Fluorescence microscopy-based quantitation of glut4 translocation: High throughput or high content?

Verena Stadlbauer, Peter Lanzerstorfer, Cathrina Neuhauser, Florian Weber, Flora Stübl, Petra Weber, Michael Wagner, Birgit Plochberger, Stefan Wieser, Herbert Schneckenburger, Julian Weghuber

Research output: Contribution to journalArticlepeer-review

Abstract

Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content.

Original languageEnglish
Article number7964
Pages (from-to)1-16
Number of pages16
JournalInternational Journal of Molecular Sciences
Volume21
Issue number21
DOIs
Publication statusPublished - 1 Nov 2020

Keywords

  • Diabetes mellitus
  • GLUT4 translocation
  • GPMV formation
  • Insulin mimetic compounds
  • TIR multiwell reader
  • TIRF microscopy
  • Cricetulus
  • Humans
  • Glucose Transporter Type 4/metabolism
  • Protein Transport
  • Animals
  • Cell Membrane/metabolism
  • Microscopy, Fluorescence/methods
  • HeLa Cells
  • CHO Cells

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