Fluorescence microscopy-based quantitation of GLUT4 translocation

Abstract

Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.

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“…The primary antibody (1° Ab) GLUT-4 was diluted in 3% BSA with 0.1% Triton-X (1 : 500), and the attached cells were incubated with 1° Ab overnight at 4 °C. After washing with PBS, the cells were further incubated with the secondary antibody (2° Ab) mouse Alexa fluor (1 : 1000) at room temperature for 1 h. 24…”

“…The observations from histology analysis were as follows. 24,28,35,36 Liver: in the control group, normal histological structure of the liver tissue was observed. The central vein and hepatocytes were arranged in cords.…”

“…The pellet was washed in phosphate buffered saline (PBS, pH = 7.4), and the cell viability was tested by mixing the cells with 0.1% trypan blue solution. 24 The cell viability was checked by counting under a bright field microscope using a hemocytometer, as viable (live) cells were unstained and unviable cells were stained blue. The experiment was performed in triplicate.…”

“…The primary antibody (1°Ab) GLUT-4 was diluted in 3% BSA with 0.1% Triton-X (1 : 500), and the attached cells were incubated with 1°Ab overnight at 4 °C. After washing with PBS, the cells were further incubated with the secondary antibody (2°Ab) mouse Alexa fluor (1 : 1000) at room temperature for 1 h. 24 Following a PBS wash, the cells attached to the coverslips were mounted on glass slides using ProLong Gold antifade reagent with DAPI. Thereafter, the protein expression was detected under a Leica confocal microscope using a 63× oil immersion objective (microscope lens zoom) with a 405 nm laser for DAPI and a 488 nm laser for Alexa-fluor-labeled GLUT-4.…”

“…The sections were deparaffinized in xylene, rehydrated in graded ethanol with distilled water, and stained with hematoxylin and eosin (H&E). 24 Images were visualized under an optical microscope at 10× magnification. Images of the vital tissues, namely, liver, pancreas, skeletal muscle, kidney and adipose tissue, were studied.…”