Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 2000 Dec 1;352(Pt 2):267–276.

Role for the microtubule cytoskeleton in GLUT4 vesicle trafficking and in the regulation of insulin-stimulated glucose uptake.

L M Fletcher 1, G I Welsh 1, P B Oatey 1, J M Tavaré 1
PMCID: PMC1221456  PMID: 11085918

Abstract

Insulin stimulates glucose uptake into adipocytes by promoting the translocation of the glucose transporter isoform 4 (GLUT4) from intracellular vesicles to the plasma membrane. In 3T3-L1 adipocytes GLUT4 resides both in an endosomal pool, together with transferrin receptors, and in a unique pool termed 'GLUT4 storage vesicles' (GSVs), which excludes endosomal proteins. The trafficking of GLUT4 vesicles was studied in living 3T3-L1 adipocytes by time-lapse confocal microscopy of GLUT4 tagged with green fluorescent protein. GLUT4 vesicles exhibited two types of motion: rapid vibrations around a point and short (generally less than 10 microm) linear movements. The linear movements were completely blocked by incubation of the cells in the presence of microtubule-depolymerizing agents. This suggests that a subpopulation of GLUT4 vesicles can exhibit motor-driven movements along microtubules. Upon further examination, microtubule depolymerization inhibited insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane by approx. 40%, but had no effect on insulin-induced translocation of the transferrin receptor to the plasma membrane from endosomes. We propose that an intact microtubule cytoskeleton may be required for optimal trafficking of GLUT4 present in the GSV pool, but not that resident in the endosomal pool.

Full Text

The Full Text of this article is available as a PDF (439.1 KB).

