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• W2058618870 abstract "Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1–5 and SGLT1. The apparentK m of DHA transport via GLUT1 and GLUT3 was 1.1 ± 0.2 and 1.7 ± 0.3 mm, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2–4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, d-glucose, and 3-O-methylglucose among other hexoses while fructose and l-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-α-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2–8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA. Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1–5 and SGLT1. The apparentK m of DHA transport via GLUT1 and GLUT3 was 1.1 ± 0.2 and 1.7 ± 0.3 mm, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2–4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, d-glucose, and 3-O-methylglucose among other hexoses while fructose and l-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-α-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2–8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA. Ascorbate (AA) 1The abbreviations used are: AA, ascorbic acid; 2-DG, 2-deoxyglucose; DHA, dehydroascorbic acid; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; HPLC, high performance liquid chromatography; 2-DG, 2-deoxyglucose; CHAPS, 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonic acid. is transported across cellular membranes by two distinct mechanisms. Ascorbate itself is transported by a sodium-dependent saturable transporter which has not been isolated (1Stevenson N.R. Gastroenterology. 1974; 67: 952-956Abstract Full Text PDF PubMed Google Scholar, 2Thorn N.A. Nielsen F.S. Jeppesen C.K. Acta Physiol. Scand. 1991; 141: 97-106Crossref PubMed Scopus (11) Google Scholar, 3Wright J.R. Castranova V. Colby H.D. Miles P.R. J. Appl. Physiol. 1981; 51: 1477-1483Crossref PubMed Scopus (16) Google Scholar, 4Washko P. Rotrosen D. Levine M. J. Biol. Chem. 1989; 264: 18996-19002Abstract Full Text PDF PubMed Google Scholar, 5Dixon S.J. Wilson J.X. J. Bone Miner. Res. 1992; 7: 675-681Crossref PubMed Scopus (30) Google Scholar, 6Welch R.W. Bergsten P. Butler J.D. Levine M. Biochem. J. 1993; 294: 505-510Crossref PubMed Scopus (63) Google Scholar, 7Welch R.W. Wang Y. Crossman Jr., A. Park J.B. Kirk K.L. Levine M. J. Biol. Chem. 1995; 270: 12584-12592Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 8Bergsten P. Yu R. Kehrl J. Levine M. Arch. Biochem. Biophys. 1995; 317: 208-214Crossref PubMed Scopus (44) Google Scholar). Ascorbate outside cells can be oxidized to dehydroascorbic acid (DHA), which is transported by a different mechanism (7Welch R.W. Wang Y. Crossman Jr., A. Park J.B. Kirk K.L. Levine M. J. Biol. Chem. 1995; 270: 12584-12592Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 9Heath H. Fiddick R. Exp. Eye Res. 1966; 5: 156-163Crossref PubMed Scopus (28) Google Scholar, 10Bianchi J. Wilson F.A. Rose R.C. Am. J. Physiol. 1986; 250: G461-G468PubMed Google Scholar, 11Helbig H. Korbmacher C. Wohlfarth J. Berweck S. Kuhner D. Wiederholt M. Am. J. Physiol. 1989; 256: C44-C49Crossref PubMed Google Scholar, 12Kern H.L. Zolot S.L. Curr. Eye Res. 1987; 6: 885-896Crossref PubMed Scopus (42) Google Scholar, 13Ingermann R.L. Stankova L. Bigley R.H. Am. J. Physiol. 1986; 250: C637-C641Crossref PubMed Google Scholar, 14Vera J.C. Rivas C.I. Zhang R.H. Farber C.M. Golde D.W. Blood. 1994; 84: 1628-1634Crossref PubMed Google Scholar). Once within cells, dehydroascorbic acid is immediately reduced to ascorbate by both chemical and protein mediated processes (15Washko P.W. Wang Y. Levine M. J. Biol. Chem. 1993; 268: 15531-15535Abstract Full Text PDF PubMed Google Scholar, 16Winkler B.S. Orselli S.M. Rex T.S. Free Rad. Biol. Med. 1994; 17: 333-349Crossref PubMed Scopus (441) Google Scholar, 17Wells W.W. Xu D.P. J. Bioenerg. Biomembr. 1994; 26: 369-377Crossref PubMed Scopus (114) Google Scholar, 18Park J.B. Levine M. Biochem. J. 1996; 315: 931-938Crossref PubMed Scopus (110) Google Scholar). Dehydroascorbic acid is structurally similar to glucose. Therefore, DHA entry has been proposed to be mediated by glucose transporters (12Kern H.L. Zolot S.L. Curr. Eye Res. 1987; 6: 885-896Crossref PubMed Scopus (42) Google Scholar, 13Ingermann R.L. Stankova L. Bigley R.H. Am. J. Physiol. 1986; 250: C637-C641Crossref PubMed Google Scholar,19Mann G.V. Newton P. Ann. N. Y. Acad. Sci. 1975; 258: 243-252Crossref PubMed Scopus (124) Google Scholar, 20Bigley R. Wirth M. Layman D. Riddle M. Stankova L. Diabetes. 1983; 32: 545-548Crossref PubMed Scopus (93) Google Scholar). Despite investigations in several cell types, this hypothesis has not been proven. The ideal means to verify it is to express glucose transporters using an expression system, and to study DHA transport activity. If any transporters were active, transport kinetics could be characterized only under conditions of 100% internal reduction to ascorbate, consistent with DHA transport into cells being rate-limiting (7Welch R.W. Wang Y. Crossman Jr., A. Park J.B. Kirk K.L. Levine M. J. Biol. Chem. 1995; 270: 12584-12592Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). If internal DHA reduction were incomplete, kinetics could not be calculated. Although one study characterized DHA transport by expressed GLUT1 (21Vera J.C. Rivas C.I. Fischbarg J. Golde D.W. Nature. 1993; 364: 79-82Crossref PubMed Scopus (448) Google Scholar), there were a number of flaws in this report. Experiments were performed using mixtures of ascorbic acid and ascorbic acid oxidase instead of pure DHA as substrate. There was insufficient data about internal DHA reduction at each external DHA concentration, and calculations of high affinity transport were based on incorrect mathematical assumptions. In addition, although DHA transport was attributed to GLUT2 and GLUT4 as well, no data were presented to support these conclusions. To characterize dehydroascorbic acid and ascorbate transport we utilized a Xenopus laevis oocyte expression system to express glucose transport isoforms GLUT1–5 (22Birnbaum M.J. Haspel H.C. Rosen O.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5784-5788Crossref PubMed Scopus (438) Google Scholar, 23Fukumoto H. Seino S. Imura H. Seino Y. Eddy R.L. Fukushima Y. Byers M.G. Shows T.B. Bell G.I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5434-5438Crossref PubMed Scopus (369) Google Scholar, 24Kayano T. Fukumoto H. Eddy R.L. Fan Y.-S. Byers M.G. Shows T.B. Bell G.I. J. Biol. Chem. 1988; 263: 15245-15248Abstract Full Text PDF PubMed Google Scholar, 25Fukumoto H. Kayano T. Buse J.B. Edwards Y. Pilch P.F. Bell G.I. Seino S. J. Biol. Chem. 1989; 264: 7776-7779Abstract Full Text PDF PubMed Google Scholar, 26Kayano T. Burant C.F. Fukumoto H. Gould G.W. Fan Y. Eddy R.L. Byers M.G. Shows T.B. Seino S. Bell G.I. J. Biol. Chem. 1990; 265: 13276-13282Abstract Full Text PDF PubMed Google Scholar) and SGLT1 (27Hediger M.A. Coady M.J. Ikeda T.S. Wright E.M. Nature. 