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. 1994 Jun 15;300(Pt 3):631–635. doi: 10.1042/bj3000631

Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.

J F Clarke 1, P W Young 1, K Yonezawa 1, M Kasuga 1, G D Holman 1
PMCID: PMC1138214  PMID: 8010944

Abstract

Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities.

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Selected References

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  1. Arcaro A., Wymann M. P. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J. 1993 Dec 1;296(Pt 2):297–301. doi: 10.1042/bj2960297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. Endemann G., Yonezawa K., Roth R. A. Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase. J Biol Chem. 1990 Jan 5;265(1):396–400. [PubMed] [Google Scholar]
  6. Fingar D. C., Hausdorff S. F., Blenis J., Birnbaum M. J. Dissociation of pp70 ribosomal protein S6 kinase from insulin-stimulated glucose transport in 3T3-L1 adipocytes. J Biol Chem. 1993 Feb 5;268(4):3005–3008. [PubMed] [Google Scholar]
  7. 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]
  8. Inoue G., Kuzuya H., Hayashi T., Okamoto M., Yoshimasa Y., Kosaki A., Kono S., Okamoto M., Maeda I., Kubota M. Effects of ML-9 on insulin stimulation of glucose transport in 3T3-L1 adipocytes. J Biol Chem. 1993 Mar 5;268(7):5272–5278. [PubMed] [Google Scholar]
  9. Kanai F., Ito K., Todaka M., Hayashi H., Kamohara S., Ishii K., Okada T., Hazeki O., Ui M., Ebina Y. Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase. Biochem Biophys Res Commun. 1993 Sep 15;195(2):762–768. doi: 10.1006/bbrc.1993.2111. [DOI] [PubMed] [Google Scholar]
  10. Klip A., Ramlal T., Young D. A., Holloszy J. O. Insulin-induced translocation of glucose transporters in rat hindlimb muscles. FEBS Lett. 1987 Nov 16;224(1):224–230. doi: 10.1016/0014-5793(87)80452-0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Kuhné M. R., Pawson T., Lienhard G. E., Feng G. S. The insulin receptor substrate 1 associates with the SH2-containing phosphotyrosine phosphatase Syp. J Biol Chem. 1993 Jun 5;268(16):11479–11481. [PubMed] [Google Scholar]
  13. Merrall N. W., Plevin R. J., Stokoe D., Cohen P., Nebreda A. R., Gould G. W. Mitogen-activated protein kinase (MAP kinase), MAP kinase kinase and c-Mos stimulate glucose transport in Xenopus oocytes. Biochem J. 1993 Oct 15;295(Pt 2):351–355. doi: 10.1042/bj2950351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Myers M. G., Jr, White M. F. The new elements of insulin signaling. Insulin receptor substrate-1 and proteins with SH2 domains. Diabetes. 1993 May;42(5):643–650. doi: 10.2337/diab.42.5.643. [DOI] [PubMed] [Google Scholar]
  15. Okada T., Kawano Y., Sakakibara T., Hazeki O., Ui M. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem. 1994 Feb 4;269(5):3568–3573. [PubMed] [Google Scholar]
  16. Robinson L. J., Pang S., Harris D. S., Heuser J., James D. E. Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: effects of ATP insulin, and GTP gamma S and localization of GLUT4 to clathrin lattices. J Cell Biol. 1992 Jun;117(6):1181–1196. doi: 10.1083/jcb.117.6.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Robinson L. J., Razzack Z. F., Lawrence J. C., Jr, James D. E. Mitogen-activated protein kinase activation is not sufficient for stimulation of glucose transport or glycogen synthase in 3T3-L1 adipocytes. J Biol Chem. 1993 Dec 15;268(35):26422–26427. [PubMed] [Google Scholar]
  18. Ruderman N. B., Kapeller R., White M. F., Cantley L. C. Activation of phosphatidylinositol 3-kinase by insulin. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1411–1415. doi: 10.1073/pnas.87.4.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Satoh S., Nishimura H., Clark A. E., Kozka I. J., Vannucci S. J., Simpson I. A., Quon M. J., Cushman S. W., Holman G. D. Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action. J Biol Chem. 1993 Aug 25;268(24):17820–17829. [PubMed] [Google Scholar]
  20. Skolnik E. Y., Lee C. H., Batzer A., Vicentini L. M., Zhou M., Daly R., Myers M. J., Jr, Backer J. M., Ullrich A., White M. F. The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. EMBO J. 1993 May;12(5):1929–1936. doi: 10.1002/j.1460-2075.1993.tb05842.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Stack J. H., Herman P. K., Schu P. V., Emr S. D. A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole. EMBO J. 1993 May;12(5):2195–2204. doi: 10.1002/j.1460-2075.1993.tb05867.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stagsted J., Ziebe S., Satoh S., Holman G. D., Cushman S. W., Olsson L. Insulinomimetic effect on glucose transport by epidermal growth factor when combined with a major histocompatibility complex class I-derived peptide. J Biol Chem. 1993 Jan 25;268(3):1770–1774. [PubMed] [Google Scholar]
  24. 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]
  25. Yang J., Clark A. E., Kozka I. J., Cushman S. W., Holman G. D. Development of an intracellular pool of glucose transporters in 3T3-L1 cells. J Biol Chem. 1992 May 25;267(15):10393–10399. [PubMed] [Google Scholar]
  26. Yang J., Holman G. D. Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells. J Biol Chem. 1993 Mar 5;268(7):4600–4603. [PubMed] [Google Scholar]
  27. Yano H., Nakanishi S., Kimura K., Hanai N., Saitoh Y., Fukui Y., Nonomura Y., Matsuda Y. Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells. J Biol Chem. 1993 Dec 5;268(34):25846–25856. [PubMed] [Google Scholar]
  28. Yonezawa K., Ueda H., Hara K., Nishida K., Ando A., Chavanieu A., Matsuba H., Shii K., Yokono K., Fukui Y. Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1. J Biol Chem. 1992 Dec 25;267(36):25958–25965. [PubMed] [Google Scholar]

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