Regulation transporter NAVA Division Bashan, and Amira
of glucose transport and GLUT1 glucose expression by O2 in muscle cells in culture BASHAN,
ELENA
BURDETT,
HARINDER
Nava, Elena Burdett, Harinder S. Hundal, Klip. Regulation of glucosetransport and GLUT1
glucosetransporter expressionby O2in musclecells in culture. 262 (Cell Physiol. 31): C682-C690, 1992.-The effect of varying cellular oxygenation on L6 muscle cell 2deoxy-D-glucosetransport, glucoseutilization, lactate production, and expressionof GLUT1 and GLUT4 transport proteins was investigated. Incubation of L6 myotubes in 3% O2 (mimicking a state of hypoxia) elevated glucoseuptake by 6.5fold over 48 h relative to cells incubated in 21% O2 (normoxia). Incubation of L6 cells in hyperoxic conditions (50% 02) significantly depressedglucoseuptake by 0.4-fold. These effects were fully reversible. Incubation in 3% O2also causedlactate accumulation and enhancedglucoseconsumptionfrom the medium. Hypoxia elevated 2-deoxy-D-glucosetransport even when the concentration of glucosein the medium was kept constant, suggestingthat glucosedeprivation alone was not responsible for increasedcellular glucoseuptake. Incubation in 3% O2also elevated 3-0-methylglucoseuptake but not amino acid uptake. Cycloheximide prevented the hypoxia-induced increasein glucoseuptake, indicating that de novo synthesisof glucosetransport-related proteins was the major meansby which cells increasedglucoseuptake. The content of GLUT1 glucosetransporter was significantly elevated in total membranesof cells incubated in 3% O2and depressedin membranesfrom cells incubated in hyperoxic conditions, whereasGLUT4 expression was not affected. These results indicate that hypoxia induces an adaptive responseof increasing cellular glucose uptake through elevated expressionof GLUT1 in an attempt to maintain supply of glucosefor utilization by nonoxidative pathways. hypoxia; glucoseuptake; ischemia;oxygen levels
Am. J. Physiol.
BEEN RECOGNIZED that diverse cells respond to inhibition of oxidative metabolism with a rapid enhancement in glucose utilization by nonoxidative pathways. This is best illustrated by the increase in glycolysis upon inhibition of mitochondrial respiration, i.e., the Pasteur effect (29). The net yield of ATP obtained per molecule of glucose through glycolysis alone (2 ATP) is l&fold lower than that through aerobic respiration (36 ATP). Thus, to maintain normal energy production through anaerobic metabolism alone, glucose must be consumed at a markedly enhanced rate. In mammalian tissues such as liver, the facilitation of glycolysis under anaerobic conditions is largely due to the regulation of phosphofructokinase, the rate-limiting enzyme of the glycolytic pathway in that tissue. Stimulation of phosphofructokinase leads to increased exogenous glucose uptake by increasing the driving force for influx, i.e., the glucose concentration gradient across the cell. Therefore this effectively increases glucose uptake only in cells where there was a preexisting intracellular pool of free glucose (6, 14). In contrast, in tissues such as skeletal muscle and fat there is no detectable free intracellular glucose because glucose transport is the rate-limiting step in glucose IT HAS LONG
C682
S. HUNDAL,
AND
AMIRA
KLIP
of Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
0363-6143/92
$2.00
utilization (14, 21). Hence, in these tissues, activation of phosphofructokinase would be insufficient to increase glucose utilization during anaerobic conditions, and it would seem a priori that regulation at the level of glucose transport would be required to procure the energy demand. Indeed, a stimulation in muscle glucose transport (measured using the nonmetabolizable glucose analogue 3-0-methylglucose, which is transported into the muscle through the glucose transport system) has been demonstrated in preparations of epitrochlearis muscle made hypoxic in vitro (7 and Refs. within; 27, 28, 30). The transport of glucose into most cells occurs by facilitated diffusion mediated by a recently identified multigene family