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Reactive Oxygen Species Activate Glucose Transport in L6 Myotubes1
Free Radical Biology & Medicine, Vol. 23, No. 6, pp. 859–869, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00
PII S0891-5849(97)00069-5
Original Contribution REACTIVE OXYGEN SPECIES ACTIVATE GLUCOSE TRANSPORT IN L6 MYOTUBES NITSAN KOZLOVSKY, ASSAF RUDICH, RUTH POTASHNIK, and NAVA BASHAN Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84103, Israel (Received 8 August 1996; Revised 17 January 1997; Accepted 17 March 1997)
Abstract—Under oxidative stress, increased energy requirements are needed To induce repair mechanisms. As glucose is a major energy source in L6 myotubes, we evaluated glucose metabolism and transport, following exposure to glucose oxidase (H2O2 generating system), or xanthine oxidase (O2· and H2O2 generating system), added to the medium. Exposure for 24 h to 5 mM glucose and 50 mU/ml glucose oxidase, or to 50 mM xanthine and 20 mU/ml xanthine oxidase resulted in significant oxidant stress indicated by increased DNA binding activity of NF-kB. Under these conditions, approximately 2-fold increase in glucose consumption, lactate production and CO2 release were observed. 2-deoxyglucose uptake into myotubes increased time and dose dependently, reaching a 2.6 { 0.4-fold and 2.2 { 0.7-fold after 24 h exposure to glucose oxidase and xanthine oxidase, respectively. Peroxidase prevented this effect, indicating the role of H2O2 in mediating glucose uptake activation. The elevation in glucose uptake under oxidative stress was associated with increased expression of GLUT1 mRNA and protein. The observed 2-deoxyglucose uptake activation by oxidants was not limited to the L6 cell line and was observed in 3T3-L1 adipocytes as well. q 1997 Elsevier Science Inc. Keywords—Oxidative stress, H2O2 , Glucose transport, GLUT1
antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase, which directly convert oxygen radicals into less offensive products.10 Though the mechanisms by which cells sense oxidative stress signals and translate them into adaptive responses are only partially characterized, it is well established that upon oxidation, an induction of antioxidant enzymes occurs.9,11 Enhanced synthesis of these and other proteins requires ATP, which can be produced by glucose metabolism. Since in skeletal muscle glucose transport limits the glycolytic rate, 12 its regulation may be important in recruiting the antioxidant defense systems. Glucose transporters, which are responsible for glucose uptake, are a family of integral glycoproteins. Of its six currently characterized members, GLUT1 is widely distributed and is expressed in most tissues.13 It is regulated under conditions which require adjustment of the metabolic rate, such as during cell division, 14 differentiation, 15 transformation 16 and in response to various stresses.16,17 . Bashan et al 18 reported that hypoxia (24 h exposure to 3% oxygen) in-
INTRODUCTION
Reactive oxygen species (ROS) such as superoxide anion (O2·), hydrogen peroxide (H2O2 ), and hydroxyl radicals (HO·), are normally produced during cellular oxidation reduction processes.1 Increased ROS generation has been reported in both physiological and pathologic conditions.2 – 4 In muscle, the significance of such accelerated ROS production has been demonstrated during exhaustive exercise, 5 as well as in ischemia-reperfussion injury.6 – 8 As ROS can damage many cell components such as DNA, lipid membranes and proteins, 1 cells possess diverse antioxidant defense systems. These include small molecules like a-tocopherol, ascorbic acid or glutathione, that intervene as sacrificial molecules in redox cycles, 9 as well as specific inducible This study was supported by a grant from the Israeli Academy of Sciences. Address correspondence to: Nava Bashan, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84103, Israel. Fax: 972-7-6403240; E-Mail: nava @bgumail.bgu.ac.il 859
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duced a 14-fold elevation in GLUT1 protein in L6 myotubes. Similarly, inhibition of oxidative metabolism by sodium azide, for 8-24 h resulted in a marked increment in GLUT1 mRNA and protein content in clone 9 cells.19,20 Based on these observations, it has been suggested that GLUT1 is co-regulated with different inducible stress proteins such as the glucose regulated protein family 21 or the heat shock proteins family.22 Indeed, the two enhancers of the GLUT1 gene include regulatory elements characteristic of immediate early genes which are activated during stress responses.23 The present study was undertaken to evaluate the effect of increased oxidative stress induced by ROS generating systems on glucose transport activity and glucose transporters in L6 skeletal muscle cell line. This cell line is a continuous clonal line that expresses biochemical properties of skeletal muscle 24 including the expression of the ubiquitous glucose transporter GLUT1, the insulin sensitive glucose transporter GLUT4, and the fetal glucose transporter GLUT3.25 Here we report that exposure of L6 myotubes to glucose/glucose oxidase (H2O2 generating system26 ), or to xanthine/xanthine oxidase (O2·, and H2O2 generating system27 ), results in elevation of glucose transport activity due to increased expression of GLUT1 protein and mRNA. We also demonstrate that similar enhancement in glucose uptake occurs in another cell line exposed to glucose/glucose oxidase or xanthine/ xanthine oxidase, the 3T3-L1 adipocytes. MATERIALS AND METHODS
Tissue culture medium, serum and antibiotic solutions were obtained from Biological Industries (Israel). Cytochalasin B, 2-deoxyglucose, cycloheximide, xanthine, xanthine oxidase, glucose oxidase, isobutyl methyl xanthine (IBMX), crude horseradish peroxidase and superoxide dismutase (SOD) were obtained from Sigma Chemical Co. Dexamethasone sodium phosphate was from Teva Pharmaceutical industries (Israel), and recombinant human insulin was from Novo Nordisk (Denmark). 2-Deoxy-[ 3H] glucose and [U- 14C]glucose were purchased from Nuclear Research Center-Negev (Israel). a-[ 32P]CTP and g[ 32P]ATP were from Rotem Industries (Israel). EcoRI and BglI were from Pharmacia LKB (Sweden). The polyclonal antibodies raised against C-terminal sequences of GLUT1, GLUT3 and GLUT4 were obtained from East Acres biological laboratories (USA). Cell culture L6 muscle cells were grown in monolayers to the stage of myotubes as previously described 18 in minimal
essential medium ( a-MEM) containing 5 mM glucose, 2% fetal calf serum and 1% antibiotic solution (final concentrations 100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin B), at 377C under an atmosphere of 5% CO2 -95% air. All experiments were carried in the stage of myotubes. The cells were grown in 24-well plates for transport determinations (1 ml medium per well), or in 10-cm diameter dishes for membranes, RNA or nuclear extraction preparations (6 ml medium / dish). 3T3-L1 preadipocytes were grown to confluence in Dulbeco’s modified Eagle’s medium (DMEM) containing 25 mM glucose, supplemented with 20% bovine serum, 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 0.25 mg/ml amphotericin B, in 6 well plates (Corning). 48 h following confluence cells were induced to differentiate to adipocytes by changing the medium to DMEM supplemented with 10% fetal calf serum, 5 mg/ml recombinant human insulin, 0.5 mM IBMX and 0.25 mM dexamethasone sodium phosphate for 48–72 h, as previously described.28 Cells were used for 2-deoxyglucose (2-DG) uptake experiments 9–10 days following differentiation induction when exhibiting ú90% adipocyte phenotype. Oxidant stress. ROS were generated continuously by two different oxidant generating systems, using the enzyme-substrate mixture of glucose/glucose oxidase or xanthine/xanthine oxidase. The first catalyzes the conversion of glucose to glucoronic acid and H2O2 , while the second catalyzes the reaction of xanthine to uric acid with reduction of O2 to O2· and H2O2· H2O2 determination. Quantitation of H2O2 generated by 50 mU/ml glucose oxidase was determined by the method of Thurman.29 Cells were incubated in a-MEM (without phenol red) containing 5 mM glucose with 50 mU/ml glucose oxidase alone or in the presence of 20 mU/ml peroxidase. At the indicated periods of time, 1 ml aliquots of medium were collected, and 0.1 ml TCA (50% wt/vol) was added. The samples were centrifuged at 500 1 g for 10 min, 0.2 ml of 10 mM ferrous ammonium sulfate and 0.1 ml of 2.5 M sodium thiocyanate were added to the supernatant. Absorption of the ferrithiocyanate complex was measured using a spectrophotometer (Thermo-Max, Molecular Devices, USA), at 480 nm, and compared to standard curves obtained from dilutions of a standard H2O2 solution. O2· determination. Quantitation of O2· generated by 50 mM xanthine/ 20 mU/ml xanthine oxidase was measured as previously described.27 Briefly, L6 myotubes were incubated in a-MEM (without phenol red) containing 50 mM xanthine and 20 mU/ml xanthine
