FoxO1-ADA expression and the knockdown of FoxO1 affect Chrebp O-glycosylation, protein stability and transcriptional activity in primary hepatocytes.

<p>(A, B, D and E) Mouse primary hepatocytes infected with adenovirus expressing FoxO1-ADA or LacZ were cultured with 5 mM or 25 mM glucose. The cell lysates were subjected to real-time RT-PCR for Chrebp or L-PK (A), immunoprecipitation with anti-Chrebp antibody followed by blotting with anti-O-GlcNAc antibody (B) or anti-ubiquitin antibody (D). The cell lysates were also subjected to chromatin-immunoprecipitation assay using anti-Chrebp antibody and the primers for L-PK promoter (E). (C) Mouse primary hepatocytes infected with adenovirus expressing specific siRNA for FoxO1 or control siRNA were cultured with 5 mM or 25 mM glucose and the cell lysates were immunoprecipitated with anti-Chrebp antibody followed by blotting with anti-O-GlcNAc antibody. Input represents expression levels of Chrebp, FoxO1-ADA, endogenous FoxO1 and Tubulin. Quantitative analyses were performed by assessment of O-glycosylation level compared with the protein level of Chrebp using densitometry, showing as a bar graph below the results of blotting (B and C). (F) Mouse primary hepatocytes co-transfected with pGL3-3Xl-PK-ChoRE and OGT or LacZ were infected with adenovirus expressing FoxO1-ADA or LacZ at indicated MOI and cultured with 5 mM or 25 mM glucose for 24 hr. The cell lysates were used for luciferase assays. Experiments were repeated at least three times. Data represent mean ± SEM. *P<0.05.