1987; 330: 379-381Crossref PubMed Scopus (809) Google Scholar). The data here indicate that DHA is transported by GLUT1 and GLUT3 but not other isoforms, while ascorbate is not transported by any of the proteins studied. Rat GLUT1 and human GLUT2, -3, -4, and -5 and mutant GLUT3 (Trp410 → Leu) were obtained as plasmid constructs from G. I. Bell (University of Chicago, Chicago, IL). Mutant GLUT1 (Gln161 → Leu) was obtained from M. Mueckler (28Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar) (Washington University, St. Louis, MO). Rabbit SGLT1 was obtained from E. M. Wright (27Hediger M.A. Coady M.J. Ikeda T.S. Wright E.M. Nature. 1987; 330: 379-381Crossref PubMed Scopus (809) Google Scholar) (University of California, Los Angeles, CA). GLUT1, -2, -4, -5, SGLT1, and mutant GLUT1 and GLUT3 plasmid constructs were described previously (25Fukumoto H. Kayano T. Buse J.B. Edwards Y. Pilch P.F. Bell G.I. Seino S. J. Biol. Chem. 1989; 264: 7776-7779Abstract Full Text PDF PubMed Google Scholar, 27Hediger M.A. Coady M.J. Ikeda T.S. Wright E.M. Nature. 1987; 330: 379-381Crossref PubMed Scopus (809) Google Scholar, 28Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar, 29Gould G.W. Lienhard G.E. Biochemistry. 1989; 28: 9447-9452Crossref PubMed Scopus (55) Google Scholar, 30Burant C.F. Bell G.I. Biochemistry. 1992; 31: 10414-10420Crossref PubMed Scopus (160) Google Scholar, 31Burant C.F. Takeda J. Brot-Laroche E. Bell G.I. Davidson N.O. J. Biol. Chem. 1992; 267: 14523-14526Abstract Full Text PDF PubMed Google Scholar). The GLUT3 construct is a 2153-base pair fragment of human GLUT3 generated by PCR and inserted into the AslI/BamHI site of pGEM4Z. mRNA was prepared from each construct in vitro by digesting plasmid vectors with appropriate restriction enzymes andin vitro transcription utilizing SP6 or T3 (mMessage mMachine, Ambion, Austin, TX). GLUT1–5 and SGLT1 were analyzed by enzymatic restriction fragment digestion (New England Biolabs, Beverly, MA). GLUT1 and GLUT3 constructs were further analyzed by PCR amplification. Primer pairs specific for GLUT1 (5′-GCCATGGAGCCCAGCAGCAAG-3′, 5′-CACTTGGGAATCAGCCCCCAG-3′) and GLUT3 (5′-ATGGGGACACAGAAGGTCACC-3′, 5′-GACATTGGTGGTGGTCTCCTT-3′) were used to amplify the coding sequence of GLUT1 and GLUT3 (1480 and 1488 base pairs, respectively). Oligonucleotides were synthesized using phosphoramidite chemistry (Lofstrand Labs, Gaithersburg MD). PCR conditions consisted of 25 cycles of 1 min at 95 °C/1 min at 56 °C/2 min at 72 °C, and 10 min at 72 °C. Primer pairs failed to amplify DNA from non-appropriate templates. Gel electrophoresis was performed utilizing 1% agarose (SeaKem, Rockland, ME) in TBE buffer. Reference markers used included 1Kb ladder (Life Technologies, Inc., Gaithersburg, MD) and φX174/HaeIII digest (New England Biolabs, Beverly, MA). Oocytes were isolated fromX. laevis and injected with mRNA using established methods (32Soreq H. Seidman S. Methods Enzymol. 1992; 207: 225-265Crossref PubMed Scopus (89) Google Scholar). Briefly, ovaries were resected from adult female frogs anesthetized with 3-aminobenzoic acid ethyl ester (2 g/750 ml) (Sigma) in ice water. Ovarian lobes were opened and incubated in two changes of OR-2 without calcium (5 mm HEPES, 82.5 mm NaCl, 2.5 mm KCl, 1 mm MgCl2, 1 mm Na2HPO4, 100 μg/ml gentamicin, pH 7.8) with collagenase (2 mg/ml) (Sigma) for 30 min each at 23 °C. Individual oocytes (stages V and VI) were isolated from connective tissue and vasculature and were transferred to calcium-containing OR-2 (1 mm CaCl2) and maintained at 18–20 °C