used therapeutically in many forms of lung injury. These exposures, however, can also produce additional lung damage due to the toxic effects of oxygen. The mechanisms whereby the lung is defended against such damage are incompletely understood. At the organ level, adaptation to hyperoxia involves complex cellular (16) and metabolic changes. Considerable attention has been focused on alterations in lung antioxidant defenses as an essential element of such adaptation. However, it is clear that the expression of a number of families of proteins, including those related to ion transport (30), oxidant-antioxidant balance (22), surfactant (31), cellular proliferation, and structural remodeling (12, 20, 21, 27), is also upregulated as the lung adapts to hyperoxic stress. Indeed, elevation of expression of some of these proteins in either transgenic animals (48) or transformed cells in culture (43) has facilitated adaptation to oxidative stress.
It is increasingly recognized that energy metabolism is perturbed during exposure of the lung to hyperoxia. Previous investigators (5-8) have identified impairment of the tricarboxylic acid (TCA; Krebs; citric acid) cycle and the concomitant elevation of glycolytic activity in the lungs of rats exposed to hyperoxia for fairly brief durations. These changes occur well in advance of gross pathological, histological, or ultrastructural effects due to such exposure. Others (40) have reported a paradoxical suppression of respiration and increased glycolysis in cells incubated in hyperoxic environments.
Recently, our laboratory (19) reported that the activity of aconitase, which is the first enzymatic step in the TCA cycle, is rapidly lost in cells and in the lungs of rats exposed to hyperoxia a long time before the decline in activity of other TCA cycle enzymes or enzymes involved in energy metabolism. Previously, it was found in prokaryotes that the loss of aconitase activity during exposure to hyperbaric hyperoxia is almost instantaneous (18). In mammalian lung cells, the activity of the mitochondrial fraction of aconitase is virtually eliminated, and a loss of respiration accompanies the loss of this activity (19).
An elegant study (46) of hyperoxia-adapted cell lines has indicated that increases in cellular glucose consumption and glycolysis are likely to be crucial events in such adaptation by allowing preservation of cellular energy charge despite substantially diminished energy production through oxidative phosphorylation. Although upregulation of lung glycolysis occurs during both adaptation to sublethal hyperoxia and during the initial phases of exposure to hyperoxia at lethal levels, the biochemical mechanisms by which glucose consumption is increased remain poorly defined. Because the first potentially limiting step in glucose use is its transport across the cell plasma membrane, we sought to determine specifically how gene expression for several proteins involved in lung glucose transport is altered after adaptation of the lung to sublethal hyperoxia.
This transport can be considered to involve two distinct steps. The first step is movement of glucose across the plasma membrane through transmembrane glucose transporters. GLUT-1, GLUT-4, and sodium-glucose cotransporter (SGLT)-1 are the primary transporters found in lung tissue (4, 17). The GLUT family of transporters is described as facilitative glucose transporters because the glucose diffuses "through" these transporters, driven by the transmembrane glucose concentration gradient. On the other hand, SGLT-1 functions by cotransport of glucose and sodium, with the cotransport driven by the sodium gradient generated primarily by Na-K-adenosinetriphosphatase. The GLUT family of glucose transporters functionally relies on the phosphorylation of intracellular glucose by hexokinase (HK) to maintain the glucose gradient across the plasma membrane.
There are four isoforms of HK in mammalian tissues (47). Only three of these, HK-I, HK-II, and HK-III, are likely to be found in the lung. Glucokinase, the fourth less-regulated form, is most prominent in the liver and pancreas, where it is felt to play a role in the sensing of plasma glucose concentration rather than as a regulator of intracellular glycolytic flux. HK-I and HK-II are usually found bound to the outer mitochondrial membrane, whereas HK-III has been localized to the nuclear membrane. Differences in allosteric and hormonal regulation of enzyme activity between each of these isoforms have also been reported (47).
We describe experiments to explore the effect of hyperoxia on mRNA encoding proteins involved in glucose transport. Because elevation of mRNA content represents a pretranslational event that usually leads to an increase in the tissue content of a given protein, those proteins in which the mRNAs were increased during oxygen exposure were given further study. We report the selective upregulation of mRNAs encoding lung HK-II and -III, without the upregulation of mRNAs encoding glucose transporters, in an established model of tolerance to sublethal hyperoxia in rats.
