FIG. 3. Diminished cortical F-actin and insulin-stimulated GLUT4 translocation in cells treated with chronic insulin are corrected by PIP₂ replenishment.

A–C, cells were treated overnight in the absence (Control) or presence of 5 nM insulin for 12 h. A, during the last 60 min of the 12-h period, the medium was replaced with the same medium enriched with either histone H1 (panels 1–4) or indicated concentrations of PIP₂/histone H1 (panels 5–10). Plasma membrane sheets (panels 1, 3, 5, 7, and 9) or whole cells (panels 2, 4, 6, 8, and 10) were labeled for PIP₂ (red) and actin (green), respectively. B, histone H1 (panels 1, 2, 4, and 5) or 1.25 μM PIP₂ (panels 3 and 6) add-back incubations were performed as described for A, sheets were prepared, and GLUT4 immunofluorescence was assessed. C, 2-[³H]Deoxyglucose (2-DG) uptake was determined as described under “Experimental Procedures.” D, cells cultured in 5.5 mM glucose were treated overnight in the absence (5.5 Glc) or presence of 5 nM insulin (5.5 Glc/12 h Ins) for 12 h. Add-back treatment parameters and GLUT4 detection was performed exactly as described for A and B. All microscopic and camera settings were identical between groups and representative images from three independent experiments are shown.