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Implications of Glucose Transporter Protein Type 1 (GLUT1)-Haplodeficiency in Embryonic Stem Cells for Their Survival in Response to Hypoxic Stress

2003; Elsevier BV; Volume: 163; Issue: 5 Linguagem: Inglês

10.1016/s0002-9440(10)63546-8

1525-2191

Charles M. Heilig, Frank C. Brosius, Brian Siu, Luis Concepcion, Richard M. Mortensen, Kathleen Heilig, Min Zhu, Richard Weldon, Guimei Wu, David A. Conner,

Renal and related cancers

Glucose transporter protein type 1 (GLUT1) is a major glucose transporter of the fertilized egg and preimplantation embryo. Haploinsufficiency for GLUT1 causes the GLUT1 deficiency syndrome in humans, however the embryo appears unaffected. Therefore, here we produced heterozygous GLUT1 knockout murine embryonic stem cells (GT1+/−) to study the role of GLUT1 deficiency in their growth, glucose metabolism, and survival in response to hypoxic stress. GT1(−/−) cells were determined to be nonviable. Both the GLUT1 and GLUT3 high-affinity, facilitative glucose transporters were expressed in GT1(+/+) and GT1(+/−) embryonic stem cells. GT1(+/−) demonstrated 49 ± 4% reduction of GLUT1 mRNA. This induced a posttranscriptional, GLUT1 compensatory response resulting in 24 ± 4% reduction of GLUT1 protein. GLUT3 was unchanged. GLUT8 and GLUT12 were also expressed and unchanged in GT1(+/−). Stimulation of glycolysis by azide inhibition of oxidative phosphorylation was impaired by 44% in GT1(+/−), with impaired up-regulation of GLUT1 protein. Hypoxia for up to 4 hours led to 201% more apoptosis in GT1(+/−) than in GT1(+/+) controls. Caspase-3 activity was 76% higher in GT1(+/−) versus GT1(+/+) at 2 hours. Heterozygous knockout of GLUT1 led to a partial GLUT1 compensatory response protecting nonstressed cells. However, inhibition of oxidative phosphorylation and hypoxia both exposed their increased susceptibility to these stresses. Glucose transporter protein type 1 (GLUT1) is a major glucose transporter of the fertilized egg and preimplantation embryo. Haploinsufficiency for GLUT1 causes the GLUT1 deficiency syndrome in humans, however the embryo appears unaffected. Therefore, here we produced heterozygous GLUT1 knockout murine embryonic stem cells (GT1+/−) to study the role of GLUT1 deficiency in their growth, glucose metabolism, and survival in response to hypoxic stress. GT1(−/−) cells were determined to be nonviable. Both the GLUT1 and GLUT3 high-affinity, facilitative glucose transporters were expressed in GT1(+/+) and GT1(+/−) embryonic stem cells. GT1(+/−) demonstrated 49 ± 4% reduction of GLUT1 mRNA. This induced a posttranscriptional, GLUT1 compensatory response resulting in 24 ± 4% reduction of GLUT1 protein. GLUT3 was unchanged. GLUT8 and GLUT12 were also expressed and unchanged in GT1(+/−). Stimulation of glycolysis by azide inhibition of oxidative phosphorylation was impaired by 44% in GT1(+/−), with impaired up-regulation of GLUT1 protein. Hypoxia for up to 4 hours led to 201% more apoptosis in GT1(+/−) than in GT1(+/+) controls. Caspase-3 activity was 76% higher in GT1(+/−) versus GT1(+/+) at 2 hours. Heterozygous knockout of GLUT1 led to a partial GLUT1 compensatory response protecting nonstressed cells. However, inhibition of oxidative phosphorylation and hypoxia both exposed their increased susceptibility to these stresses. The first facilitative glucose transporter identified, characterized, and cloned was the HepG2 cell/erythrocyte/brain glucose transporter protein type 1 (GLUT1).1Fukumoto H Seino S Hiro I Seino Y Bell G Characterization and expression of human HepG2 /erythrocyte glucose transporter gene.Diabetes. 