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METHODS |
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Chemicals. All reagents were obtained from Sigma (St. Louis, MO).
Animal exposures. Ten-week-old male
(250-265 g) and female (180-215 g) Sprague-Dawley rats were
obtained from Taconic Farms (Germantown, NY) and housed in plastic
cages. Male and female rats were studied in separate experiments. For
exposure to sublethal hyperoxia for up to 7 days, the cages were placed
in cylindrical Plexiglas exposure chambers (0.6 × 1 m) that were
flushed with humidified 99% oxygen at 10 l/min. This provided the rats
with a tracheal PO2 of ~593 mmHg at
Denver's altitude (1,609 m), which is 83% of the tracheal
PO2 that theoretically would be
achieved by using the same gas mixture at sea level (713 mmHg; tracheal
PO2 = PB 47, where
PB is the atmospheric pressure
and 47 is the vapor pressure of water at 37°C). Exposure of rats to
85% oxygen at altitudes close to sea level for this duration is a
well-established model for pulmonary tolerance to otherwise lethal
hyperoxia (16). The rats were given fresh food and water daily, with
exposure of the oxygen-treated group to ambient atmosphere during
changes of food and water never exceeding 5 min. The control rats
received room air at Denver's atmospheric pressure. In the first
series of experiments, rats of each sex were exposed to either air or
100% oxygen for 168 h. In the studies of the time course of the
effects seen in the first series of experiments, female rats were
exposed to 99% oxygen for either 0, 24, 48, 72, or 168 h.
At these time intervals, the rats were anesthetized with
pentobarbital sodium (65 mg/kg ip) and injected with sodium heparin (1,000 U/kg ip). The trachea was cannulated, and the lungs were ventilated at a rate of 60 breaths/min. After a thoracotomy was performed, the lungs were briefly examined for gross appearance and
pleural effusion fluid volume was measured. The lungs were then
perfused with phosphate-buffered saline (PBS) through a pulmonary arterial cannula to remove blood. The left atrium was removed to allow
escape of the pulmonary effluent. Once the pulmonary vasculature was
rinsed, the lungs and heart were removed en bloc from the chest. To
isolate and preserve RNA, the right lung was placed in 10 ml of 4 M
guanidine isothiocyanate-0.5% sodium laural sarcosinate-1%
2-mercaptoethanol in 25 mM sodium citrate buffer (pH 7.5) as described
by Chirgwin et al. (14) and homogenized at a maximum setting with a
Virtis homogenizer. The homogenate was separated into aliquots in
cryovials, rapidly frozen in an ethanol-dry ice bath, and then stored
at 70°C. The left lung was weighed and then homogenized in a
measured volume of buffered solution containing a mixture of
antiproteases [25 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 7.0, 0.1 mM EDTA, 1% Triton X-100,
40 mM KCl, 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 10 µg/ml
of leupeptin, and 0.2 U/ml of aprotinin]. This homogenate was
centrifuged (1,500 g for 10 min) to
remove cellular debris. The supernatant was collected and then
separated into aliquots in cryovials that were rapidly frozen in
ethanol-dry ice and stored at
70°C. The pellet was also
frozen in ethanol-dry ice and stored at
70°C for later
determination of pellet DNA content.
