FIGURE 3.

Effects of expressing constitutively active (CA) or dominant negative (DN) forms of AMPK on biguanide or AICAR-induced AMPK and ACC-2 phosphorylation. A, L6 myoblasts were infected with increasing concentrations of adenovirus encoding the CA form of AMPK. Infected myoblasts were permitted to fully differentiate and lysed 48 h post-infection. Lysates (50 μg of protein) were immunoblotted with antibodies against GFPα and anti-AMPKα (which cross-react with N-terminal domains of both AMPKα1 and AMPKα2). B, L6 myoblasts were infected with 150 pfu/cell of adenovirus expressing a DN form of AMPK. Muscle cells were lysed 48 h post-infection, and lysates (50 μg of protein) were immunoblotted with antibodies against the c-Myc tag, phospho-AMPK Thr¹⁷², and AMPK. C and D, L6 cells were infected as described above with 150 pfu/cell of either control adenovirus expressing GFPα (Con), adenovirus encoding the CA-AMPK (CA), or the DN-AMPK (DN). After 48 h, cells were incubated with or without phenformin (Phen) (200 μm), metformin (Met) (1 mm), or AICAR (2 mm) for a further 16 h. Cells were lysed and immunoblotted for antibodies targeting phospho-AMPK Thr¹⁷², α-AMPK, phospho-ACC-2 Ser⁷⁹, or PKB, which was used here as a gel loading control. Immunoblots are representative of two separate experiments.