until injection with mRNA. Oocytes were injected utilizing a pressure controlled injector (Eppendorf Transjector model #5246, Eppendorf, Hamburg, Germany). mRNA was backloaded into a capillary glass pipette, which had been pulled to a fine point using a micropipette puller (P-77, Sutter, Novato, CA). Injection volume was calibrated initially utilizing radiolabeled mRNA preparations. Injection volume was 30–50 nl and mRNA concentration was 0.07–1.0 mg/ml as indicated. After injection, oocytes were placed at 20 °C in OR-2 containing 1 mm pyruvate with daily media changes. Experiments were performed on day 3 after mRNA injection unless indicated. Chinese hamster ovary cells (CHO) transfected with rat GLUT1 (CHO:G15) or rat GLUT5 (CHO:F20) were obtained from Y. Oka (33Shibasaki Y. Asano T. Lin J.L. Tsukuda K. Katagiri H. Ishihara H. Yazaki Y. Oka Y. Biochem. J. 1992; 281: 829-834Crossref PubMed Scopus (36) Google Scholar, 34Asano T. Katagiri H. Takata K. Tsukuda K. Lin J.L. Ishihara H. Inukai K. Hirano H. Yazaki Y. Oka Y. Biochem. J. 1992; 288: 189-193Crossref PubMed Scopus (36) Google Scholar, 35Inukai K. Katagiri H. Takata K. Asano T. Anai M. Ishihara H. Nakazaki M. Kikuchi M. Yazaki Y. Oka Y. Endocrinology. 1995; 136: 4850-4857Crossref PubMed Scopus (41) Google Scholar) (University of Tokyo, Tokyo, Japan). Human GLUT3 transfected (CHO:G3) and wild-type non-transfected CHO cells (CHO:K1) were obtained from J. Takeda (36Maher F. Vannucci S. Takeda J. Simpson I.A. Biochem. Biophys. Res. Commun. 1992; 182: 703-711Crossref PubMed Scopus (123) Google Scholar) (University of Chicago, Chicago, IL). Wild-type, CHO:G15, and CHO:F20 cells were maintained in Ham's F-12 with 10% fetal calf serum and 1000 mg/ml penicillin/streptomycin. CHO:G3 cells were maintained in α-minimal essential medium containing 10% dialyzed fetal calf serum, 1000 mg/ml penicillin/streptomycin, 2 mm glutamine, and 100 nm methotrexate. [14C]DHA was prepared from crystalline [14C]ascorbic acid (NEN Life Sciences Products Inc., 6.6 mCi/mmol) as described (15Washko P.W. Wang Y. Levine M. J. Biol. Chem. 1993; 268: 15531-15535Abstract Full Text PDF PubMed Google Scholar). Briefly, 5 μl of bromine solution (Fluka, Ronkonkoma, NY) was added to 600 μl of [14C]ascorbic acid solubilized in ultrapure water at a concentration of 20 mm, vortexed briefly and immediately purged with nitrogen on ice in the dark for 10 min. HPLC with radiomatic detection confirmed 100% conversion of AA to DHA, which could be completely recovered upon reduction with 2,3-dimercapto-1-propanol (37Dhariwal K.R. Washko P.W. Levine M. Anal. Biochem. 1990; 189: 18-23Crossref PubMed Scopus (56) Google Scholar). Transport of [14C]AA, [14C]DHA, 2-deoxy-d-[1,2-3H]glucose (NEN Life Products, 26.2 Ci/mmol), d-[U-14C]fructose (NEN Life Sciences Products, 302 mCi/mmol), andd-[U-14C]glucose (NEN Life Sciences Products, 265 mCi/mmol) was examined by incubating groups of 10–20 oocytes at 23 °C in OR-2 containing different concentrations of freshly prepared [14C]AA or [14C]DHA (0.6–5.5 μCi/ml) or sugar (0.5–1.0 μCi/ml labeled sugar with added non-labeled sugar) for 15 s to 10 min. After incubation, oocytes were washed immediately 4 times with 200–400 volumes of ice-cold phosphate-buffered saline containing 0.1 mm phloretin. Inhibitors or competitors were added to the incubation as described in the text. Individual oocytes were either dissolved in 500 μl of 10% SDS and internalized radioactivity was measured using scintillation spectrometry, or oocytes