FIG. 2.

C₂-ceramide inhibits insulin-stimulated GLUT4 and IRAP translocation. Plasma membrane GLUT4 (A and B) and IRAP (C and D) levels were measured as described in Materials and Methods. In both assays, C₂-ceramide (C2) (100 μM) or C₂-dihydroceramide (C₂H₂) (100 μM) was added 2 h prior to initiation of the assay. Insulin (20 nM) was present for the last 10 min. Immunofluorescence detection of GLUT4 on plasma membrane sheets was performed with polyclonal sheep anti-GLUT4 primary antibodies followed by rhodamine-conjugated anti-sheep secondary antibodies. Images were captured with a digital camera (A) and quantitated as described in Materials and Methods (B). Biotinylated IRAP was detected with streptavidin-HRP and visualized with enhanced chemifluorescence (C), and the results were quantitated on a phosphorimager (D). Each asterisk denotes that the difference from the value obtained in the presence of insulin alone was statistically significant at a P value of <0.05. Data presented are the averages ± standard errors of the means of three independent measurements.