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Fig. 7. Expression of Rabip4 unable to bind Rab4 does not affect insulin-induced glucose transport, but induces the translocation of Glut 4-myc-DsRed to the plasma membrane. (A-B) 3T3-L1 Rabip4 507-517 Tet-off adipocytes were cultivated for 3 days with or without tetracycline (tetra) to induce the expression of Rabip4507-517. (A) Serum-deprived 3T3-L1 Rabip4 507-517 Tet-off adipocytes were untreated or treated with the indicated concentrations of insulin for 20 minutes. Deoxyglucose uptake was then measured and the results of three independent experiments are shown, expressed, as the fold increase compared with basal levels in tetracycline-treated cells. The inset shows the expression of Rabip4 507-517 in adipocytes cultivated with or without tetracycline by western blot using anti-Rabip4 antibodies. (B) Serum-deprived cells were untreated or treated with 100 nM insulin for 20 minutes and used to prepare plasma membrane sheets as described in the Materials and Methods. The graph represents the mean ± s.e.m. of three experiments of the fold increase in insulin stimulation over the basal condition of cells cultivated with tetracycline (no Rabip4 expression). The amounts of Glut 4 and of transferrin receptors were quantified. (C-D) 3T3-L1 adipocytes expressing Glut 4-myc-DsRed in combination with GFP or GFP-Rabip4 507-517 were untreated or treated with 100 nM insulin for 20 minutes. Fixed cells were incubated with anti-myc mAb that binds the extracellular myc epitope of Glut 4-myc-DsRed incorporated into the plasma membrane. The anti-myc antibody is then detected using Cy5-coupled anti mouse antibodies to visualize the plasma membrane Glut 4 (mGlut4). (C) Typical labeling for GFP and GFP-Rabip4 507-517 (in green), Glut 4-myc-DsRed (Glut 4, in red) and membrane Glut 4-myc-DsRed (mGlut 4) (in blue) are shown for basal and insulin-stimulated conditions. (D) The number of adipocytes with Glut 4-myc-DsRed detected at the plasma membrane was counted. 50-100 co-transfected cells were counted in each experimental condition and the figure shows the mean ± s.e.m. of the numbers obtained in six independent experiments. ^*P<0.001 compared with levels in the +tetra control.

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