were frozen to −70 °C in 50 μl of 60% MeOH, 1 mm EDTA for later HPLC analysis. To measure [14C]DHA or 2-[3H]deoxyglucose uptake in CHO cells, confluent cells in 12-well plates were washed 2 times with Krebs buffer (30 mm HEPES, 130 mmNaCl, 4 mm KH2PO4, 1 mmMgSO4, 1 mm CaCl2, pH 7.4), and incubated at 23 °C for 5 min with Krebs buffer containing different concentrations of substrate. Afterward, cells were washed 4 times with ice-cold phosphate-buffered saline containing 10 mmglucose, solubilized in 0.1 n NaOH, 1% CHAPS (Calbiochem-Novabiochem, La Jolla, CA) and radioactivity was measured by scintillation spectrometry. Protein content of wells was measured by spectrophotometry using bicinchoninic acid (BCA Protein Assay Reagent Kit, Pierce, Rockford IL). Oocyte total AA mass and internalized AA and DHA radioactivity were measured using HPLC. AA mass was determined using electrochemical detection (4Washko P. Rotrosen D. Levine M. J. Biol. Chem. 1989; 264: 18996-19002Abstract Full Text PDF PubMed Google Scholar). Internalized radioactivity was determined using the same HPLC system followed by on-line scintillation spectrometry (Packard Series A-120 Flo-one radiomatic detector (Downers Grove, IL)). Prior to HPLC analysis, oocytes previously frozen in 60% MeOH, 1 mm EDTA were thawed on ice, lysed by agitation with a pipette tip, and centrifuged at 14,000 rpm for 10 min. The supernatant was removed and analyzed by HPLC. MeOH was confirmed to extract 100% of AA and DHA from oocytes (data not shown). Data are expressed as the arithmetic mean ± S.D. of 10–20 oocytes at each data point, unless otherwise indicated. S.D. is not displayed when smaller than the symbol size. Transport kinetics were analyzed by best-fit analysis of data points utilizing curve-fitting (Jandel Scientific, San Rafael, CA) or Eadie-Hofstee transformation. IC50 values for DHA inhibition were determined by fitting data to a logit-log plot. We investigated DHA and ascorbate transport by GLUT1–5 and SGLT1. mRNA coding for the individual isoforms was injected intoXenopus oocytes and concentration-dependent DHA transport activity was assessed (Fig. 1). Radiolabeled 2-DG, fructose, or glucose uptake performed within the same experiment was a positive control for transporter activity. [14C]DHA transport in GLUT1 and GLUT3 expressing oocytes was over 100-fold greater than control sham-injected oocytes and similar to 2-[3H]DG transport on a mole for mole basis. DHA transport by oocytes expressing GLUT2, GLUT5, and SGLT1 did not differ from sham-injected controls. Oocytes expressing GLUT4 transported 2–4-fold more DHA than control, but 2-DG transport was an order of magnitude greater. Uptake of radiolabeled sugars for the different transporters was in the range expected (38Gould G.W. Holman G.D. Biochem. J. 1993; 295: 329-341Crossref PubMed Scopus (654) Google Scholar, 39Keller K. Mueckler M. Biomed. Biochim. Acta. 1990; 49: 1201-1203PubMed Google Scholar). Ascorbate transport by GLUT1–5 and SGLT1 was not different from sham-injected controls (<1 pmol/oocyte/10-min incubation) (data not shown). We verified the identity of the GLUT1 and GLUT3 cDNA constructs utilizing restriction digestion and PCR (Fig. 2). Restriction digestion of GLUT1 and GLUT3 insert DNA usingHindIII and EcoRI gave predicted fragments based on known sequences of GLUT1 and GLUT3 cDNA (22Birnbaum M.J. Haspel H.C. Rosen O.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5784-5788Crossref PubMed Scopus (438) Google Scholar, 24Kayano T. Fukumoto H. Eddy R.L. Fan Y.