Introduction

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Owing to its situation, alveolar epithelium is in situ exposed to high O₂ tension: alveolar PO₂ ~ 100 mm Hg. However, a decrease in alveolar O₂ tension is observed under diverse conditions. Alveolar hypoxia may be the consequence of a decrease in O₂ tension in the inspired gas (as observed during high-altitude ascent) or may result from localized or generalized ventilatory defect in various respiratory disorders. Alveolar type II cells are the most abundant alveolar epithelial cells (AEC) and they play a vital role in respiratory mechanics and gas exchange because they synthesize and secrete surfactant, replace alveolar type I cells in injured alveolar epithelium, and maintain alveolar space free of fluid by transporting Na actively from the alveolar to the interstitial space. Maintenance of these functions during alveolar hypoxia depends on the ability of AEC to develop adaptative strategies to overcome O₂ deprivation. Little information is available about the cellular adaptation of AEC to hypoxia. There is some evidence that AEC are fairly tolerant to severe hypoxia, surviving at least 24 h without cellular damage (1, 2). However, the demonstrated decrease in functional expression of Na transport proteins in AEC after hypoxia (1) raises the question of their ability to cope with O₂ deprivation.

In diverse tissues and cell types, tolerance to hypoxia has been assumed to be the result of maintenance of adequate energy supply under hypoxia despite the reduction of oxidative phosphorylation (3). To do so, the cells dramatically increased anaerobic glycolysis by upregulating the expression and the activity of glycolytic enzymes (4) and increasing glucose transport at the membrane level. AEC under normoxic conditions display a very high aerobic and anaerobic glycolytic (2). Because the rate of glycolytic enzymes largely exceeds glucose utilization, glucose transport is, in normoxia, the main rate-limiting step for its metabolism (5). Under hypoxia, even though the activity of glycolytic enzymes is upregulated, the increase in glycolysis will be efficient only if a parallel increase in glucose transport occurs. The goal of this study was to evaluate whether, in AEC, hypoxia regulates activity and expression of glucose transport, and to determine the mechanisms involved in this regulation. Our results indicate that hypoxia increases activity of the Na-independent glucose transport in an O₂ concentration-dependent and time-dependent manner, an effect that is reversible after reoxygenation. The increase in glucose transport was dependent on protein synthesis and associated with an increase in GLUT1 protein units at the membrane level. Concomitantly, hypoxia upregulated GLUT1 messenger RNA (mRNA) transcript through two distinct O₂ deprivation regulatory pathways and increased the DNA binding activity of hypoxia-inducible factor (HIF), which suggest that this gene regulation occurred at the transcriptional level.

	Materials and Methods

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Cell Isolation

Alveolar type II cells were isolated from pathogen-free, male Sprague-Dawley rats (200 to 250 g) as previously described (6). Briefly, pooled cells from three rats were prepared as follows. Rats were injected with 30 mg/kg pentobarbital sodium intraperitoneally and 1 U/g heparin sodium intravenously. After a tracheotomy was performed, the animal was exsanguinated. Solution II (40 to 50 ml), which contained (in mM) 140 NaCl, 5 KCl, 2.5 sodium phosphate buffer, 10 N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepes), 2 CaCl₂, and 1.3 MgSO₄, pH 7.40, at 22°C, was perfused through the air-filled lungs via the pulmonary artery to clear the vascular space of blood. The lungs were removed from the thorax and lavaged to total lung capacity (8 to 10 ml) five times with Solution I, which contained (in mM) 140 NaCl, 5 KCl, 2.5 sodium phosphate buffer, 10 Hepes, 6 D-glucose, and 0.2 ethylene glycol- bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, to remove macrophages; and two times with Solution II. Then, lungs were filled with 12 to 15 ml of elastase solution (porcine pancreas, twice-crystallized 40 U/ml, prepared in Solution II) and incubated in a shaking water bath in air for 10 min at 37°C, after which additional elastase solution was instilled for another 10-min incubation. The lungs were minced in the presence of DNAse I, and 5 ml of fetal bovine serum (FBS) was added to stop the effect of elastase. The lungs were then sequentially filtered through 150- and 30-mm nylon mesh. The filtrate was centrifuged at 300 × g for 8 min. The cell pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 25 mM D-glucose at 37°C. The cell supension was plated at a density of 10⁶ cells/cm² in 25 cm² bacteriologic plastic dishes to aid in the removal of macrophages by differential adherence. After incubation at 37°C in a 5% CO₂ incubator for 1 h, the unattached cells in suspension were removed and centrifuged at 300 × g for 8 min. The resulting cell pellet (70% purity, > 95% viability, 8 to 10 × 10⁶ cells/rat) was plated at a density of 7 to 10 × 10⁵ cells/cm² in 6-, 12-, or 24-well culture dishes. Culture medium consisted of DMEM containing 25 mM D-glucose, 10 mM Hepes, 23.8 mM NaHCO₃, 2 mM L-glutamine, 10% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10 µg/ml gentamycin, and the pellet was incubated in a 5% CO₂-95% air atmosphere. The cell purity after 24 h was 90 ± 2%, as assessed by a characteristic fluorescence with phosphine 3R as previously described (6). Contaminating cells were essentially macrophages. Culture medium was changed 24 h after isolation and then on alternate days. Cells were used after 48 h in culture and were likely in transition to type I AEC, thus they are designated AEC (7).

