Figure 6.

AMPK Mediates the Inhibitory Effect of Adiponectin on LH Secretion

A, In vitro kinase activity of mutant AMPKs. LβT2 cells were infected with adenoviruses expressing either a dominant-negative form of AMPKα1 (AMPK-DN) or a kinase-dead form of AMPKα2 (AMPK-KD) for 48 h. Cells were also treated with 20 μm compound C (Cpd.C) or 1 mm AICAR for 4 h. AMPK activity was determined by kinase assay using SAMS peptide. A kinase reaction in the presence of 300 μm AMP serves as a positive control. B, Mutant AMPKs block endogenous AMPK activity in cells. CHO cells were infected with AMPK-DN or AMPK-KD. Cells were treated with 1 mm AICAR for 2 h, and phospho-ACC was detected by immunoblotting whole-cell extracts. Extracts of uninfected and infected cells were immunoblotted for the HA or c-Myc tags to demonstrate mutant protein expression (lower panels). C, A dominant-negative AMPK mutant increases LH secretion and synthesis. LβT2 cells were infected with dominant-negative AMPK adenovirus (AMPK-DN) or control virus (GFP). Cells were serum starved 32 h later for an additional 16 h. Cells were then washed and subjected to secretion in a 30-min period with or without 100 nm GnRH. Secreted LH and cellular LH were determined, and LH levels (ng) per mg of cell lysates are shown. D, A dominant-negative AMPK mutant blocks the acute effect of adiponectin. Infected cells were treated with 20 μg/ml adiponectin (fAd) for 30 min. Secreted and cellular LH levels were determined. Experiments were performed in triplicate. Data are shown as mean ± sd from three experiments. Asterisks indicate statistical significance (P < 0.05) vs. control or between conditions connected by bars. HA, Hemagglutinin; Ctl, control.