| Title | A robust assay measuring GLUT4 translocation in rat myoblasts overexpressing Glut4myc and AS160_v2. | | Author(s) | Baus D, Yan Y, Li Z, Garyantes T, Hoop MD, Tennagels N | | Institution | Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany. | | Source | Anal Biochem 2009 Oct 22. | | Abstract | Muscle and fat cells translocate the glucose transporter 4, GLUT4, to the plasma membrane when stimulated by insulin. Usually this event is measured in differentiated adipocytes, myotubes, or cell lines overexpressing tagged GLUT4, by immunostaining. However, measurement of the translocation in differentiated adipocytes or myotubes or GLUT-4 overexpressing cell lines is difficult because of high assay variability caused by either the differentiation protocol or low assay sensitivity. We recently reported the identification of a novel splice variant of AS160 (AS160_v2) and showed that its co-expression with GLUT4 in L6 myoblasts increased insulin-stimulated glucose uptake rate due to an increased amount of GLUT4 on the cell surface. L6 cells, which co-express myc-tagged GLUT4 and AS160_v2 can be efficiently used to generate an assay useful for identifying compounds that affect cellular responses to insulin. We compared the EC(50) values for radioactive glucose uptake and GLUT4 translocation of different insulins and several small molecules to validate the assay. The use of L6 cells overexpressing AS160_v2 can be considered as a novel tool for the characterization of molecules modulating insulin signaling and GLUT4 translocation and an image based assay increase our confidence in the MOA of the compounds identified. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 19854150 |
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