Hypoxic Exposure

Two days after plating, growth medium was removed and replaced by a thin layer of fresh medium (0.15 ml/cm²) with 10% FBS to decrease the diffusion distance of the ambient gas. To achieve hypoxic exposure, culture dishes were placed in a humidified, airtight incubator with inflow and outflow valves, and the hypoxic gas mixture (0% O₂- 5% CO₂-95% N₂) was delivered at 10 liters/min for 20 min. The airtight incubator was kept at 37°C for 3, 6, 12, or 18 h and control normoxic cells were placed in a 5% CO₂-21% O₂-74% N₂ humidified incubator for the same period of time. In additional experiments, exposure to a mild hypoxia mixture (5% O₂-5% CO₂-90% N₂) was performed as described previously over an 18-h period. Oxygen tensions assayed in the culture medium were 30, 60, and 140 mm Hg after 18 h 0% O₂ (severe hypoxia), 5% O₂ (mild hypoxia), and 21% O₂ (normoxia), respectively. In culture medium, pH measured at the end of exposure was not significantly different under normoxic and hypoxic conditions. Trypan blue exclusion measured in cells exposed to hypoxia did not decrease, as compared with normoxic controls. For hypoxia-reoxygenation experiments, cells were exposed to 18 h hypoxia, then placed in a 5% CO₂-21% O₂-74% N₂ humidified incubator with the normoxic counterparts for 24 or 48 additional hours. Culture medium was changed at the end of hypoxia and at 24 h of reoxygenation. In additional experiments, cells were exposed for 18 h in normoxic conditions to cobalt chloride (100 µM) or to inhibitor of oxydative phosphorylation Na azide (1 mM).

Measurement of Glucose Transport

The measurement of glucose uptake was done as previously described (6). Uptake was determined immediately after hypoxic exposure. The assays were performed at 37°C in a buffered solution of the following composition (in mM): 137 NaCl, 5.4 KCl, 2.8 CaCl₂, 1.2 MgSO₄, and 14 Hepes (pH 7.4). In the sodium-free medium, sodium was replaced by N-methyl-glucamine. After removal of the culture medium, cells were washed twice with 1 ml/well of the uptake solution, and were incubated for adequate periods of time in the presence of either [³H]deoxy-D-glucose (DG) (0.5 µCi/ml), [¹⁴C]-methyl-glucopyranoside (MGP), or 3-O-[methyl-³H] glucose (1 µCi/ml) with appropriate concentrations of DG, MGP, or 3-O-methyl glucose (3 OMG). At the end of the incubation, uptake was stopped by washing the cells three times with 1 ml/well of ice-cold solution containing (in mM) 137 NaCl and 15 Hepes, pH 7.4. After incubations, cells were solubilized in 0.5% Triton X-100. Tracer activities were determined by liquid scintillation counting and the remaining volume of each sample was used for assessing the protein content per well.

Measurement of Adenosine Triphosphate Contents

Adenosine triphosphate (ATP) content of cells exposed to normoxia and hypoxia was determined enzymatically. After removal of the culture medium, cells were washed with ice-cold solution containing (in mM) 14 Tris and 137 NaCl, pH 7.4, and extracted with ice-cold 0.3 N perchloric acid. Extracts were neutralized to pH 7 with potassium hydroxide. ATP was measured in the supernatant by bioluminescence using an ATP determination kit (Calbiochem Biochemicals, San Diego, CA) based on the light generation reaction with luciferin and firefly luciferase. Measurements were performed in a luminometer (Hewlett Packard Picolite Luminometer). Standard curves of log photons versus log ATP were linear over the range 10⁸ to 10⁵ M ATP.