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oxidase alone or in the presence of 200 mU/ml SOD. At the indicated incubation periods, 100 ml medium was transferred to 96 well plate and 5 mg/ml cytochrome C was added to each well. Cytochrome C reduction was continuously monitored for 20 min, using a spectrophotometer at 550 nm absorbance, and generation rate was calculated, using the extinction coefficient of 2.1 1 10 04 (M·cm) 01 . CO2 production. L6 myotubes grown in 24-well plates were incubated in a medium containing 50 mU/ml glucose oxidase or 50 mM xanthine and 20 mU/ml xanthine oxidase for 24 h. At the end of the incubation time, [U- 14C] glucose (1 mCi/well) was added. Each well was covered with glass fiber filters soaked in hydroxide of hyamine (Packard) to collect the CO 2 released. After 2 or 4 h incubation at 377C, the filters were removed and counted in a scintillation counter (Packard TriCarb, USA). Hexose transport determinations. All studies were carried out in fused L6 myotubes or in differentiated 3T3-L1 adipocytes. After the appropriate treatments as indicated in the figure legends, cells were rinsed with glucose-free N-2-hydroxyethylpiperazine- N * *2-ethanesulfonic acid ( HEPES ) -buffered saline ( 140 mM NaCl, 20 mM HEPES-Na pH 7.4, 2.5 mM mgSO4 , 5 mM KCl, 1 mM CaCl2 ) . Hexose uptake was measured for 10 min using 10 mM 2-deoxy- [ 3H ] glucose ( 1 mCi /ml ) for L6 myotubes or 50 mM 2deoxy- [ 3H ] glucose ( 1 mCi /ml ) for 3T3-L1 adipocytes. Nonspecific uptake was determined in the presence of cytochalasin B ( 10 mM for L6 and 50 mM for 3T3-L1 adipocytes ) , and was subtracted from the total uptake. The nonspecific component could account for less than 15% of the total uptake. After the uptake period, the radioactive solution was aspirated and the cells were rinsed three times with ice-cold isotonic saline solution, disrupted with 0.05 N NaOH and radioactivity was determined by liquid scintillation counter. Within each uptake experiment at least duplicate assays of each condition were performed. Results of glucose transporter mediated uptake are expressed as mean { SE in pmol /mg protein·min. Total membrane isolation. L6 cell monolayers at the myotube stage were incubated under the indicated experimental conditions (10 dishes per condition), then scraped gently with a rubber policeman and concentrated by centrifugation (700 1 g for 10 min). All remaining steps were performed at 47C. Cells were resuspended in 20 ml of buffer A [250 mM sucrose, 5 mM NaN3 , 2 mM ethylene glycol-bis (b-aminoethyl-
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ether)- N,N,N *,N *-tetraacetic acid, 200 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM pepstatin A, 1 mM leupeptin, 1 mM transepoxysuccinyl-leucymido (4guanidino) butane, 20 mM HEPES-Na pH 7.4], and homogenized using a motor-driven Potter-Elvehjem homogenizer (35 strokes). The homogenates were centrifuged at 760 1 g for 10 min to remove nuclei and unbroken cells, and the supernatant was centrifuged 60 min at 190,000 1 g to obtain the total cell membranes (TM). TM were resuspended in solution A and frozen at 0707C until used. Western Blot Analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10% polyacrylamide gels and Western blot analysis were carried out as previously described, 18 using a 1:500 dilution of anti-GLUT4 antibody, 1:5000 dilution of antiGLUT1 antibody or 1:300 of anti-GLUT3 antibody, followed by incubation with Horseradish Peroxide conjugated Protein A (Bio-Rad), which was detected by a luminescence technique (ECL; Amersham International plc, Amersham UK). Density values of the different bands were obtained after scanning the blots with video densitometry using the UVP-GDF 5000 system (UVP Inc, San Gabriel, CA) and thereafter quantitated using quant program, version 3.3, (Phosphoimager Multiscan HG Molecular Dynamics USA). RNA preparation and Northern Blot Analysis. Total cellular RNA was extracted from L6 myotubes, using the acid guanidium thiocyanate-phenol-chloroform method of Chomczynski et. al.30 For Northern-blot analysis, 30 mg of total RNA were separated under denaturing conditions on 1% (wt/vol) agarose gels containing 8% (vol/vol) formaldehyde with trace amount of ethidium bromide. The RNA was then transferred onto Hybond N / (Amersham international plc, UK) nylon membranes as describe by Maniatis.31 The RNA was cross-linked to the membrane with a UV cross linker (Hoefer Scientific instrument, San Francisco). GLUT1 cDNA in an PuK 19 plasmid was digested with the restriction enzymes EcoRI and BglI, the 2.6 Kb GLUT1 insert isolated by agarose gel electrophoresis, followed by electroelution and 32P-labeling with random prime DNA labeling Kit (Boehringer Mannheim GmbH, Germany). RNA Hybridization was performed in a hybridization oven (Micro-4 Hybaid limited) at 657C for 20 h in a hybridization solution [0.2 mM Na2HPO4 pH 7.2, 7% (vol/vol) SDS, 1% (wt/vol) BSA and 1 mM EDTA]. The washings were done in 0.4 1 SSC, and 0.1% SDS at 657C. Autoradiographs were performed with Bio-Imagining analyzer BAS 1000 (FUJI photo film CO., LTD Japan).
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Electrophoretic gel mobility shift assay. L6 myotubes were treated as describes in the figure legend. Nuclear extracts were isolated as previously described.31 Briefly, cells were scraped and pelleted in ice cold PBS buffer. The pellet was resuspended in 10 mM Tris-HCl pH 7.8, 1.5 mM MgCl2 , 10 mM KCl, 0.5 mM dithiothreitol ( DTT ) , 0.2 mM PMSF, 10 mg /ml leupeptin and 1 mg /ml pepstatin. The cells were allowed to swell on ice for 10 min and then were pelleted again and resuspended for 10 min in 20 mM Hepes-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 10 mg /ml leupeptin and 1 mg /ml pepstatin. Cellular debris was removed by centrifugation in 1000 1 g for 2 min, and the supernatant fraction containing the nuclear proteins were stored at 0707C. Binding reaction was performed for 20 min on ice with 10 mg total protein in 20 ml of 100 mM TrisHCl pH 7.5, 25 mM MgCl2 , 2 mM EDTA, 40% glycerol, 0.2 M KCl, 2 mM DTT, 0.2% NP-40 and 2 mg poly ( dI-dC ) and 150,000 c.p.m. of 32P-labeled oligonucleotides. DNA-protein complexes were separated from unbound DNA probe on native 4.5% polyacrylamide gels at 30 mA in 1 M Tris / HCl, pH 7.5, 50 mM boric acid and 0.5 mM EDTA. Gels were vacuum dried and autoradiographs were analyzed with Bio-Imaging analyzer BAS 1000 ( FUJI photo film CO., LTD Japan ) . The sequences of the oligo-
nucleotides were as follows ( factor binding sites are underlined ) : NF-kB: 5 *-TCGACTGGAAAGTCCCCAGCGGAAAGTCCCTTG-3 3 *-AGCTGACCTTT CAGGGGTCGCCTTTCAGGGAAC-5 * Other assays. Glucose and lactic acid concentrations in the medium were determined by the glucose oxidase method and spectrophotometric analysis using lactate dehydrogenase, respectively.32,33 Cell viability was measured by MTT test as previously described.34 Oxygen level in the medium was analyzed in a gas analyzer (Nova-Biomedical, Stat-profile). Protein concentration was determined by the Bio-Rad- Bradford procedure.35 Statistical analysis. Data are expressed as mean { standard error (SE). Statistical analysis was performed using Student’s t-test. RESULTS
H2O2 and O2· production by the oxidants generating system. To investigate the regulation of glucose trans-
Fig. 1. H2O2 and O2· production by glucose/glucose oxidase or xanthine/xanthine oxidase. (A) At time zero, 50 mU/ml glucose oxidase alone ( h ) or with 20 mU/ml peroxidase ( j ) were added to cells. H2O2 concentration was measured in the medium as described in ‘‘Methods.’’ (B) At time zero, 50 mM xanthine and 20 mU/ml xanthine oxidase alone ( s ), or with 200 mU/ml SOD ( l ) were added. O2· production rate was determined as described in ‘‘Methods.’’ Results are presented as mean values { SE of three independent experiments each performed in triplicate.
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ing in the system at a given time.36 The addition of 200 mU/ml superoxide dismutase (SOD) resulted in 80% inhibition in O2· production rate. No significant cell viability loss (as determined by light-phase microscopy, MTT test and by protein/well content), was found when cells were incubated up to 24 h in both systems under these conditions (data not shown).
Fig. 2. Effect of Xa/XO and G/GO on the DNA-binding activity of NF-kB. Nuclear extract were prepared from L6 myotubes exposed for 8 h to 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/Xo), 50 mU/ml glucose oxidase in a medium containing 5 mM glucose (G/GO), or to 200 mM H2O2 for 2 h. Extract were incubated with 32 P-labeled NF-kB oligonucleotide. Electophoretic mobility shift assay (EMSA) was preformed as describe in ‘‘Methods.’’ On the right panel, nuclear extract from cells exposed to Xa/XO for 8 h were preincubated with 100 fold molar excess of unlabeled NF-kB (specific), or with 100 fold molar excess of ROA I (non specific) oligonucleotide followed by incubation with the labeled NF-kB oligonucleotide and analyzed by (EMSA). Autoradiograph of a native gel is shown.
port in L6 myotubes by ROS, we incubated the cells with two different ROS generating systems: glucose and glucose oxidase which produces H2O2 , 26 or xanthine and xanhine oxidase which generates O2· and H2O2.27 Addition of 50 mU/ml glucose oxidase to L6 myotubes grown in a medium containing 5 mM glucose, resulted in elevated medium H2O2 concentration, which was completely prevented by peroxidase (20 mU/ml), (Fig. 1A). H2O2 reached 38.0 { 4.6 nmole/ ml at 4 h, after which further incubation resulted in a progressive decline in its concentration. Yet, after 24 h the level of H2O2 was increased about 4-fold compared to incubation in the absence of glucose oxidase. The rate of cytochrome C reduction was used to determine O2· production rate following addition of 50 mM xanthine and 20 mU/ml xanthine oxidase to the medium of L6 myotubes. The activity of this enzymatic system remained relatively constant for 24 h, yielding an O2· production rate of 0.6 nmoles/ml.minute (Fig. 1B). As the half life of O2· is 10 05 s, its production rate approximately represents the actual concentration exist-
ROS generating system activate NF-kB binding. In various cell types oxidative stress has been shown to result in increased DNA binding activity of the transcription factor NF-kB.37,38 To assess whether exposure of L6 myotubes to both ROS generating systems result in an intracellular response typical of oxidant stress, we evaluated NF-kB DNA binding activity. Nuclear extracts were prepared from L6 myotubes exposed to ROS generating systems for 8 h. Binding of extract protein to an oligonucleotide containing the consensus binding sequence of NF-kB was evaluated by gel mobility shift assay. As demonstrated in Fig. 2, NF-kB binding was increased in nuclear extracts prepared from cells treated with both 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/XO), or 5 mM glucose and 50 mU/ml glucose oxidase (G/GO) as compared to control. Exposure of cells to 200 mM H2O2 , a known activator of NF-kB, 37 served as a positive control. The specificity of the observed DNA binding activity was confirmed, as a 100-fold excess of the unlabeled oligonucleotide, but not nonspecific oligonucleotide, eliminated binding activity. ROS generating system increase 2-deoxyglucose (2DG) uptake. Incubation of L6 myotubes in the presence of glucose/glucose oxidase or xanthine/xanthine oxidase, activated glucose uptake in a time and dose dependent manner. The rate of 2-DG uptake into L6 myotubes incubated with 5 mM glucose and increasing concentrations of glucose oxidase caused progressive 2-DG uptake stimulation, reaching 2.6 { 0.4 fold increase in the presence of 50 mU/ml glucose oxidase, after 24 h (Fig. 3A and C). Similarly, in the presence of 50 mM xanthine and 20 mU/ml xanthine oxidase, 2DG uptake into L6 myotubes increased progressively reaching 2.2 { 0.7 fold after 24 h incubation (Fig. 3B and D). 2-DG uptake in cells incubated with 50 mM xanthine in the absence of xanthine oxidase did not significantly differ from control (data not shown). Longer incubation periods or higher concentrations of either glucose oxidase or xanthine oxidase resulted in viability loss as detected by MTT test (data not shown). Thus, all further experiments reported herein, were performed in conditions which produced maximal biologic effect without causing viability loss, i.e. 24 h incubation with either 50 mU/ml glucose oxidase in a
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Fig. 3. Dose and time- dependent changes in 2-deoxyglucose uptake in L6 myotubes exposed to glucose/glucose oxidase or xanthine/xanthine oxidase. L6 myotubes in 24 well plates, were exposed to: (A) varying concentrations of glucose oxidase in a medium containing 5 mM glucose, for 24 h, (B) varying concentrations of xanthine oxidase in medium containing 50 mM xanthine, for 24 h, (C) 50 mU/ml glucose oxidase in a medium containing 5 mM glucose for various periods of time, or (D), 50 mM xanthine and 20 mU/ml xanthine oxidase for the indicated periods of time. At the end of the incubation periods the plates were rinsed 3 times with HEPES-buffer, and 2-deoxyglucose transport was measured for 10 min, as described in ‘‘Methods.’’ Results are expressed as mean { SE of five independent experiments, each performed in triplicate. *p õ 0.03 vs. untreated cells (A,B), or ’time 0’ (C,D).
medium containing 5 mM glucose, or 50 mM xanthine and 20 mU/ml xanthine oxidase. Oxidation of glucose by glucose oxidase requires glucose and atmospheric oxygen. O2 is also required for the oxidation of xanthine by xanthine oxidase. Thus, hypoxia or reduction in medium glucose concentration could cause the stimulated glucose transport observed, as previously reported.39,40 To eliminate these possibilities we measured glucose and oxygen levels in the medium of cells after 24 h incubation with either G/GO or Xa/XO. Partial oxygen pressure (pO2 ) in the medium of control, G/GO or Xa/XO treated cells was not statistically different (156 { 18, 170 { 15 and 165 { 21 mmHg, respectively). Medium glucose concen-
tration did not differ significantly whether cells were incubated in the absence or presence of glucose oxidase for 24 h (4.0 { 0.5 and 3.5 { 0.8 mM, respectively). Thus, the increased stimulation in 2-DG uptake appears to result specifically from the exposure of the cells to ROS generating system. In order to determine the respective role of H2O2 in activating glucose uptake, we evaluate the effect of peroxidase. L6 myotubes were coincubated with peroxidase during incubation with G/GO or Xa/XO for 24 h. Table 1 shows that full inhibition of 2-DG uptake stimulation was observed when 20 mU/ml peroxidase were added to cells treated with G/GO, indicating the role of H2O2 in 2-DG uptake activation. Both peroxidase
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Oxidation activates glucose uptake Table 1. Effect of Peroxidase and Superoxide Dismutase (SOD) on 2-deoxyglucose Uptake into L6 Myotubes During Incubation with G/GO or Xa/XO
Table 2. Effect of G/GO and Xa/XO on Glucose Consumption, Lactic Acid Production, and [14]CO2 Release in L6 Myotubes Glucose consumption
2-deoxyglucose uptake (pmol/mg protein·min) Conditions Control G/GO Xa/XO
No addition
Peroxidase (20 mU/ml)
SOD (200 mU/ml)
20.2 { 7.5 (33) 51.2 { 9.6* (17) 41.9 { 7.5* (23)
25.5 { 6.0 (6) 22.4 { 7.2Ú (7) 32.4 { 7.2§ (6)
23.5 { 2.2 (6) ND 33.8 { 2.4 (5)
L6 myotubes were incubated for 24 h in a-MEM medium in the absence (control), or presence of 50 mU/ml glucose oxidase (G/GO) or 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/XO), with either 20 mU/ml peroxidase or 200 mU/ml superoxide dismutase (SOD). 2-deoxyglucose uptake was measured for 10 min as described in ‘‘Methods.’’ Each value is a mean { SE of number of independent experiments (n), each performed in triplicates. *p õ 0.001 vs. control, Úp õ 0.03 vs. G/GO, §p õ 0.05 vs. Xa/XO, ND-not determined.
and SOD exerted only a partial inhibitory effect on the activation of glucose uptake observed with Xa/XO. This is in agreement with the work of Fridovich et. al., 27 who reported that xanthine oxidase produces both H2O2 and O2·. These results emphasize the role of H2O2 in the activation of 2-DG uptake in L6 myotubes. Oxidant generating system increases glucose metabolism. To evaluate the metabolic consequences of the observed increase in glucose uptake, we investigated the effect of ROS generating systems on glucose metabolism in L6 myotubes. Cells incubated for 24 h in the presence of G/GO, exhibited increased lactate production and CO2 release (2.2 { 0.2, 2.0 { 0.8 -fold increase vs. control, respectively) (Table 2). Similarly, in the presence of Xa/XO, glucose consumption, lactate production and CO2 release were increased by 2.1 { 0.1, 2.0 { 0.2 and 2.3 { 0.9-fold vs. control, respectively (Table 2). This stimulation in the metabolic rate suggests that the exposure of the cells to ROS may enhance ATP production. 2-DG uptake stimulation is caused by elevation of GLUT1 protein and mRNA. The involvement of ongoing protein synthesis in the observed activation of glucose uptake by ROS was investigated with the protein synthesis inhibitor cycloheximide. When L6 were incubated for 24 h in the presence of cycloheximide (5 mg/ml), 2-DG uptake stimulation elicited by G/GO or Xa/XO was totally inhibited, without altering 2-DG uptake in control cells. (Table 3). These observations suggest that synthesis of either glucose transporters or activator proteins may be responsible for the stimulation of transport activity seen in the presence of the oxidants. To directly investigate whether de novo glucose transporter protein synthesis is triggered when
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[14]
CO2 release
(mmole/mg protein·h)
Treatment 0.16 { 0.04 ND 0.34 { 0.02*
None G/GO Xa/XO
Lactic acid production
0.21 { 0.01 0.47 { 0.05* 0.42 { 0.05*
0.004 { 0.001 0.008 { 0.001** 0.009 { 0.002**
L6 myotubes were grown in 24-well plates, in the absence or presence of 50 mU/ml glucose oxidase (G/GO) or 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/XO). After 24 h the medium was changed to HEPES-buffer containing 5 mM [U-14C]-glucose (1 mCi/ ml) in the absence or presence of the oxidants generating systems. Glucose consumption, lactic acid production and [14]CO2 release, were determined as described in ‘‘Methods.’’ Results are mean { SE of 2 independent experiments carried out in triplicate. *p õ 0.05 vs. untreated cells. **p õ 0.01 vs. untreated cells. ND-not determined.
cells are exposed to ROS generating systems, we assessed the amount of immunoreactive glucose transporters in total membranes prepared from L6 myotubes incubated for 24 h with G/GO or Xa/XO. 30 mg of membrane protein from each treatment were probed by Western-Blot analysis using antibodies directed against either GLUT1 GLUT3 or GLUT4 transporters. As shown in Fig. 4A, the intensity of GLUT1 immunoreactive protein in total membranes of either G/GO or Xa/XO treated cells was increased compared to control. Densitometric analysis indicate that GLUT1 content in total membrane of cells treated with either G/ GO or Xa/XO was increased by 2.0 { 0.2 and 2.4 { 0.4 -fold, respectively, as compared to GLUT1 from untreated cells. The amount of GLUT4 and GLUT3 were analyzed similarly, revieling a reduction in both transporter isoforms. While the reduction in GLUT4 Table 3. Effect of Cycloheximide (CHX) on 2deoxyglucose Uptake Stimulated by G/GO or Xa/XO in L6 Myotubes 2-Deoxyglucose uptake (pmol/mg protein·min) Treatment Control G/GO Xa/XO
0CHX
/CHX
18.5 { 4.0 51.2 { 10.0* 42.3 { 8.0*
17.1 { 4.0 23.3 { 2.0ÚÚ 20.2 { 5.0Ú
L6 myotubes, in 24 well plates, were incubated for 24 h with 5 mM glucose and 50 mU/ml glucose oxidase (G/ GO), or with 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/XO), in the absence or presence of 5 mg/ml cycloheximide. Following the incubation period, the plates were rinsed 3 times with HEPES-buffer, and 2deoxyglucose uptake was measured for 10 min, as described in ‘‘Methods.’’ Results of seven independent experiments each performed in triplicate are expressed as mean { SE. *p õ 0.001 vs. control 0CHX, Úp õ 0.05 vs. Xa/XO 0CHX, ÚÚp õ 0.001 vs. G/GO 0CHX.