of transmembrane glycoproteins (3). Skeletal muscle expresses two glucose transporter isoforms of this family, denominated GLUT1 and GLUT4 (9, 11, 21). The GLUT1 transporters are distributed ubiquitously in most tissues; in skeletal muscle they appear to be located primarily in the plasma membrane and are thought largely to be responsible for basal glucose uptake (9). Conversely, GLUT4 transporters are expressed only in tissues where glucose uptake is insulin sensitive (muscle and fat) (3) and are predominantly located in an intracellular compartment whose precise identity presently remains unknown (8). Insulin stimulates glucose transport into skeletal muscle, in part, by promoting the translocation of GLUT4 transporters from this intracellular location to the plasma membrane (9, 16, 22). The mechanism by which hypoxia stimulates glucose uptake into skeletal muscle has only recently begun to be addressed. This question is of paramount importance to the physiology of muscle in vivo, since Po2 varies considerably within this tissue, depending on muscle thickness and fiber distance from the capillaries. The Pop of moist inspired air (containing 21% 02) is 150 mmHg. The Paz falls to just over 100 mmHg in the lungs and blood and can be as low as 40 mmHg in peripheral tissues. Despite these large changes, the effect of variations in tissue oxygenation on glucose uptake and energy production has not been documented at the molecular level. In a collaborative study, we have recently reported that short-term (up to 45 min) exposure of isolated epitrochlearis muscle to hypoxic conditions induces an increase in GLUT4 glucose transporters in the plasma membrane, as determined by subcellular fractionation of skeletal muscle (7). Isolated muscle preparations, however, are unsuitable for studying the effects of prolonged exposure to hypoxic conditions, since they become metabolically compromised and show marked changes in glucose transport upon incubation of the tissue in vitro (1% In contrast, muscle cell lines are stable during prolonged incubation periods and offer the advantage that
Copyright 0 1992 the American Physiological Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
GLUCOSE TRANSPORT AND OXYGEN
C683
isotonic saline solution (containing 1 mM HgClz in the caseof 3-0-[methy13H]methyl-D-glucose uptake assays). Cells were disrupted with 0.05 N NaOH, and cellular radioactivity was quantitated by liquid scintillation counting usingan LKB 1217 beta counter. Each uptake experiment was performed in quadruplicate, and resultsof specific uptake are expressedasmeans t SE (in pmol . mg protein’min .-‘). Amino acid transport. L6 myotubes, incubated under identical conditions as for hexose uptake, were used to assessthe transport of MeAIB, a nonmetabolizable amino acid analogue specific for the system A amino acid transporter (17). Cells were routinely depleted of intracellular amino acids for 1 h by preincubation in HBS to avoid truns-inhibition during uptake measurements.Subsequently, the experimental procedure for measuringuptake in L6 cellswasas describedabovefor hexose uptake. Initial rates of 10 PM [1-14C]MeAIB (0.1 &i/ml) uptake were measuredfor 10 min in HBS. Uptake was linear over a period of 20 min, and transport in choline-substituted medium (i.e., the Na’-independent component of uptake) was negligible (~4%). Experiments were performed three times in quadruplicate, with uptake experiments being terminated and cells being processedfor liquid scintillation counting as describedabove for hexoseuptake. Total membrane isolation and Western blots. L6 cell monolayers at the myotube stagewere incubated under the indicated experimental conditions (10 dishes/condition), then scraped gently with a rubber policeman, and concentrated by centrifuMATERIALS AND METHODS gation (700 g for 10 min). All remaining stepswere performed at 4°C. Cells were resuspendedin 20 ml of solution A [250 mM Materials. Tissue culture medium, serum, and reagents were sucrose,5 mM NaN3, 2 mM ethylene glycol-bis(P-aminoethyl obtained from GIBCO. Cytochalasin B, insulin, 2-deoxy-Dglucose, and 3-0-methylglucose were obtained from Sigma ether)-N,N,N’,N’-tetraacetic acid, 100 PM phenylmethylsulfonyl fluoride, 10 PM E-64, 1 PM pepstatin A, 1 PM leupeptin, Chemical. 2-[3H]deoxy-D-glucose and 3-0-[methyl-3H]methyland 20 mM HEPES-Na (pH 7.4)] and homogenizedwith a D-glucose were purchased from ICN. a-[ 1-14C]methylaminoisomotor-driven Potter-Elvehjem homogenizer (15 strokes). The butyric acid (MeAIB) was purchased from New England Nuhomogenateswere centrifuged at 760 g for 3 min to remove clear (Boston, MA). Antisera containing polyclonal antibodies raised against COOH-terminal sequences of GLUT1 and nuclei and unbroken cells, and the supernatants were centrifuged at 190,000g for 60 min. The resulting pellet contained GLUT4 were obtained from East Acres Biologicals Laboratototal cell membranesand was routinely resuspendedin a small ries. Monoclonal antibody against the al-subunit of the Na+volume of solution A. K+-adenosinetriphosphatase (ATPase) was a kind gift from Dr. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Katherine Sweadner, Harvard University (10). Cell cultures. L6 muscle cells were grown in monolayers to (using 10% polyacrylamide gels) and Western blot analysis the stage of myotubes as previously described (23) in alpha were carried out as previously described (26), using a 1:500 minimum essential medium containing 5 mM glucosein the dilution of anti-GLUT4 antiserum, a 1:5,000 dilution of antipresenceof 2% fetal bovine serum and 1% antibiotic solution GLUT1 antiserum, or a 1:500dilution of the monoclonalanti(final concn 10 mg/ml penicillin, 10 mg/ml streptomycin, and body to the al-subunit of Na’-K’-ATPase (lo), followed by A in 25 mg/ml amphotericin B) at 37°C in an atmosphere of 5% radioactive labeling of bound antibodieswith 1251-protein C02-95% air. The cells were grown in 3.5cm-diameter wells the first two casesand sheepanti-mouse 1251-immunoglobulin (6-well plates) for transport determinations (4 ml medium/ G in the latter case.Labeled proteins were visualized by autowell) or in lo-cm-diameter dishesfor isolation of total mem- radiography with Kodak XAR-5 film and quantitated by laser branes (10 ml medium/dish). Two to 3 days after cell fusion, scanningdensitometry. Other metubolite assays.Glucose and lactate concentrations cells were further incubated under the sameatmospheric conditions describedabove or were transferred to low-02 (3%) or in the medium were determined by the glucoseoxidasemethod high-02 (50%) incubators. The O2concentration in the low-O:! and spectrophotometric analysis using lactate dehydrogenase, incubator was reducedto 3% by blending NZ with ambient air respectively (15, 33). Protein was determined by the Bio-Rad by useof a multiplex gascontrol system.The CO:!concentration Bradford procedure (5). remainedat 5% in all cases. Hexose transport determinations. After the appropriate treat- RESULTS ments as indicated in the figure legends,cells were rinsed with glucose-free N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic To investigate the regulation of glucose transport in acid (HEPES)-buffered‘ saline [HBS: 140 mM NaCl, 20 mM L6 myotubes by O2 availability, we incubated L6 myoHEPES-Na (pH 7.4), 2.5 mM MgS04, 5 mM KCl, and 1 mM tubes under different O2 concentrations: 21% 02, which CaCIZ] as previously described (23, 26). Hexose uptake was is the ambient concentration of 02 in air; 3% O2 (simumeasuredusing either 10 PM 2-[3H]deoxy-D-glucose(1 &i/ml) lating hypoxia); and 50% 02 (inducing a state of hyperfor 5 min or 10 PM 3-@[methyZ-3H]methyl-D-glucose(3 &i/ ml) for 15 s. Nonspecificuptake wasdeterminedin the presence oxia). Incubation of L6 myotubes in 3% 02 for increasing of 10 PM cytochalasin B and was subtracted from the total periods of time ranging from 4 to 48 h resulted in an in the medium, uptake. After the uptake period, the radioactive solution was elevation in lactic acid concentration aspirated and the cells were rinsed three times with ice-cold concomitant to a reduction in glucose concentration (Fig.
