FIG. 2.

Activity of α1³¹² in hepatocytes. Hepatocytes were grown in the presence of 25 mM glucose and 100 nM insulin and infected with either Ad.null or Ad.α1³¹² (30 PFU/cell). Twenty-four hours postinfection, hepatocytes were incubated in the presence or absence of 250 μM AICA riboside (AICAR) for 1 h before harvesting. (A) Total activity (endogenous AMPK and expressed α1³¹²) was measured in cell lysates, without prior immunoprecipitation, using the SAMS peptide assay. (B) Endogenous AMPK activity was measured in immune complexes isolated by immunoprecipitation using an anti-γ antibody bound to protein G-Sepharose. (C) AMPK activity present in either anti-γ (endogenous) or anti-Myc (expressed α1³¹²) immunoprecipitates was measured in the absence (shaded bars) or presence (open bars) of 0.2 mM AMP. Activities shown are the mean ± the SEM from four experiments (A and B) or the average of two independent experiments assayed in duplicate (C). ∗∗∗ denotes a significant difference from the Ad.null value (P < 0.0005), and ∗∗ indicates that P was <0.005. Activities are plotted as picomoles of ³²P incorporation/minutes per milliliter of lysate.