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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To determine whether glucose depletion is a principal determinant of hyperoxic cell death in vitro, human lung epithelial-like cells (A549) were exposed to hyperoxia (95% O2) in either 10, 30, or 50 ml of medium (Ham's F-12K). Glucose was depleted in the medium after 36, 60, or 96 h, respectively. Medium lactate dehydrogenase (LDH) activity increased only after glucose was depleted. To confirm that glucose depletion was critical to cell death, cells exposed to 95% O2 were supplemented with glucose at regular intervals to reestablish initial medium glucose concentrations. Other cells received no supplements. Without supplementation, glucose was depleted within 48 h, followed within 12 h by an almost complete loss of cell ATP and elevated medium LDH activity. Glucose-supplemented cells appeared normal microscopically and did not release LDH activity despite an extracellular pH of 6.5 due to fermentation. Additional experiments at sea-level pressure confirmed that glucose supplementation prevents extensive cell death in hyperoxia in cultured A549 cells.
adenosine 5'-triphosphate; oxygen toxicity; culture; necrosis; lung; epithelium
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INTRODUCTION |
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INCREASED GLUCOSE CONSUMPTION has been observed both in isolated perfused lungs from rats exposed to hyperoxia and in a number of cell types exposed to hyperoxia under culture conditions. Because necrotic cell death is widely reported in cultured cells exposed to a high O2 concentration, we questioned whether limited glucose transport capacity contributed to this process. Before questions concerning glucose transport were addressed, preliminary experiments were performed to determine the rates of glucose consumption to ensure that adequate glucose was available for the transport experiments. Surprisingly, cells consumed all of the medium glucose within a relatively short period during hyperoxic exposure. Through the series of experiments reported here, we have determined that extensive cell death does not occur under hyperoxic conditions as long as glucose is provided from the culture medium.
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METHODS |
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Medium glucose consumption in hyperoxia. A549 cells (an adenocarcinoma cell line from human lungs; American Type Culture Collection, Rockville, MD; see Refs. 7, 10) were seeded at 1.5 million/plate on 100-mm tissue culture plates (Becton Dickinson, Franklin Lakes, NJ) and allowed to attach for 3 days under 10 ml of Ham's F-12K medium (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum and penicillin-streptomycin (100 U/ml and 100 µg/ml, respectively) (complete medium). The medium on the plates was then replaced with either 10, 30, or 50 ml of fresh complete medium, placed in polystyrene exposure chambers (Billups-Rothenberg, Del Mar, CA), and equilibrated with 95% O2-5% CO2. A set of A549 plates incubated with 50 ml of medium was maintained in 95% air-5% CO2 in a cell incubator (Forma Scientific, Marietta, OH). The exposure chambers were then maintained at 37°C. At regular intervals, the chamber was opened, and small aliquots of medium were collected for glucose and lactate dehydrogenase (LDH) activity measurements. During this collection period, the plates were exposed to room air for no more than 2 min. In these preliminary studies, medium glucose and LDH activity (a marker of cellular injury) were measured in the National Jewish Center's Clinical Laboratory (Denver, CO) with a Paramax 720 automated analyzer (Baxter, Irvine, CA) and appropriate reagent kits (Baxter Diagnostics, Wilmington, DE; Dade Diagnostics, Aguada, PR).
Glucose supplementation in hyperoxia.
To further confirm that glucose depletion leads to cell death in
hyperoxia, A549 cells were seeded onto 100-mm tissue culture plates as
before. After the 3-day attachment-equilibration period under normoxic
conditions (95% air-5% CO2),
the medium on each plate was replaced with 20 ml of fresh complete
medium. Eighteen plates were assigned to a glucose-supplemented group
while another 18 plates were assigned to a nonsupplemented group. These
plates were placed in the polystyrene exposure chambers, equilibrated
with 95% O2-5%
CO2, and placed in a 37°C
incubator. Six additional plates of cells were used to establish
baseline ATP and protein levels in normoxia. To preserve cellular ATP
for subsequent assay, the cells were rinsed on the plate with sterile
phosphate-buffered saline and then harvested into 75°C 80%
methanol, 5 mM 2-(N-morpholino)ethanesulfonic acid, pH 5.0, and 0.5 mM EDTA in an adaptation of the method of Shryock et al. (16).