-S. Byers M.G. Shows T.B. Bell G.I. J. Biol. Chem. 1988; 263: 15245-15248Abstract Full Text PDF PubMed Google Scholar). In addition, PCR performed using cDNA primers specific for either GLUT1 or GLUT3 cDNA, produced DNA products only when the appropriate primers were used. The identities of the other glucose transporter constructs were also confirmed by restriction digest mapping (data not shown). Injection conditions for GLUT1 and GLUT3 mRNA were determined based on post-injection time and amount of mRNA injected. Variation in injection amount of either mRNA from 2 to 10 ng/oocyte resulted in a linear increase in transport activity for both DHA and 2-DG, and activity achieved plateau at 20–40 ng mRNA/oocyte (data not shown). Transport activity increased over time post-injection, with maximal activity occurring at 3–5 days (data not shown). To determine DHA transport kinetics internal reduction of DHA to AA must be complete and efflux of DHA should not occur. To establish these conditions, we first examined concentration-dependent [14C]DHA uptake (0.1–8 mm) over 10 min into oocytes injected with 30 ng of GLUT1 mRNA (21Vera J.C. Rivas C.I. Fischbarg J. Golde D.W. Nature. 1993; 364: 79-82Crossref PubMed Scopus (448) Google Scholar). Total radiolabeled uptake was measured, representing the sum of AA, DHA, and metabolites. The percentage of label present intracellularly as DHA, AA, or metabolites was also analyzed by HPLC and is displayed as % reduction to ascorbate. The results of total radiolabel data suggest that uptake saturated at 4 mm external DHA (Fig. 3). However, these observations can be explained by incomplete internal reduction of DHA to ascorbate (Fig. 3). Complete internal reduction occurred at [14C]DHA external concentrations ≤1 mm but reduction was incomplete at higher concentrations. [14C]DHA metabolites were only present when DHA reduction was incomplete (not shown). Under conditions of incomplete reduction transport kinetics cannot be calculated because reduction rather than transport becomes limiting. Similar results were obtained for GLUT3 (data not shown). Consistent with these observations, DHA efflux occurred from oocytes expressing GLUT1 or GLUT3 only when reduction was incomplete (data not shown). To achieve complete internal reduction, incubation time with substrates was decreased to ≤1 min for oocytes expressing either GLUT1 or GLUT3. For GLUT1 expressing oocytes, injected mRNA was also decreased to 2 ng/oocyte. Using these conditions, concentration-dependent DHA transport occurred in GLUT1 and GLUT3 expressing oocytes (Fig.4, A and B). At all concentrations of DHA, HPLC analysis confirmed 100% reduction of internalized label to AA (data not shown). For each concentration selected, uptake was linear with respect to time. Kinetic parameters of GLUT1- and GLUT3-mediated DHA transport were calculated using best-fit analysis and Eadie-Hofstee transformation (Fig. 4, A and B, inset). Using best-fit analysis, apparent K mwas 1.1 ± 0.2 mm and V max was 108 pmol/min/oocyte for GLUT1, and apparent K m was 1.7 ± 0.3 mm with V max of 241 pmol/min/oocyte for GLUT3. Eadie-Hofstee transformation yielded similar results. For GLUT1, apparent K m was 1.2 mm and V max was 124 pmol/min/oocyte, and for GLUT3 apparent K m was 1.1 mm andV max was 201 pmol/min/oocyte. We examined the ability of different sugars, ascorbic acid, and cytochalasins B and E to inhibit [14C]DHA transport in both GLUT1 and GLUT3 expressing oocytes (Table