1988; 37: 657-661Crossref PubMed Scopus (125) Google Scholar This is now the best characterized of 12 identified GLUT isoforms.2Joost HG Bell GI Best JD Birnbaum M Charron M Chen Y Doege H James D Lodish H Moley K Moley J Mueckler M Rogers S Schurmann A Seino S Thorens B Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators.Am J Physiol. 2002; 282: E974-E976Google Scholar GLUT1 is believed to play a key role in maintaining basal glucose uptake for metabolism in many cell types.3Bell GI Burant CF Takeda J Gould GW Structure and function of mammalian facilitative sugar transporters.J Biol Chem. 1993; 268: 19161-19164Abstract Full Text PDF PubMed Google Scholar, 4Devaskar S Mueckler M The mammalian glucose transporters.Pediatr Res. 1992; 31: 1-13Crossref PubMed Scopus (127) Google Scholar, 5Gould GW Holman GD The glucose transporter family: structure, function and tissue-specific expression.Biochem J. 1993; 295: 329-341Crossref PubMed Scopus (659) Google Scholar, 6Thorens B Charron MJ Lodish HF Molecular physiology of glucose transporters.Diabetes Care. 1990; 13: 209-218Crossref PubMed Scopus (182) Google Scholar Its expression is detectable throughout preimplantation development from the oocyte through the blastocyst stage,7Morita Y Tsutsumi O Oka Y Taketani Y Glucose transporter GLUT1 mRNA expression in the ontogeny of glucose incorporation in mouse preimplantation embryos.Biochem Biophys Res Commun. 1994; 199: 1525-1531Crossref PubMed Scopus (48) Google Scholar, 8Hogan A Heyner S Charron MJ Copeland N Gilbert D Jenkins N Thorens B Schultz G Glucose transporter gene expression in early mouse embryos.Development. 1991; 113: 363-372PubMed Google Scholar, 9Aghayan M Rao LV Smith RM Jarett L Charron M Thorens B Heyner S Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development.Development. 1992; 115: 305-312PubMed Google Scholar and it increases 11-fold in developing embryos from the two-cell stage to the blastocyst.7Morita Y Tsutsumi O Oka Y Taketani Y Glucose transporter GLUT1 mRNA expression in the ontogeny of glucose incorporation in mouse preimplantation embryos.Biochem Biophys Res Commun. 1994; 199: 1525-1531Crossref PubMed Scopus (48) Google Scholar This suggests it plays an important nutritional role in development, and previous studies indicated GLUT1 may be important in supplying glucose for the glycolytic pathway.10Bashan N Burdett E Hundal H Klip A Regulation of glucose transport and GLUT1 glucose transporter expression by O2 in muscle cells in culture.Am J Physiol. 1992; 262: C682-C690PubMed Google Scholar, 11Shetty M Ismail-Beigi N Loeb JN Ismail-Beigi F Induction of GLUT1 mRNA in response to inhibition of oxidative phosphorylation.Am J Physiol. 1993; 265: C1224-C1229PubMed Google Scholar, 12Shetty M Loeb JN Vikstrom K Ismail-Beigi F Rapid activation of GLUT-1 glucose transporter following inhibition of oxidative phosphorylation in clone 9 cells.J Biol Chem. 1993; 268: 17225-17232Abstract Full Text PDF PubMed Google Scholar, 13Shetty M Loeb JN Ismail-Beigi F Enhancement of glucose transport in response to inhibition of oxidative metabolism: pre- and posttranslational mechanisms.Am J Physiol. 1992; 262: C527-C532PubMed Google Scholar, 14Heilig C Zaloga C Lee M Zhao X Riser B Cortes P Immunogold localization of high affinity GLUT isoforms in normal rat kidney.Lab Invest. 