Skip to main content
SpringerLink
Log in

Tracking GLUT2 Translocation by Live-Cell Imaging

  • Protocol
  • First Online:
Book cover Glucose Transport

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1713))

Abstract

The facilitative glucose transporter (GLUT) family plays a key role in metabolic homeostasis, controlling the absorption rates and rapid response to changing carbohydrate levels. The facilitative GLUT2 transporter is uniquely expressed in metabolic epithelial cells of the intestine, pancreas, liver, and kidney. GLUT2 dysfunction is associated with several pathologies, including Fanconi-Bickel syndrome, a glycogen storage disease, characterized by growth retardation and renal dysfunction. Interestingly, GLUT2 activity is modulated by its cellular localization. Membrane translocation specifically regulates GLUT2 activity in enterocytes, pancreatic β-cells, hepatocytes, and proximal tubule cells. We have established a system to visualize and quantify GLUT2 translocation, and its dynamics, by live imaging of a mCherry-hGLUT2 fusion protein in polarized epithelial cells. This system enables testing of putative modulators of GLUT2 translocation, which are potential drugs for conditions of impaired glucose homeostasis and associated nephropathy.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Thorens B, Mueckler M (2010) Glucose transporters in the 21st century. Am J Phys Endocrinol Metab 298(2):E141–E145. https://doi.org/10.1152/ajpendo.00712.2009

    Article  CAS  Google Scholar 

  2. Mueckler M, Thorens B (2013) The SLC2 (GLUT) family of membrane transporters. Mol Asp Med 34(2–3):121–138. https://doi.org/10.1016/j.mam.2012.07.001

    Article  CAS  Google Scholar 

  3. Thorens B (2015) GLUT2, glucose sensing and glucose homeostasis. Diabetologia 58(2):221–232. https://doi.org/10.1007/s00125-014-3451-1

    Article  CAS  PubMed  Google Scholar 

  4. Thorens B, Weir GC, Leahy JL, Lodish HF, Bonner-Weir S (1990) Reduced expression of the liver/beta-cell glucose transporter isoform in glucose-insensitive pancreatic beta cells of diabetic rats. Proc Natl Acad Sci U S A 87(17):6492–6496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Seyer P, Vallois D, Poitry-Yamate C, Schutz F, Metref S, Tarussio D, Maechler P, Staels B, Lanz B, Grueter R, Decaris J, Turner S, da Costa A, Preitner F, Minehira K, Foretz M, Thorens B (2013) Hepatic glucose sensing is required to preserve beta cell glucose competence. J Clin Invest 123(4):1662–1676. https://doi.org/10.1172/JCI65538

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Souza-Menezes J, Morales MM, Tukaye DN, Guggino SE, Guggino WB (2007) Absence of ClC5 in knockout mice leads to glycosuria, impaired renal glucose handling and low proximal tubule GLUT2 protein expression. Cell Physiol Biochem 20(5):455–464. https://doi.org/10.1159/000107529

    Article  CAS  PubMed  Google Scholar 

  7. Guillemain G, Loizeau M, Pincon-Raymond M, Girard J, Leturque A (2000) The large intracytoplasmic loop of the glucose transporter GLUT2 is involved in glucose signaling in hepatic cells. J Cell Sci 113(Pt 5):841–847

    CAS  PubMed  Google Scholar 

  8. Santer R, Schneppenheim R, Dombrowski A, Gotze H, Steinmann B, Schaub J (1997) Mutations in GLUT2, the gene for the liver-type glucose transporter, in patients with Fanconi-Bickel syndrome. Nat Genet 17(3):324–326. https://doi.org/10.1038/ng1197-324

    Article  CAS  PubMed  Google Scholar 

  9. Santer R, Schneppenheim R, Suter D, Schaub J, Steinmann B (1998) Fanconi-Bickel syndrome—the original patient and his natural history, historical steps leading to the primary defect, and a review of the literature. Eur J Pediatr 157(10):783–797

    Article  CAS  PubMed  Google Scholar 

  10. Manz F, Bickel H, Brodehl J, Feist D, Gellissen K, Geschollbauer B, Gilli G, Harms E, Helwig H, Nutzenadel W, Waldherr R (1987) Fanconi-Bickel syndrome. Pediatr Nephrol 1(3):509–518

    Article  CAS  PubMed  Google Scholar 

  11. Leturque A, Brot-Laroche E, Le Gall M (2009) GLUT2 mutations, translocation, and receptor function in diet sugar managing. Am J Phys Endocrinol Metab 296(5):E985–E992. https://doi.org/10.1152/ajpendo.00004.2009

    Article  CAS  Google Scholar 

  12. Suzuki K, Kono T (1980) Evidence that insulin causes translocation of glucose-transport activity to the plasma-membrane from an intracellular storage site. P Natl Acad Sci-Biol 77(5):2542–2545. https://doi.org/10.1073/Pnas.77.5.2542

    Article  CAS  Google Scholar 

  13. Cushman SW, Wardzala LJ (1980) 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 255(10):4758–4762

    CAS  PubMed  Google Scholar 

  14. Uldry M, Ibberson M, Hosokawa M, Thorens B (2002) GLUT2 is a high affinity glucosamine transporter. FEBS Lett 524(1–3):199–203

    Article  CAS  PubMed  Google Scholar 

  15. Zheng Y, Scow JS, Duenes JA, Sarr MG (2012) Mechanisms of glucose uptake in intestinal cell lines: role of GLUT2. Surgery 151(1):13–25. https://doi.org/10.1016/j.surg.2011.07.010