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Fig. 4. Western-Blot and densitometric analysis of glucose transporters, prepared from L6 myotubes total membranes after 24 h exposure to Xa/XO or G/GO. L6 myotubes were incubated in a-MEM for 24 h without treatment (cont), with 50 mU/ml glucose oxidase (G/GO) or 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/XO). Total membranes were prepared as described in ‘‘Methods.’’ (A) 30 mg protein/lane were separated by SDS-PAGE on 10% polyacrylamide gels, transferred to nitrocellulose membrane and immunoassayed using anti-GLUT1, anti-GLUT4 or anti-GLUT3 antibodies. (B) Results of scanning densitometry analysis performed on 4 independent autoradiographs for GLUT1 and GLUT4 and 2 autoradiographs for GLUT3 are presented. Values (mean { SE) are in relative units as compared to the corresponding GLUTs from untreated cells. *p õ 0.03 vs. control. ND -not determined.
protein did not reach statistical significance, GLUT3 was markedly reduced in G/GO treated cells by approximately 70% (Fig. 4B). To assess the involvement of GLUT1 gene expression in this response, Northern-Blot analysis was performed using GLUT1 radio labeled cDNA probes. Cells were treated for 24 h with either 5 mM glucose and different concentrations of glucose oxidase (Fig. 5A) or 50 mM xanthine and different concentrations of xanthine oxidase (Fig. 5B). Scanning densitometry of four independent experiments revealed a progressive increase in GLUT1 mRNA, resulting in a 3.5 { 0.3 and 2.3 { 0.7 -fold increase in the presence of 50 mU/ml glucose oxidase or 20 mU/ml xanthine oxidase, respectively (Fig. 5C).
ROS generating systems induce increased 2-DG uptake in 3T3-L1 adipocytes. To address the question of whether the observed 2-DG uptake activation by ROS generating system is limited to the L6 cell line, or rather represents a more generalized phenomenon, we utilized a different cell line, the 3T3-L1 adipocytes, which contains GLUT1 and GLUT4.18 As can be seen in Table 4, cells incubated for 18 h with 2% serum and either 50 mU/ml glucose oxidase, or 50 mM xanthine and 20 mU/ml xanthine oxidase, exhibited an elevation in glucose transport activity (40% and 60% for G/GO and Xa/XO, respectively). In serum deprived cells, a 4.0 { 0.2 -fold increase in 2-DG uptake was observed when cells were treated for 18 h with 50 mU/ml glucose oxidase compared to control. These data support
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Fig. 5. Northern-Blot and densitometry analysis of GLUT1 mRNA in L6 myotubes incubated with different concentrations of oxidant generating systems. L6 myotubes were incubated for 24 h, with either (A) 0, 20, 50 mU/ml glucose oxidase in medium containing 5 mM glucose, or (B) 50 mM xanthine and 0, 10, 20 mU/ml xanthine oxidase. Total RNA was isolated as described in ‘‘Methods.’’ Thirty micrograms/lane were loaded and, after fractionation in a 1% agarose-formaldehyde gel, the resulting blot was probed with a full length rat GLUT1 cDNA. Presented are representative autoradiographs. (C) Scanning densitometry analysis of four independent experiments with similar results. Values (mean { SE) are in relative units, with a value of 1.00 assigned to corresponding mRNA from each control cells. *p õ 0.05 vs. control.
the notion that ROS generating systems induced glucose uptake stimulation, is not limited to muscle cells in culture, and presumably represents a generalized adaptation mechanism to oxidative stress. DISCUSSION
This study was undertaken To investigate the role of glucose transport regulation as an adaptive response to low-grade, prolonged oxidative stress. Since ROS have an extremely short half-life, 36 we utilized two well characterized ROS generating systems which continu-
ously produce fairly low and constant concentrations of H2O2 and O2· over a 24 h period. In both L6 myotubes and in 3T3-L1 adipocytes, glucose uptake limits cellular metabolism, rendering its regulation crucial in the adaptation to diverse extracellular conditions. The results obtained in this study demonstrate that exposure of L6 cells to ROS generating system increases the steady state levels of GLUT1 mRNA (Fig. 5), by either increased transcription rate and/or by increased stability, resulting in increased glucose uptake and metabolism (Fig. 3; Table 2). The increase in GLUT1 trans-
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N. KOZLOVSKY et al. Table 4. Effect of G/GO and Xa/XO on 2Deoxyglucose Uptake in 3T3-L1 Adipocytes 2-Deoxyglucose uptake (pmole/mg protein·min) Treatment Control G/GO Xa/XO
0.2% BSA
2% Serum
87 { 15 (6) 345 { 80* (4) ND
139 { 11 (7) 195 { 19** (6) 226 { 18** (5)
Differentiated 3T3-L1 adipocytes were exposed for 18 h, to a medium containing 25 mM glucose and 50 mU/ ml glucose oxidase, or 50 mM xanthine and 20 mU/ml xanthine oxidase in the absence (0.2% BSA) or presence of 2% serum. 2-deoxyglucose uptake was measured for 10 min as described in ‘‘õMethods.’’ The number of independent experiments performed at least in duplicates is shown in parenthesis. *p õ 0.007 vs. control with 0.2% BSA, **p õ 0.05 vs. control with 2% serum. ND-not determined.
porter isoform (Fig. 4) correlates well to the degree of enhancement in glucose transport and metabolism. Under the same conditions, GLUT4 and GLUT3 protein content were reduced, reaching statistical significance in the case of GLUT3 in G/GO treated cells (Fig. 4B). These findings further suppport the predominant role of GLUT1 isoform in the adaptive response to oxidant stress in L6 myotubes. Our work suggests that oxidative stress imposed upon L6 muscle cells causes an adaptive response which results in increased energy production, presumably to recruit cellular defense mechanisms. Enhanced glucose uptake in muscle cells can be achieved by rapid translocation of preexisting transporters from intracellular pools to the plasma membrane, as seen following insulin stimulation 41 or by a secondary late phase response.40 In the case of ROS generating system, glucose transport stimulation occurs beginning at 6 h of continuous exposure (Fig. 3), and is therefore a late phase response similar to that observed following anoxia, glucose deprivation or iron chelators, which involves GLUT1 gene induction.18,40,42 In all these conditions the response is dependent upon de-novo protein synhesis as demonstrated by the inhibitory effect of cycloheximide (Table 3). This further suggests that redistribution of preexisting transporters, is proboably relativiely minor compared to the increased GLUT1 protein synthesis observed in the response to oxidant stress. Late phase response to Xa/XO or G/GO were reported in other systems such as the cystine uptake stimulation in endothelial cells, 43 or MnSOD mRNA elevation in epithelial cells.44 As GLUT1 is the ubiquitous glucose transporter, regulatory responses similar to those describe here in L6 myotubes, are likely to occur in various cell types under oxidative stress, as demonstrated by the enhanced glucose transport
observed under similar conditions with 3T3-L1 (Table 4). The role of H2O2 produced by both G/GO and Xa/ XO in mediating GLUT1 gene transcription is demonstrated by the effect peroxidase had in preventing glucose uptake stimulation (Table 1). H2O2 has been implicated to play a role in transcriptional induction of several genes such as the oxy-R encoded protein in bacteria, 45 the heat-shock factor in Drosophila, 46 the iron responsive elements binding proteins in mammalian cells, 47 and the immediate early genes c-fos and cJun.48 The products of these genes are presumably involved in the defense mechanisms of various cells to stress conditions. The signals and sequence of events mediating H2O2 stimulated induction of these genes are unknown, but there are accumulating reports pointing to the involvement of transcriptional factors such as AP-1 and NF-kB in these responses 37,38 In the 5 * untranslated region of GLUT1 gene there are several important regulatory elements, including AP-1 and serum responsive element binding sites23 which may be regulated by oxidants. Preliminary results in our laboratory, indicate increased binding activity of AP-1 and increase in c-fos mRNA following exposure to the oxidant generating systems. Whether this is relevant to the induction of GLUT1 gene during the response to oxidative stress, remains to be established. Acknowledgement — We wish to thank the S. Daniel Abraham International Center for Health and Nutrition, Ben-Gurion University of the Negev.
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ROS— Reactive oxygen species TM—total membranes Xa/XO—xanthine/xanthine oxidase G/GO—glucose/glucose oxidase SOD—superoxide dismutase 2-DG—2-deoxyglucose BSA—bovine serum albumin mRNA—messenger RNA
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Original Contribution REACTIVE OXYGEN SPECIES ACTIVATE GLUCOSE TRANSPORT IN L6 MYOTUBES NITSAN KOZLOVSKY, ASSAF RUDICH, RUTH POTASHNIK, and NAVA BASHAN Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84103, Israel (Received 8 August 1996; Revised 17 January 1997; Accepted 17 March 1997)
Abstract—Under oxidative stress, increased energy requirements are needed To induce repair mechanisms. As glucose is a major energy source in L6 myotubes, we evaluated glucose metabolism and transport, following exposure to glucose oxidase (H2O2 generating system), or xanthine oxidase (O2· and H2O2 generating system), added to the medium. Exposure for 24 h to 5 mM glucose and 50 mU/ml glucose oxidase, or to 50 mM xanthine and 20 mU/ml xanthine oxidase resulted in significant oxidant stress indicated by increased DNA binding activity of NF-kB. Under these conditions, approximately 2-fold increase in glucose consumption, lactate production and CO2 release were observed. 2-deoxyglucose uptake into myotubes increased time and dose dependently, reaching a 2.6 { 0.4-fold and 2.2 { 0.7-fold after 24 h exposure to glucose oxidase and xanthine oxidase, respectively. Peroxidase prevented this effect, indicating the role of H2O2 in mediating glucose uptake activation. The elevation in glucose uptake under oxidative stress was associated with increased expression of GLUT1 mRNA and protein. The observed 2-deoxyglucose uptake activation by oxidants was not limited to the L6 cell line and was observed in 3T3-L1 adipocytes as well. q 1997 Elsevier Science Inc. Keywords—Oxidative stress, H2O2 , Glucose transport, GLUT1
antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase, which directly convert oxygen radicals into less offensive products.10 Though the mechanisms by which cells sense oxidative stress signals and translate them into adaptive responses are only partially characterized, it is well established that upon oxidation, an induction of antioxidant enzymes occurs.9,11 Enhanced synthesis of these and other proteins requires ATP, which can be produced by glucose metabolism. Since in skeletal muscle glucose transport limits the glycolytic rate, 12 its regulation may be important in recruiting the antioxidant defense systems. Glucose transporters, which are responsible for glucose uptake, are a family of integral glycoproteins. Of its six currently characterized members, GLUT1 is widely distributed and is expressed in most tissues.13 It is regulated under conditions which require adjustment of the metabolic rate, such as during cell division, 14 differentiation, 15 transformation 16 and in response to various stresses.16,17 . Bashan et al 18 reported that hypoxia (24 h exposure to 3% oxygen) in-
INTRODUCTION
Reactive oxygen species (ROS) such as superoxide anion (O2·), hydrogen peroxide (H2O2 ), and hydroxyl radicals (HO·), are normally produced during cellular oxidation reduction processes.1 Increased ROS generation has been reported in both physiological and pathologic conditions.2 – 4 In muscle, the significance of such accelerated ROS production has been demonstrated during exhaustive exercise, 5 as well as in ischemia-reperfussion injury.6 – 8 As ROS can damage many cell components such as DNA, lipid membranes and proteins, 1 cells possess diverse antioxidant defense systems. These include small molecules like a-tocopherol, ascorbic acid or glutathione, that intervene as sacrificial molecules in redox cycles, 9 as well as specific inducible This study was supported by a grant from the Israeli Academy of Sciences. Address correspondence to: Nava Bashan, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84103, Israel. Fax: 972-7-6403240; E-Mail: nava @bgumail.bgu.ac.il 859
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duced a 14-fold elevation in GLUT1 protein in L6 myotubes. Similarly, inhibition of oxidative metabolism by sodium azide, for 8-24 h resulted in a marked increment in GLUT1 mRNA and protein content in clone 9 cells.19,20 Based on these observations, it has been suggested that GLUT1 is co-regulated with different inducible stress proteins such as the glucose regulated protein family 21 or the heat shock proteins family.22 Indeed, the two enhancers of the GLUT1 gene include regulatory elements characteristic of immediate early genes which are activated during stress responses.23 The present study was undertaken to evaluate the effect of increased oxidative stress induced by ROS generating systems on glucose transport activity and glucose transporters in L6 skeletal muscle cell line. This cell line is a continuous clonal line that expresses biochemical properties of skeletal muscle 24 including the expression of the ubiquitous glucose transporter GLUT1, the insulin sensitive glucose transporter GLUT4, and the fetal glucose transporter GLUT3.25 Here we report that exposure of L6 myotubes to glucose/glucose oxidase (H2O2 generating system26 ), or to xanthine/xanthine oxidase (O2·, and H2O2 generating system27 ), results in elevation of glucose transport activity due to increased expression of GLUT1 protein and mRNA. We also demonstrate that similar enhancement in glucose uptake occurs in another cell line exposed to glucose/glucose oxidase or xanthine/ xanthine oxidase, the 3T3-L1 adipocytes. MATERIALS AND METHODS
Tissue culture medium, serum and antibiotic solutions were obtained from Biological Industries (Israel). Cytochalasin B, 2-deoxyglucose, cycloheximide, xanthine, xanthine oxidase, glucose oxidase, isobutyl methyl xanthine (IBMX), crude horseradish peroxidase and superoxide dismutase (SOD) were obtained from Sigma Chemical Co. Dexamethasone sodium phosphate was from Teva Pharmaceutical industries (Israel), and recombinant human insulin was from Novo Nordisk (Denmark). 2-Deoxy-[ 3H] glucose and [U- 14C]glucose were purchased from Nuclear Research Center-Negev (Israel). a-[ 32P]CTP and g[ 32P]ATP were from Rotem Industries (Israel). EcoRI and BglI were from Pharmacia LKB (Sweden). The polyclonal antibodies raised against C-terminal sequences of GLUT1, GLUT3 and GLUT4 were obtained from East Acres biological laboratories (USA). Cell culture L6 muscle cells were grown in monolayers to the stage of myotubes as previously described 18 in minimal
essential medium ( a-MEM) containing 5 mM glucose, 2% fetal calf serum and 1% antibiotic solution (final concentrations 100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin B), at 377C under an atmosphere of 5% CO2 -95% air. All experiments were carried in the stage of myotubes. The cells were grown in 24-well plates for transport determinations (1 ml medium per well), or in 10-cm diameter dishes for membranes, RNA or nuclear extraction preparations (6 ml medium / dish). 3T3-L1 preadipocytes were grown to confluence in Dulbeco’s modified Eagle’s medium (DMEM) containing 25 mM glucose, supplemented with 20% bovine serum, 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 0.25 mg/ml amphotericin B, in 6 well plates (Corning). 48 h following confluence cells were induced to differentiate to adipocytes by changing the medium to DMEM supplemented with 10% fetal calf serum, 5 mg/ml recombinant human insulin, 0.5 mM IBMX and 0.25 mM dexamethasone sodium phosphate for 48–72 h, as previously described.28 Cells were used for 2-deoxyglucose (2-DG) uptake experiments 9–10 days following differentiation induction when exhibiting ú90% adipocyte phenotype. Oxidant stress. ROS were generated continuously by two different oxidant generating systems, using the enzyme-substrate mixture of glucose/glucose oxidase or xanthine/xanthine oxidase. The first catalyzes the conversion of glucose to glucoronic acid and H2O2 , while the second catalyzes the reaction of xanthine to uric acid with reduction of O2 to O2· and H2O2· H2O2 determination. Quantitation of H2O2 generated by 50 mU/ml glucose oxidase was determined by the method of Thurman.29 Cells were incubated in a-MEM (without phenol red) containing 5 mM glucose with 50 mU/ml glucose oxidase alone or in the presence of 20 mU/ml peroxidase. At the indicated periods of time, 1 ml aliquots of medium were collected, and 0.1 ml TCA (50% wt/vol) was added. The samples were centrifuged at 500 1 g for 10 min, 0.2 ml of 10 mM ferrous ammonium sulfate and 0.1 ml of 2.5 M sodium thiocyanate were added to the supernatant. Absorption of the ferrithiocyanate complex was measured using a spectrophotometer (Thermo-Max, Molecular Devices, USA), at 480 nm, and compared to standard curves obtained from dilutions of a standard H2O2 solution. O2· determination. Quantitation of O2· generated by 50 mM xanthine/ 20 mU/ml xanthine oxidase was measured as previously described.27 Briefly, L6 myotubes were incubated in a-MEM (without phenol red) containing 50 mM xanthine and 20 mU/ml xanthine
