Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1994 Nov 15;304(Pt 1):307–311. doi: 10.1042/bj3040307

Expression of the liver-type glucose transporter (GLUT2) in 3T3-L1 adipocytes: analysis of the effects of insulin on subcellular distribution.

A M Brant 1, S Martin 1, G W Gould 1
PMCID: PMC1137487  PMID: 7998950

Abstract

We have expressed the liver-type facilitative glucose transporter, GLUT2, in the insulin-sensitive 3T3-L1 adipocyte clonal cell line in an effort to address the importance of transporter isoform and cellular environment on the ability of insulin to mediate glucose-transporter translocation. Analysis of non-differentiated fibroblastic cell clones transfected with the GLUT2 cDNA identified the presence of this isoform in several independent clones. These clones exhibited increased deoxyglucose and fructose transport rates compared with control cells. Upon differentiation, the fibroblastic clones selected for study achieved > 95% phenotypic conversion into adipocytes. Expression of the GLUT2 protein was maintained throughout the differentiation protocol. Subcellular fractionation revealed that in response to insulin, unlike the native GLUT4, GLUT2 protein did not undergo significant translocation to the plasma membrane; furthermore, the subcellular distribution of the expressed GLUT2 was quite distinct from that of the endogenous GLUT4. 3T3-L1 adipocytes expressing GLUT2 only exhibited a 2-fold increase in insulin-stimulated fructose uptake, further suggesting that GLUT2 does not undergo insulin-stimulated translocation.

Full text

PDF
307

Images in this article

308Figure 1
on p.308
310Figure 2
on p.310

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baur H., Heldt H. W. Transport of hexoses across the liver-cell membrane. Eur J Biochem. 1977 Apr 1;74(2):397–403. doi: 10.1111/j.1432-1033.1977.tb11404.x. [DOI] [PubMed] [Google Scholar]
  2. Bell G. I., Burant C. F., Takeda J., Gould G. W. Structure and function of mammalian facilitative sugar transporters. J Biol Chem. 1993 Sep 15;268(26):19161–19164. [PubMed] [Google Scholar]
  3. Bloch R. Inhibition of glucose transport in the human erythrocyte by cytochalasin B. Biochemistry. 1973 Nov 6;12(23):4799–4801. doi: 10.1021/bi00747a036. [DOI] [PubMed] [Google Scholar]
  4. Blok J., Gibbs E. M., Lienhard G. E., Slot J. W., Geuze H. J. Insulin-induced translocation of glucose transporters from post-Golgi compartments to the plasma membrane of 3T3-L1 adipocytes. J Cell Biol. 1988 Jan;106(1):69–76. doi: 10.1083/jcb.106.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brant A. M., Jess T. J., Milligan G., Brown C. M., Gould G. W. Immunological analysis of glucose transporters expressed in different regions of the rat brain and central nervous system. Biochem Biophys Res Commun. 1993 May 14;192(3):1297–1302. doi: 10.1006/bbrc.1993.1557. [DOI] [PubMed] [Google Scholar]
  6. Brant A. M., McCoid S., Thomas H. M., Baldwin S. A., Davies A., Parker J. C., Gibbs E. M., Gould G. W. Analysis of the glucose transporter content of islet cell lines: implications for glucose-stimulated insulin release. Cell Signal. 1992 Nov;4(6):641–650. doi: 10.1016/0898-6568(92)90045-a. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Colville C. A., Seatter M. J., Jess T. J., Gould G. W., Thomas H. M. Kinetic analysis of the liver-type (GLUT2) and brain-type (GLUT3) glucose transporters in Xenopus oocytes: substrate specificities and effects of transport inhibitors. Biochem J. 1993 Mar 15;290(Pt 3):701–706. doi: 10.1042/bj2900701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Czech M. P., Chawla A., Woon C. W., Buxton J., Armoni M., Tang W., Joly M., Corvera S. Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization. J Cell Biol. 1993 Oct;123(1):127–135. doi: 10.1083/jcb.123.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Fukumoto H., Seino S., Imura H., Seino Y., Eddy R. L., Fukushima Y., Byers M. G., Shows T. B., Bell G. I. Sequence, tissue distribution, and chromosomal localization of mRNA encoding a human glucose transporter-like protein. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5434–5438. doi: 10.1073/pnas.85.15.5434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gould G. W., Brant A. M., Kahn B. B., Shepherd P. R., McCoid S. C., Gibbs E. M. Expression of the brain-type glucose transporter is restricted to brain and neuronal cells in mice. Diabetologia. 1992 Apr;35(4):304–309. doi: 10.1007/BF00401196. [DOI] [PubMed] [Google Scholar]
  14. Gould G. W., Derechin V., James D. E., Tordjman K., Ahern S., Gibbs E. M., Lienhard G. E., Mueckler M. Insulin-stimulated translocation of the HepG2/erythrocyte-type glucose transporter expressed in 3T3-L1 adipocytes. J Biol Chem. 1989 Feb 5;264(4):2180–2184. [PubMed] [Google Scholar]
  15. Gould G. W., Holman G. D. The glucose transporter family: structure, function and tissue-specific expression. Biochem J. 1993 Oct 15;295(Pt 2):329–341. doi: 10.1042/bj2950329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gould G. W., Thomas H. M., Jess T. J., Bell G. I. Expression of human glucose transporters in Xenopus oocytes: kinetic characterization and substrate specificities of the erythrocyte, liver, and brain isoforms. Biochemistry. 1991 May 28;30(21):5139–5145. doi: 10.1021/bi00235a004. [DOI] [PubMed] [Google Scholar]
  17. Gunning P., Leavitt J., Muscat G., Ng S. Y., Kedes L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4831–4835. doi: 10.1073/pnas.84.14.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Haney P. M., Slot J. W., Piper R. C., James D. E., Mueckler M. Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type. J Cell Biol. 1991 Aug;114(4):689–699. doi: 10.1083/jcb.114.4.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hudson A. W., Fingar D. C., Seidner G. A., Griffiths G., Burke B., Birnbaum M. J. Targeting of the "insulin-responsive" glucose transporter (GLUT4) to the regulated secretory pathway in PC12 cells. J Cell Biol. 1993 Aug;122(3):579–588. doi: 10.1083/jcb.122.3.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jordan N. J., Holman G. D. Photolabelling of the liver-type glucose-transporter isoform GLUT2 with an azitrifluoroethylbenzoyl-substituted bis-D-mannose. Biochem J. 1992 Sep 1;286(Pt 2):649–656. doi: 10.1042/bj2860649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Merrall N. W., Wakelam M. J., Plevin R., Gould G. W. Insulin and platelet-derived growth factor acutely stimulate glucose transport in 3T3-L1 fibroblasts independently of protein kinase C. Biochim Biophys Acta. 1993 Jun 6;1177(2):191–198. doi: 10.1016/0167-4889(93)90040-v. [DOI] [PubMed] [Google Scholar]
  24. Piper R. C., Hess L. J., James D. E. Differential sorting of two glucose transporters expressed in insulin-sensitive cells. Am J Physiol. 1991 Mar;260(3 Pt 1):C570–C580. doi: 10.1152/ajpcell.1991.260.3.C570. [DOI] [PubMed] [Google Scholar]
  25. Piper R. C., Tai C., Slot J. W., Hahn C. S., Rice C. M., Huang H., James D. E. The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus. J Cell Biol. 1992 May;117(4):729–743. doi: 10.1083/jcb.117.4.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Slot J. W., Geuze H. J., Gigengack S., James D. E., Lienhard G. E. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7815–7819. doi: 10.1073/pnas.88.17.7815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Thomas H. M., Takeda J., Gould G. W. Differential targeting of glucose transporter isoforms heterologously expressed in Xenopus oocytes. Biochem J. 1993 Mar 15;290(Pt 3):707–715. doi: 10.1042/bj2900707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Verhey K. J., Birnbaum M. J. A Leu-Leu sequence is essential for COOH-terminal targeting signal of GLUT4 glucose transporter in fibroblasts. J Biol Chem. 1994 Jan 28;269(4):2353–2356. [PubMed] [Google Scholar]
  32. Williams T. F., Exton J. H., Park C. R., Regen D. M. Stereospecific transport of glucose in the perfused rat liver. Am J Physiol. 1968 Nov;215(5):1200–1209. doi: 10.1152/ajplegacy.1968.215.5.1200. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES