Journal: The Biochemical journal
Article Title: Regulation of basal expression of hepatic PEPCK and G6Pase by AKT2
Figure Lengend Snippet: CREB phosphorylation mediates AKT2-regulated induction of PEPCK. ( A ) CREB phosphorylation induced by PTEN loss is regulated by AKT2. Liver lysates from mice with different genotypes were analysis for p-CREB. PM, Pten deleted; DM, Pten and Akt2 deleted; AM, Akt2 deleted. Two biological replicas are included for each genotype group. Protein levels of p-AKT, PTEN and p-GSK3 are analyzed and showed up-regulation of AKT and GSK-3 with PTEN loss and inhibition when AKT2 is lost. Phosphorylation of CREB is only observed when PTEN is lost. AKT2 loss together with PTEN blocked this phosphorylation in the DM livers. ( B ) Akt2 +/+ and Akt2 − / − cell were treated with forskolin (100 μM) for 24 h to induce cAMP and CREB phosphorylation or vehicle as controls. Phosphorylation of CREB is detected and showed to be lower when AKT2 is lost. When forskolin is added to induce cAMP, protein expression of p-CREB is evaluated in both Akt2 +/+ and Akt2 − / − cells even though its levels remains lower in the Akt2 − / − cells. Vinculin is probed as loading control. ( C ) Introduction of CREB-133A and wtCREB to Akt2 +/+ and Akt2 −/− hepatocytes. Expression of PEPCK is analyzed using quantitative PCR analysis. Left panel, CREB-S133A is introduced to Akt2 +/+ cells. Middle panel, wtCREB is introduced to Akt2 −/− cells; Right panel, wtCREB is introduced to Akt2 +/+ cells. n = 3. Data presented as mean ± SEM. * P ≤ 0.05. Different from vector transfected cells of the same genotype. Experiments repeated at least three times.
Article Snippet: The medium was replaced with fresh medium with 100 μM Forskolin (Alomone Labs Cat# F-500) or vehicle and incubated for 24 h before harvesting.
Techniques: Mouse Assay, Inhibition, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection