Glucose Uptake and Glycolysis Reduce Hypoxia-induced Apoptosis in Cultured Neonatal Rat Cardiac Myocytes
1999; Elsevier BV; Volume: 274; Issue: 18 Linguagem: Inglês
10.1074/jbc.274.18.12567
ISSN1083-351X
AutoresRicky Malhotra, Frank C. Brosius,
Tópico(s)Cell death mechanisms and regulation
ResumoMyocardial ischemia/reperfusion is well recognized as a major cause of apoptotic or necrotic cell death. Neonatal rat cardiac myocytes are intrinsically resistant to hypoxia-induced apoptosis, suggesting a protective role of energy-generating substrates. In the present report, a model of sustained hypoxia of primary cultures of Percoll-enriched neonatal rat cardiac myocytes was used to study specifically the modulatory role of extracellular glucose and other intermediary substrates of energy metabolism (pyruvate, lactate, propionate) as well as glycolytic inhibitors (2-deoxyglucose and iodoacetate) on the induction and maintenance of apoptosis. In the absence of glucose and other substrates, hypoxia (5% CO2 and 95% N2) caused apoptosis in 14% of cardiac myocytes at 3 h and in 22% of cells at 6–8 h of hypoxia, as revealed by sarcolemmal membrane blebbing, nuclear fragmentation, and chromatin condensation (Hoechst staining), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and DNA laddering. This was accompanied by translocation of cytochrome c from the mitochondria to the cytosol and cleavage of the death substrate poly(ADP-ribose) polymerase. Cleavage of poly(ADP-ribose) polymerase and DNA laddering were prevented by preincubation with the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (zDEVD-fmk), indicating activation of caspases in the apoptotic process. The caspase inhibitor zDEVD-fmk also partially inhibited cytochrome c translocation. The presence of as little as 1 mm glucose, but not pyruvate, lactate, or propionate, before hypoxia prevented apoptosis. Inhibiting glycolysis by 2-deoxyglucose or iodoacetate, in the presence of glucose, reversed the protective effect of glucose. This study demonstrates that glycolysis of extracellular glucose, and not other metabolic pathways, protects cardiac myocytes from hypoxic injury and subsequent apoptosis. Myocardial ischemia/reperfusion is well recognized as a major cause of apoptotic or necrotic cell death. Neonatal rat cardiac myocytes are intrinsically resistant to hypoxia-induced apoptosis, suggesting a protective role of energy-generating substrates. In the present report, a model of sustained hypoxia of primary cultures of Percoll-enriched neonatal rat cardiac myocytes was used to study specifically the modulatory role of extracellular glucose and other intermediary substrates of energy metabolism (pyruvate, lactate, propionate) as well as glycolytic inhibitors (2-deoxyglucose and iodoacetate) on the induction and maintenance of apoptosis. In the absence of glucose and other substrates, hypoxia (5% CO2 and 95% N2) caused apoptosis in 14% of cardiac myocytes at 3 h and in 22% of cells at 6–8 h of hypoxia, as revealed by sarcolemmal membrane blebbing, nuclear fragmentation, and chromatin condensation (Hoechst staining), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and DNA laddering. This was accompanied by translocation of cytochrome c from the mitochondria to the cytosol and cleavage of the death substrate poly(ADP-ribose) polymerase. Cleavage of poly(ADP-ribose) polymerase and DNA laddering were prevented by preincubation with the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (zDEVD-fmk), indicating activation of caspases in the apoptotic process. The caspase inhibitor zDEVD-fmk also partially inhibited cytochrome c translocation. The presence of as little as 1 mm glucose, but not pyruvate, lactate, or propionate, before hypoxia prevented apoptosis. Inhibiting glycolysis by 2-deoxyglucose or iodoacetate, in the presence of glucose, reversed the protective effect of glucose. This study demonstrates that glycolysis of extracellular glucose, and not other metabolic pathways, protects cardiac myocytes from hypoxic injury and subsequent apoptosis. Apoptosis, a form of cell death characterized by cell shrinkage, plasma membrane blebbing, chromatin condensation, and genomic DNA fragmentation, is essential for development, maintenance of tissue homeostasis, and elimination of harmful and diseased cells in metazoan organisms (1Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2419) Google Scholar, 2Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2383) Google Scholar). On the other hand, dysregulated apoptosis has been implicated in many human diseases such as cancer and neurodegenerative diseases (e.g. Alzheimer's disease, AIDS encephalopathy, and ischemic stroke) (3Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6159) Google Scholar, 4Cheng Y. Deshmukh M. D'Costa A. Demaro J.A. Gidday J.M. Shah A. Sun Y. Jacquin M.F. Johnson E.M. Holtzman D.M. J. Clin. Invest. 1998; 101: 1992-1999Crossref PubMed Scopus (474) Google Scholar, 5Cotman C.W. 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Some of this relative resistance to hypoxia may be caused by the high levels of endogenous apoptosis inhibitors in these cells, such as the cellular FLIPS (cFLIP) (cellular FLICE-inhibitory proteins) and ARC (apoptosis repressor with caspase recruitment domain), which have been reported to interact selectively with the death domain of the Fas (CD95/Apo-1) death receptor and upstream initiator caspases 2 and 8, respectively (33Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.-L. Schroter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Crossref PubMed Scopus (2205) Google Scholar, 34Koseki T. Inohara N. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5156-5160Crossref PubMed Scopus (305) Google Scholar). However, the precise role of these inhibitors in heart cells has not been documented. Under normoxic conditions, cardiac myocytes produce most of their ATP by oxidative phosphorylation, making the regulation of this process central to balancing cardiac energy metabolism (35Heineman F.W. Balaban R.S. Fozzard H.A. Haber E. Jenings R.B. Katz A.M. Morgan H.E. The Heart and Cardiovascular System: Scientific Foundations. 2nd Ed. Raven Press, New York1992: 1641-1656Google Scholar). It is estimated that approximately 60–70% of myocardial energy is obtained from the metabolism of fatty acids, and the remaining is derived from non-lipid sources including carbohydrates, ketone bodies, and amino acids (36Tahiliani A.G. Fozzard H.A. Haber E. Jenings R.B. Katz A.M. Morgan H.E. The Heart and Cardiovascular System: Scientific Foundations. 2nd Ed. Raven Press, New York1992: 1599-1620Google Scholar). This fact is evident from the observation that cardiac myocytes cultured under glucose-free normoxic conditions remain viable and beat synchronously for several days. However, during ischemia or hypoxia, glucose uptake and glycolysis become critical to the maintenance of myocardial viability. Multiple studies have suggested that glucose uptake and glycolysis can prevent cardiac myocytes from ischemic or hypoxic injury in vivo or in vitro (37Eberli F.R. Weinberg E.O. Grice W.N. Horowitz G.L. Apstein C.S. Circ. Res. 1991; 68: 466-481Crossref PubMed Scopus (203) Google Scholar, 38Owen P. Dennis S. Opie L.H. Circ. Res. 1990; 66: 344-354Crossref PubMed Scopus (179) Google Scholar, 39Bekheit S. Isber N. Jani H. Butrous G. Boutjdir M. El-Sherif N. J. Am. Coll. Cardiol. 1993; 22: 1214-1222Crossref PubMed Scopus (8) Google Scholar). Hypoxia and ischemia induce increased glucose uptake in part by induction of translocation of the major glucose transporter GLUT-4 to the sarcolemma, and prolonged ischemia also increases the expression of GLUT-1 (40Sun D.Q. Nguyen N. DeGrado T.R. Schwaiger M. Brosius III, F.C. Circulation. 1994; 89: 793-798Crossref PubMed Scopus (215) Google Scholar, 41Brosius III, F.C. Yannan L. Nguyen N. Sun D.Q. Bartlett J. Schwaiger M. J. Mol. Cell. Cardiol. 1997; 29: 1675-1685Abstract Full Text PDF PubMed Scopus (70) Google Scholar). However, no studies have documented the role that glucose uptake and glycolysis may play either in the induction or prevention of apoptosis in the heart. Therefore, in the present study we investigated whether glucose uptake and glycolysis prevented hypoxia-induced apoptosis of neonatal rat cardiac myocytes. We also investigated the potential role of various intermediaries of energy metabolism, namely fatty acids (propionate), pyruvate, and lactate, in modulating hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes. In the present study we demonstrate that hypoxia induced substantial apoptosis by 3–8 h in cultured neonatal rat cardiac myocytes in the absence of glucose. This process was accompanied by the translocation of cytochrome c to the cytosol in a caspase 3 activation-dependent manner. Only glucose uptake and glycolysis protected cultured neonatal rat cardiac myocytes from hypoxia-induced apoptosis. Other substrates were ineffectual. Primary cultures of neonatal rat cardiac myocytes were prepared by a modification of a protocol reported previously (42Sadoshima J. Jahn L. Takahashi T. Kulik T.J. Izumo S. J. Biol. Chem. 1992; 267: 10551-10560Abstract Full Text PDF PubMed Google Scholar). Briefly, cardiac myocytes were obtained from ventricular tissue of 1-day-old Wistar rats by six to seven 15-min digestions at 37 °C in HEPES-buffered saline solution containing 0.1% collagenase IV, 0.1% trypsin, 15 μg/ml DNase I, and 1.0% chicken serum. The dissociated cells were collected by centrifugation and resuspended in ADS buffer (in g/liter: 6.8 NaCl, 4.76 HEPES, 0.138 NaH2PO4, 0.6 glucose, 0.4 KCl, 0.205 MgSO4, 0.002 phenol red, pH 7.4). The cells were then selectively enriched by differential centrifugation through a discontinuous Percoll (Amersham Pharmacia Biotech) gradient of densities 1.050, 1.062, and 1.082 g/ml (43Sheng Z. Pennica D. Wood W.I. Chien K.R. Development. 1996; 122: 419-428Crossref PubMed Google Scholar). The band at the 1.062/1.082 density interface was collected and used as the source of cardiac myocytes. The cardiac myocytes were washed and suspended in Dulbecco's modified Eagle's medium (DMEM) 1The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; PARP, poly(ADP-ribose) polymerase; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; Z-DEVD-fmk, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone; Z-FA-fmk, benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone/F-12 medium (Life Technologies, Inc.) (1:1, v/v) supplemented with 5% horse serum, 3 mm pyruvic acid, 100 μm ascorbic acid, 1 μg/ml transferrin, 10 ng/ml selenium, and 100 μg/ml ampicillin. Cardiac myocytes were plated on gelatin-precoated 60-mm dishes at a density of 2.5 × 106 cells/dish. The cells were also plated on gelatin-coated Falcon culture slides at a density of 1.5 × 104 cells/cm (2Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2383) Google Scholar). Bromodeoxyuridine at a final concentration of 0.1 mm was added during the first 36 h to prevent proliferation of cardiac fibroblasts. Cardiac myocyte purity was monitored by immunofluorescence after staining with monoclonal antibodies specific for cardiac α-sarcomeric actin (Sigma). Myocyte purity averaged 96 ± 3% 48 h after plating. Cardiac fibroblasts were collected from the upper density gradient of 1.050/1.062 and cultured on separate untreated 100-mm plates in DMEM supplemented with 10% fetal calf serum and antibiotics. All experiments of cardiac myocytes and hypoxia were done after 4 days of plating. The cells were initially plated in DMEM/F-12 medium (1:1) which contained 17.5 mmglucose. This was replaced with glucose- and pyruvate-free DMEM for hypoxia experiments; however, serum was not removed because of a report suggesting that serum withdrawal can induce apoptosis in primary cultures of cardiac myocytes (44Sheng Z. Knowlton K. Chen J. Hoshijima M. Brown J.H. Chien K.R. J. Biol. Chem. 1997; 272: 5783-5791Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). For hypoxia experiments, the cells were placed in a Plexiglas chamber, and a constant stream of water-saturated 95% N2 and 5% CO2was maintained over the culture. To lower the Po2 to < 5 mm Hg, Oxyrase, a mixture of bacterial membrane monooxygenases and dioxygenases (Oxyrase Inc., Ashland, OH) was added to the culture medium at a final concentration of 2–6%. As shown in Fig. 1 cardiac myocytes cultured in 6% Oxyrase for 24 h under normoxic conditions do not show any DNA laddering or morphologic signs of apoptosis. For morphological studies the cardiac myocytes were grown in eight-well gelatin-coated Falcon glass culture slides (Becton Dickinson Labware, Franklin Lakes, NJ).The cells were rinsed in phosphate-buffered saline (PBS), pH 7.4, and fixed for 30 min in 4% paraformaldehyde in PBS, pH 7.4, at room temperature. After a rinse in PBS, the cells were permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate. The cells were rinsed twice in PBS and then stained with the karyophilic dye Hoechst 33258 (5 μg/ml) for 10 min at room temperature. After a final rinse in PBS, the cells were mounted in moiwol, an antifade agent, and visualized under ultraviolet light with a Leitz Orthoplan microscope. Because this dye stains both apoptotic and non-apoptotic cells we could specifically count the percentage of apoptotic cells displaying chromatin condensation and nuclear fragmentation. For statistical analysis, 100 cells were counted in five different fields. Further characterization of apoptosis was achieved using a commercially available in situ cell death detection kit to find DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reagent according to the manufacturer's protocol (Boehringer Mannheim). The number of TUNEL-positive cells were also counted in five different fields. Cardiac myocytes were rinsed with cold PBS, and mitochondrial, and cytosolic (S100) fractions were prepared (45Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1229) Google Scholar). Briefly, cells were resuspended in 0.25 ml of ice-cold isotonic buffer A (250 mm sucrose, 20 mm HEPES, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mmdithiothreitol, 17 μg/ml phenylmethylsulfonyl fluoride, 8 μg/ml aprotonin, 2 μg/ml leupeptin, 5 μg/ml pepstatin, pH 7.4). The cells were disrupted by two successive sonications on ice for 20 s each with a pause of 1–2 min using a microtip generator set at 40% duty cycle (Sonicator Vibracell, Sonics and Materials, CT). A pellet highly enriched in mitochondria was prepared by centrifugation at 10,000 × g for 30 min. This pellet was resuspended in the same buffer A, and the resulting supernatant was further spun at 160,000 × g for 1 h in a TLA-100 rotor in a Beckman table top ultracentrifuge. The supernatant from this final ultracentrifugation represented the cytosolic fraction. Equivalent amounts of mitochondrial and cytosolic fractions were subjected to Western blot analysis as described previously (46Boise L.H. Thompson C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3759-3764Crossref PubMed Scopus (207) Google Scholar). Briefly, the proteins were electrophoresed on 15% SDS-polyacrylamide gels, transferred to Hybond nylon membranes (Amersham Pharmacia Biotech), and immunoblotted with monoclonal antibodies specific for cytochrome c (monoclonal antibody 7H8.2C12 at 1.5 μg/ml; Pharmingen, San Diego). To ensure that cytochrome c release was not caused by a physical disruption of mitochondria, both the mitochondrial and cytosolic fractions were probed with monoclonal antibodies to cytochrome oxidase (subunit IV) (monoclonal antibody 20E8-C12 at a dilution of 0.1 μg/ml; Molecular Probes, Eugene, OR), an enzyme complex bound to the outer leaflet of the inner mitochondrial membrane. Visualization of the signal was by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech). For Western blot analysis of PARP (poly(ADP-ribose) polymerase), both floating and attached cells were rinsed in cold PBS, pH 7.4, and then collected into a defined volume of lysis buffer (62.5 mm Tris, pH 6.8, 8 m deionized urea, 10% glycerol, 2% SDS, and protease inhibitors). The cells were then sonicated on ice for 20 s. After the addition of loading buffer the samples were incubated at 65 °C for 15 min, and equal amounts of protein were resolved on a 7.5% SDS-polyacrylamide gel. Immunoblotting for PARP was performed (47Jacobson M.D. Weil M. Raff M.C. J. Cell Biol. 1996; 133: 1041-1051Crossref PubMed Scopus (365) Google Scholar) using a monoclonal antibody that specifically detects rat, mouse, or human PARP (monoclonal antibody SA-250; clone C-2–10, BIOMOL Research Labs, Inc., Plymouth Meeting, PA) at a 1:5,000 dilution. Visualization of the signal was by ECL. Genomic DNA was isolated and detected (48Chinnaiyan A.M. Orth K. O'Rourke K. Duan H. Poirier G.G. Dixit V.M. J. Biol. Chem. 1996; 271: 4573-4576Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). Cardiac myocytes were harvested and resuspended in 0.5 ml of lysis buffer (100 mm NaCl, 10 mm Tris, pH 8.0, 1 mmEDTA, 0.5% SDS, 0.20 mg/ml proteinase K, 200 μg/ml RNase A). The cell lysate was incubated at 37 °C for 2 h, and the genomic DNA was extracted twice with phenol/chloroform and once with chloroform. The DNA was precipitated with ethanol and then dissolved in 50 μl of TE (10 mm Tris-HCl, pH 8, 1 mm EDTA). After spectrophotometric determination of the nucleic acid content, DNA samples (10 μg) were electrophoresed on 1% agarose gels to visualize laddering. Data are presented as the means ± S.E. The means of numbers of cells undergoing apoptosis were subjected to analysis of variance for multiple comparisons. Paired analysis between two groups was performed by Student's t test. Cardiac myocytes were subjected to hypoxia in the presence of 17.5 mm glucose for 24 h. Genomic DNA was isolated, and 10 μg of DNA was run on a 1% agarose gel. The cardiac myocytes cultured under normoxic conditions in the presence or absence of Oxyrase showed no fragmentation of DNA into oligonucleosomes. However, cardiac myocytes cultured under hypoxic conditions for 24 h showed characteristic internucleosomal DNA fragmentation of approximately 200 base pairs consistent with apoptosis. Preincubation of the cells with 100 μm Z-VAD-fmk, a broad spectrum caspase inhibitor, followed by 24 h of hypoxia, completely prevented DNA laddering (Fig. 1 A). Cultured cardiac myocytes also show strongly