FIGURE 3.

Effect of AMPK α subunit knockdown on UGCG activity and GlcCer levels. A, immunoblot analysis showing the expression and phosphorylation levels of AMPK α subunit in siRNA-mediated AMPK α subunit-disrupted cells. Short interfering RNAs against AMPK α1 or α2 subunits were transfected into 3T3 cells and incubated for 72 h, and then AMPK α1-attenuated cells were treated with (−) or without (+) AICAR for 3 h. B, intracellular UGCG activity of AMPK α₁ subunit-attenuated cells. After treatment with AMPK α₁ siRNA, cells were incubated with (+) or without (−) AICAR (1 mm) for 8 h, and then NBD C6-ceramide conjugated with BSA was added to cells and incubated for 90 min and analyzed by TLC. C, quantification of cellular GlcCer and LacCer levels of AMPK-attenuated cells. Lipids were extracted from cells treated with (+) or without (−) AICAR (1 mm) for 8 h and then applied to LC-ESI MS, as described under “Experimental Procedures.” Bars, means ± S.D. (error bars) of at least three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.