Measurement of Lactate Content and Glucose Consumption

Glucose concentrations in the medium and lactate content of cells were determined by the glucose oxidase method and spectrophotometric analysis using lactate dehydrogenase, respectively (Sigma Chemical, St. Louis, MO). Protein was determined by the Bradford method.

Total Membrane Isolation and Western Blots

Confluent monolayers were rinsed twice with iced phosphate-buffered saline (PBS), pH 7.4, and scraped gently with a rubber policeman in a Solution A containing 3 mM Tris HCl and 1 mM ethylenediaminetetraacetic acid (EDTA). All remaining steps were performed at 4°C. Cells were homogenized in a Dounce potter homogenizer (15 strokes). The homogenates were then centrifuged at 750 × g for 5 min to remove nuclei and unbroken cells, and the supernatants were centrifuged at 150,000 × g for 60 min. The resulting pellets containing total cell membranes were routinely resuspended in a small volume of Solution A. The supernatants were frozen at 80°C until used. Protein was determined by the method of Bradford. Samples (30 µg) were resolved in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE), electrically transferred to nitrocellulose paper, and blocked for 1 h at 37°C in PBS- milk (137 mM NaCl, 15.5 Na₂PO₄, pH 7.4, containing 50 mg/ml nonfat dry milk). Blots were then washed in PBS- Tween (in mM: 137 NaCl, 15.4 Na₂PO₄, 1.4 KH₂PO₄, and 2.7 KCl) as well as 0.1% Tween-20, pH 7.4, and incubated with antibody in PBS-milk (3 mg/ml) for 16 h at 4°C. The following antibodies and dilutions were used: rabbit polyclonal anti-GLUT1 transporter directed against the C-terminus of rat GLUT1 coupled to KLH (Chemicon, Temecula, CA) 1:1,000, and actin, 1:5,000. Blots were washed and incubated with an antirabbit antibody for 2 h at 23°C. Blots were washed three times in PBS-Tween for 15 min each and subjected to autoradiography using Kodak film. Quantification of GLUT1 and actin levels was obtained using NYH software.

Ribonuclease Protection Assay

Cells in 35-mm plastic dishes were lysed in a buffer containing 4 M guanidium thiocyanate, 25 mM sodium citrate (pH 7.0), and 0.5% sarcosyl, and directly used for ribonuclease (RNase) protection assay (RPA) as previously described (1). Total RNA equivalent of 10⁶ cells or 20 mg of yeast transfer RNA (Boehringer Mannheim, Indianapolis, IN) were cohybridized with 5 × 10⁵ counts per minute (cpm) for GLUT1, and 5 × 10⁴ cpm for -actin probes in 80% formamide, 40 mM 1,4-piperazine-diethanesulfonic acid (pH 7.4), 400 mM NaCl, and 1 mM EDTA at 50°C overnight. RNase digestion (RNAse A, 40 mg/ml, and T1, 2 mg/ml; Boehringer) was performed at 30°C for 60 min, then digestion with proteinase K (12.5 mg/ml; Boehringer) was done at 37°C for 30 min. After phenol extraction and ethanol precipitation, protected fragments were separated by urea-PAGE. Gels were fixed with 10% acetic acid and vacuum-dried before exposure to Kodak X-OMAT AR 5 film, and signal was quantitated from the gel using direct radioactivity measurement with an Instant Imager (Packard Instrument Co., Meriden, CT). Actin expression was used as an internal standard because neither hypoxia nor reoxygenation significantly modified the level of actin mRNA measured in six independent experiments (1). Results were expressed as the ratio of expression of the mRNA of interest to actin mRNA (arbitrary units [AU]).

Cellular RNA Probes

The GLUT1 probe was synthesized by reverse transcription polymerase chain reaction from rat kidney mRNA from pGEM Easy Vector Plasmid (Promega, Madison, WI). The length of the GLUT1 probe was 336 bp (protected fragment 272 bp, corresponding to bp 486-758). The probe was located in the 3'-untranslated region. Mouse -actin was synthesized using a complementary DNA insert in pGEM-3. The probe was 190 bp long with a protected fragment of 135 bp (bp 696-831). Antisense RNA probes were synthesized using a T7 in vitro synthesis kit (Promega) in the presence of [³²P]uridine triphosphate (15 TBq/mmol).