Northern blot analysis. Lung RNA was purified by centrifugation through CsCl as described by Chirgwin et al. (14). The RNA pellet was resuspended in Tris-EDTA buffer (10 mM Tris · HCl, pH 8.0, and 1 mM EDTA), and the RNA concentration and purity were assessed spectrophotometrically at 260 and 280 nm (38). Lung RNA (20 µg) was then fractionated by electrophoresis in 1% agarose gel. RNA was transferred to nylon membrane as described by Sambrook et al. (38) and cross-linked to the membrane by ultraviolet irradiation. cDNA probes for the rat glucose transporters GLUT-1 and GLUT-4 were provided by Dr. Graeme Bell. The GLUT-1 probe was a tandem copy of a 436-base pair (bp) cDNA fragment (11). The insert was released from plasmid prGT-B436x2 by BamH I restriction. The GLUT-4 probe was a 1.5-kilobase (kb) cDNA fragment (10) released from plasmid pSM1D2 by EcoR I-Sac I restriction. The cDNA probes for the rat HK isoforms HK-I, HK-II, and HK-III were provided by Dr. John Wilson. The HK-I probe included the entire 3.6-kb cDNA (41). The HK-II probe included bases 1-3104 (44), whereas that for HK-III consisted of bases 525-3029 of the published cDNA sequence (42). The probe for SGLT-1 was provided by Dr. Matthias Hediger and included the entire 2.6-kb cDNA (26). These probes were labeled with [32P]dCTP by random priming (RadPrime Kit, GIBCO BRL, Life Technologies, Grand Island, NY). The probe for 28S rRNA was a 30-nucleotide oligomer (Clontech, Palo Alto, CA) labeled with [32P]UTP with a 5'-terminus DNA labeling kit (GIBCO BRL). A separate electrophoretic separation, transfer, and hybridization procedure was performed for each cDNA probe used. The nylon membranes were prehybridized in heat-sealed bags for 2 h at 42°C in 50% deionized formamide, 5× saline-sodium phosphate-EDTA (SSPE; 1× SSPE is 0.15 M NaCl, 0.01 M Na2HPO4, and 0.001 M EDTA, pH 7.4), 5× Denhardt's solution, 100 µg/ml of salmon sperm DNA, and 0.1% sodium dodecyl sulfate (SDS). The hybridization solution contained 50% deionized formamide, 5× SSPE, 5× Denhardt's solution, 100 µg/ml of salmon sperm DNA, 0.1% SDS, 100 mg/ml of dextran sulfate (mol wt ~500,000), and 1-10 × 106 counts/min of the 32P-labeled cDNA probe. The prehybridization solution was removed, 10 ml of the hybridization solution containing the 32P-labeled cDNA probe were added to the bag, and the bag was then resealed. The probes were allowed to hybridize for 16-24 h at 42°C. At the end of the hybridization period, the nylon membrane was removed from the hybridization solution and rinsed for 5 min at room temperature in 400 ml of 2× saline-sodium citrate (SSC; 1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) containing 0.1% SDS. The membrane was then washed four times in 2× SSC containing 0.1% SDS at 42°C, with each wash lasting 30 min. To increase the stringency of the hybridization, the membrane was then washed for 30 min in 0.2× SSC with 0.1% SDS at 42°C. A 2-min final wash in 0.2× SSC was then performed at room temperature. Next, the membranes were placed in storage phosphor cassettes (Molecular Dynamics, Sunnyvale, CA) and, after a period of time to maximize the signal-to-background ratio, were examined with a Molecular Dynamics phosphorimager. Quantitation of the acquired images was achieved with ImageQuant software (Molecular Dynamics). Each membrane was subsequently stripped of the hybridized probe by incubation in 2% glycerol in water for 4 min at 82°C. After the membranes were washed in cold water, the efficiency of stripping was assessed by autoradiography. The stripped membranes were then probed for 28S rRNA with the same methods as above.
It is a common practice to normalize the levels of mRNA coding for proteins of interest to the levels of mRNA for other constitutively expressed or "housekeeping" genes. In the case of exposure to hyperoxia, choosing such constitutive mRNAs has been problematic. A number of mRNAs that have been used by others for mRNA normalization have been found to be increased during adaptation to 85% oxygen (22). Furthermore, all of the candidate constitutive probes we tested showed marked differences in expression between male and female rats. During the studies reported herein, we found that the levels of mRNA for 28S rRNA (3) were very consistent in the lungs of both air- and oxygen-exposed rats of the same sex (data not shown). Therefore, the relative levels of all other mRNAs in each lung sample were obtained by comparing the hybridization density of each mRNA to the hybridization density for 28S rRNA, with the understanding that such normalization would confound interpretation of the effect of sex on these normalized values.