all cells in the monolayer are evenly exposed to both solutes and gases in the medium. The present work was designed to investigate the influence of long-term hypoxia on glucose transport in L6 skeletal muscle cells. The L6 cell line is a continuous clonal line of myoblasts originally derived from the thigh muscle of neonatal rats. These cells can divide indefinitely as myoblasts, but if allowed to grow to confluence, they undergo terminal differentiation into multinucleated skeletal muscle myotubes (38). Myotubes, but not myoblasts, express many morphological, biochemical, electrical, and contractile properties of skeletal muscle (32, 38). We have recently shown that L6 muscle cells express the two glucose transporters of skeletal muscle, GLUT1 and GLUT4 (23, 26). GLUT4 transporters appear only after L6 cells myogenic differentiation, concomitant to the ability to display insulin-stimulated glucose transport. In the present study we test the effect of prolonged incubation in hypoxic, normoxic, and hyperoxic atmospheres on glucose transport activity and glucose transporters of L6 myotubes in an attempt to understand the mechanisms by which skeletal muscle ceils adapt to different oxygenation levels.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
C684
GLUCOSE
TRANSPORT
AND
OXYGEN
hexose transport, its effect on the uptake of 3-O-methylglucose (a nonphosphorylatable analogue of glucose) was also tested. Table 1 shows that after 24 h of incubation in low O2 levels, 3-0-methylglucose transport was increased about four times relative to the uptake in cells incubated in 21% OZ. The increases in the uptake of 2deoxy-D-glucose and 3-0-methylglucose were similar, although not identical. This is probably due to the difficulty in measuring absolute initial rates of uptake of 30-methylglucose by cell monolayers, which requires very short times of uptake (
of glucose transport and GLUT1 glucose expression by O2 in muscle cells in culture BASHAN,
ELENA
BURDETT,
HARINDER
Nava, Elena Burdett, Harinder S. Hundal, Klip. Regulation of glucosetransport and GLUT1
glucosetransporter expressionby O2in musclecells in culture. 262 (Cell Physiol. 31): C682-C690, 1992.-The effect of varying cellular oxygenation on L6 muscle cell 2deoxy-D-glucosetransport, glucoseutilization, lactate production, and expressionof GLUT1 and GLUT4 transport proteins was investigated. Incubation of L6 myotubes in 3% O2 (mimicking a state of hypoxia) elevated glucoseuptake by 6.5fold over 48 h relative to cells incubated in 21% O2 (normoxia). Incubation of L6 cells in hyperoxic conditions (50% 02) significantly depressedglucoseuptake by 0.4-fold. These effects were fully reversible. Incubation in 3% O2also causedlactate accumulation and enhancedglucoseconsumptionfrom the medium. Hypoxia elevated 2-deoxy-D-glucosetransport even when the concentration of glucosein the medium was kept constant, suggestingthat glucosedeprivation alone was not responsible for increasedcellular glucoseuptake. Incubation in 3% O2also elevated 3-0-methylglucoseuptake but not amino acid uptake. Cycloheximide prevented the hypoxia-induced increasein glucoseuptake, indicating that de novo synthesisof glucosetransport-related proteins was the major meansby which cells increasedglucoseuptake. The content of GLUT1 glucosetransporter was significantly elevated in total membranesof cells incubated in 3% O2and depressedin membranesfrom cells incubated in hyperoxic conditions, whereasGLUT4 expression was not affected. These results indicate that hypoxia induces an adaptive responseof increasing cellular glucose uptake through elevated expressionof GLUT1 in an attempt to maintain supply of glucosefor utilization by nonoxidative pathways. hypoxia; glucoseuptake; ischemia;oxygen levels
Am. J. Physiol.
