Insulin stimulates Rac1 activation through PI3K-Akt in insulin-induced keratinocyte migration. (A-H). Cells were seeded in 8 well chamber slides for at least 24 h before the various treatments and then fixed and stained for either RhoA or Rac1 to visualize these molecules in the cell. (A-F) Cultures were either left untreated or treated with 10-7 M insulin for 3 or 5 min. (A-C) Staining for RhoA: (A), control, (B), insulin treatment 3 min (C), insulin treatment 5 min. Insulin does not stimulate translocation of RhoA to the cell membrane. (D-F) Staining for Rac1: (D), control. (E, F), insulin treatment at 3 and 5 min. Arrowhead shows membrane ruffles. Insulin induces Rac1 membrane translocation as well as membrane ruffling in migrating cells. (G) Cells were treated with insulin for 5 min and a Rac1 pull-down assay performed. Insulin induced Rac1 activation. (H) Cells were transfected using lipofectin with plasmids expressing the constitutively active form of Rac1 (V12, Rac1-CA), the dominant-negative mutant of Rac1 (N17, Rac1-DN) and the wild type Rac1 (Rac1-WT). Forty-eight hours after transfection, scratch wounds were made, and cells were treated with 10-7 M insulin. Cell migration was monitored for 24 h after insulin. Insulin-induced keratinocyte migration was eliminated by Rac1-DN. Each treatment group was performed in triplicate. Data is shown as the mean value +/- SD. ***P < 0.001 vs control treatment. (I, J) Cells were pre-treated with 50 μM LY294002 for 1 h followed by 10-7 M insulin treatment for 5 min. (I) Rac1 pull-down assay was then performed as mentioned above. Results are representative of three independent experiments. Levels of the active form of Rac1 were quantified by determining the ratio of the integrated density of the GTP binding form of Rac1 to GAPDH, the loading control, using ImageJ software. Data are shown as the mean value +/- SD. *P < 0.05 vs control treatment. Both Rac1 activation (I) and translocation to the plasma membrane (J) were inhibited by LY294002 pre-treatment.