Figure 7. Effects of metformin on AMPK activation in WT and Lkb1-KO hepatocytes.

After attachment, WT and LKB1-deficient primary hepatocytes were cultured for 16 hours in M199 medium containing 100 nM dex. Hepatocytes were then incubated in glucose-free DMEM containing lactate/pyruvate (10:1 mM) and 100 nM dex alone or with 100 μM Bt₂-cAMP and with or without 0.25, 0.5, 1, or 2 mM metformin. After 8 hours, cells were harvested for Western blot analysis and measurement of LKB1 activity. (A) The level of LKB1 protein was assessed by immunoblot analysis using anti-LKB1 antibodies. β-Actin was immunoblotted as a loading control. (B) LKB1 activity was assessed following its immunoprecipitation and assayed with the LKBtide peptide. Assays were performed on hepatocyte extracts from 3 independent experiments. (C) Immunoblots were performed against phospho-AMPKα (Thr172), AMPKα, phospho-ACC (Ser79), ACC, CRTC2, G6Pase, and PEPCK. Blots are representative of 3 independent experiments.