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oxidase alone or in the presence of 200 mU/ml SOD. At the indicated incubation periods, 100 ml medium was transferred to 96 well plate and 5 mg/ml cytochrome C was added to each well. Cytochrome C reduction was continuously monitored for 20 min, using a spectrophotometer at 550 nm absorbance, and generation rate was calculated, using the extinction coefficient of 2.1 1 10 04 (M·cm) 01 . CO2 production. L6 myotubes grown in 24-well plates were incubated in a medium containing 50 mU/ml glucose oxidase or 50 mM xanthine and 20 mU/ml xanthine oxidase for 24 h. At the end of the incubation time, [U- 14C] glucose (1 mCi/well) was added. Each well was covered with glass fiber filters soaked in hydroxide of hyamine (Packard) to collect the CO 2 released. After 2 or 4 h incubation at 377C, the filters were removed and counted in a scintillation counter (Packard TriCarb, USA). Hexose transport determinations. All studies were carried out in fused L6 myotubes or in differentiated 3T3-L1 adipocytes. After the appropriate treatments as indicated in the figure legends, cells were rinsed with glucose-free N-2-hydroxyethylpiperazine- N * *2-ethanesulfonic acid ( HEPES ) -buffered saline ( 140 mM NaCl, 20 mM HEPES-Na pH 7.4, 2.5 mM mgSO4 , 5 mM KCl, 1 mM CaCl2 ) . Hexose uptake was measured for 10 min using 10 mM 2-deoxy- [ 3H ] glucose ( 1 mCi /ml ) for L6 myotubes or 50 mM 2deoxy- [ 3H ] glucose ( 1 mCi /ml ) for 3T3-L1 adipocytes. Nonspecific uptake was determined in the presence of cytochalasin B ( 10 mM for L6 and 50 mM for 3T3-L1 adipocytes ) , and was subtracted from the total uptake. The nonspecific component could account for less than 15% of the total uptake. After the uptake period, the radioactive solution was aspirated and the cells were rinsed three times with ice-cold isotonic saline solution, disrupted with 0.05 N NaOH and radioactivity was determined by liquid scintillation counter. Within each uptake experiment at least duplicate assays of each condition were performed. Results of glucose transporter mediated uptake are expressed as mean { SE in pmol /mg protein·min. Total membrane isolation. L6 cell monolayers at the myotube stage were incubated under the indicated experimental conditions (10 dishes per condition), then scraped gently with a rubber policeman and concentrated by centrifugation (700 1 g for 10 min). All remaining steps were performed at 47C. Cells were resuspended in 20 ml of buffer A [250 mM sucrose, 5 mM NaN3 , 2 mM ethylene glycol-bis (b-aminoethyl-
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ether)- N,N,N *,N *-tetraacetic acid, 200 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM pepstatin A, 1 mM leupeptin, 1 mM transepoxysuccinyl-leucymido (4guanidino) butane, 20 mM HEPES-Na pH 7.4], and homogenized using a motor-driven Potter-Elvehjem homogenizer (35 strokes). The homogenates were centrifuged at 760 1 g for 10 min to remove nuclei and unbroken cells, and the supernatant was centrifuged 60 min at 190,000 1 g to obtain the total cell membranes (TM). TM were resuspended in solution A and frozen at 0707C until used. Western Blot Analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10% polyacrylamide gels and Western blot analysis were carried out as previously described, 18 using a 1:500 dilution of anti-GLUT4 antibody, 1:5000 dilution of antiGLUT1 antibody or 1:300 of anti-GLUT3 antibody, followed by incubation with Horseradish Peroxide conjugated Protein A (Bio-Rad), which was detected by a luminescence technique (ECL; Amersham International plc, Amersham UK). Density values of the different bands were obtained after scanning the blots with video densitometry using the UVP-GDF 5000 system (UVP Inc, San Gabriel, CA) and thereafter quantitated using quant program, version 3.3, (Phosphoimager Multiscan HG Molecular Dynamics USA). RNA preparation and Northern Blot Analysis. Total cellular RNA was extracted from L6 myotubes, using the acid guanidium thiocyanate-phenol-chloroform method of Chomczynski et. al.30 For Northern-blot analysis, 30 mg of total RNA were separated under denaturing conditions on 1% (wt/vol) agarose gels containing 8% (vol/vol) formaldehyde with trace amount of ethidium bromide. The RNA was then transferred onto Hybond N / (Amersham international plc, UK) nylon membranes as describe by Maniatis.31 The RNA was cross-linked to the membrane with a UV cross linker (Hoefer Scientific instrument, San Francisco). GLUT1 cDNA in an PuK 19 plasmid was digested with the restriction enzymes EcoRI and BglI, the 2.6 Kb GLUT1 insert isolated by agarose gel electrophoresis, followed by electroelution and 32P-labeling with random prime DNA labeling Kit (Boehringer Mannheim GmbH, Germany). RNA Hybridization was performed in a hybridization oven (Micro-4 Hybaid limited) at 657C for 20 h in a hybridization solution [0.2 mM Na2HPO4 pH 7.2, 7% (vol/vol) SDS, 1% (wt/vol) BSA and 1 mM EDTA]. The washings were done in 0.4 1 SSC, and 0.1% SDS at 657C. Autoradiographs were performed with Bio-Imagining analyzer BAS 1000 (FUJI photo film CO., LTD Japan).
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Electrophoretic gel mobility shift assay. L6 myotubes were treated as describes in the figure legend. Nuclear extracts were isolated as previously described.31 Briefly, cells were scraped and pelleted in ice cold PBS buffer. The pellet was resuspended in 10 mM Tris-HCl pH 7.8, 1.5 mM MgCl2 , 10 mM KCl, 0.5 mM dithiothreitol ( DTT ) , 0.2 mM PMSF, 10 mg /ml leupeptin and 1 mg /ml pepstatin. The cells were allowed to swell on ice for 10 min and then were pelleted again and resuspended for 10 min in 20 mM Hepes-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 10 mg /ml leupeptin and 1 mg /ml pepstatin. Cellular debris was removed by centrifugation in 1000 1 g for 2 min, and the supernatant fraction containing the nuclear proteins were stored at 0707C. Binding reaction was performed for 20 min on ice with 10 mg total protein in 20 ml of 100 mM TrisHCl pH 7.5, 25 mM MgCl2 , 2 mM EDTA, 40% glycerol, 0.2 M KCl, 2 mM DTT, 0.2% NP-40 and 2 mg poly ( dI-dC ) and 150,000 c.p.m. of 32P-labeled oligonucleotides. DNA-protein complexes were separated from unbound DNA probe on native 4.5% polyacrylamide gels at 30 mA in 1 M Tris / HCl, pH 7.5, 50 mM boric acid and 0.5 mM EDTA. Gels were vacuum dried and autoradiographs were analyzed with Bio-Imaging analyzer BAS 1000 ( FUJI photo film CO., LTD Japan ) . The sequences of the oligo-
nucleotides were as follows ( factor binding sites are underlined ) : NF-kB: 5 *-TCGACTGGAAAGTCCCCAGCGGAAAGTCCCTTG-3 3 *-AGCTGACCTTT CAGGGGTCGCCTTTCAGGGAAC-5 * Other assays. Glucose and lactic acid concentrations in the medium were determined by the glucose oxidase method and spectrophotometric analysis using lactate dehydrogenase, respectively.32,33 Cell viability was measured by MTT test as previously described.34 Oxygen level in the medium was analyzed in a gas analyzer (Nova-Biomedical, Stat-profile). Protein concentration was determined by the Bio-Rad- Bradford procedure.35 Statistical analysis. Data are expressed as mean { standard error (SE). Statistical analysis was performed using Student’s t-test. RESULTS
H2O2 and O2· production by the oxidants generating system. To investigate the regulation of glucose trans-
Fig. 1. H2O2 and O2· production by glucose/glucose oxidase or xanthine/xanthine oxidase. (A) At time zero, 50 mU/ml glucose oxidase alone ( h ) or with 20 mU/ml peroxidase ( j ) were added to cells. H2O2 concentration was measured in the medium as described in ‘‘Methods.’’ (B) At time zero, 50 mM xanthine and 20 mU/ml xanthine oxidase alone ( s ), or with 200 mU/ml SOD ( l ) were added. O2· production rate was determined as described in ‘‘Methods.’’ Results are presented as mean values { SE of three independent experiments each performed in triplicate.
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ing in the system at a given time.36 The addition of 200 mU/ml superoxide dismutase (SOD) resulted in 80% inhibition in O2· production rate. No significant cell viability loss (as determined by light-phase microscopy, MTT test and by protein/well content), was found when cells were incubated up to 24 h in both systems under these conditions (data not shown).
Fig. 2. Effect of Xa/XO and G/GO on the DNA-binding activity of NF-kB. Nuclear extract were prepared from L6 myotubes exposed for 8 h to 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/Xo), 50 mU/ml glucose oxidase in a medium containing 5 mM glucose (G/GO), or to 200 mM H2O2 for 2 h. Extract were incubated with 32 P-labeled NF-kB oligonucleotide. Electophoretic mobility shift assay (EMSA) was preformed as describe in ‘‘Methods.’’ On the right panel, nuclear extract from cells exposed to Xa/XO for 8 h were preincubated with 100 fold molar excess of unlabeled NF-kB (specific), or with 100 fold molar excess of ROA I (non specific) oligonucleotide followed by incubation with the labeled NF-kB oligonucleotide and analyzed by (EMSA). Autoradiograph of a native gel is shown.