Electrophoretic Mobility Shift Assays

Nuclear extracts were prepared as described by Semenza and Wang (8). After exposure to 0% or 21% O₂ for 6 h, cells were washed twice with cold PBS, scraped into 5 ml of PBS, and pelleted by centrifugation at 1,500 rpm for 5 min at 4°C. Nuclear extracts were prepared with Buffers A and C containing 0.5 mM dithiothreitol (DTT), 0.4 mM phenylmethylsulfonyl fluoride, 2 µg of leupeptin/ml, 2 µg of aprotinin/ml, 2 µg of pepstatin/ml, and 1 mM sodium vanadate. The cell pellet was washed with 4 packed-cell volume (PCV) of Buffer A (10 mM Tris-HCl [pH 7.8], 1.5 mM MgCl₂, and 10 mM KCl), resuspended in 4 PCV of Buffer A, and incubated on ice for 10 min. The cell suspension was homogenized with a type B pestle and the nuclei were pelleted by centrifugation at 3,000 rpm for 5 min, resuspended in 3 PCV of Buffer C (0.42 M KCl, 20 mM Tris-HCl [pH 7.8], 1.5 mM MgCl₂, and 20% glycerol), and mixed on a rotator at 4°C for 30 min. Nuclear debris was pelleted by centrifugation for 30 min at 13,500 rpm. The supernatant was dialyzed against one change of Buffer D (20 mM Tris-HCl [pH 7.8], 0.1 M KCl, 0.2 mM EDTA, and 20% glycerol) for a total of 3 h at 4°C. The dialysate was centrifuged for 10 min at 13,500 rpm, and aliquots were stored at 80°C. Protein concentration was determined by the method of Bradford.

The oligonucleotide sequence for rat GLUT1 5' enhancer was used as probe and competitors. The oligonucleotide sequence was 5'-TCCACAGGCGTGCTGGCTGACACGCA-3' (9). Oligonucleotide probe was generated by the 5' labeling of the sense strand with -[³²P]ATP and T4 polynucleotide kinase, annealing to a 4-fold excess of unlabeled antisense strand. Binding reactions were carried out in a total volume of 20 µl containing 5 µg of nuclear extract and 0.1 µg of denaturated calf thymus DNA in 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 50 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, 5 mM DTT, and 5% glycerol. After incubation at room temperature, probe (10⁴ cpm) was added and the incubation was continued for an additional 15 min, after which the reaction mixture was loaded onto 5% nondenaturating polyacrylamide gels. Electrophoresis was performed at 185 V in 0.3× Tris boric acid (TBE) (1 × TBE is 89 mM Tris-HCl, 89 mM boric acid, and 5 mM EDTA) at 4°C. Gels were vacuum-dried and autoradiographed with intensifying screens at 80°C for 10 d. Competitor DNAs were preincubated with nuclear extract and calf thymus DNA for 5 min before addition of the labeled probe.

Materials

All chemicals were purchased from Sigma Chemical. Radioactive tracers were provided by Amersham (Aylesbury, UK). Culture media and reagents were from GIBCO BRL (Cergy-Pontoise, France). Plasticware was from Costar (Cambridge, MA).

Presentation of Data and Statistical Analysis

Uptakes of DG were expressed as nanomoles per milligram of protein. Results are presented as means ± standard error (SE) of three to six separate experiments in which triplicates were obtained. One-way or two-way variance analyses were performed and, when allowed by the F value, results were compared by the modified least significant difference.

	Results

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Effect of Hypoxia on ATP and Lactic Acid Contents

In AEC exposed to 0% O₂ for increasing times, ATP content fell by about 60% of the control value after 3 h of exposure, then progressively increased, reaching normoxic values at 6 h of exposure to hypoxia. That AEC exposed to hypoxic conditions retained substantial store of ATP suggested a role for anaerobic glycolysis. Therefore, we examined lactic acid content of AEC in the same conditions. The increase in lactic acid content was apparent at 6 h of exposure and was maximal at 18 h (Figure 1).

View larger version (14K): [in this window] [in a new window]   Figure 1.   ATP content (A) and lactate content (B) of AEC exposed to 0% hypoxia. Cells were exposed for increasing incubation times (3, 6, 12, and 18 h) to hypoxia, then ATP and lactate contents were determined as described in MATERIALS AND METHODS. Results represent means ± SE of four experiments in which duplicates were obtained. *Significantly different from corresponding normoxic control values.