1995; 73: 674-684PubMed Google Scholar Another high-affinity glucose transporter expressed in embryos at the blastocyst stage is GLUT 3, although this transporter is not present at the beginning of gestation in rodents. GLUT3 may also supply glucose for maintenance of cell nutrition.3Bell GI Burant CF Takeda J Gould GW Structure and function of mammalian facilitative sugar transporters.J Biol Chem. 1993; 268: 19161-19164Abstract Full Text PDF PubMed Google Scholar, 5Gould GW Holman GD The glucose transporter family: structure, function and tissue-specific expression.Biochem J. 1993; 295: 329-341Crossref PubMed Scopus (659) Google Scholar, 15Kayano T Fukumoto H Eddy RL Fan Y Byers M Shows T Bell G Evidence for a family of human glucose transporter-like proteins. Sequence and gene localization of a protein expressed in fetal skeletal muscle and other tissues.J Biol Chem. 1988; 263: 15245-15248Abstract Full Text PDF PubMed Google Scholar, 16Chih CP Lipton P Roberts Jr, EL Do active cerebral neurons really use lactate rather than glucose?.Trends Neurosci. 2001; 24: 573-578Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar The tissue-specific expressions and different kinetic properties of GLUT isoforms indicate their potential for unique roles in cell glucose metabolism, and this has stimulated further research to determine their roles in normal and neoplastic growth, diabetes, apoptosis, and other conditions.3Bell GI Burant CF Takeda J Gould GW Structure and function of mammalian facilitative sugar transporters.J Biol Chem. 1993; 268: 19161-19164Abstract Full Text PDF PubMed Google Scholar, 5Gould GW Holman GD The glucose transporter family: structure, function and tissue-specific expression.Biochem J. 1993; 295: 329-341Crossref PubMed Scopus (659) Google Scholar, 17Boden G Murer E Mozzoli M Glucose transporter proteins in human insulinoma.Ann Intern Med. 1994; 121: 109-112Crossref PubMed Scopus (35) Google Scholar, 18Flier JS Mueckler MM Usher P Lodish HF Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes.Science. 1987; 235: 1492-1495Crossref PubMed Scopus (693) Google Scholar, 19Kahn B Facilitative glucose transporters: regulatory mechanisms and dysregulation by diabetes.J Clin Invest. 1992; 13: 548-564Google Scholar, 20Vander Heiden MG Plas DR Rathmell JC Fox CJ Harris MH Thompson CB Growth factors can influence cell growth and survival through effects on glucose metabolism.Mol Cell Biol. 2001; 21: 5899-5912Crossref PubMed Scopus (436) Google Scholar, 21Greene D Lattimer S Altered sorbitol and myo-inositol metabolism as the basis for defective protein kinase C and sodium-potassium ATPase regulation in diabetic neuropathy.Ann NY Acad Sci. 1986; : 334-340Crossref PubMed Google Scholar In the GLUT1 deficiency syndrome (GLUT1-DS), also known as glucose transporter protein syndrome,22Klepper J Wang D Fischbarg J Vera J Jarjour I O'Driscoll K De Vivo D Defective glucose transport across brain tissue barriers: a newly recognized neurological syndrome.Neurochem Res. 1999; 24: 587-594Crossref PubMed Scopus (89) Google Scholar affected individuals have haploinsufficiency for GLUT123Seidner G Alvarez MG Yeh JI O'Driscoll K Klepper J Stump T Wang D Spinner N Birnbaum M De Vivo D GLUT-1 deficiency syndrome caused by haploinsufficiency of the blood-brain barrier hexose carrier.Nat Genet. 1998; 18: 188-191Crossref PubMed Scopus (317) Google Scholar, 24Klepper J Voit T Facilitated glucose transporter protein type 1 (GLUT1) deficiency syndrome: impaired glucose transport into brain—a review.Eur J Pediatr. 2002; 161: 295-304Crossref PubMed Scopus (152) Google Scholar because of missense, nonsense, splice site, insertional, or deletional mutations.25Wang D Kranz-Eble P De Vivo DC Mutational analysis of GLUT1 (SLC2A1) in Glut-1 deficiency syndrome.Hum Mutat. 2000; 16: 224-231Crossref PubMed Scopus (131) Google Scholar The disorder manifests from childhood onwards, with developmental impairment and seizures because of hypoglycorrachia.24Klepper J Voit T Facilitated glucose transporter protein type 1 (GLUT1) deficiency syndrome: impaired glucose transport into brain—a review.Eur J Pediatr. 2002; 161: 295-304Crossref PubMed Scopus (152) Google Scholar The ability of the GLUT1-DS embryos to survive gestation without obvious impairment suggests a compensatory mechanism exists to allow this. Although GLUT1-DS infants typically appear normal at birth, they may later exhibit childhood seizures of multiple different types, developmental delay, acquired microcephaly, and a variety of motor disturbances with impaired coordination.23Seidner G Alvarez MG Yeh JI O'Driscoll K Klepper J Stump T Wang D Spinner N Birnbaum M De Vivo D GLUT-1 deficiency syndrome caused by haploinsufficiency of the blood-brain barrier hexose carrier.Nat Genet. 1998; 18: 188-191Crossref PubMed Scopus (317) Google Scholar, 26De Vivo DC Trifiletti RR Jacobson RI Ronen GM Behmand RA Harik SI Defective glucose transport across the blood-brain barrier as a cause of persistent hypoglycorrhachia, seizures, and developmental delay.N Engl J Med. 1991; 325: 703-709Crossref PubMed Scopus (628) Google Scholar Therefore, in the current report we have produced heterozygous GLUT1 deficiency (gene knockout) in murine embryonic stem (ES) cells derived from the blastocyst stage embryo to characterize the responses of these ES cells to the deficiency. We identified a compensatory mechanism that could minimize damage to the nonstressed ES cells during gestation, and an impaired capacity of these ES cells to adapt to chemical inhibition of oxidative phosphorylation or hypoxia, exposing their vulnerability to such stresses. A partial digest SauI mouse genomic library from strain DBA-2J in phage vector EMBL-3 (Clontech, Palo Alto, CA) was screened by hybridization with a 32P-labeled human GLUT1 cDNA probe to isolate four partial GLUT1 clones, two overlapping. One of these DNA clones, later used to build gene-targeting constructs, was partially sequenced by the Sanger method (35S) with 5′ and 3′ 25-mer synthetic oligonucleotide primers, to confirm its identity as GLUT1. The GLUT1 genomic fragments were mapped by restriction fragment analysis to determine the plan for construction. HindIII restriction sites inside and 3′ to exon 6 were identified, which could be used to excise the 3′ portion of the exon, and create a mutant, nonfunctional coding sequence. The gene fragment was first subcloned into a plasmid vector pBS SKII+ (Stratagene, La Jolla, CA) that was modified by removing the HindIII cloning site. This allowed for excision of the HindIII fragment in the GLUT1 genomic insert, which is the 3′ portion of exon 6, without excising other portions of the genomic insert from the plasmid. A blunt-end phosphoglycerate kinase promoter-neomycin resistance gene (PGKneoR) cassette was ligated into the site where the 3′ end of exon 6 had been removed. Phosphoglycerate kinase promoter-thymidine kinase gene (PGKTK) was then excised by XbaI from its carrier plasmid pBS SKII+. This PGKTK cassette was then blunted and ligated to the 3′ end of the GLUT1 fragment. The entire construct then consisted of pBS SKII+ containing a GLUT1 genomic fragment with 5.7 kb of homology, plus a deletion mutation in exon 6, PGKneoR disrupting exon 6, and PGKTK at the 3′ end of the fragment. The construct was then linearized by XhoI at the 5′ end, leaving the carrier plasmid as a 3′ cap over PGKTK. This linearized gene-targeting construct was used to transfect ES cells by electroporation. A 1.66-kb human GLUT1 cDNA probe that binds to genomic GLUT1 DNA immediately 3′ to the point of insertion of the targeting construct was used as an adjacent probe to confirm insertion of the targeting construct inside the endogenous GLUT1 gene of ES cells after homologous recombination. HindIII restriction sites inside the construct and 3′ to the construct allowed excision of a 5.3-kb fragment containing both a portion of the targeting construct with PGKneoR inside it, and a 1.92-kb 3′ fragment of endogenous, ES cell genomic GLUT1 DNA. Both