    Article  PubMed  Google Scholar 

  16. Au A, Gupta A, Schembri P, Cheeseman CI (2002) Rapid insertion of GLUT2 into the rat jejunal brush-border membrane promoted by glucagon-like peptide 2. Biochem J 367(Pt 1):247–254. https://doi.org/10.1042/BJ20020393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Marks J, Carvou NJ, Debnam ES, Srai SK, Unwin RJ (2003) Diabetes increases facilitative glucose uptake and GLUT2 expression at the rat proximal tubule brush border membrane. J Physiol 553(Pt 1):137–145. https://doi.org/10.1113/jphysiol.2003.046268

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ohtsubo K, Takamatsu S, Minowa MT, Yoshida A, Takeuchi M, Marth JD (2005) Dietary and genetic control of glucose transporter 2 glycosylation promotes insulin secretion in suppressing diabetes. Cell 123(7):1307–1321. https://doi.org/10.1016/j.cell.2005.09.041

    Article  CAS  PubMed  Google Scholar 

  19. Eisenberg ML, Maker AV, Slezak LA, Nathan JD, Sritharan KC, Jena BP, Geibel JP, Andersen DK (2005) Insulin receptor (IR) and glucose transporter 2 (GLUT2) proteins form a complex on the rat hepatocyte membrane. Cell Physiol Biochem 15(1–4):51–58. https://doi.org/10.1159/000083638

    Article  CAS  PubMed  Google Scholar 

  20. Fletcher LM, Welsh GI, Oatey PB, Tavare JM (2000) Role for the microtubule cytoskeleton in GLUT4 vesicle trafficking and in the regulation of insulin-stimulated glucose uptake. Biochem J 352(Pt 2):267–276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hirayama S, Hori Y, Benedek Z, Suzuki T, Kikuchi K (2016) Fluorogenic probes reveal a role of GLUT4 N-glycosylation in intracellular trafficking. Nat Chem Biol 12(10):853–859. https://doi.org/10.1038/nchembio.2156

    Article  CAS  PubMed  Google Scholar 

  22. Cohen M, Kitsberg D, Tsytkin S, Shulman M, Aroeti B, Nahmias Y (2014) Live imaging of GLUT2 glucose-dependent trafficking and its inhibition in polarized epithelial cysts. Open Biol 4(7). https://doi.org/10.1098/rsob.140091

  23. Elia N, Lippincott-Schwartz J (2009) Culturing MDCK cells in three dimensions for analyzing intracellular dynamics. Curr Protoc Cell Biol Chapter 4:Unit 4.22. https://doi.org/10.1002/0471143030.cb0422s43

    PubMed  Google Scholar 

  24. O’Brien LE, Zegers MM, Mostov KE (2002) Opinion: building epithelial architecture: insights from three-dimensional culture models. Nat Rev Mol Cell Biol 3(7):531–537. https://doi.org/10.1038/nrm859

    Article  PubMed  Google Scholar 

  25. Nissim-Rafinia M, Meshorer E (2011) Photobleaching assays (FRAP & FLIP) to measure chromatin protein dynamics in living embryonic stem cells. J Vis Exp (52):2696. https://doi.org/10.3791/2696

  26. Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS (1986) Phenol red in tissue-culture media is a weak estrogen—implications concerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci U S A 83(8):2496–2500. https://doi.org/10.1073/Pnas.83.8.2496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Schwimmer R, Ojakian GK (1995) The alpha 2 beta 1 integrin regulates collagen-mediated MDCK epithelial membrane remodeling and tubule formation. J Cell Sci 108(Pt 6):2487–2498

    CAS  PubMed  Google Scholar 

  28. Ettinger A, Wittmann T (2014) Fluorescence live cell imaging. Methods Cell Biol 123:77–94. https://doi.org/10.1016/B978-0-12-420138-5.00005-7

    Article  PubMed  PubMed Central  Google Scholar 

  29. Purschke M, Rubio N, Held KD, Redmond RW (2010) Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy. Photochem Photobiol Sci 9(12):1634–1639. https://doi.org/10.1039/c0pp00234h

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the European Research Council Consolidator Grant (OCLD 681870), and through the generous gift of Sam and Rina Frankel. Resources were provided by the Silberman Institute of Life Sciences and the Alexander Grass Center for Bioengineering of the Hebrew University of Jerusalem.

Author information

Authors and Affiliations

  1. Alexander Grass Center for Bioengineering, The Rachel and Selim Benin School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem, 9190401, Israel

    Sabina Tsytkin-Kirschenzweig, Merav Cohen & Yaakov Nahmias

  2. Department of Cell and Developmental Biology, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, 9190401, Israel

    Merav Cohen & Yaakov Nahmias

Authors
  1. Sabina Tsytkin-Kirschenzweig
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Merav Cohen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yaakov Nahmias
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yaakov Nahmias .

Editor information

Editors and Affiliations

  1. Department of Experimental Medical Science, Lund University, Lund, Sweden

    Karin Lindkvist-Petersson

  2. Department of Experimental Medical Science, Lund University, Lund, Sweden

    Jesper S Hansen

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Tsytkin-Kirschenzweig, S., Cohen, M., Nahmias, Y. (2018). Tracking GLUT2 Translocation by Live-Cell Imaging. In: Lindkvist-Petersson, K., Hansen, J. (eds) Glucose Transport. Methods in Molecular Biology, vol 1713. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7507-5_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7507-5_18

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7506-8

  • Online ISBN: 978-1-4939-7507-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation