FIGURE 5.

Effects of IRS-2 knockdown on Akt activation and glucose uptake. A, 3T3-L1 adipocytes were electroporated with siRNA against nonrelevant control (NC), against IRS-1 (siIRS-1), or against IRS-2 (siIRS-2) as described under “Experimental Procedures.” Twenty four hours after electroporation, cells were serum-starved for 24 h. After washing, cells were treated with or without 100 nm insulin for 20 min. Cells were solubilized with Tris/Triton lysis buffer. Whole cell lysates were separated by SDS-PAGE and immunoblotted (IB) with anti-IRS-1 antibody, anti-IRS-2 antibody, anti-phospho-Akt (Ser-473), or anti-β-actin antibody. B, 3T3-L1 adipocytes were electroporated with siRNA against nonrelevant control (NC), against IRS-1 (siIRS-1), or against IRS-2 (siIRS-2) along with pGLUT4-myc-GFP. Twenty four hours after electroporation, cells were serum-starved for 2 h and then treated with or without 100 nm insulin for 20 min. Cells were then fixed without permeabilization and immunostained with anti-Myc antibody to detect cells with GLUT4 fused to plasma membrane. Average of percentage of cells showing GLUT4-myc-GFP rim on the cell surface was calculated. The ratio of GLUT4-myc on the cell surface in insulin-stimulated nonrelevant control cells was used as control. C, 3T3-L1 adipocytes were electroporated with siRNA against nonrelevant control (NC), against IRS-1 (siIRS-1), or against IRS-2 (siIRS-2). Cells were assayed for glucose uptake as described under “Experimental Procedures.” The results are presented as the means ± S.E. of five wells. Glucose uptake by the nonrelevant control cells without insulin was used as control. The difference between insulin-stimulated cells electroporated with siRNA against nonrelevant control and IRS-2 is significant with p < 0.001 (*).