Electrophoretic separation of HK isoforms. The lung homogenate was supplemented with 2.5 mM dithioerythritol, 2 mM glucose 6-phosphate, and 10 mM glucose and incubated at 30°C for 30 min both to reactivate HK activity lost during homogenization and freezing (34) and to free HK bound to the mitochondrial membranes (15, 36). Approximately 3 µl of this treated homogenate were then applied to cellulose acetate plates (Titan III plates, Helena Laboratories, Beaumont, TX) with a Sepratek-8 applicator (Gelman Sciences, Ann Arbor, MI). The proteins were separated electrophoretically (45) in Tris-borate-EDTA buffer (36 mM Tris, 20 mM boric acid, 4 mM Na2HPO4, and 2 mM EDTA, pH 8.4) containing either 100 or 0.5 mM glucose at 200 V and 2 mA for 30 min. The cellulose acetate plates were then stained for HK enzymatic activity by covering the plate with 20 ml of a solution containing 1% ultra-low-melt agarose (type IX-A, Sigma), 100 mM Tris, pH 8.0, 0.5 mM EDTA, 10 mM MgCl2, either 100 or 0.5 mM glucose, 20 mM ATP, 2 mM NAD, 3.2 U glucose-6-phosphate dehydrogenase (from Leuconostoc mesentroides), 65.3 µM phenazine methosulfate, and 628 µM nitro blue tetrazolium. The use of a low-glucose (0.5 mM) concentration in both the electrophoresis and detection buffers allows detection of HK-III that is inhibited by higher glucose concentrations (24, 47). The plates were cooled at 4°C until the agarose solidified. The plates were then allowed to develop at room temperature until maximum color development was achieved. In this assay, HK converts glucose to glucose 6-phosphate, the reducing equivalents of which are transferred to NAD to form NADH through the action of glucose-6-phosphate dehydrogenase. Reducing equivalents from NADH are then transferred through phenazine methosulfate to nitro blue tetrazolium, resulting in formation of a purple formazan precipitate. Rat heart and liver homogenates were prepared and examined with identical procedures to allow identification of the HK isoforms detected in the lung samples.
Lung immunohistochemistry. The lungs
were perfused with PBS at 25°C. The trachea was infused with 1%
ultra-low-melt agarose in PBS at 25°C and 25 cmH2O pressure. The heart and
lungs were removed from the thorax en bloc while this airway pressure
was maintained and were submerged in chilled PBS (4°C) to harden
the agarose. The lungs were cut into 1-mm slices, embedded in optimum cutting temperature compound (Sakura Finetek USA, Torrance, CA), and
immediately frozen in isopentane. Cryosections were made at 4-µm
thickness, air-dried, and mounted on
3-aminopropyltriethoxysilane-coated glass slides. The sections were
fixed in cold acetone for 20 min (20°C). These were then
washed in PBS for 10 min and then permeabilized with 0.5% Triton X-100
for 30 min (25°C). Next, slides were washed with PBS, treated with
0.3% hydrogen peroxide in PBS for 30 min (25°C) to block
endogenous peroxidase activity, and then washed three times with PBS (5 min each). The slides were then incubated in 10% normal horse serum
(NHS) in PBS for 20 min to block nonspecific binding, followed by
exposure to 5% NHS with 0.1% saponin in PBS for permeabilization. The
sections were then incubated for 16 h with a monoclonal antibody (C7C3,
1:400, 4°C; Chemicon, Temecula, CA) developed by Preller and Wilson
(32) against rat HK-III. The slides were then washed with PBS three
times (5 min each), placed in 5% NHS with 0.1% saponin for 10 min,
and then incubated with biotinylated horse anti-mouse immunoglobulin G
(1:250; Boehringer Mannheim, Indianapolis, IN). The slides were
subsequently incubated with horseradish peroxidase-streptavidin
conjugate in 5% NHS with 0.1% saponin for 60 min at room temperature.