Effect of Hypoxia and Hypoxia-Reoxygenation on Glucose Uptake

Exposure to hypoxia caused a large stimulation of glucose transport activity in AEC. The effect of hypoxia on glucose transport was O₂ concentration-dependent: exposure to 5% and 0% O₂ for the same period of time (18 h) increased DG uptake by 150 and 263%, respectively, as compared with normoxic control values (Figure 2A). As shown in Figure 2B, hypoxia-induced increase in DG transport was time-dependent: apparent at 6 h and maximal at 12 h with about a 4-fold increase above the baseline. Reoxygenation of the cells exposed to 0% O₂ for 18 h resulted in a progressive decrease of DG uptake that reached the normoxic control values in 24 h. Hypoxia-induced stimulation of glucose transport was not related to a decrease of protein content because protein content was not different in normoxic and hypoxic cells except for cells exposed to 0% O₂ 18 h, in which protein content decreased 20 ± 2.4% when compared with normoxic cells. The hypoxia-induced stimulation of glucose transport was further examined by the kinetic analysis of the DG uptake in normoxia and hypoxia (Figure 3). Analysis of the Lineweaver- Burk plot showed that hypoxia increased the maximal velocity (V_max) (100.06 nmol/mg protein/10 min compared with 41.06 nmol/mg protein/10 min in normoxia) without affecting the Michaelis content (K_m) (4.34 and 3.44 mM in hypoxia and normoxia, respectively).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Effect of hypoxia and hypoxia- reoxygenation on DG uptake in AEC. (A) Effect of decreasing oxygen concentration on DG uptake in AEC. AEC grown on plastic dishes for 3 d were exposed to either 21, 5, or 0% for 18 h. Oxygen tensions assayed in the culture medium were approximately 140, 60, and 30 mm Hg, respectively. Immediately at the end of the exposure, DG uptake was performed over a 10-min period. (B) Effect of exposure to various times of hypoxia. AEC from the same primary culture grown on plastic dishes for 3 d were exposed to either normoxia (21% O2; open squares) or hypoxia (0% O2; solid squares) for 1, 3, 6, 12, or 18 h; or to hypoxia followed by reoxygenation (18 h 0% O2 + 24 or 48 h 21% O2). Immediately at the end of the experiments, DG uptake was performed over a 10-min period. Values are means ± SE of three to four experiments run in duplicate. Statistical differences of values from the 21% group are indicated by *(P < 0.05) and **(P < 0.01).

View larger version (19K): [in this window] [in a new window]   Figure 3.   Effect of hypoxia on the kinetic parameters of DG transport in AEC. Cells were incubated in normoxic (21% O2; open squares) or hypoxic (0% O2; solid squares) atmosphere for 18 h before uptake measurement. Michaelis constant (Km) and maximal velocity (Vmax) were calculated using Lineweaver plot. Data represent means ± SE of four independent experiments in which duplicates were obtained.

To be certain that the increase in DG uptake reflected an increase in transport and not a change in the efficiency of glucose phosphorylation, we measured initial rates of 3 OMG uptake, a glucose analog that is not phosphorylated by cells. In 2 min incubation, AEC accumulated 248 ± 10.2 pmol/mg protein in hypoxia, compared with 84.7 ± 4.8 pmol/mg protein in normoxia. Because glucose transport in AEC was also shown to occur through a Na-dependent pathway, we examined the uptake of MGP, a nonmetabolized analog of D-glucose, which competes with the latter on the Na-D-glucose cotransport system. As previously reported, MGP uptake was low in AEC exposed to normoxia (75.1 ± 2.5 pmol/mg protein/10 min) and did not change in cells exposed to 0% O₂ for 18 h (85 ± 3.4 pmol/ mg protein/10 min).

Effect of Hypoxia on Glucose Consumption

AEC maintained for 18 h in 0% O₂ consumed more glucose than in normoxia, inasmuch as the glucose concentration in the medium of hypoxic cells fell from 25 to 17 mM, whereas no significant change occurred in the medium of normoxic cells. To test whether, in AEC, hypoxia-induced increase in DG uptake might be ascribed to the decrease of extracellular glucose concentration, we compared DG uptake in cells cultured for 18 h in a low- (5 mM) or high- (25 mM) glucose concentration medium. No significant change in DG uptake was noted either in normoxia (9.35 ± 1.81 versus 7.28 ± 0.74 nmol/mg protein/10 min) or in hypoxia (21.18 ± 3.94 versus 28.51 ± 5.8 nmol/mg protein/10 min).

Effect of Protein Synthesis Inhibitor on Hypoxia-Induced DG Transport Increase

To test the possible involvement of de novo protein synthesis in upregulation of glucose transport, AEC were incubated for 18 h with 1 µM cycloheximide, a protein synthesis inhibitor, under either normoxia or hypoxia (0% O₂). Cycloheximide completely blunted hypoxia-induced stimulation of DG uptake, suggesting that synthesis of glucose transporters or activator proteins is required for expression of hypoxia-induced stimulation of glucose transport (Figure 4).

