Quantitation of GLUT4 translocation and glucose uptake in 3T3-L1 cells.

<p>(A) TIRF microscopy based quantitation of GLUT4 translocation in 3T3-L1 GLUT4-GFP adipocytes. Cells were grown in 96-well plates (20,000 cells/well) for 24 hours and then starved overnight in serum-free medium. Images before and 10 minutes after stimulation with indicated substances are shown. Scale bar = 20 μm. (B) GLUT4-GFP signal intensity increase was analyzed after 10 and 30 minutes of stimulation. Error bars are based on the standard error of the mean. **P < 0.01, ***P < 0.001 and ****P < 0.0001, significant increase with respect to KRPH treated cells. (C) For glucose uptake measurements 3T3-L1 cells were grown in 96-well plates (2,000 cells/well) and differentiated to adipocytes. Cells were starved in serum-free medium overnight, glucose deprived in KRPH buffer for 40 minutes, and then stimulated with the indicated substances for 20 minutes followed by addition of 2-DG (20 minutes). Cell extracts were prepared and 2-DG uptake was measured after a colorimetric reaction using a plate reader device. Samples were measured in duplicates at least in three individual experiments. Error bars are based on the standard error of the mean. *P < 0.05, **P < 0.01 and ****P < 0.0001, significant increase with respect to KRPH (Ins, PP60, PUR) or 0.25% DMSO in KRPH (TIN, GIN, BIL, JIA, MT) treated cells.