After three washes in PBS (5 min each), the slides were placed in 5%
NHS with 0.1% saponin for 10 min. The slides were washed once in PBS,
then immersed in a solution containing 1.4 mM
3,3'-diaminobenzidine and 0.03% hydrogen peroxide in
Tris · HCl buffer (0.05 M, pH 7.4) to develop peroxidase activity. No counterstain was applied. Immunostained sections were dehydrated, cleared, and mounted with coverslips, then
examined by light microscopy for peroxidase localization.
Biochemical assay of homogenate DNA and HK activities. Soluble lung DNA and lung pellet DNA were estimated in the homogenates of the left lung by Richards' (33) modification of the diphenylamine method described by Burton (13). Left lung total DNA content was then calculated as the sum of the supernatant DNA and the pellet DNA contents. HK activity in the tissue homogenate was estimated by a spectrophotometric method described by Beutler (9).
Statistical analysis. Significant
treatment effects were identified by using two-way analysis of variance
procedures provided by the JMP software package (39). Linear contrasts
were used to identify significant gas effects within each sex group
when a significant sex-by-gas interaction was found. For the
time-course data, analysis was by one-way analysis of variance with a
post hoc Dunnett's test for multiple comparisons, with "zero
time" as the control group. Significance was accepted in all
comparisons when P was 0.05.
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RESULTS |
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Gross appearance. The appearance of the lungs of rats exposed to sublethal hyperoxia for 1-7 days showed no gross difference from that of lungs from air-exposed rats. Furthermore, neither oxygen- nor air-exposed rats had measurable pleural fluid at any time point.
mRNA and DNA. After 7 days of exposure, lung total RNA increased nearly threefold in oxygen-exposed rats, whereas a smaller increase in soluble protein (57%) was observed relative to the air-exposed control rats. Total lung DNA was not affected significantly by a sublethal oxygen exposure (Table 1).
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As an initial step in learning the effects of hyperoxia on gene expression pertinent to glucose transport, we measured the levels of various lung mRNAs of adult rats after exposure to sublethal hyperoxia for 7 days. Animals treated in this fashion have become resistant to a subsequent hyperoxic challenge that would normally be lethal (16). Figure 1 shows that, on a per lung basis, mRNA for all 3 HKs was increased. However, relative to lung total RNA content, as indexed by 28S rRNA content of the samples, HK-1 activity remained unchanged, whereas both HK-II and HK-III mRNA increased two- to threefold.
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The concentration of GLUT-1 mRNA in the lung relative to lung RNA content was slightly increased in male rats exposed to the high oxygen environment (Fig. 2). No significant change in the relative concentration of GLUT-1 mRNA was observed in the female rats. However, the whole lung content of GLUT-1 mRNA was increased in the oxygen-treated rats of both sexes due to the striking increase in lung content of total RNA. Furthermore, the relative content of both GLUT-4 and SGLT-1 mRNA was markedly decreased in hyperoxia. Interestingly, the level of GLUT-4 mRNA in the lungs from the hyperoxic rats increased 43 and 30% in the male and female rats, respectively, when expressed on a per lung basis, whereas SGLT-1 mRNA content normalized in this way was unaffected by oxygen exposure in either sex.
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Time course of HK mRNA changes. Little change in HK-I mRNA was observed in Northern blots from rats exposed to hyperoxia for periods of time from 24 to 168 h. However, specific expression of HK-II mRNA (per 28S rRNA) increased gradually during the first 72 h of exposure to hyperoxia. At both 72 and 168 h of hyperoxic exposure, specific expression of lung HK-II mRNA was significantly greater than that in the lungs of air-exposed control rats (Fig. 3).
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In comparison, lung HK-III mRNA (per 28S rRNA) also increased gradually during the first 72 h of adaptation to hyperoxia. On this basis, lung HK-III mRNA specific expression only became significantly different from the air-exposed control lungs after 168 h of exposure.