View larger version (32K): [in this window] [in a new window]   Figure 4.   Effect of protein synthesis inhibitor on hypoxia-induced DG uptake increase. AEC were exposed to normoxia (21% O2) or hypoxia (0% O2) for 18 h in the absence or presence of cycloheximide 1 µM before DG uptake measurement. Results represent means ± SE of four independent experiments. Statistical difference of values from normoxia group is indicated by *(P < 0.01).

Effect of Hypoxia on GLUT1 Protein Level

A recent study has shown that AEC maintained more than 2 d in culture mainly expressed the facilitated glucose transporter GLUT1 (10). The effect of hypoxia on the GLUT1 transporter was assessed by evaluation of the amount of immunoreactive glucose transporter in total membranes prepared from AEC incubated 18 h in 0% O₂. Thirty micrograms of membrane protein of normoxic and hypoxic cells were probed by Western blot analysis using antibodies directed against GLUT1 transporter. As seen in Figure 5, the amount of GLUT1 transporter in hypoxic cells was markedly increased compared with that of cells exposed to 21% O₂. The quantification by laser densitometry of GLUT1 transporter in three independent experiments, using -actin as standard, showed an average 3-fold increase in GLUT1 transporter in hypoxia as compared with the corresponding normoxic values.

View larger version (22K): [in this window] [in a new window]   Figure 5.   Effect of hypoxia on GLUT1 protein steady-state levels in AEC. Rat AEC from two different preparations were exposed to either normoxia (21% O2) or hypoxia (0% O2) for 18 h. At the end of the exposure, Western blot analysis was performed on cell membranes using polyclonal antibody specific for GLUT1 as described in MATERIALS AND METHODS. The molecular mass of GLUT1 is 50 kD.

Effect of Hypoxia and Hypoxia-Reoxygenation on GLUT1 mRNA Level

RPAs were performed in order to determine the level of GLUT1 mRNA transcript in normoxic and hypoxic cells. As shown in Figure 6, transcript encoding GLUT1 was detectable in AEC. Exposure of the cells to 0% O₂ for 18 h induced a 3-fold increase in the GLUT1 mRNA level. When hypoxic cells were allowed to recover in 21% O₂, a complete recovery in GLUT1 mRNA level was achieved in 24 h.

View larger version (49K): [in this window] [in a new window]   Figure 6.   Effect of hypoxia and hypoxia-reoxygenation on GLUT1 mRNA level. mRNA expression of GLUT1 in AEC exposed to hypoxia and hypoxia-reoxygenation. Rat AEC from the same primary culture were grown on plastic dishes and exposed to either normoxia (21% O2) or hypoxia (0% O2) for 18 h; or to hypoxia-reoxygenation (18 h 0% O2 + 48 h 21% O2). At the end of the exposure, RPAs were performed on cell lysates (RNA equivalent to 106 cells) as described in MATERIAL AND METHODS. Quantification was performed using an Instant Imager. Data were normalized for the corresponding actin signal for each lane. Results are expressed as the ratio of GLUT1 mRNA/actin mRNA and represent means ± SE of three independent experiments. Statistical difference of values from normoxia group is indicated by *(P < 0.01).

Effect of Cobalt Chloride and Inhibitor of Oxidative Phosphorylation on Glucose Transport and GLUT1 mRNA Levels

To determine whether upregulation of GLUT1 mRNA and glucose transport by hypoxia was directly related to O₂ deprivation per se, or ascribed to hypoxia-induced inhibition of oxidative phosphorylation, we examined the effects of cobalt chloride and of Na azide, an inhibitor of oxidative phosphorylation, respectively. AEC incubated in the presence of 21% O₂ with 100 µM cobalt chloride for 18 h demonstrated an ~ 2-fold stimulation in the 2DG uptake and an ~ 4-fold increase in GLUT1 mRNA level compared with control cells incubated under normoxic conditions. Incubation of the cells with 1 mM azide for 18 h produced a 3-fold increase in 2DG uptake and an ~ 2-fold increase in GLUT1 mRNA level (Figure 7).

View larger version (26K): [in this window] [in a new window]   Figure 7.   Effects of hypoxia, cobalt chloride, and Na azide on DG uptake (A) and GLUT1 mRNA levels (B) in AEC. Cells were incubated in hypoxic atmosphere (0%, O2), or in normoxic conditions in the presence of cobalt chloride (100 µM) or Na azide (1 mM) for 24 h before DG uptake and isolation of RNA as described in MATERIALS AND METHODS. Results represent means ± SE of three to five independent experiments. Statistical difference of values from normoxia group is indicated by *(P < 0.01) and from hypoxia group §(P < 0.01).

