Effects of recombiant adiponectin and AICAR treatments on AMP kinase activation in MC3T3-E1 cells. MC3T3-E1 cells were starved in serum-free medium for 2 h and then stimulated by addition of serum-free medium with 3.0 μg/ml recombinant adiponectin or 0.5–2.0 mM AICAR. Whole cell lysates were collected after 5 min incubation for Western blotting. Immunoprecipitation and immunoblotting were performed as described in MATERIALS AND METHODS. The lower panels show the intensity of phosphorylated AMP kinase (p-AMPK) that was measured using the NIH Image Program and was normalized using the total AMP kinase (AMPK) signal. Recombinant adiponectin (A) and AICAR (B) activated AMP kinase rapidly. The result was the representative of three different experiments.