The cell extracts prepared in this manner were maintained at 4°C
for 24 h and then stored at 20°C until assayed for ATP
content.
After 24, 36, 42, 48, 60, and 84 h of hyperoxic exposure, three plates
from each group were removed from the chamber, and the chamber was
immediately refilled with 95%
O2-5%
CO2 and placed in the incubator.
The volume of medium was measured in each of the six plates being
harvested, and the media were then centrifuged at 1,500 g to remove any cellular debris. The
cells were again extracted for ATP measurement with the hot-methanol
method as above. Aliquots of medium were frozen at 70°C for
subsequent assays.
Medium glucose concentrations were determined at the 24-, 48-, 60-, and 84-h time points, and the glucose-supplemented plates were supplemented with a volume of sterile glucose solution (100 mg/ml) calculated to reestablish baseline glucose levels (120 mg/dl) in those plates. The plates were then regassed with 95% O2-5% CO2 and immediately returned to the incubator.
Cell exposures to hyperoxia at sea-level atmospheric pressure. The calculated PO2 value achievable in medium exposed to 95% O2 at Denver's altitude is ~83% of that achievable using the same gas concentrations at sea level. It seemed possible, then, that the failure of cells to die during hyperoxic exposure at Denver's atmospheric pressure may have been due to this reduced PO2. To determine whether cell death in hyperoxia was due to glucose depletion even at sea level, an apparatus was constructed that allowed sterile incubation of culture plates in a gaseous environment pressurized to an absolute pressure of 760 mmHg (sea level; +120 mmHg relative to Denver).
A549 cells were seeded on 100-mm cell culture plates at 500,000/plate. The seeding density was decreased for these experiments to preclude comparison of data from confluent cells in air with those from subconfluent cells in hyperoxia. The cells were allowed to attach at Denver's altitude in an air-5% CO2 incubator for 3 days, then were placed in the exposure chambers and pressurized to sea-level pressure. Cells and medium were harvested daily for the determination of medium glucose concentration and LDH activity along with cellular protein, DNA content, and LDH activity.
Assays. Cell DNA was estimated with the Hoechst dye method described by Labarca and Paigen (12). Protein was measured with bicinchoninic acid (Micro BCA Protein Assay Kit, Pierce, Rockford, IL). Medium glucose was determined with a kit (Sigma, St. Louis, MO), incorporating a modification of the hexokinase method of Bondar (6). Cellular ATP content was measured with a luciferase-luciferin kit (Calbiochem, San Diego, CA). LDH activity was assayed as described by Bergmeyer and Garvehn (5). Percent LDH release was calculated as (increase in medium LDH activity)/(increase in medium LDH activity + LDH activity present in cells), where increase in medium LDH activity refers to the level in excess of the background activity contributed by the serum present in complete medium.
Statistics. Student's
t-test was used to compare the gas
effects at each time point. In the supplementation study, Dunnett's test was used to compare the effects of hyperoxia at multiple times
with normoxic control values at time
0. Significance was accepted when
P 0.05. All statistical
calculations were performed with JMP software (SAS Institute, Cary,
NC).
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RESULTS |
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Glucose consumption was increased greatly in cells exposed to hyperoxia (Fig. 1A). During the period between 24 and 48 h of hyperoxia, the rate of glucose consumption in the hyperoxic plates containing 50 ml of medium was increased more than fourfold (0.75 vs. 0.18 mg/h) over that in the normoxic plates at the same volume. In hyperoxia, 10, 30, and 50 ml of medium appear to be completely depleted of glucose after ~36, 60, and 96 h, respectively. On the other hand, only about a 44% decline in glucose content was noted in 50 ml of medium after 96 h of normoxic exposure. Furthermore, the medium LDH activity was greatly elevated at 96 h in the 10-ml group, whereas there was only a slight elevation in LDH activity in the medium at 96 h in the 30-ml group (Fig. 1B). No elevation in medium LDH activity was detected at any time point in the 50-ml group in either hyperoxia or normoxia. Because several hours elapsed between the time glucose was fully depleted and the time at which elevation in LDH activity was first observed, it was necessary to determine that glucose depletion was responsible for the increased LDH activity.