Analysis of HIF DNA Binding Activity

HIF DNA binding activity was determined by electrophoretic mobility shift assay (EMSA) with probe GLUT1, a double-stranded oligonucleotide containing a 24-base pair sequence from the rat GLUT1 gene enhancer that mediates hypoxia-inducible transcription. Nuclear extracts obtained from AEC exposed to 21 and 0% O₂ for 6 h, and cells incubated with 100 µM cobalt chloride for 6 h, were incubated with ³²P-labeled probe. EMSA revealed that AEC exhibited HIF DNA binding activity in 21% O₂, which increased in hypoxia and after cobalt chloride treatment (Figure 8).

View larger version (79K): [in this window] [in a new window]   Figure 8.   Analysis of HIF DNA binding activity in AEC. Aliquots (5 µg) of nuclear extracts prepared from AEC exposed to normoxia (21% O2), hypoxia (0% O2), and cobalt chloride (100 µM) for 6 h were incubated with a radiolabeled oligonucleotide probe corresponding to the HIE sequence upstream from the GLUT1 gene. Lane 1: no protein; lane 2: nuclear extracts of normoxic AEC; lanes 3-5: nuclear extracts of hypoxic cells. Unlabeled oligonucleotide probes (50- and 100-fold) were used as competitors in lanes 4 and 5, respectively; and in lanes 6-8, nuclear extracts of normoxic cells incubated with cobalt chloride. Unlabeled oligonucleotide probes (50- and 100-fold) were used as competitors in lanes 7 and 8, respectively; and in lane 9, nuclear extracts of hypoxic cells. Unlabeled oligonucleotide probe corresponding to the activator protein-1 sequence was used as a nonspecific competitor. C: constitutive binding.

	Discussion

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Our data show that AEC were fairly tolerant to prolonged hypoxia, inasmuch as they maintained their energy status at a level close to that of normoxic cells. Indeed, ATP content showed an early drop followed by a rapid return to normoxic control values. This ATP recovery roughly paralleled lactate content increase, suggesting that a drop in ATP content triggered increased anaerobic glycolysis which, in turn, put back the ATP level of hypoxic cells. These results agree with in vivo studies showing that rat lung tissue was not damaged by alveolar hypoxia and that it maintained a normal energy status under important oxygen deprivation (11).

In AEC, the first limiting step in glucose use is its transport across the membrane because glucose concentration is low and activity of glycolytic enzymes largely exceeds the rate of glucose utilization. This study demonstrated that in AEC, glucose transport was upregulated in response to hypoxia. This stimulation involved only the Na-independent glucose transport, as shown by the dramatic increase in 2DG, but did not change the Na-dependent glucose transport, evaluated by MGP uptake, a glucose analog that is transported only by the Na-dependent glucose transporter. Hypoxia-induced increase in glucose transport increase was (1) time-dependent: observed for prolonged periods of hypoxia ( 6 h); (2) O₂ concentration- dependent: induced by moderate oxygen deprivation (5% O₂) and maximal for severe hypoxia (0% O₂); and (3) reversible when the cells were allowed to reoxygenate in normoxic atmosphere for 24 h. Hypoxia-induced stimulation of DG uptake was not the consequence of an increase in intracellular phosphorylation because hypoxia induced a similar increase in 3 OMG uptake, a glucose analog that is not phosphorylated. In mammalian tissues, facilitative glucose transporters consist in five isoforms. The ubiquitous glucose transporter GLUT1, which is responsible in most of the tissues for the basal glucose uptake, is largely prominent in cultured AEC (10). In our study, prolonged hypoxia induced an increase in the GLUT1 protein expression and in GLUT1 mRNA levels. A causative link between increases in mRNA levels and protein expression of GLUT1, and stimulation of glucose transport, was supported by the following evidence: (1) the time course of hypoxia effect, with no substantial change before 6 h exposure; (2) the prevention of hypoxia-induced DG stimulation by cycloheximide, an inhibitor of translation; and (3) the parallel recovery during reoxygenation in DG uptake and GLUT1 mRNA level. Comparison of AEC with other hypoxic-tolerant cells showed that they resemble endothelial cells (12) but differ from heart and skeletal muscle, in which the upregulation of glucose transport is an early event ( 1 h) with no change of GLUT1 mRNA level (13, 14).