GLUT1 and PGKneoR 32P-labeled probes bound to this 5.3-kb band on the Southern blots, confirming the targeting construct had inserted within the GLUT1 gene (not shown). The relative quantities of targeted and nontargeted GLUT1 alleles as determined by optical scanning densitometry of the 3.4- and 5.3-kb bands, were identical as would be expected for a single copy gene with one normal allele and one targeted allele. Two additional targeted clones were identified similarly, for a targeting frequency of 3 of 450 or 0.67%. Examination of cell and colony morphology revealed no visible change in targeted ES cells at the light microscopic level. No visible evidence of cell differentiation was present in either nontargeted or targeted cells, and the latter continued to form the rounded, raised colonies typical of normal, totipotent ES cells. CC1.2 mouse ES cells (2 × 107) were transferred to 0.8 ml of Capecchi buffer27Thomas KR Capecchi MR Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.Cell. 1987; 51: 503-512Abstract Full Text PDF PubMed Scopus (1839) Google Scholar containing 1 pmol of the linearized HR construct. This mixture was transferred to a sterile electroporation cuvette (Bio-Rad Gene Pulser; Bio-Rad, Richmond, CA), where the cells were shocked with 400 V. They were then seeded to gelatinized culture plates and grown in selective media for isolation of G418- and Ganciclovir-resistant clones (positive to negative selection). Individual, G418- and Ganciclovir-resistant colonies were picked and transferred to separate wells of 24-well culture plates. The clones were grown until nearly confluent and then transferred to individual wells of six-well culture plates. Again the clones were allowed to become nearly confluent, whereupon they were divided into two fractions, one to be frozen, and one to be passed to new six-well plates. DNA was isolated from each clone by high-salt precipitation.28Ausebel F Brent R Kingston R Moore D Sedman J Smith J Struhl K Current Protocols in Molecular Biology. John Wiley & Sons, New York1994Google Scholar Southern analysis using a 32P-labeled human GLUT1 cDNA probe that bound to genomic GLUT1 DNA adjacent (3′) to the insertion site of the targeting construct, resulted in the appearance of a new band of larger size (5.3 kb versus 3.4 kb), which was used to verify homologous recombination within the GLUT1 gene. The remaining, nontargeted GLUT1 allele at 3.4 kb was also identified by the probe as expected. The corresponding targeted cell clones were retrieved from the freezer and their DNA reanalyzed by Southern analysis to confirm the targeting events. Cells were seeded at a density of 5 × 105 cells per 150-mm culture plate, and grown until 90 to 100% confluent (7 days). On the final day of growth total RNA was harvested using a commercial preparation of guanidinium and phenol (RNA STAT-60; Tel-Test Inc., Friendswood, TX). The total RNA from each sample was isolated by following the manufacturer's instructions. RNA was resuspended in diethyl pyrocarbonate-treated double-distilled water, and stored at −80°C until use. RNA samples (20 μg each) were denatured in glyoxal/dimethyl sulfoxide at 55°C for 1 hour, then loaded to individual wells of a 10 mmol/L sodium phosphate/1% agarose gel. Gels were run at 90 V overnight. Subsequently, they were stained with ethidium bromide, destained, and photographed. Integrity and equal lane loading of RNA was confirmed by inspection of ribosomal RNA bands. Gels were blotted to Genescreen membranes (Perkin Elmer Life Sciences, Boston, MA) for 36 hours with 10× standard saline citrate using a standard method28Ausebel F Brent R Kingston R Moore D Sedman J Smith J Struhl K Current Protocols in Molecular Biology. John Wiley & Sons, New