Selected References

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

  1. Bandyopadhyay G., Standaert M. L., Zhao L., Yu B., Avignon A., Galloway L., Karnam P., Moscat J., Farese R. V. Activation of protein kinase C (alpha, beta, and zeta) by insulin in 3T3/L1 cells. Transfection studies suggest a role for PKC-zeta in glucose transport. J Biol Chem. 1997 Jan 24;272(4):2551–2558. doi: 10.1074/jbc.272.4.2551. [DOI] [PubMed] [Google Scholar]
  2. Chen F., Foran P., Shone C. C., Foster K. A., Melling J., Dolly J. O. Botulinum neurotoxin B inhibits insulin-stimulated glucose uptake into 3T3-L1 adipocytes and cleaves cellubrevin unlike type A toxin which failed to proteolyze the SNAP-23 present. Biochemistry. 1997 May 13;36(19):5719–5728. doi: 10.1021/bi962331n. [DOI] [PubMed] [Google Scholar]
  3. Clark S. F., Martin S., Carozzi A. J., Hill M. M., James D. E. Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: potential involvement of a membrane skeleton. J Cell Biol. 1998 Mar 9;140(5):1211–1225. doi: 10.1083/jcb.140.5.1211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cole N. B., Sciaky N., Marotta A., Song J., Lippincott-Schwartz J. Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites. Mol Biol Cell. 1996 Apr;7(4):631–650. doi: 10.1091/mbc.7.4.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Foran P. G., Fletcher L. M., Oatey P. B., Mohammed N., Dolly J. O., Tavaré J. M. Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin. J Biol Chem. 1999 Oct 1;274(40):28087–28095. doi: 10.1074/jbc.274.40.28087. [DOI] [PubMed] [Google Scholar]
  6. Holman G. D., Kasuga M. From receptor to transporter: insulin signalling to glucose transport. Diabetologia. 1997 Sep;40(9):991–1003. doi: 10.1007/s001250050780. [DOI] [PubMed] [Google Scholar]
  7. Holman G. D., Lo Leggio L., Cushman S. W. Insulin-stimulated GLUT4 glucose transporter recycling. A problem in membrane protein subcellular trafficking through multiple pools. J Biol Chem. 1994 Jul 1;269(26):17516–17524. [PubMed] [Google Scholar]
  8. Huby R. D., Weiss A., Ley S. C. Nocodazole inhibits signal transduction by the T cell antigen receptor. J Biol Chem. 1998 May 15;273(20):12024–12031. doi: 10.1074/jbc.273.20.12024. [DOI] [PubMed] [Google Scholar]
  9. Jordan M. A., Wilson L. Use of drugs to study role of microtubule assembly dynamics in living cells. Methods Enzymol. 1998;298:252–276. doi: 10.1016/s0076-6879(98)98024-7. [DOI] [PubMed] [Google Scholar]
  10. Kandror K. V., Pilch P. F. Compartmentalization of protein traffic in insulin-sensitive cells. Am J Physiol. 1996 Jul;271(1 Pt 1):E1–14. doi: 10.1152/ajpendo.1996.271.1.E1. [DOI] [PubMed] [Google Scholar]
  11. Lane J., Allan V. Microtubule-based membrane movement. Biochim Biophys Acta. 1998 Jun 29;1376(1):27–55. doi: 10.1016/s0304-4157(97)00010-5. [DOI] [PubMed] [Google Scholar]
  12. Lee W., Ryu J., Souto R. P., Pilch P. F., Jung C. Y. Separation and partial characterization of three distinct intracellular GLUT4 compartments in rat adipocytes. Subcellular fractionation without homogenization. J Biol Chem. 1999 Dec 31;274(53):37755–37762. doi: 10.1074/jbc.274.53.37755. [DOI] [PubMed] [Google Scholar]
  13. Livingstone C., James D. E., Rice J. E., Hanpeter D., Gould G. W. Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes. Biochem J. 1996 Apr 15;315(Pt 2):487–495. doi: 10.1042/bj3150487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Loten E. G., Jeanrenaud B. Effects of cytochalasin B, colchicine and vincristine on the metabolism of isolated fat-cells. Biochem J. 1974 May;140(2):185–192. doi: 10.1042/bj1400185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Malide D., Cushman S. W. Morphological effects of wortmannin on the endosomal system and GLUT4-containing compartments in rat adipose cells. J Cell Sci. 1997 Nov;110(Pt 22):2795–2806. doi: 10.1242/jcs.110.22.2795. [DOI] [PubMed] [Google Scholar]
  16. Malide D., Dwyer N. K., Blanchette-Mackie E. J., Cushman S. W. Immunocytochemical evidence that GLUT4 resides in a specialized translocation post-endosomal VAMP2-positive compartment in rat adipose cells in the absence of insulin. J Histochem Cytochem. 1997 Aug;45(8):1083–1096. doi: 10.1177/002215549704500806. [DOI] [PubMed] [Google Scholar]
  17. Martin S., Tellam J., Livingstone C., Slot J. W., Gould G. W., James D. E. The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells. J Cell Biol. 1996 Aug;134(3):625–635. doi: 10.1083/jcb.134.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Oatey P. B., Van Weering D. H., Dobson S. P., Gould G. W., Tavaré J. M. GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein. Biochem J. 1997 Nov 1;327(Pt 3):637–642. doi: 10.1042/bj3270637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pessin J. E., Thurmond D. C., Elmendorf J. S., Coker K. J., Okada S. Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. Location! Location! Location! J Biol Chem. 1999 Jan 29;274(5):2593–2596. doi: 10.1074/jbc.274.5.2593. [DOI] [PubMed] [Google Scholar]
  20. Presley J. F., Cole N. B., Schroer T. A., Hirschberg K., Zaal K. J., Lippincott-Schwartz J. ER-to-Golgi transport visualized in living cells. Nature. 1997 Sep 4;389(6646):81–85. doi: 10.1038/38001. [DOI] [PubMed] [Google Scholar]
  21. Rapp S., Saffrich R., Anton M., Jäkle U., Ansorge W., Gorgas K., Just W. W. Microtubule-based peroxisome movement. J Cell Sci. 1996 Apr;109(Pt 4):837–849. doi: 10.1242/jcs.109.4.837. [DOI] [PubMed] [Google Scholar]
  22. Rea S., James D. E. Moving GLUT4: the biogenesis and trafficking of GLUT4 storage vesicles. Diabetes. 1997 Nov;46(11):1667–1677. doi: 10.2337/diab.46.11.1667. [DOI] [PubMed] [Google Scholar]
  23. Robinson L. J., James D. E. Insulin-regulated sorting of glucose transporters in 3T3-L1 adipocytes. Am J Physiol. 1992 Aug;263(2 Pt 1):E383–E393. doi: 10.1152/ajpendo.1992.263.2.E383. [DOI] [PubMed] [Google Scholar]
  24. Shepherd P. R., Soos M. A., Siddle K. Inhibitors of phosphoinositide 3-kinase block exocytosis but not endocytosis of transferrin receptors in 3T3-L1 adipocytes. Biochem Biophys Res Commun. 1995 Jun 15;211(2):535–539. doi: 10.1006/bbrc.1995.1846. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Smas C. M., Sul H. S. Control of adipocyte differentiation. Biochem J. 1995 Aug 1;309(Pt 3):697–710. doi: 10.1042/bj3090697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Soifer D., Braun T., Hechter O. Insulin and microtubules in rat adipocytes. Science. 1971 Apr 16;172(3980):269–271. doi: 10.1126/science.172.3980.269. [DOI] [PubMed] [Google Scholar]
  28. Tanner L. I., Lienhard G. E. Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization. J Biol Chem. 1987 Jul 5;262(19):8975–8980. [PubMed] [Google Scholar]
  29. Tsakiridis T., Vranic M., Klip A. Disassembly of the actin network inhibits insulin-dependent stimulation of glucose transport and prevents recruitment of glucose transporters to the plasma membrane. J Biol Chem. 1994 Nov 25;269(47):29934–29942. [PubMed] [Google Scholar]
  30. Tsakiridis T., Vranic M., Klip A. Phosphatidylinositol 3-kinase and the actin network are not required for the stimulation of glucose transport caused by mitochondrial uncoupling: comparison with insulin action. Biochem J. 1995 Jul 1;309(Pt 1):1–5. doi: 10.1042/bj3090001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wang Q., Bilan P. J., Tsakiridis T., Hinek A., Klip A. Actin filaments participate in the relocalization of phosphatidylinositol3-kinase to glucose transporter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 adipocytes. Biochem J. 1998 May 1;331(Pt 3):917–928. doi: 10.1042/bj3310917. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary figure 1
bj3520267add01.gif (1.9MB, gif)
Supplementary figure 2a - panel A
bj3520267add02a1.gif (210.6KB, gif)
Supplementary figure 2a - panel B
bj3520267add02a2.gif (822.3KB, gif)
Supplementary figure 2b
bj3520267add02b.gif (1.8MB, gif)
Supplementary figure 5a - basal
bj3520267add05a1.gif (1.4MB, gif)
Supplementary figure 5a - Colchicine
bj3520267add05a2.gif (2.2MB, gif)
Supplementary figure 5b - basal
bj3520267add05b1.gif (1.7MB, gif)
Supplementary figure 5b - Colchicine
bj3520267add05b2.gif (1.3MB, gif)

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

RESOURCES