HK proteins. Hyperoxic adaptation was associated with an increase in the lung activities of different HK isoforms. Lung total HK activity more than doubled in the lungs of the male rats, whereas it increased fourfold in the lungs of female oxygen-exposed rats relative to their air-exposed counterparts (Table 2). Electrophoretic separation of the proteins on cellulose acetate plates followed by detection of HK enzymatic activity allowed qualitative comparison of individual HK isoform activities in the lung homogenates. Total HK-I activity increased in the lungs of both male and female rats adapted to hyperoxia (Fig. 4). Relative to the minor change in HK-I activity, HK-II activity in the lungs from oxygen-adapted rats showed a considerably greater increase over that in the lungs of the air-exposed rats. The latter contained little detectable HK-II activity. Regardless of glucose concentration present (0.5 or 100 mM), we did not detect HK-III activity in the lung homogenates subjected to electrophoresis (data not shown). However, HK-III protein was detected in microscopic sections of the lungs from both the air-exposed and oxygen-adapted rats (Fig. 5). HK-III immunostaining appeared in a circular pattern similar to the previously described perinuclear location (32). Subjectively, there was no change in either cellular distribution or intensity of HK-III staining. Similarly, the pulmonary distribution of cells staining positively for HK-III did not appear different, although the cellularity of the lung periphery in the hyperoxia-adapted rat lungs was subjectively slightly increased after 7 days.
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DISCUSSION |
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We report here that the lungs from rats exposed to sublethal hyperoxia for up to 7 days exhibit several changes in mRNAs for the predominant glucose transporters and HK isoforms and in HK enzymatic activities. It should be recognized that the total lung content of a particular mRNA species represents the number of mRNA templates available in the lung for translation of its respective protein. Calculation of the lung content of the mRNA species of interest, then, provides insight into the regulation of nuclear events within the changing cellular milieu of the hyperoxic lung. For example, although total lung concentrations of most of the mRNAs studied increased in the oxygen-exposed rats, it is notable that the total lung concentration of SGLT-1 mRNA remained unchanged.
Alternatively, mRNA levels in the lung can be compared after normalization to lung total RNA, as indexed here by 28S rRNA. When lung mRNA levels are expressed in this fashion, it becomes clear that the mRNAs for the HK isoforms and for the glucose transporters are differentially regulated. Specifically, although the relative lung content of mRNAs for GLUT-4 and SGLT-1 was decreased in the lungs of rats exposed to hyperoxia, the relative content of mRNAs for HK-II and HK-III was increased in the hyperoxic lungs. The relative content of GLUT-1 and HK-I mRNAs was unaffected by oxygen exposure. When these changes in relative mRNA content are considered in the context of the striking increase in lung total RNA, the existence of a complex network of adaptive strategies employed by the cells of the lung to survive hyperoxic injury is indicated.
The advantages and disadvantages of standardization of lung enzyme activities against total lung protein or DNA content have been well reviewed (22). Both overestimation and underestimation of actual enzyme levels are possible. To avoid biased interpretation in either direction, enzyme activities are presented here using both methods of standardization. Regardless of how lung HK activities are expressed (per lung, per mg DNA, or per mg protein), a significant increase in lung HK activity was found in rats of both sexes after hyperoxic adaptation.
Previous investigators (29) have suggested that sex hormones can affect the spontaneous acquisition of tolerance to extreme hyperoxia in adult rats, and they found that a higher percentage of females than males survive such exposure. Therefore, we examined all mRNAs and HK activities in both sexes before and after adaptation to hyperoxia. The patterns of changes in mRNA expression for all glucose transporters and HKs were similar in the two sexes. Although baseline total and specific activities of lung HK were lower in females, both these values were similar in males and females after adaptation to hyperoxia. However, the magnitude of elevation in HK activity was considerably greater in females (3.4-fold) than in males (1.7-fold) when expressed per milligram of lung DNA. Furthermore, the content of lung HK activity in normoxia, expressed per milligram of DNA, was 50% greater in females than in males. These findings may be pertinent to sexual differences in acquired tolerance to hyperoxia.