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Glucose supplementation by itself prevented the elevation in medium LDH activity observed in the unsupplemented cells exposed to hyperoxia. Glucose in the medium was depleted in the unsupplemented group after ~48 h of hyperoxic exposure, whereas the medium of the glucose-supplemented group never contained <6.75 mg of glucose (34 mg/dl in 20 ml; Fig. 2). Medium LDH activity was slightly, but significantly, increased after 60 h of hyperoxia in the unsupplemented group. This was ~12 h after all of the glucose in the medium was consumed. Medium LDH activity was never increased from baseline levels in the glucose-supplemented group despite a decrease in pH from 7.5 to 6.5 due to glucose fermentation.
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Glucose supplementation also prevented the nearly complete loss of ATP observed in the unsupplemented cells. Cellular ATP contents of both groups, normalized to cell protein, were similar through 42 h of exposure (Fig. 3). Thereafter, the ATP content of the unsupplemented cells rapidly declined. The ATP content of glucose-supplemented cells remained greater than or equal to that present in normoxic cells (time 0) until the 60-h timepoint, at which time those cells contained 72% of the ATP present in the normoxic cells. The ATP content of supplemented cells was further reduced at the 84-h timepoint to 54% of that of the normoxic cells.
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Light-microscopic examination of these cells demonstrated that despite the acidosis present in the glucose-supplemented medium in hyperoxia, the cells retained normal morphology. In comparison, the unsupplemented cells exhibited normal morphology until the 84-h time point, at which time these cells appeared pyknotic, with many floating cells and abundant sheets of cells lifting off the culture plate (Fig. 4).
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Much like the A549 cells exposed to hyperoxia at Denver's altitude,
A549 cells exposed to hyperoxia at sea-level pressure did not
demonstrate extensive cell death when incubated in high volumes of
medium (30 ml) and with daily changes of that medium. The cells exposed
to hyperoxia under these conditions ceased growing as assessed by both
protein and DNA content (Fig. 5,
A and
B, respectively). There was a
transient increase in percent LDH release (Fig.
5C) in the hyperoxic cells at
sea-level pressure that appeared to reach a maximal value at
3 days of O2 exposure. Furthermore, the rate of glucose
consumption increased to a maximum rate of 231 ± 14 (SD)
µg · mg
protein1 · h
1
after 3 days of hyperoxic exposure (Fig.
5D) in comparison with a glucose
consumption rate of 103 ± 7 µg · mg
protein
1 · h
1
in air at the same time. There was no indication of cytolysis when the
cells were examined by phase-contrast microscopy (data not shown).
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DISCUSSION |
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The results of the experiments described here reveal that cultured A549
cells exposed to normobaric hyperoxia increase their rate of glucose
consumption approximately two- to fourfold relative to normoxic cells,
depending on experimental conditions. A number of other investigators
have found increases in glucose consumption in response to hyperoxic
exposure. Schoonen et al. (15) described such an increase in Chinese
hamster ovary cells, whereas Balin et al. (1) found that WI-38 cells (a
human diploid cell line) increased glucose consumption four- to sixfold
after exposure to high O2
concentrations. One cause of this increased glucose consumption might
be an increased dependence on anaerobic glycolysis for ATP production
subsequent to inactivation of the mitochondrial enzymes involved in
aerobic energy production. Hyperoxic exposure of Chinese hamster ovary
cells has been shown to reduce the activity of several enzymes involved
in intermediary metabolism, including 2-oxoglutarate dehydrogenase
(-ketoglutarate dehydrogenase), succinate dehydrogenase, and NADH
dehydrogenase (15). More recently, Gardner et al. (7) have described a
more rapid loss of aconitase activity in A549 cells during hyperoxic
exposure. Additional increases in glucose consumption might result from
an increased flux through the hexose monophosphate shunt pathway.
