A novel quantitative assay for analysis of GLUT4 translocation using high content screening

• Published in: Biomedicine & pharmacotherapy Vol. 133; p. 111032
• Main Authors: Komakula, SaiSantosh Babu, Tiwari, Ashok Kumar, Singh, Shashi
• Format: Journal Article
• Language: English
• Published: France Elsevier Masson SAS 01-01-2021; Elsevier
• Subjects: 3T3 Cells; Adipocytes - drug effects; Adipocytes - metabolism; Animals; Biological Assay; Cell based assay; CHO Cells; Cricetulus; Glucose Transporter Type 4 - genetics; Glucose Transporter Type 4 - metabolism; GLUT4-GFP; Hep G2 Cells; Hepatocytes - drug effects; Hepatocytes - metabolism; High-Throughput Screening Assays; Humans; Hypoglycemic Agents - pharmacology; Insulin - pharmacology; Insulin Resistance; Insulin sensitizers; Insulinomimetics; Membrane translocation; Mice; Phytochemicals; Protein Transport - drug effects; Reproducibility of Results; Screening; Time Factors; Screening; Insulin sensitizers; Phytochemicals; Cell based assay; GLUT4-GFP; Membrane translocation; Insulinomimetics
• DOI: 10.1016/j.biopha.2020.111032
• EISSN: 1950-6007
• ISSN: 0753-3322

•High throughput screening methods to test small molecules/extracts efficiency to act as an insulinomimetics is established.•The process uses real time analysis of GLUT4 translocation.•Method validated by downstream glucose uptake by adipose cells.•Screening of dietary phytoextracts.
Insulin resistance is associated with obesity and can lead to several metabolic disorders including type II diabetes, nonalcoholic fatty liver disease and cardiovascular problems. Search for the small molecules which can either induce or mimic the insulin action are of great interest and can be utilized to manage insulin resistance. There are several dietary phytochemicals which can potentially have insulinomimetic action. Nevertheless, high throughput screening methods to test efficiency of small molecules to act as an insulinomimetic are not fully established. In this paper we have performed chemical screen analysis based on GLUT4 translocation using a cell line CHO-HIRC-myc-GLUT4 eGFP that expresses GLUT4-GFP in association with human Insulin receptor. We have established a high content screening-based method which can track and quantify the GLUT4 translocation from perinuclear area to the cell membrane. The assay involves measuring fluorescence intensity in a defined perinuclear area and a defined area along the cell membrane; and the results are expressed as the ratio of fluorescence intensity in the perinuclear to membrane area. The assay could collect real time data of GLUT4 translocation from thousand of cells/ sample and from many such samples in one experiment. We validated the assay using Insulin, insulin mimics/sensitizers and insulin inhibitors. The agonist or antagonists were analyzed for their ability to enhance or block the GLUT4 translocation independent of insulin. The outcome of the assay was correlated by performing glucose uptake assay using differentiated 3T3L1 cells. Using this platform we further identified several plant extracts which had the insulin mimetic action. We confirmed that these plant extracts were non-toxic to the beta cells using RIN mf5cells and 3T3L1 cells. We have identified plant extracts with the potential insulinomimetic action using novel high-content screening approach; these can be further tested for their efficiency in-vivo in pre-clinical trials.