The nature of the signal by which hypoxia triggers the increase in GLUT1 mRNA level has been addressed. GLUT1 belongs to the glucose-regulated protein family of stress proteins, and both GLUT1 protein and mRNA levels have been shown to be upregulated by glucose starvation (15, 16). In our study, upregulation of GLUT1 transporter was not the consequence of hypoxia-induced glucose deprivation because (1) cells were cultured in a high glucose concentration medium, and after 18 h of exposure to 0% O₂ the glucose concentration in the medium did not fall below 17 mM; and (2) glucose transport activity was not different in cells cultured in low glucose concentration (5 mM) medium compared with cells grown in high glucose concentration (25 mM) medium. The glucose transporter GLUT1 gene also belongs to the family of hypoxia-regulated genes. In different nonepithelial cell lines, GLUT1 mRNA expression was reported to be upregulated by O₂ deprivation (9, 12, 17). In our study GLUT1 mRNA accumulation was induced by hypoxia and reversed by a following exposure to normoxia, suggesting that in primary alveolar epithelial cells, as well as in nonepithelial cells, upregulation of GLUT1 expression was regulated by O₂ deprivation. The O₂ sensing mechanisms whereby hypoxia regulates GLUT1 mRNA transcripts are not univocal and may result from either reduced O₂ concentration per se or inhibition of oxidative phosphorylation (9, 17). This distinction can be made through employment of specific chemical agents that mimic the actions of the different components of the hypoxic response. Cobalt chloride, employed in the presence of oxygen, mimics the effects of lowered oxygen concentration per se. In normoxic AEC, cobalt chloride induced an increase of both GLUT1 mRNA levels and DG uptake. Na azide, an inhibitor of oxidative phosphorylation used in the presence of oxygen, was as effective as hypoxia to stimulate glucose transport but induced a lesser increase in GLUT1 mRNA levels. Further studies are required to determine the temporal and spatial contributions of these two mechanisms in hypoxia-induced upregulation of GLUT1 mRNA level.

Recent studies have clearly demonstrated that hypoxia-inducible GLUT1 expression is critically dependent on the binding of a nuclear protein, HIF-1, to the hypoxia-inducible DNA element (HIE) upstream of the GLUT1 gene (9, 17). HIF-1 is a heterodimer composed of HIF1- and HIF1- subunits, both of which are members of the basic-helix-loop-helix-PAS (bHLH/PAS) family of proteins (18): HIF1- is a protein unique to HIF-1; whereas HIF1- is identical to the aryl hydrocarbon nuclear translocator that can heterodimerize with several other proteins, including the aryl hydrocarbon receptor (19) and other bHLH/PAS proteins such as endothelial PAS protein-1 (EPAS₁) (20, 21). Both HIF1- and HIF1- subunits are instrumental in activating the GLUT1 gene in response to O₂ deprivation (22, 23). In our study AEC expressed a constitutive level of HIF-1 DNA binding, the activity of which increased under hypoxic exposure and after cobalt chloride treatment. In most of the cell lines, HIF-1 DNA binding was clearly dependent on O₂ concentrations with low or undetectable activity in normoxia and with a maximum increase for very low O₂ concentrations (0.5% O₂) (24). However, the presence of high constitutive levels of HIF DNA binding has already been reported in a few cell types, such as pulmonary arterial smooth muscle cell (25), J1 embryonic stem cells (22), and v-Src-transformed rat fibroblasts (26), but its significance is not clearly understood. One possibility is that cultured cells exposed to normoxic atmosphere were in relatively hypoxic conditions due to the presence of culture medium above them. The second possibility is that the high basal HIF-1 DNA binding may be adaptive in cells with increased metabolic demands and, in this case, a signal transduction pathway that if not hypoxia-driven could upregulate HIF-1 expression in a cell type-specific manner. Finally, it could not be excluded that the nuclear protein that bound to the probe was not HIF-1 but an HIF-like factor. EPAS is an HIF that exhibits very similar characteristics to HIF1- in the properties of dimerization, DNA binding, and transcriptional activation (20, 27). EPAS was less ubiquitously expressed than HIF1- and mainly regulated the transcription of vascular endothelial growth factor (21). In AEC, EPAS might be a good candidate because: (1) EPAS-1 was more active in normoxia and less inducible by hypoxia than HIF-1 (20); and (2) EPAS mRNA transcript was expressed in mice alveolar epithelium at a higher level than HIF1- mRNA (21). Further studies are required to determine whether EPAS is involved in the transcriptional regulation of the GLUT1 gene.

In conclusion, AEC regulate the GLUT1 transporter at the transcriptional level in response to hypoxia, a phenomenon that allows them to maintain their main functions. This regulation involves, at least in part, HIFs, the nature of which remains to be determined. Further studies should be useful in determining their physiologic roles and their specific regulation.