In the experiments reported here, complete exhaustion of medium glucose was followed within a few hours by a nearly complete depletion of cell ATP. The effect of glucose depletion on cell ATP content appears to be partially dependent on the cell type being studied. For example, when rat islet insulinoma cells (RINm5F) were grown on microcarrier beads and the effect of glucose starvation on cellular ATP content was examined by nuclear magnetic resonance spectroscopy, it was found that the cell ATP was completely depleted within 20 min of glucose removal (11). However, in CEM cells (a lymphoblastic leukemia CD4+ cell line), ATP content gradually declined 60% in the 24 h after the onset of glucose starvation, with ATP being further depleted to 4.5% of the baseline value after 48 h of glucose starvation (13). In the experiments presented here, we found that ATP was almost completely absent in the unsupplemented cells within 12 h of glucose depletion. We also found that the ATP content of the glucose-supplemented cells declined during the hyperoxic exposure, reaching ~54% of the level present in the normoxic cells. This finding of decreased ATP content while glucose remained present in the medium was not unexpected because similar reductions in cellular ATP content have been described in studies (9, 14) in which decreased, but not absent, ATP was observed in cultured mammalian cells exposed to fluorocitrate, an inhibitor of aconitase.
The relationship between cellular ATP content and glucose concentration in the medium apparently depends on the cell type and tissue of origin. The exquisite sensitivity of rat insulinoma cells to glucose starvation is likely an expression of the physiological role of the islet cells in sensing blood glucose levels. In this cell type, the rapid changes in cellular ATP content in response to changing glucose concentrations is thought to be an integral step in regulating insulin secretion (8). The ATP content of other cell types in which the physiological function is not directly related to sensing glucose concentrations may be less affected by changes in glucose concentration in the medium.
Few of the articles describing studies of hyperoxic effects on cultured cells reveal sufficient information to know the glucose availability. Furthermore, inadvertent glucose starvation in hyperoxic cell models might lead to misinterpretation of experimental findings. For example, a recent report (10) concluded that cultured A549 cells exposed to hyperoxia died via necrosis after several days, whereas exposure to hydrogen peroxide or paraquat resulted in apoptotic death within a matter of hours. It was reported in the same article that cells from the lungs of mice exposed to hyperoxia for 48 h showed signs of apoptotic cell death. Without information concerning medium volumes or culture plate sizes, it is difficult to know whether the cultured cells exposed to hyperoxia also were deprived of glucose during these experiments.
Cells in culture are used as models of physiological events occurring in vivo. Although little is known about the effects of hyperoxic exposure on lung metabolism in vivo, a number of investigations have been conducted in vitro with isolated perfused rat lungs. Bassett and colleagues (2-4) have described the rate of glucose flux through the glycolytic pathway and through the tricarboxylic acid cycle in isolated perfused lungs from rats. These investigators found that glucose consumption was increased 53% in the lungs of rats exposed to 100% O2 for 24 h. However, direct comparison of these findings with those from cell culture or in vivo conditions would be difficult because the lungs were perfused with a buffered salt solution lacking any amino acids that might have altered metabolic rates or provided alternative energetic substrates.
The time required to deplete medium glucose altogether in hyperoxia depends on the initial glucose concentration, the volume of medium provided to the cells, the frequency of medium changes, the number of cells being studied along with their state of confluency, and the effect of other experimental elements. Without this information, it is difficult to ascertain whether the cell death described in many reports results from glucose starvation or O2 toxicity. However, our findings indicate that cell death in the pulmonary adenocarcinoma cell line A549 is markedly inhibited by the provision of unconventionally large quantities of glucose. These findings may have pertinence to a variety of other in vitro models for pulmonary O2 toxicity and oxidative stress.
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ACKNOWLEDGEMENTS |
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We are grateful to Jacque Guthrie for preparing the manuscript.
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FOOTNOTES |
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Research support was provided by National Heart, Lung, and Blood Institute Grants HL-46481, HL-52732 (both to C. W. White), and HL-07670 (to C. B. Allen).
Address for reprint requests: C. W. White, J-101, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206.
Received 6 October 1997; accepted in final form 16 October 1997.
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