BEEN RECOGNIZED that diverse cells respond to inhibition of oxidative metabolism with a rapid enhancement in glucose utilization by nonoxidative pathways. This is best illustrated by the increase in glycolysis upon inhibition of mitochondrial respiration, i.e., the Pasteur effect (29). The net yield of ATP obtained per molecule of glucose through glycolysis alone (2 ATP) is l&fold lower than that through aerobic respiration (36 ATP). Thus, to maintain normal energy production through anaerobic metabolism alone, glucose must be consumed at a markedly enhanced rate. In mammalian tissues such as liver, the facilitation of glycolysis under anaerobic conditions is largely due to the regulation of phosphofructokinase, the rate-limiting enzyme of the glycolytic pathway in that tissue. Stimulation of phosphofructokinase leads to increased exogenous glucose uptake by increasing the driving force for influx, i.e., the glucose concentration gradient across the cell. Therefore this effectively increases glucose uptake only in cells where there was a preexisting intracellular pool of free glucose (6, 14). In contrast, in tissues such as skeletal muscle and fat there is no detectable free intracellular glucose because glucose transport is the rate-limiting step in glucose IT HAS LONG
C682
S. HUNDAL,
AND
AMIRA
KLIP
of Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
0363-6143/92
$2.00
utilization (14, 21). Hence, in these tissues, activation of phosphofructokinase would be insufficient to increase glucose utilization during anaerobic conditions, and it would seem a priori that regulation at the level of glucose transport would be required to procure the energy demand. Indeed, a stimulation in muscle glucose transport (measured using the nonmetabolizable glucose analogue 3-0-methylglucose, which is transported into the muscle through the glucose transport system) has been demonstrated in preparations of epitrochlearis muscle made hypoxic in vitro (7 and Refs. within; 27, 28, 30). The transport of glucose into most cells occurs by facilitated diffusion mediated by a recently identified multigene family of transmembrane glycoproteins (3). Skeletal muscle expresses two glucose transporter isoforms of this family, denominated GLUT1 and GLUT4 (9, 11, 21). The GLUT1 transporters are distributed ubiquitously in most tissues; in skeletal muscle they appear to be located primarily in the plasma membrane and are thought largely to be responsible for basal glucose uptake (9). Conversely, GLUT4 transporters are expressed only in tissues where glucose uptake is insulin sensitive (muscle and fat) (3) and are predominantly located in an intracellular compartment whose precise identity presently remains unknown (8). Insulin stimulates glucose transport into skeletal muscle, in part, by promoting the translocation of GLUT4 transporters from this intracellular location to the plasma membrane (9, 16, 22). The mechanism by which hypoxia stimulates glucose uptake into skeletal muscle has only recently begun to be addressed. This question is of paramount importance to the physiology of muscle in vivo, since Po2 varies considerably within this tissue, depending on muscle thickness and fiber distance from the capillaries. The Pop of moist inspired air (containing 21% 02) is 150 mmHg. The Paz falls to just over 100 mmHg in the lungs and blood and can be as low as 40 mmHg in peripheral tissues. Despite these large changes, the effect of variations in tissue oxygenation on glucose uptake and energy production has not been documented at the molecular level. In a collaborative study, we have recently reported that short-term (up to 45 min) exposure of isolated epitrochlearis muscle to hypoxic conditions induces an increase in GLUT4 glucose transporters in the plasma membrane, as determined by subcellular fractionation of skeletal muscle (7). Isolated muscle preparations, however, are unsuitable for studying the effects of prolonged exposure to hypoxic conditions, since they become metabolically compromised and show marked changes in glucose transport upon incubation of the tissue in vitro (1% In contrast, muscle cell lines are stable during prolonged incubation periods and offer the advantage that