York1994Google Scholar (1× standard saline citrate = 150mmol/L NaCl, 15mmol/L sodium citrate, pH 7.0). Blots were then UV-fixed, prehybridized, and probed for individual GLUT isoforms and β-tubulin, using the respective cDNAs. The latter were 32P-labeled by the random hexamer priming method (PRIME-1 kit; Sigma-Aldrich, St. Louis, MO). Blots were washed in 2× standard saline citrate with 1% sodium dodecyl sulfate at room temperature for 30 minutes for two times, then in 0.2× standard saline citrate with 1% sodium dodecyl sulfate at 55°C for 20 minutes for two times. They were then exposed to Kodak XAR-5 film (Eastman-Kodak, Rochester, NY) for periods of 3 to 14 days, and autoradiograms were analyzed by optical scanning densitometry (Howtek, ScanMaster 3+; Howtek, Inc., Hudson, NH) with the NIH Image gel plotting software (version 1.52; National Technical Information Service, Springfield, VA). Relative quantities of GLUT mRNAs in nontargeted versus targeted ES cells were compared after normalization to mRNA for the housekeeping gene, β-tubulin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (10%) were used to run 100 μg of total protein per lane, as determined for each sample by the Lowry assay.29Lowry O Rosebrough N Farr A Randall R Protein measurement with the folin phenol reagent.J Biol Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar Equal lane loading was further confirmed on some immunoblots with antibody to β-tubulin or by quantitative copper/silver staining. A chemiluminescent immunoblot assay (ECL Western blot kit; Amersham Life Sciences, Buckinghamshire, UK) was used in which luminol was used to detect GLUT protein bands identified by specific, rabbit anti-rat GLUT1 (reacts also with mouse GLUT1) and rabbit anti-mouse GLUT3 polyclonal antibodies (East Acres Biologicals, Southbridge, MA). These antibodies are directed against the 12 C-terminal peptides of the GLUT1 and GLUT3 proteins. Identification of GLUT1 and GLUT3 bands on immunoblots was confirmed by determination of band size, followed by competition experiments in which preadsorption of either the GLUT1 or GLUT3 antibody with the respective C-terminal peptide antigen was observed to block appearance of the band. Additional antibodies for GLUTs 4, 8, 9, and 12 were also obtained for immunoblotting in GT1(+/+) and GT1(+/−) cells. The GLUT4 and GLUT8 antibodies were obtained from Dr. Maureen Charron, Albert Einstein School of Medicine, Bronx, New York; the GLUT9 antibody was obtained from Dr.'s Jeffrey and Kelle Moley, Washington University, St. Louis, MO; and the GLUT12 antibody was obtained from A.D.I., Inc. (Alpha Diagnostics Inc., San Antonio, TX). Detection of GLUT8 protein required modification of our standard immunoblotting protocol as described by Dr. M. Charron's laboratory. Addition of a combination of protease inhibitors to the extraction buffer was important because of the potential for rapid degradation of GLUT8 protein: aprotinin (2 μg/ml), leupeptin (5 μg/ml), and pepstatin (0.5 μg/ml) were included along with phenylmethyl sulfonyl fluoride (34 mg/ml) in this solution. In addition, dithiothreitol was added to the loading dye solution (400 μl dithiothreitol per 600 μl loading dye solution to make a 4× loading mix), to take advantage of the stabilizing effect reducing agents have on GLUT8. Samples for GLUT8 were incubated at 55°C for 1 hour in the presence of the loading mix. GLUT8 detection was performed with a rabbit polyclonal GLUT8 antibody directed against the C-terminal 11 amino acids,30Reagan LP Gorovits N Hoskin EK Alves S Katz E Grillo C Piroli G Localization and regulation of GLUTx1 glucose transporter in the hippocampus of streptozotocin diabetic rats.Proc Natl Acad Sci USA. 2001; 98: 2820-2825Crossref PubMed Scopus (91) Google Scholar using the Pierce ECL Detection Kit following the manufacturer's instructions (Pierce, Rockford, IL). Cycloheximide (10 μg/ml = 36 μmol/L; Sigma-Aldrich) was used to inhibit protein synthesis as previously described.31Karnieli E Garvey WT Olefsky JM Hueckstead T Harel C Maianu L Armoni M Potential role for insulin and cycloheximide in regulating the intrinsic activity of glucose transporters in isolated rat adipocytes.Endocrinology. 1993; 133: 2943-2950Crossref PubMed Scopus (13) Google Scholar The decay curves and half-lives of GLUT1 in targeted and nontargeted cells were determined by quantitating changes in GLUT1 protein on immunoblots at preselected time points after addition of cycloheximide to the standard growth medium. The 3H2-DOG uptake rates were determined in targeted and nontargeted ES cells using a modification of the method of Haneda and colleagues.32Haneda M Kikkawa R Togawa M Koya D Kajiwara N Shigeta Y Metabolic actions of insulin-like growth factor-1 in cultured glomerular mesangial cells.Metabolism. 1991; 40: 1311-1316Abstract Full Text PDF PubMed Scopus (9) Google Scholar ES cells were seeded to 35-mm culture plates at 50% confluence and allowed to attach for 24 hours. They were then washed twice with phosphate-buffered saline (PBS), and 1 ml of 3H2-DOG (Perkin Elmer Life Sciences) in PBS (3.27 pmol, 0.1 μCi/ml) was added to each plate for 5 minutes, followed by rapid aspiration of the isotope, and two washes with PBS. Cells from individual plates were then dissolved in 1 ml of 1 N NaOH, one third of which went for protein measurements, and two thirds of which were transferred to scintillation vials for counting. Preliminary time course experiments indicated that the 5-minute time point was within the linear portion of the uptake curve. Dose-response experiments of the 5-minute glucose uptake rates versus medium glucose concentration were used to generate Lineweaver-Burk plots from which Km and Vmax values were determined for ES cell glucose transporters in the two cell types. Cells (1 × 104) were seeded to 0.79-cm2 wells of gelatinized eight-well glass chamber slides and allowed to grow from 1 to 15 days. Cell counts were performed and averaged on four wells for each cell type and day, on multiple days throughout this 15-day period. The growth curves of nontargeted and targeted cells were then compared. Sodium azide (5 mmol/L, Sigma-Aldrich) was added to media of cells for 12 hours as previously described.11Shetty M Ismail-Beigi N Loeb JN Ismail-Beigi F Induction of GLUT1 mRNA in response to inhibition of oxidative phosphorylation.Am J Physiol. 1993; 265: C1224-C1229PubMed Google Scholar, 12Shetty M Loeb JN Vikstrom K Ismail-Beigi F Rapid activation of GLUT-1 glucose transporter following inhibition of oxidative phosphorylation in clone 9 cells.J Biol Chem. 1993; 268: 17225-17232Abstract Full Text PDF PubMed Google Scholar This compound inhibits oxidative phosphorylation and thereby stimulates glycolysis. A modification of the method of Shetty and colleagues12Shetty M Loeb JN Vikstrom K Ismail-Beigi F Rapid activation of GLUT-1 glucose transporter following inhibition of oxidative phosphorylation in clone 9 cells.J Biol Chem. 1993; 268: 17225-17232Abstract Full Text PDF PubMed Google Scholar was used, and media was collected after 12 hours for measurements of lactate concentration. Equal numbers of cells were seeded to each 150-mm plate (5 × 105). Lactate release to the medium was determined from total lactate measurements by subtracting the baseline concentration of lactate in the medium. Lactate measurements were expressed as μmol/mg cell protein. All lactate measurements were performed with a standard assay kit (Sigma-Aldrich), following the manufacturer's