Figure 4. Effects of A-769662 on gluconeogenesis in WT and AMPK-KO hepatocytes.

After attachment, WT and AMPK-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 1, 10, or 100 μM A-769662. After 8 hours, medium was collected for glucose measurement and cells were harvested for Western blot and gluconeogenic gene expression analyses. (A) Glucose production was normalized to protein content and expressed as a percentage of glucose production by WT hepatocytes incubated in the absence of both Bt₂-cAMP and A-769662. Results are representative of 5 independent experiments. (B) Immunoblots were performed against phospho-AMPKα (Thr172), AMPKα, phospho-ACC (Ser79), ACC, CRTC2, and PEPCK. Blots are representative of least 5 independent experiments. (C) Relative mRNA levels of Pgc-1α, Pepck, and G6Pase expressed as fold activation relative to levels in WT hepatocytes incubated in the absence of both Bt₂-cAMP and A-769662. Results are representative of 5 independent experiments. Data are mean ± SEM. ^§P < 0.01, ^‡P < 0.01 compared with WT and AMPK-KO hepatocytes incubated without Bt₂-cAMP; *P < 0.01, ^†P < 0.01 compared with WT and AMPK-KO hepatocytes incubated with Bt₂-cAMP alone.