Copyright 0 1992 the American Physiological Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
GLUCOSE TRANSPORT AND OXYGEN
C683
isotonic saline solution (containing 1 mM HgClz in the caseof 3-0-[methy13H]methyl-D-glucose uptake assays). Cells were disrupted with 0.05 N NaOH, and cellular radioactivity was quantitated by liquid scintillation counting usingan LKB 1217 beta counter. Each uptake experiment was performed in quadruplicate, and resultsof specific uptake are expressedasmeans t SE (in pmol . mg protein’min .-‘). Amino acid transport. L6 myotubes, incubated under identical conditions as for hexose uptake, were used to assessthe transport of MeAIB, a nonmetabolizable amino acid analogue specific for the system A amino acid transporter (17). Cells were routinely depleted of intracellular amino acids for 1 h by preincubation in HBS to avoid truns-inhibition during uptake measurements.Subsequently, the experimental procedure for measuringuptake in L6 cellswasas describedabovefor hexose uptake. Initial rates of 10 PM [1-14C]MeAIB (0.1 &i/ml) uptake were measuredfor 10 min in HBS. Uptake was linear over a period of 20 min, and transport in choline-substituted medium (i.e., the Na’-independent component of uptake) was negligible (~4%). Experiments were performed three times in quadruplicate, with uptake experiments being terminated and cells being processedfor liquid scintillation counting as describedabove for hexoseuptake. Total membrane isolation and Western blots. L6 cell monolayers at the myotube stagewere incubated under the indicated experimental conditions (10 dishes/condition), then scraped gently with a rubber policeman, and concentrated by centrifuMATERIALS AND METHODS gation (700 g for 10 min). All remaining stepswere performed at 4°C. Cells were resuspendedin 20 ml of solution A [250 mM Materials. Tissue culture medium, serum, and reagents were sucrose,5 mM NaN3, 2 mM ethylene glycol-bis(P-aminoethyl obtained from GIBCO. Cytochalasin B, insulin, 2-deoxy-Dglucose, and 3-0-methylglucose were obtained from Sigma ether)-N,N,N’,N’-tetraacetic acid, 100 PM phenylmethylsulfonyl fluoride, 10 PM E-64, 1 PM pepstatin A, 1 PM leupeptin, Chemical. 2-[3H]deoxy-D-glucose and 3-0-[methyl-3H]methyland 20 mM HEPES-Na (pH 7.4)] and homogenizedwith a D-glucose were purchased from ICN. a-[ 1-14C]methylaminoisomotor-driven Potter-Elvehjem homogenizer (15 strokes). The butyric acid (MeAIB) was purchased from New England Nuhomogenateswere centrifuged at 760 g for 3 min to remove clear (Boston, MA). Antisera containing polyclonal antibodies raised against COOH-terminal sequences of GLUT1 and nuclei and unbroken cells, and the supernatants were centrifuged at 190,000g for 60 min. The resulting pellet contained GLUT4 were obtained from East Acres Biologicals Laboratototal cell membranesand was routinely resuspendedin a small ries. Monoclonal antibody against the al-subunit of the Na+volume of solution A. K+-adenosinetriphosphatase (ATPase) was a kind gift from Dr. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Katherine Sweadner, Harvard University (10). Cell cultures. L6 muscle cells were grown in monolayers to (using 10% polyacrylamide gels) and Western blot analysis the stage of myotubes as previously described (23) in alpha were carried out as previously described (26), using a 1:500 minimum essential medium containing 5 mM glucosein the dilution of anti-GLUT4 antiserum, a 1:5,000 dilution of antipresenceof 2% fetal bovine serum and 1% antibiotic solution GLUT1 antiserum, or a 1:500dilution of the monoclonalanti(final concn 10 mg/ml penicillin, 10 mg/ml streptomycin, and body to the al-subunit of Na’-K’-ATPase (lo), followed by A in 25 mg/ml amphotericin B) at 37°C in an atmosphere of 5% radioactive labeling of bound antibodieswith 1251-protein C02-95% air. The cells were grown in 3.5cm-diameter wells the first two