Impairment of TCA cycle in hyperoxia. Studies both in other laboratories and in our own have demonstrated sites of inhibition of the pathways of intermediary metabolism by hyperoxia. Bassett and Bowen-Kelly (5) found that the concentration of citrate, the initial substrate of aconitase, increased in the lungs of rats exposed to 100% oxygen (24 h), whereas the lung concentrations of both 2-oxoglutarate and glutamate, metabolites downstream from aconitase in the TCA cycle, were decreased. Loss of aconitase activity also has been measured directly in our laboratory both in the lungs of rats exposed to hyperoxia (19) and in the lungs of premature newborn baboons ventilated with high concentrations of oxygen during the evolution of bronchopulmonary dysplasia (28). Similar observations have been made in cultured cells. Gardner et al. (19) described a rapid loss of aconitase activity in A549 cells (a lung adenocarcinoma cell line of human origin) during hyperoxic exposure. In those experiments, the decline in aconitase activity occurred before any decline in activity of 2-oxoglutarate dehydrogenase. Working with Chinese hamster ovary (CHO) cells, Schoonen et al. (40) found that 2-oxoglutarate dehydrogenase activity declined during oxygen exposure earlier than the activity of either succinate dehydrogenase or NADH dehydrogenase. It seems likely, then, that aconitase is inactivated in oxygen more rapidly than the other TCA cycle enzymes. In A549 cells, loss of respiration and respiratory capacity occurred in parallel with loss of aconitase activity and could be reproduced by specific aconitase inhibitors, fluorocitrate or fluoroacetate. This loss of aconitase activity in hyperoxia is likely to be mediated by the effects on iron redox status (35). Thus loss of aconitase activity has an important role in the early decline in the lung TCA cycle and respiratory capacity caused by hyperoxia and very likely contributes, in large part, to the increased reliance on anaerobic glycolysis for energy production in hyperoxic cells.
Adaption to hyperoxia involves increased glucose utilization. Rats exposed to hyperoxia in this study exhibited striking increases in lung HK activity. The significant role of HK in regulating lung glucose metabolism was illustrated by Salotra and Singh (37). Working with rat lung slices and using 2-deoxyglucose to measure uptake and phosphorylation, these investigators demonstrated that, at physiological glucose concentrations, glucose metabolism in the lung is limited by the rate of glucose phosphorylation rather than by glucose transport into cells. In addition, exposure to hyperoxia has been shown to increase glucose consumption in isolated perfused lungs from rats. Bassett et al. (6) have described the rate of glucose flux through the glycolytic pathway in this preparation and reported that glucose consumption was increased 53% in the lungs of rats exposed to 100% oxygen for 24 h. From these results, it is reasonable to conclude that increased HK activity contributes, in large part, to the increased glucose consumption in hyperoxic lungs.
Using cultured cells, other investigators (2, 40) also have reported that glucose consumption is increased during exposure to hyperoxia. This increase in glucose consumption can impact cell survival in hyperoxia. Recently, we found that A549 cells increased their glucose consumption two- to fourfold in hyperoxia and that, with adequate glucose supplementation, progressive necrotic cell death in 95% oxygen, even at sea-level pressures, could be avoided. Nonetheless, cells maintained under these conditions showed severe growth impairment (1). However, in an impressive series of experiments, Van der Valk et al. (46) showed that CHO cells adapted to continuous hyperoxia (99% oxygen-1% carbon dioxide) over a period of months to years not only showed a fivefold increased reliance on glycolysis for ATP production but also retained the ability to proliferate. Thus it appears that a shift from oxidative to glycolytic metabolism is an important, perhaps critical, adaptive strategy exercised by a variety of cells in response to hyperoxic challenge.
Upregulation of glucose transport. Any increase in glucose consumption by cells necessarily requires increased transport of glucose molecules. This is a coordinated function of the membrane glucose transporters and cellular HK activity. In the studies on rats adapted to sublethal hyperoxia reported here, lung HK activity increased substantially. This increase in HK facilitates both glucose metabolism and glucose transport. Furthermore, the lung content of mRNA encoding GLUT-1 was increased in oxygen-exposed rats along with a similar trend in GLUT-4 mRNA, whereas the lung content of SGLT-1 mRNA remained unchanged. Because the lung content of the glucose transporter mRNAs relative to total RNA did not increase, it is likely that the increases in lung content of these glucose transporter mRNAs are part of a generalized increase in production of a broad spectrum of mRNA species rather than a specific adaptive response to hyperoxia.
