FIG. 3.

Induction of SHP by AMPK inhibits GH-induced PDK4 expression in primary hepatocytes. A: Rat primary hepatocytes were infected with 30, 60 multiplicity of infection (MOI) of adenoviral vector expressing CA-AMPK for 36 h. After infection with Ad-CA-AMPK, cells were treated with GH (500 ng/mL) for 1 h. Total RNA extracts were isolated and analyzed by RT-PCR analysis with the indicated primers and then normalized to an internal control (β-actin level). *P < 0.05 and **P < 0.05 compared with untreated control, GH-treated cells. B: Rat primary hepatocytes were infected with Ad-DN-AMPK for 36 h and then treated with GH (500 ng/mL) for 1 h in the presence or absence of metformin (Met) for 12 h. Total RNA were isolated from hepatocytes and used by RT-PCR analysis. SHP, PDK2, and PDK4 mRNA levels were normalized to an internal control with β-actin level. *P < 0.05 and **P < 0.01 compared with untreated control, GH-treated cells. Rat primary hepatocytes were infected with Ad-CA-AMPK (C) and Ad-DN-AMPK (D) for 30 or 60 MOI for 36 h. After infection, cells were treated with GH (500 ng/mL) for 1 h with or without metformin for 12 h. Specific proteins were determined by Western blot analysis with the indicated antibodies, and then normalized to an internal control (β-actin level and/or total forms). E: HepG2 cells were cotransfected with CA-AMPK and DN-AMPK and then treated with GH (500 ng/mL) for 1 h in the presence of metformin for 12 h. F: Cells were cotransfected with TEL-JAK2, STAT5, CA-AMPK, and DN-AMPK and then treated with metformin for 12 h. Luciferase (Luc) activity was normalized to β-galactosidase activity to correct for variations in transfection efficiency. All data are representative of at least three independent experiments. All data are shown as fold activations relative to the control (± SEM).