casesand sheepanti-mouse 1251-immunoglobulin (6-well plates) for transport determinations (4 ml medium/ G in the latter case.Labeled proteins were visualized by autowell) or in lo-cm-diameter dishesfor isolation of total mem- radiography with Kodak XAR-5 film and quantitated by laser branes (10 ml medium/dish). Two to 3 days after cell fusion, scanningdensitometry. Other metubolite assays.Glucose and lactate concentrations cells were further incubated under the sameatmospheric conditions describedabove or were transferred to low-02 (3%) or in the medium were determined by the glucoseoxidasemethod high-02 (50%) incubators. The O2concentration in the low-O:! and spectrophotometric analysis using lactate dehydrogenase, incubator was reducedto 3% by blending NZ with ambient air respectively (15, 33). Protein was determined by the Bio-Rad by useof a multiplex gascontrol system.The CO:!concentration Bradford procedure (5). remainedat 5% in all cases. Hexose transport determinations. After the appropriate treat- RESULTS ments as indicated in the figure legends,cells were rinsed with glucose-free N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic To investigate the regulation of glucose transport in acid (HEPES)-buffered‘ saline [HBS: 140 mM NaCl, 20 mM L6 myotubes by O2 availability, we incubated L6 myoHEPES-Na (pH 7.4), 2.5 mM MgS04, 5 mM KCl, and 1 mM tubes under different O2 concentrations: 21% 02, which CaCIZ] as previously described (23, 26). Hexose uptake was is the ambient concentration of 02 in air; 3% O2 (simumeasuredusing either 10 PM 2-[3H]deoxy-D-glucose(1 &i/ml) lating hypoxia); and 50% 02 (inducing a state of hyperfor 5 min or 10 PM 3-@[methyZ-3H]methyl-D-glucose(3 &i/ ml) for 15 s. Nonspecificuptake wasdeterminedin the presence oxia). Incubation of L6 myotubes in 3% 02 for increasing of 10 PM cytochalasin B and was subtracted from the total periods of time ranging from 4 to 48 h resulted in an in the medium, uptake. After the uptake period, the radioactive solution was elevation in lactic acid concentration aspirated and the cells were rinsed three times with ice-cold concomitant to a reduction in glucose concentration (Fig.
all cells in the monolayer are evenly exposed to both solutes and gases in the medium. The present work was designed to investigate the influence of long-term hypoxia on glucose transport in L6 skeletal muscle cells. The L6 cell line is a continuous clonal line of myoblasts originally derived from the thigh muscle of neonatal rats. These cells can divide indefinitely as myoblasts, but if allowed to grow to confluence, they undergo terminal differentiation into multinucleated skeletal muscle myotubes (38). Myotubes, but not myoblasts, express many morphological, biochemical, electrical, and contractile properties of skeletal muscle (32, 38). We have recently shown that L6 muscle cells express the two glucose transporters of skeletal muscle, GLUT1 and GLUT4 (23, 26). GLUT4 transporters appear only after L6 cells myogenic differentiation, concomitant to the ability to display insulin-stimulated glucose transport. In the present study we test the effect of prolonged incubation in hypoxic, normoxic, and hyperoxic atmospheres on glucose transport activity and glucose transporters of L6 myotubes in an attempt to understand the mechanisms by which skeletal muscle ceils adapt to different oxygenation levels.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
C684
GLUCOSE
TRANSPORT
AND
OXYGEN
hexose transport, its effect on the uptake of 3-O-methylglucose (a nonphosphorylatable analogue of glucose) was also tested. Table 1 shows that after 24 h of incubation in low O2 levels, 3-0-methylglucose transport was increased about four times relative to the uptake in cells incubated in 21% OZ. The increases in the uptake of 2deoxy-D-glucose and 3-0-methylglucose were similar, although not identical. This is probably due to the difficulty in measuring absolute initial rates of uptake of 30-methylglucose by cell monolayers, which requires very short times of uptake (