I). The relative ability of different sugars to inhibit DHA uptake was similar for both transport proteins (2-DG ≥ glucose ≥ 3-O-methylglucose > maltose > mannose > xylose). IC50 values were lower for GLUT3 for all of the sugars with an inhibitory effect. Cytochalasin B strongly inhibited DHA transport via both isoforms with an IC50 similar to that seen with transport of 2-[3H]DG under similar conditions (data not shown), while cytochalasin E had no effect. As expected, AA did not inhibit DHA uptake through either transporter.Table IInhibition (IC50(mm) of DHA transport in GLUT1 and GLUT3 expressing oocytesInhibitorTransporter isoformGLUT1GLUT32-Deoxyglucose7.14.9d-Glucose10.13.73-O-Methylglucose9.55.4Mannose24.58.5Xylose26.39.9Maltose>5020.9l-GlucoseNo effectNo effectFructoseNo effectNo effectAscorbic acidNo effectNo effectCytochalasin B0.00260.0018Cytochalasin ENo effectNo effectOocytes expressing either GLUT1 or GLUT3 were incubated with 150 μm [14C]DHA for 10 min at 23 °C in the presence of individual sugars, ascorbic acid (0.1–100 mm) or cytochalasin B or E (0.001–100 μm). Oocytes were then washed and internalized radioactivity was quantified as described under “Experimental Procedures.” IC50 was calculated by fitting data to a logit-lot plot. Open table in a new tab Oocytes expressing either GLUT1 or GLUT3 were incubated with 150 μm [14C]DHA for 10 min at 23 °C in the presence of individual sugars, ascorbic acid (0.1–100 mm) or cytochalasin B or E (0.001–100 μm). Oocytes were then washed and internalized radioactivity was quantified as described under “Experimental Procedures.” IC50 was calculated by fitting data to a logit-lot plot. Glucose transporters possess both endofacial and exofacial substrate-binding sites (40Carruthers A. Helgerson A.L. Biochemistry. 1991; 30: 3907-3915Crossref PubMed Scopus (70) Google Scholar). When DHA is present externally, internal DHA is absent under conditions of complete internal reduction. Therefore, we anticipated that exofacial and endofacial inhibitors of glucose transport would behave differently with DHA as the substrate. We predicted that the endofacial glucose transport inhibitor cytochalasin B (41Basketter D.A. Widdas W.F. J. Physiol. 1978; 278: 389-401Crossref PubMed Scopus (98) Google Scholar, 42Wang J. Falke J.J. Chan S.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3277-3281Crossref PubMed Scopus (25) Google Scholar) would behave as a non-competitive inhibitor of DHA transport, and that the exofacial inhibitor 4,6-O-ethylidene-α-glucose (43Barnett J.E.G. Holman G.D. Chalkley R.A. Munday K.A. Biochem. J. 1975; 145: 417-429Crossref PubMed Scopus (128) Google Scholar, 44Gorga F.R. Lienhard G.E. Biochemistry. 1981; 20: 5108-5113Crossref PubMed Scopus (106) Google Scholar) would behave as a competitive inhibitor. Oocytes expressing either GLUT1 or GLUT3 were incubated with increasing concentrations of DHA in the presence of either inhibitor under conditions of complete internal reduction (Fig.5). The results show ethylidene glucose was a competitive inhibitor of DHA uptake by GLUT1 and GLUT3. These data suggest that the external binding sites for ethylidene glucose and DHA are identical. Cytochalasin B inhibited DHA transport by GLUT1 and GLUT3 non-competitively. Glucose transporter mutants, previously constructed with single amino acid substitutions in domains believed to be important for glucose transport, were demonstrated to be defective in their ability to transport 2-DG (28Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar, 30Burant C.F. Bell G.I. Biochemistry. 