port in L6 myotubes by ROS, we incubated the cells with two different ROS generating systems: glucose and glucose oxidase which produces H2O2 , 26 or xanthine and xanhine oxidase which generates O2· and H2O2.27 Addition of 50 mU/ml glucose oxidase to L6 myotubes grown in a medium containing 5 mM glucose, resulted in elevated medium H2O2 concentration, which was completely prevented by peroxidase (20 mU/ml), (Fig. 1A). H2O2 reached 38.0 { 4.6 nmole/ ml at 4 h, after which further incubation resulted in a progressive decline in its concentration. Yet, after 24 h the level of H2O2 was increased about 4-fold compared to incubation in the absence of glucose oxidase. The rate of cytochrome C reduction was used to determine O2· production rate following addition of 50 mM xanthine and 20 mU/ml xanthine oxidase to the medium of L6 myotubes. The activity of this enzymatic system remained relatively constant for 24 h, yielding an O2· production rate of 0.6 nmoles/ml.minute (Fig. 1B). As the half life of O2· is 10 05 s, its production rate approximately represents the actual concentration exist-
ROS generating system activate NF-kB binding. In various cell types oxidative stress has been shown to result in increased DNA binding activity of the transcription factor NF-kB.37,38 To assess whether exposure of L6 myotubes to both ROS generating systems result in an intracellular response typical of oxidant stress, we evaluated NF-kB DNA binding activity. Nuclear extracts were prepared from L6 myotubes exposed to ROS generating systems for 8 h. Binding of extract protein to an oligonucleotide containing the consensus binding sequence of NF-kB was evaluated by gel mobility shift assay. As demonstrated in Fig. 2, NF-kB binding was increased in nuclear extracts prepared from cells treated with both 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/XO), or 5 mM glucose and 50 mU/ml glucose oxidase (G/GO) as compared to control. Exposure of cells to 200 mM H2O2 , a known activator of NF-kB, 37 served as a positive control. The specificity of the observed DNA binding activity was confirmed, as a 100-fold excess of the unlabeled oligonucleotide, but not nonspecific oligonucleotide, eliminated binding activity. ROS generating system increase 2-deoxyglucose (2DG) uptake. Incubation of L6 myotubes in the presence of glucose/glucose oxidase or xanthine/xanthine oxidase, activated glucose uptake in a time and dose dependent manner. The rate of 2-DG uptake into L6 myotubes incubated with 5 mM glucose and increasing concentrations of glucose oxidase caused progressive 2-DG uptake stimulation, reaching 2.6 { 0.4 fold increase in the presence of 50 mU/ml glucose oxidase, after 24 h (Fig. 3A and C). Similarly, in the presence of 50 mM xanthine and 20 mU/ml xanthine oxidase, 2DG uptake into L6 myotubes increased progressively reaching 2.2 { 0.7 fold after 24 h incubation (Fig. 3B and D). 2-DG uptake in cells incubated with 50 mM xanthine in the absence of xanthine oxidase did not significantly differ from control (data not shown). Longer incubation periods or higher concentrations of either glucose oxidase or xanthine oxidase resulted in viability loss as detected by MTT test (data not shown). Thus, all further experiments reported herein, were performed in conditions which produced maximal biologic effect without causing viability loss, i.e. 24 h incubation with either 50 mU/ml glucose oxidase in a
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Fig. 3. Dose and time- dependent changes in 2-deoxyglucose uptake in L6 myotubes exposed to glucose/glucose oxidase or xanthine/xanthine oxidase. L6 myotubes in 24 well plates, were exposed to: (A) varying concentrations of glucose oxidase in a medium containing 5 mM glucose, for 24 h, (B) varying concentrations of xanthine oxidase in medium containing 50 mM xanthine, for 24 h, (C) 50 mU/ml glucose oxidase in a medium containing 5 mM glucose for various periods of time, or (D), 50 mM xanthine and 20 mU/ml xanthine oxidase for the indicated periods of time. At the end of the incubation periods the plates were rinsed 3 times with HEPES-buffer, and 2-deoxyglucose transport was measured for 10 min, as described in ‘‘Methods.’’ Results are expressed as mean { SE of five independent experiments, each performed in triplicate. *p õ 0.03 vs. untreated cells (A,B), or ’time 0’ (C,D).
medium containing 5 mM glucose, or 50 mM xanthine and 20 mU/ml xanthine oxidase. Oxidation of glucose by glucose oxidase requires glucose and atmospheric oxygen. O2 is also required for the oxidation of xanthine by xanthine oxidase. Thus, hypoxia or reduction in medium glucose concentration could cause the stimulated glucose transport observed, as previously reported.39,40 To eliminate these possibilities we measured glucose and oxygen levels in the medium of cells after 24 h incubation with either G/GO or Xa/XO. Partial oxygen pressure (pO2 ) in the medium of control, G/GO or Xa/XO treated cells was not statistically different (156 { 18, 170 { 15 and 165 { 21 mmHg, respectively). Medium glucose concen-
tration did not differ significantly whether cells were incubated in the absence or presence of glucose oxidase for 24 h (4.0 { 0.5 and 3.5 { 0.8 mM, respectively). Thus, the increased stimulation in 2-DG uptake appears to result specifically from the exposure of the cells to ROS generating system. In order to determine the respective role of H2O2 in activating glucose uptake, we evaluate the effect of peroxidase. L6 myotubes were coincubated with peroxidase during incubation with G/GO or Xa/XO for 24 h. Table 1 shows that full inhibition of 2-DG uptake stimulation was observed when 20 mU/ml peroxidase were added to cells treated with G/GO, indicating the role of H2O2 in 2-DG uptake activation. Both peroxidase
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Oxidation activates glucose uptake Table 1. Effect of Peroxidase and Superoxide Dismutase (SOD) on 2-deoxyglucose Uptake into L6 Myotubes During Incubation with G/GO or Xa/XO
Table 2. Effect of G/GO and Xa/XO on Glucose Consumption, Lactic Acid Production, and [14]CO2 Release in L6 Myotubes Glucose consumption
2-deoxyglucose uptake (pmol/mg protein·min) Conditions Control G/GO Xa/XO
No addition
Peroxidase (20 mU/ml)
SOD (200 mU/ml)
20.2 { 7.5 (33) 51.2 { 9.6* (17) 41.9 { 7.5* (23)
25.5 { 6.0 (6) 22.4 { 7.2Ú (7) 32.4 { 7.2§ (6)
23.5 { 2.2 (6) ND 33.8 { 2.4 (5)
L6 myotubes were incubated for 24 h in a-MEM medium in the absence (control), or presence of 50 mU/ml glucose oxidase (G/GO) or 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/XO), with either 20 mU/ml peroxidase or 200 mU/ml superoxide dismutase (SOD). 2-deoxyglucose uptake was measured for 10 min as described in ‘‘Methods.’’ Each value is a mean { SE of number of independent experiments (n), each performed in triplicates. *p õ 0.001 vs. control, Úp õ 0.03 vs. G/GO, §p õ 0.05 vs. Xa/XO, ND-not determined.
and SOD exerted only a partial inhibitory effect on the activation of glucose uptake observed with Xa/XO. This is in agreement with the work of Fridovich et. al., 27 who reported that xanthine oxidase produces both H2O2 and O2·. These results emphasize the role of H2O2 in the activation of 2-DG uptake in L6 myotubes. Oxidant generating system increases glucose metabolism. To evaluate the metabolic consequences of the observed increase in glucose uptake, we investigated the effect of ROS generating systems on glucose metabolism in L6 myotubes. Cells incubated for 24 h in the presence of G/GO, exhibited increased lactate production and CO2 release (2.2 { 0.2, 2.0 { 0.8 -fold increase vs. control, respectively) (Table 2). Similarly, in the presence of Xa/XO, glucose consumption, lactate production and CO2 release were increased by 2.1 { 0.1, 2.0 { 0.2 and 2.3 { 0.9-fold vs. control, respectively (Table 2). This stimulation in the metabolic rate suggests that the exposure of the cells to ROS may enhance ATP production. 2-DG uptake stimulation is caused by elevation of GLUT1 protein and mRNA. The involvement of ongoing protein synthesis in the observed activation of glucose uptake by ROS was investigated with the protein synthesis inhibitor cycloheximide. When L6 were incubated for 24 h in the presence of cycloheximide (5 mg/ml), 2-DG uptake stimulation elicited by G/GO or Xa/XO was totally inhibited, without altering 2-DG uptake in control cells. (Table 3). These observations suggest that synthesis of either glucose transporters or activator proteins may be responsible for the stimulation of transport activity seen in the presence of the oxidants. To directly investigate whether de novo glucose transporter protein synthesis is triggered when
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[14]
CO2 release
(mmole/mg protein·h)
Treatment 0.16 { 0.04 ND 0.34 { 0.02*
None G/GO Xa/XO
Lactic acid production
0.21 { 0.01 0.47 { 0.05* 0.42 { 0.05*
0.004 { 0.001 0.008 { 0.001** 0.009 { 0.002**
L6 myotubes were grown in 24-well plates, in the absence or presence of 50 mU/ml glucose oxidase (G/GO) or 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/XO). After 24 h the medium was changed to HEPES-buffer containing 5 mM [U-14C]-glucose (1 mCi/ ml) in the absence or presence of the oxidants generating systems. Glucose consumption, lactic acid production and [14]CO2 release, were determined as described in ‘‘Methods.’’ Results are mean { SE of 2 independent experiments carried out in triplicate. *p õ 0.05 vs. untreated cells. **p õ 0.01 vs. untreated cells. ND-not determined.
cells are exposed to ROS generating systems, we assessed the amount of immunoreactive glucose transporters in total membranes prepared from L6 myotubes incubated for 24 h with G/GO or Xa/XO. 30 mg of membrane protein from each treatment were probed by Western-Blot analysis using antibodies directed against either GLUT1 GLUT3 or GLUT4 transporters. As shown in Fig. 4A, the intensity of GLUT1 immunoreactive protein in total membranes of either G/GO or Xa/XO treated cells was increased compared to control. Densitometric analysis indicate that GLUT1 content in total membrane of cells treated with either G/ GO or Xa/XO was increased by 2.0 { 0.2 and 2.4 { 0.4 -fold, respectively, as compared to GLUT1 from untreated cells. The amount of GLUT4 and GLUT3 were analyzed similarly, revieling a reduction in both transporter isoforms. While the reduction in GLUT4 Table 3. Effect of Cycloheximide (CHX) on 2deoxyglucose Uptake Stimulated by G/GO or Xa/XO in L6 Myotubes 2-Deoxyglucose uptake (pmol/mg protein·min) Treatment Control G/GO Xa/XO
0CHX
/CHX
18.5 { 4.0 51.2 { 10.0* 42.3 { 8.0*
17.1 { 4.0 23.3 { 2.0ÚÚ 20.2 { 5.0Ú
L6 myotubes, in 24 well plates, were incubated for 24 h with 5 mM glucose and 50 mU/ml glucose oxidase (G/ GO), or with 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/XO), in the absence or presence of 5 mg/ml cycloheximide. Following the incubation period, the plates were rinsed 3 times with HEPES-buffer, and 2deoxyglucose uptake was measured for 10 min, as described in ‘‘Methods.’’ Results of seven independent experiments each performed in triplicate are expressed as mean { SE. *p õ 0.001 vs. control 0CHX, Úp õ 0.05 vs. Xa/XO 0CHX, ÚÚp õ 0.001 vs. G/GO 0CHX.
