FIGURE 5.

Effect of AICAR on Tbc1d1 inhibition of GLUT4 translocation. 3T3-L1 adipocytes were electroporated with HA-GLUT4-GFP and either control vector plasmid (V) or the plasmid for 3× FLAG-tagged human Tbc1d1. The cells were untreated (basal, B) or treated with insulin (I), 1 mm AICAR (A), or 1 mm AICAR plus insulin (Ins). Treatment with AICAR was 70 min. Insulin was added during the final 30 min. A, cell surface HA-GLUT4-GFP was measured as described under “Experimental Procedures.” The values are the averages ± S.E. for four independent experiments. They have been normalized to a value of 1.0 for the vector (Vec) control in the insulin state. The significance of the difference between surface GLUT4 in the presence of insulin versus AICAR plus insulin for cells overexpressing Tbc1d1 was calculated by the Student's two-tailed, paired t test. B, SDS samples of the cells in A were immunoblotted for pThr172 AMPK, AMPK, pThr56 eEF2, eEF2, and 3× FLAG-tagged Tbc1d1. The 1× load contained 30 μg of protein. A repetition of these blots with the samples from a second experiment gave similar results.