1992; 31: 10414-10420Crossref PubMed Scopus (160) Google Scholar). If similar sites are involved in both DHA and 2-DG transport, transport of either substrate by these mutant constructs should be similar. We measured the ability of two mutants, GLUT1161 (Gln161 → Leu) and GLUT3410 (Trp410 → Leu), to transport 2-[3H]DG or [14C]DHA (TableII). Transport of both substrates via GLUT1 and GLUT3 was >100-fold higher than control and was virtually eliminated by the mutations. Western blotting demonstrated that both mutant proteins were present in the plasma membrane (data not shown).Table IIDHA and 2-DG transport in oocytes expressing glucose isoform mutants (pmol/oocyte/10 min)[14C]DHA2-[3H]DeoxyglucoseControl0.57 ± 0.50.65 ± 0.4GLUT1197.5 ± 61.1139.3 ± 18.6GLUT11611.8 ± 0.72.1 ± 0.5GLUT3182.4 ± 42.9155.1 ± 26.0GLUT34100.27 ± 0.30.65 ± 0.7Xenopus oocytes were injected with mRNA transcribed from normal or mutant (GLUT1161 and GLUT3410) cDNA constructs. Oocytes were incubated on the third day after injection for 10 min at 23 °C with either 200 μm [14C]DHA or 2-[3H]deoxyglucose, washed, and internalized radioactivity was quantified as described under “Experimental Procedures.” Results are the mean ± S.D. of 15–20 oocytes. Open table in a new tab Xenopus oocytes were injected with mRNA transcribed from normal or mutant (GLUT1161 and GLUT3410) cDNA constructs. Oocytes were incubated on the third day after injection for 10 min at 23 °C with either 200 μm [14C]DHA or 2-[3H]deoxyglucose, washed, and internalized radioactivity was quantified as described under “Experimental Procedures.” Results are the mean ± S.D. of 15–20 oocytes. To confirm that GLUT1 and GLUT3 also transport DHA in mammalian cells, we examined DHA transport in cells overexpressing these proteins. DHA transport was measured in Chinese hamster ovary cells stably transfected with rat GLUT1, human GLUT3, or rat GLUT5. GLUT1 and GLUT3 overexpressing cells demonstrated a 2–8-fold increase in [14C]DHA uptake and a 2–18-fold increase in 2-DG uptake compared with control cells (Fig. 6). Increased transport activity of both substrates was inhibited by up to 95% by cytochalasin B (data not shown). GLUT5 overexpressing cells showed higher [14C]fructose transport but [14C]DHA uptake was no different from control (data not shown). In the present report we demonstrate that glucose transporter isoforms GLUT1 and GLUT3 mediate the transport of dehydroascorbic acid. Transport activity was demonstrated both in the Xenopusoocyte expression system and in CHO cells overexpressing these transport proteins. Mutant constructs of both GLUT1 and GLUT3 failed to transport DHA. Determination of DHA transport kinetics was performed under conditions of complete internal reduction of DHA to AA. This was confirmed at all external concentrations of DHA by HPLC. Without complete reduction, kinetics cannot be calculated because efflux of substrate occurs and the reduction process becomes rate-limiti" @default.
• W2058618870 created "2016-06-24" @default.
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• W2058618870 date "1997-07-01" @default.
• W2058618870 modified "2023-10-11" @default.
• W2058618870 title "Glucose Transporter Isoforms GLUT1 and GLUT3 Transport Dehydroascorbic Acid" @default.
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• W2058618870 doi "https://doi.org/10.1074/jbc.272.30.18982" @default.
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