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. 1992 May 15;284(Pt 1):275–282. doi: 10.1042/bj2840275

Kinetic resolution of the separate GLUT1 and GLUT4 glucose transport activities in 3T3-L1 cells.

R W Palfreyman 1, A E Clark 1, R M Denton 1, G D Holman 1, I J Kozka 1
PMCID: PMC1132727  PMID: 1599406

Abstract

A bis-mannose-photolabel-displacement method has been developed for resolving the separate kinetic properties of the glucose transporters GLUT1 and GLUT4, which are both present in 3T3-L1 cells. We have quantified the cell-surface transporter abundance (Bmax.) for the two isoforms by displacing radiolabelled 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D- mannos-4-yloxy)-2-propylamine (ATB-BMPA) by non-labelled ATB-BMPA. In cells acutely treated with insulin, the GLUT1 Bmax. was 0.19 microM and the GLUT4 Bmax. was 0.17 microM. In cells which were chronically treated with insulin, the GLUT1 Bmax. was increased by approximately 4-fold to 0.7 microM, whereas the GLUT4 was decreased by approximately 50% (Bmax. = 0.1 microM). However, this large increase in total concentrations of cell-surface transporters (the sum of GLUT1 and GLUT4 concentrations) was not reflected in a large increase in 3-O-methyl-D-glucose transport, suggesting that GLUT1 makes a smaller contribution to transport than does GLUT4. In acutely insulin-treated cells at 37 degrees C, the apparent kinetic parameters for 3-O-methyl-D-glucose transport were Vapp.max. = 0.52 mM.s-1 and Kapp.m = 12.3 mM. In chronically insulin-treated cells the Vapp.max. = 1.24 mM.s-1 and Kapp.m = 23.0 mM. We have measured the displacement of ATB-BMPA by different concentrations of 3-O-methyl-D-glucose to resolve the separate affinity constants of GLUT1 and GLUT4 for this transported ligand. In acute- and chronic-insulin-treated cells the GLUT1 Km for 3-O-methyl-D-glucose was approximately 20 mM, and the GLUT4 Km for 3-O-methyl-D-glucose was approximately 7 mM. An analysis of these data and the 3-O-methyl-D-glucose transport rates was carried out to calculate transport capacity (TK values) for the two isoforms at 37 degrees C. In acute- and chronic-insulin-treated cells the TK values were 0.36 x 10(4) mM-1.min-1 for GLUT1 and 1.13 x 10(4) mM-1.min-1 for GLUT4. Thus GLUT1 has an approximately 3-fold lower transport capacity than GLUT4 at low concentrations of transported sugar. The lower GLUT1 transport capacity was shown to be mainly due to the high Km of GLUT1. The calculated turnover numbers were 7.2 x 10(4) min-1 for GLUT1 and 7.9 x 10(4) min-1 for GLUT4.

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Selected References

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  1. Bird T. A., Davies A., Baldwin S. A., Saklatvala J. Interleukin 1 stimulates hexose transport in fibroblasts by increasing the expression of glucose transporters. J Biol Chem. 1990 Aug 15;265(23):13578–13583. [PubMed] [Google Scholar]
  2. Birnbaum M. J., Haspel H. C., Rosen O. M. Transformation of rat fibroblasts by FSV rapidly increases glucose transporter gene transcription. Science. 1987 Mar 20;235(4795):1495–1498. doi: 10.1126/science.3029870. [DOI] [PubMed] [Google Scholar]
  3. Birnbaum M. J. Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell. 1989 Apr 21;57(2):305–315. doi: 10.1016/0092-8674(89)90968-9. [DOI] [PubMed] [Google Scholar]
  4. Calderhead D. M., Kitagawa K., Tanner L. I., Holman G. D., Lienhard G. E. Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes. J Biol Chem. 1990 Aug 15;265(23):13801–13808. [PubMed] [Google Scholar]
  5. Carruthers A. Facilitated diffusion of glucose. Physiol Rev. 1990 Oct;70(4):1135–1176. doi: 10.1152/physrev.1990.70.4.1135. [DOI] [PubMed] [Google Scholar]
  6. Charron M. J., Brosius F. C., 3rd, Alper S. L., Lodish H. F. A glucose transport protein expressed predominately in insulin-responsive tissues. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2535–2539. doi: 10.1073/pnas.86.8.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clancy B. M., Czech M. P. Hexose transport stimulation and membrane redistribution of glucose transporter isoforms in response to cholera toxin, dibutyryl cyclic AMP, and insulin in 3T3-L1 adipocytes. J Biol Chem. 1990 Jul 25;265(21):12434–12443. [PubMed] [Google Scholar]
  8. Clancy B. M., Harrison S. A., Buxton J. M., Czech M. P. Protein synthesis inhibitors activate glucose transport without increasing plasma membrane glucose transporters in 3T3-L1 adipocytes. J Biol Chem. 1991 Jun 5;266(16):10122–10130. [PubMed] [Google Scholar]
  9. Clark A. E., Holman G. D. Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose. Biochem J. 1990 Aug 1;269(3):615–622. doi: 10.1042/bj2690615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clark A. E., Holman G. D., Kozka I. J. Determination of the rates of appearance and loss of glucose transporters at the cell surface of rat adipose cells. Biochem J. 1991 Aug 15;278(Pt 1):235–241. doi: 10.1042/bj2780235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cleland W. W. Statistical analysis of enzyme kinetic data. Methods Enzymol. 1979;63:103–138. doi: 10.1016/0076-6879(79)63008-2. [DOI] [PubMed] [Google Scholar]
  12. Cornelius P., Marlowe M., Lee M. D., Pekala P. H. The growth factor-like effects of tumor necrosis factor-alpha. Stimulation of glucose transport activity and induction of glucose transporter and immediate early gene expression in 3T3-L1 preadipocytes. J Biol Chem. 1990 Nov 25;265(33):20506–20516. [PubMed] [Google Scholar]
  13. Cushman S. W., Wardzala L. J. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem. 1980 May 25;255(10):4758–4762. [PubMed] [Google Scholar]
  14. Devés R., Krupka R. M. The relationship between substrate dissociation constants derived from transport experiments and from equilibrium binding assays. Implications of the conventional carrier model. Biochim Biophys Acta. 1984 Jan 25;769(2):455–460. doi: 10.1016/0005-2736(84)90330-4. [DOI] [PubMed] [Google Scholar]
  15. Eilam Y., Stein W. D. A simple resolution of the kinetic anomaly in the exchange of different sugars across the membrane of the human red blood cell. Biochim Biophys Acta. 1972 Apr 14;266(1):161–173. doi: 10.1016/0005-2736(72)90132-0. [DOI] [PubMed] [Google Scholar]
  16. Flier J. S., Mueckler M. M., Usher P., Lodish H. F. Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes. Science. 1987 Mar 20;235(4795):1492–1495. doi: 10.1126/science.3103217. [DOI] [PubMed] [Google Scholar]
  17. Frost S. C., Lane M. D. Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes. J Biol Chem. 1985 Mar 10;260(5):2646–2652. [PubMed] [Google Scholar]
  18. Fukumoto H., Kayano T., Buse J. B., Edwards Y., Pilch P. F., Bell G. I., Seino S. Cloning and characterization of the major insulin-responsive glucose transporter expressed in human skeletal muscle and other insulin-responsive tissues. J Biol Chem. 1989 May 15;264(14):7776–7779. [PubMed] [Google Scholar]
  19. Gould G. W., Bell G. I. Facilitative glucose transporters: an expanding family. Trends Biochem Sci. 1990 Jan;15(1):18–23. doi: 10.1016/0968-0004(90)90125-u. [DOI] [PubMed] [Google Scholar]
  20. Gould G. W., Lienhard G. E. Expression of a functional glucose transporter in Xenopus oocytes. Biochemistry. 1989 Nov 28;28(24):9447–9452. doi: 10.1021/bi00450a030. [DOI] [PubMed] [Google Scholar]
  21. Harrison S. A., Buxton J. M., Clancy B. M., Czech M. P. Insulin regulation of hexose transport in mouse 3T3-L1 cells expressing the human HepG2 glucose transporter. J Biol Chem. 1990 Nov 25;265(33):20106–20116. [PubMed] [Google Scholar]
  22. Haspel H. C., Wilk E. W., Birnbaum M. J., Cushman S. W., Rosen O. M. Glucose deprivation and hexose transporter polypeptides of murine fibroblasts. J Biol Chem. 1986 May 25;261(15):6778–6789. [PubMed] [Google Scholar]
  23. Hiraki Y., Rosen O. M., Birnbaum M. J. Growth factors rapidly induce expression of the glucose transporter gene. J Biol Chem. 1988 Sep 25;263(27):13655–13662. [PubMed] [Google Scholar]