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Fig. 4. Western-Blot and densitometric analysis of glucose transporters, prepared from L6 myotubes total membranes after 24 h exposure to Xa/XO or G/GO. L6 myotubes were incubated in a-MEM for 24 h without treatment (cont), with 50 mU/ml glucose oxidase (G/GO) or 50 mM xanthine and 20 mU/ml xanthine oxidase (Xa/XO). Total membranes were prepared as described in ‘‘Methods.’’ (A) 30 mg protein/lane were separated by SDS-PAGE on 10% polyacrylamide gels, transferred to nitrocellulose membrane and immunoassayed using anti-GLUT1, anti-GLUT4 or anti-GLUT3 antibodies. (B) Results of scanning densitometry analysis performed on 4 independent autoradiographs for GLUT1 and GLUT4 and 2 autoradiographs for GLUT3 are presented. Values (mean { SE) are in relative units as compared to the corresponding GLUTs from untreated cells. *p õ 0.03 vs. control. ND -not determined.
protein did not reach statistical significance, GLUT3 was markedly reduced in G/GO treated cells by approximately 70% (Fig. 4B). To assess the involvement of GLUT1 gene expression in this response, Northern-Blot analysis was performed using GLUT1 radio labeled cDNA probes. Cells were treated for 24 h with either 5 mM glucose and different concentrations of glucose oxidase (Fig. 5A) or 50 mM xanthine and different concentrations of xanthine oxidase (Fig. 5B). Scanning densitometry of four independent experiments revealed a progressive increase in GLUT1 mRNA, resulting in a 3.5 { 0.3 and 2.3 { 0.7 -fold increase in the presence of 50 mU/ml glucose oxidase or 20 mU/ml xanthine oxidase, respectively (Fig. 5C).
ROS generating systems induce increased 2-DG uptake in 3T3-L1 adipocytes. To address the question of whether the observed 2-DG uptake activation by ROS generating system is limited to the L6 cell line, or rather represents a more generalized phenomenon, we utilized a different cell line, the 3T3-L1 adipocytes, which contains GLUT1 and GLUT4.18 As can be seen in Table 4, cells incubated for 18 h with 2% serum and either 50 mU/ml glucose oxidase, or 50 mM xanthine and 20 mU/ml xanthine oxidase, exhibited an elevation in glucose transport activity (40% and 60% for G/GO and Xa/XO, respectively). In serum deprived cells, a 4.0 { 0.2 -fold increase in 2-DG uptake was observed when cells were treated for 18 h with 50 mU/ml glucose oxidase compared to control. These data support
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Fig. 5. Northern-Blot and densitometry analysis of GLUT1 mRNA in L6 myotubes incubated with different concentrations of oxidant generating systems. L6 myotubes were incubated for 24 h, with either (A) 0, 20, 50 mU/ml glucose oxidase in medium containing 5 mM glucose, or (B) 50 mM xanthine and 0, 10, 20 mU/ml xanthine oxidase. Total RNA was isolated as described in ‘‘Methods.’’ Thirty micrograms/lane were loaded and, after fractionation in a 1% agarose-formaldehyde gel, the resulting blot was probed with a full length rat GLUT1 cDNA. Presented are representative autoradiographs. (C) Scanning densitometry analysis of four independent experiments with similar results. Values (mean { SE) are in relative units, with a value of 1.00 assigned to corresponding mRNA from each control cells. *p õ 0.05 vs. control.
the notion that ROS generating systems induced glucose uptake stimulation, is not limited to muscle cells in culture, and presumably represents a generalized adaptation mechanism to oxidative stress. DISCUSSION
This study was undertaken To investigate the role of glucose transport regulation as an adaptive response to low-grade, prolonged oxidative stress. Since ROS have an extremely short half-life, 36 we utilized two well characterized ROS generating systems which continu-
ously produce fairly low and constant concentrations of H2O2 and O2· over a 24 h period. In both L6 myotubes and in 3T3-L1 adipocytes, glucose uptake limits cellular metabolism, rendering its regulation crucial in the adaptation to diverse extracellular conditions. The results obtained in this study demonstrate that exposure of L6 cells to ROS generating system increases the steady state levels of GLUT1 mRNA (Fig. 5), by either increased transcription rate and/or by increased stability, resulting in increased glucose uptake and metabolism (Fig. 3; Table 2). The increase in GLUT1 trans-
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0.2% BSA
2% Serum
87 { 15 (6) 345 { 80* (4) ND
139 { 11 (7) 195 { 19** (6) 226 { 18** (5)
Differentiated 3T3-L1 adipocytes were exposed for 18 h, to a medium containing 25 mM glucose and 50 mU/ ml glucose oxidase, or 50 mM xanthine and 20 mU/ml xanthine oxidase in the absence (0.2% BSA) or presence of 2% serum. 2-deoxyglucose uptake was measured for 10 min as described in ‘‘õMethods.’’ The number of independent experiments performed at least in duplicates is shown in parenthesis. *p õ 0.007 vs. control with 0.2% BSA, **p õ 0.05 vs. control with 2% serum. ND-not determined.
porter isoform (Fig. 4) correlates well to the degree of enhancement in glucose transport and metabolism. Under the same conditions, GLUT4 and GLUT3 protein content were reduced, reaching statistical significance in the case of GLUT3 in G/GO treated cells (Fig. 4B). These findings further suppport the predominant role of GLUT1 isoform in the adaptive response to oxidant stress in L6 myotubes. Our work suggests that oxidative stress imposed upon L6 muscle cells causes an adaptive response which results in increased energy production, presumably to recruit cellular defense mechanisms. Enhanced glucose uptake in muscle cells can be achieved by rapid translocation of preexisting transporters from intracellular pools to the plasma membrane, as seen following insulin stimulation 41 or by a secondary late phase response.40 In the case of ROS generating system, glucose transport stimulation occurs beginning at 6 h of continuous exposure (Fig. 3), and is therefore a late phase response similar to that observed following anoxia, glucose deprivation or iron chelators, which involves GLUT1 gene induction.18,40,42 In all these conditions the response is dependent upon de-novo protein synhesis as demonstrated by the inhibitory effect of cycloheximide (Table 3). This further suggests that redistribution of preexisting transporters, is proboably relativiely minor compared to the increased GLUT1 protein synthesis observed in the response to oxidant stress. Late phase response to Xa/XO or G/GO were reported in other systems such as the cystine uptake stimulation in endothelial cells, 43 or MnSOD mRNA elevation in epithelial cells.44 As GLUT1 is the ubiquitous glucose transporter, regulatory responses similar to those describe here in L6 myotubes, are likely to occur in various cell types under oxidative stress, as demonstrated by the enhanced glucose transport
observed under similar conditions with 3T3-L1 (Table 4). The role of H2O2 produced by both G/GO and Xa/ XO in mediating GLUT1 gene transcription is demonstrated by the effect peroxidase had in preventing glucose uptake stimulation (Table 1). H2O2 has been implicated to play a role in transcriptional induction of several genes such as the oxy-R encoded protein in bacteria, 45 the heat-shock factor in Drosophila, 46 the iron responsive elements binding proteins in mammalian cells, 47 and the immediate early genes c-fos and cJun.48 The products of these genes are presumably involved in the defense mechanisms of various cells to stress conditions. The signals and sequence of events mediating H2O2 stimulated induction of these genes are unknown, but there are accumulating reports pointing to the involvement of transcriptional factors such as AP-1 and NF-kB in these responses 37,38 In the 5 * untranslated region of GLUT1 gene there are several important regulatory elements, including AP-1 and serum responsive element binding sites23 which may be regulated by oxidants. Preliminary results in our laboratory, indicate increased binding activity of AP-1 and increase in c-fos mRNA following exposure to the oxidant generating systems. Whether this is relevant to the induction of GLUT1 gene during the response to oxidative stress, remains to be established. Acknowledgement — We wish to thank the S. Daniel Abraham International Center for Health and Nutrition, Ben-Gurion University of the Negev.
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ROS— Reactive oxygen species TM—total membranes Xa/XO—xanthine/xanthine oxidase G/GO—glucose/glucose oxidase SOD—superoxide dismutase 2-DG—2-deoxyglucose BSA—bovine serum albumin mRNA—messenger RNA
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