Changes in the activities of other glycolytic enzymes may contribute to the increase in glycolytic flux and, through allosteric effects on upstream enzymes, facilitate glucose transport. For example, Ho et al. (22) have reported recently that expression of glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase mRNAs are both elevated in the lungs of rats breathing 85% oxygen. Because glucose 6-phosphate is the common substrate for both the glycolytic and pentose phosphate pathways, the upregulation of proteins involved in glucose transport during the development of tolerance to oxygen may be part of a coordinated response to provide glucose for energy, for ribonucleotide production, and for antioxidant protection through reducing equivalents provided to glutathione reductase.
Potential contribution of shifting cell populations. It is possible that some of the changes observed here are related to changes in cell populations within the lung consequent to hyperoxic exposure. Complex changes in lung cellularity occur after day 3 of adaptation to 85% oxygen in adult rats (16). Because the various cells within the lung likely show considerable type-specific differences in the contents of the proteins and mRNAs studied here, a changing distribution of cell types in hyperoxia might have contributed, at least in part, to the changes in whole lung content of the mRNAs and proteins that we observed after 7 days of adaptation. It also can be speculated that the changes in lung cell populations that occurred during hyperoxic adaptation are related, in part, to successful energy adaptation. That is, those that cannot adapt rapidly enough to accommodate critical cell functions may not survive, whereas those that do so may thrive and even proliferate (23, 25).
It is unlikely, however, that all of the changes in gene expression reported here can be linked to shifts in cell populations. During adaptation to 85% oxygen, it was reported that all of the major cellular components of the alveolus (type I and II pneumocytes, interstitial cells, endothelial cells, and macrophages) were unchanged in number, volume, surface area, and relative alveolar representation during the first 3 days of exposure (16). Hence the early (24-72 h) elevation of HK-II mRNA expression and the similar trend in HK-III mRNA are probably not related to changing cell populations.
In conclusion, we have identified a number of changes in mRNAs and proteins related to glucose transport in the lungs during adaptation to hyperoxia. From these observations, we can identify those mechanisms that are likely to be of greatest importance in increasing glucose consumption in the lung. The selective elevations of relative lung content of mRNAs for HK-II and -III along with the finding of increased HK enzymatic activity suggest that the expression of HK is relatively more important in the adaptation process. By contrast, hyperoxic adaptation was accompanied by mixed changes in the lung content of mRNAs encoding glucose transporters relative to total RNA. Although the relative content of GLUT-1 mRNA remained unchanged in the hyperoxic rats, the relative content of both GLUT-4 and SGLT-1 mRNAs decreased substantially. Indeed, whole lung SGLT-1 mRNA content was unchanged despite the nearly threefold increase in total lung RNA content.
The findings of the present study implicate the elevation of gene expression for proteins involved in glucose movement into cells as an important component of adaptation to hyperoxia. Such strategies may be pertinent to other cellular oxidant injuries in which the TCA cycle and/or respiratory function are compromised. Further studies are required to clarify the mechanisms by which the molecular events described here are regulated and to determine additional downstream steps in the adaptive process pertinent to energy metabolism.
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ACKNOWLEDGEMENTS |
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We acknowledge many helpful discussions with Dr. John Wilson and Dr. Robert Mason, the outstanding technical assistance of Lynn Cunningham in performing the immunohistochemistry, and Jacque Guthrie in preparing the manuscript. In addition, we are grateful to Dr. Wilson (Michigan State University, East Lansing) for providing cDNAs for the hexokinases, to Dr. Matthias Hediger (Brigham and Women's Hospital, Boston, MA) for the sodium-glucose cotransporter-1 cDNA, and to Dr. Graeme Bell (Howard Hughes Medical Institute, University of Chicago, IL) for providing the cDNAs for rat GLUT-1 and GLUT-4.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-52732, HL-46481 (both to C. W. White), and HL-07670 (to C. B. Allen).
Address for reprint requests: C. B. Allen, D-301, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206.
Received 29 August 1997; accepted in final form 2 December 1997.
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