  24. Holman G. D., Busza A. L., Pierce E. J., Rees W. D. Evidence for negative cooperativity in human erythrocyte sugar transport. Biochim Biophys Acta. 1981 Dec 21;649(3):503–514. doi: 10.1016/0005-2736(81)90153-x. [DOI] [PubMed] [Google Scholar]
  25. Holman G. D., Kozka I. J., Clark A. E., Flower C. J., Saltis J., Habberfield A. D., Simpson I. A., Cushman S. W. Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. J Biol Chem. 1990 Oct 25;265(30):18172–18179. [PubMed] [Google Scholar]
  26. Holman G. D., Parkar B. A., Midgley P. J. Exofacial photoaffinity labelling of the human erythrocyte sugar transporter. Biochim Biophys Acta. 1986 Feb 13;855(1):115–126. doi: 10.1016/0005-2736(86)90195-1. [DOI] [PubMed] [Google Scholar]
  27. James D. E., Strube M., Mueckler M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature. 1989 Mar 2;338(6210):83–87. doi: 10.1038/338083a0. [DOI] [PubMed] [Google Scholar]
  28. Kaestner K. H., Christy R. J., McLenithan J. C., Braiterman L. T., Cornelius P., Pekala P. H., Lane M. D. Sequence, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3-L1 adipocytes. Proc Natl Acad Sci U S A. 1989 May;86(9):3150–3154. doi: 10.1073/pnas.86.9.3150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kayano T., Burant C. F., Fukumoto H., Gould G. W., Fan Y. S., Eddy R. L., Byers M. G., Shows T. B., Seino S., Bell G. I. Human facilitative glucose transporters. Isolation, functional characterization, and gene localization of cDNAs encoding an isoform (GLUT5) expressed in small intestine, kidney, muscle, and adipose tissue and an unusual glucose transporter pseudogene-like sequence (GLUT6). J Biol Chem. 1990 Aug 5;265(22):13276–13282. [PubMed] [Google Scholar]
  30. Kayano T., Fukumoto H., Eddy R. L., Fan Y. S., Byers M. G., Shows T. B., Bell G. I. 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 Oct 25;263(30):15245–15248. [PubMed] [Google Scholar]
  31. Keller K., Strube M., Mueckler M. Functional expression of the human HepG2 and rat adipocyte glucose transporters in Xenopus oocytes. Comparison of kinetic parameters. J Biol Chem. 1989 Nov 15;264(32):18884–18889. [PubMed] [Google Scholar]
  32. Kozka I. J., Clark A. E., Holman G. D. Chronic treatment with insulin selectively down-regulates cell-surface GLUT4 glucose transporters in 3T3-L1 adipocytes. J Biol Chem. 1991 Jun 25;266(18):11726–11731. [PubMed] [Google Scholar]
  33. Mueckler M. Family of glucose-transporter genes. Implications for glucose homeostasis and diabetes. Diabetes. 1990 Jan;39(1):6–11. doi: 10.2337/diacare.39.1.6. [DOI] [PubMed] [Google Scholar]
  34. Pilch P. F. Glucose transporters: what's in a name? Endocrinology. 1990 Jan;126(1):3–5. doi: 10.1210/endo-126-1-3. [DOI] [PubMed] [Google Scholar]
  35. Rollins B. J., Morrison E. D., Usher P., Flier J. S. Platelet-derived growth factor regulates glucose transporter expression. J Biol Chem. 1988 Nov 15;263(32):16523–16526. [PubMed] [Google Scholar]
  36. Simpson I. A., Yver D. R., Hissin P. J., Wardzala L. J., Karnieli E., Salans L. B., Cushman S. W. Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions. Biochim Biophys Acta. 1983 Dec 19;763(4):393–407. doi: 10.1016/0167-4889(83)90101-5. [DOI] [PubMed] [Google Scholar]
  37. Slot J. W., Geuze H. J., Gigengack S., Lienhard G. E., James D. E. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol. 1991 Apr;113(1):123–135. doi: 10.1083/jcb.113.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Suzuki K., Kono T. Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc Natl Acad Sci U S A. 1980 May;77(5):2542–2545. doi: 10.1073/pnas.77.5.2542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Taylor L. P., Holman G. D. Symmetrical kinetic parameters for 3-O-methyl-D-glucose transport in adipocytes in the presence and in the absence of insulin. Biochim Biophys Acta. 1981 Apr 6;642(2):325–335. doi: 10.1016/0005-2736(81)90449-1. [DOI] [PubMed] [Google Scholar]
  40. Thorens B., Sarkar H. K., Kaback H. R., Lodish H. F. Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and beta-pancreatic islet cells. Cell. 1988 Oct 21;55(2):281–290. doi: 10.1016/0092-8674(88)90051-7. [DOI] [PubMed] [Google Scholar]
  41. Tordjman K. M., Leingang K. A., James D. E., Mueckler M. M. Differential regulation of two distinct glucose transporter species expressed in 3T3-L1 adipocytes: effect of chronic insulin and tolbutamide treatment. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7761–7765. doi: 10.1073/pnas.86.20.7761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tordjman K. M., Leingang K. A., Mueckler M. Differential regulation of the HepG2 and adipocyte/muscle glucose transporters in 3T3L1 adipocytes. Effect of chronic glucose deprivation. Biochem J. 1990 Oct 1;271(1):201–207. doi: 10.1042/bj2710201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yang J., Clark A. E., Harrison R., Kozka I. J., Holman G. D. Trafficking of glucose transporters in 3T3-L1 cells. Inhibition of trafficking by phenylarsine oxide implicates a slow dissociation of transporters from trafficking proteins. Biochem J. 1992 Feb 1;281(Pt 3):809–817. doi: 10.1042/bj2810809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zorzano A., Wilkinson W., Kotliar N., Thoidis G., Wadzinkski B. E., Ruoho A. E., Pilch P. F. Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations. J Biol Chem. 1989 Jul 25;264(21):12358–12363. [PubMed] [Google Scholar]

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