instructions. Cell-culture dishes (60 mm) were coated with 0.1% gelatin for 30 minutes, then the excess gelatin was removed and the dishes allowed to dry for 30 minutes. ES cells (8 × 104) were then seeded to the dishes with 2 ml of media. They were allowed to grow to 30 to 50% confluence, then the media was changed and 1 ml of fresh media added. Hypoxia was induced by placing the cells in a Plexiglas chamber with a continuous flow of water-saturated 95% N2 and 5% CO2 maintained over the cells at 37°C.33Malhotra R Brosius FC Glucose uptake and glycolysis reduce hypoxia-induced apoptosis in cultured neonatal rat cardiac myocytes.J Biol Chem. 1999; 274: 12567-12575Crossref PubMed Scopus (199) Google Scholar The PO2 was lowered to less than 5 mmHg by adding Oxyrase (bacterial monooxygenases and dioxygenases; Oxyrase Inc., Ashland, OH) to the culture medium at a final concentration of 6% as previously described.33Malhotra R Brosius FC Glucose uptake and glycolysis reduce hypoxia-induced apoptosis in cultured neonatal rat cardiac myocytes.J Biol Chem. 1999; 274: 12567-12575Crossref PubMed Scopus (199) Google Scholar The control cells were placed in the regular cell-culture incubator at 37°C where they were exposed to air plus 5% CO2 without oxyrase. The cells were incubated for 1, 2, and 4 hours, before propidium iodide staining and the terminal dUTP nick-end labeling (TUNEL) assay to assess them for apoptosis. At the selected time points (1, 2, and 4 hours), the media was collected from the dishes and transferred to a 15-ml tube on ice. Then 0.5 ml of trypsin was added to the dishes for 3 to 5 minutes at room temperature to release the cells. One ml of media was then added to the cells to inhibit the trypsin effect, and the cells were transferred to the 15-ml tube on ice to be combined with the previous media. The cells were then centrifuged at 1000 rpm for 1 minute at 4°C in a Beckman centrifuge with a GS-6R rotor. Next, they were resuspended in 30% methanol with PBS for 5 minutes. The cells were then centrifuged again at 1000 rpm for 1 minute at 4°C. They were resuspended in 100 μl of 1:1000 propidium iodide (PI) (no. P-4170, Sigma Chemical Co.) in PBS and stained for 1 minute. Subsequently, 20 to 30 μl of this mix was placed on a glass slide with coverslip and the edges sealed with Permount (ProSciTech, Kelso, Australia). The cells were then counted and the percentage of cells with apoptotic nuclear morphology, including condensed and fragmented nuclei as previously described,34Welham SJ Wade A Woolf AS Protein restriction in pregnancy is associated with increased apoptosis of mesenchymal cells at the start of rat metanephrogenesis.Kidney Int. 2002; 61: 1231-1242Crossref PubMed Scopus (154) Google Scholar was determined for GLUT1-targeted (GT1+/−) and nontargeted (GT1+/+) ES cells exposed to hypoxic and normoxic conditions. The TUNEL assay was performed using a commercial assay on adherent cells after paraformaldehyde fixation and permeabilization, following the manufacturer's instructions (Roche Diagnostics, Basel, Switzerland). The incorporated fluorescein-dUTP in the assay allowed detection of TUNEL-positive ES cells by fluorescence microscopy. The percentage of TUNEL-positive cells was determined for GLUT1-targeted (GT1 +/−) and nontargeted (GT1 +/+) ES cells with a 1-hour exposure to hypoxia or normoxia. Measurements of ES cell caspase-3 activity were performed using a previously published method35Zou H Henzel WJ Liu X Lutschg A Wang X Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3.Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2748) Google Scholar to complement the propidium iodide and TUNEL apoptosis assays. ES cells were plated into 60-mm tissue-culture dishes (0.1% gelatin-coated). Sub