positive TUNEL staining. The TUNEL-positive cells showed distinct condensation of the chromatin and fragmented nuclei (Fig. 1 B). After 24 h of hypoxia, 16 ± 4% (mean ± S.E.) of cardiac myocytes were apoptotic by these criteria compared with only 4 ± 3% apoptotic cells observed in control, normoxic conditions. To determine if short term hypoxia in the absence of glucose also resulted in induction of apoptosis in the cultured neonatal rat cardiac myocytes, we first examined cell morphology by phase-contrast microscopy and nuclear morphology by Hoechst and TUNEL staining (Fig. 2). Cardiac myocytes cultured in the presence of 17.5 mmglucose were switched to glucose- and pyruvate-free DMEM/F-12 medium and allowed to equilibrate under normoxic conditions for 30 min. Subsequently, these cells were placed in the hypoxia chamber for 1, 3, and 8 h. 3 h of hypoxia resulted in the appearance of a substantial number of apoptotic cells as determined by membrane blebbing, chromatin condensation, and nuclear fragmentation revealed by Hoechst staining (14 ± 3% versus 3 ± 2% apoptotic cells in control, normoxic conditions, n = 5,p < 0.05) (Fig. 3). These cells were also strongly positive by TUNEL (quantitative data not shown). After 8 h of hypoxia, 22 ± 4% cells were apoptotic (n = 5, p < 0.001) (Fig. 3). The cardiac myocytes cultured either in the presence or absence of glucose and pyruvate, under normoxic conditions, showed basal levels of apoptosis of 3 ± 2%.Figure 3Quantitative analyses of time-dependent induction of apoptosis. Neonatal rat cardiac myocytes were cultured under normoxia (N) or hypoxia (H) in the presence (G+P+) or absence (G−P−) of glucose and pyruvate. The percentage of dead cells was determined by Hoechst 33258 staining and counted as described under "Materials and Methods." Values are mean ± S.E. Data shown are from five independent experiments. *p < 0.05, ** p < 0.001.View Large Image Figure ViewerDownload (PPT) We next examined whether morphologic features of apoptosis were accompanied by the cleavage of the DNA repair enzyme, PARP, a known substrate for activated caspase 3. Neonatal rat cardiac myocytes cultured in the presence of glucose containing DMEM/F-12 medium were switched to glucose- and pyruvate-free DMEM/F-12 medium and then subjected to hypoxia for 1, 3, or 8 h. Under normoxic conditions only the uncleaved, proform of the enzyme was detected. Hypoxia gradually resulted in the appearance of the characteristic cleaved fragment of approximately 85 kDa, with a dramatic reduction in the amount of proform by 8 h (Fig.4 A). To test the possibility that PARP cleavage under hypoxic conditions was caspase-dependent, the cardiac myocytes were preincubated with either 100 μm Z-VAD-fmk, a broad spectrum caspase inhibitor, or 100 μm Z-DEVD-fmk, a more caspase 3-specific inhibitor. As expected, cleavage of PARP was significantly prevented by either caspase inhibitor, with a greater protective effect observed with Z-DEVD-fmk. The nonspecific control peptide Z-FA-fmk had no effect (Fig. 4 A). Genomic DNA isolated from cardiac myocytes subjected to hypoxia for 8 h in the absence of glucose showed distinct laddering confirming the presence of significant apoptosis. Preincubation of cells with the caspase inhibitors Z-VAD-fmk and Z-DEVD-fmk completely prevented DNA laddering (Fig. 4 B). The fact that glucose withdrawal led to significant apoptosis in the cultured cardiac myocytes in response to hypoxia prompted us to examine the role this substrate was playing in the apoptotic process. Readdition of glucose to the medium at a final concentration of 17.5 mm and then preincubation of cells under normoxic conditions for 30 min followed by hypoxia of 1, 3, and 8 h prevented apoptosis as demonstrated by the lack of PARP cleavage (Fig. 5), DNA laddering (Fig.4 B), as well as cell morphology (data not shown). To elucidate further the precise concentration at which glucose achieved its protective effect, a dose-response experiment was performed. Glucose at a concentration of ∼1.0 mm prevented PARP cleavage with a maximal effect reached at 7.5 mm glucose (Fig. 6).Figure 6Dose response of glucose protection of PARP cleavage in cardiac myocytes exposed to 8 h of hypoxia.Cardiac myocytes were preincubated with 0, 1, 3, 5, 7.5, 10, 12.5, 15, or 17.5 mm glucose for 30 min and then subjected to hypoxia for 8 h. Total cell lysates were analyzed for PARP cleavage by Western blot analyses. The data shown are representative of three independent experiments.View Large Image Figure ViewerDow
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