A. 832/13 cells were treated for 6 h with 2 or 20 mM glucose in the presence or absence of 10 µM forskolin. Total RNA was isolated, and RT-PCR was performed using primers specific for L-PK. Values are means ± SEM from three independent experiments. *P 0.001 vs. 20 mM glucose. (B–C, F) 832/13 cells were transduced with adenovirus expressing either GFP, wild-type ChREBP or a dominant- negative ChREBP protein for 24 h. B. Cell lysates were harvested, the nuclear fraction was isolated and immunoblots performed with antibodies for either ChREBP or β Actin serving as a loading control. The immunoblot is a representative of 2 independent experiments. C, F. 24 h following viral transduction, cells were then treated with 2 or 20 mM glucose for 6 h, (C) total RNA was isolated and relative abundance of L-PK mRNA was examined and (F) chromatin immunoprecipitation assays was performed using an antibody against CBP; a segment of the L-PK gene promoter containing the ChoRE site and a portion of the coding region were targeted for amplification. Data are means ± SEM from three individual experiments. *P < 0.05 vs. 20 mM glucose, ‡P < 0.01 vs. 20 mM glucose. (D–E, G) 832/13 cells were transfected with either a negative siRNA control (siScramble) or an siRNA duplex targeting the coding region of CBP. D. After a 48 h incubation with siRNA duplexes, nuclear extracts were prepared, and immunoblots performed with antibodies against CBP with β-Actin as a loading control. The immunoblot is a representative of 2 independent experiments. E, G. Following exposure to siRNA duplexes for 48 h, cells were treated with 2 or 20 mM glucose (E) total RNA was isolated and relative abundance of L-PK mRNA was examined via RT-PCR and (G) chromatin immunoprecipitation assays were performed using an antibody against ChREBP with the L-PK gene promoter and coding region targeted for amplification via RT-PCR. Data represent means ± SEM from three individual experiments. **P < 0.05 vs. siScramble at 2 mM glucose, *P < 0.001 vs. 20 mM glucose, ‡ P < 0.05 vs 20 mM glucose.

Cells were treated with 2 or 20 mM glucose in the presence or absence of 10 µM forskolin for 6 h. Chromatin immunoprecipitation assays was performed using antibodies against Pol II pCTD^Ser5 (A), or Pol II pCTD^Ser2 (B), and normalized to control IgG. The L-PK promoter and coding region were targeted for amplification via real-time RT-PCR. Data shown are means ± SEM from five independent experiments. *P < 0.05 vs. 20 mM glucose, ‡ P < 0.05 vs 20 mM glucose.

A, C, E. 832/13 cells were treated with 2 or 20 mM glucose in the presence or absence of 10 µM forskolin for 6 h. B, C, F. Recombinant adenoviruses expressing GFP, wild-type ChREBP or dominant-negative ChREBP were transduced into cells for 24 h, cells were then cultured in 2 or 20 mM glucose in the presence or absence of 10 µM forskolin for an additional 6 h. Chromatin immunoprecipitation assays were employed to determine relative promoter occupancy of methylated histone H3-K4 (A–D) and H3-K9 (E–F), and normalized to control IgG. The L-PK promoter and a segment of the L-PK coding region were targeted for amplification by RT-PCR. Data represent means ± SE from three to five independent experiments. *P < 0.05 vs. 20 mM glucose samples, ‡ P < 0.05 vs 20 mM glucose.

A. 832/13 cells were incubated in 2 or 20 mM glucose in the presence or absence of 10 mM forskolin for 6 h. B. Cells were transduced with the indicated adenoviruses for 24 h, followed by a 6 h incubation in 2 or 20 mM glucose. C. Cells were transfected with an siScramble or siCBP duplex for 48 h, then cultured in 2 or 20 mM glucose for 6 h. Using an anti-Pol II antibody, relative promoter binding of Pol II was determined via ChIP assay. Gene promoter (A–D) and coding region (A) for L-PK was targeted for amplification, and the data were normalized to control IgG. Data are expressed as means ± SEM from four to five independent experiments. *P < 0.05 vs. 20 mM glucose. D. Cells were treated as in (A), CBP was then immunoprecipitated with anti-CBP antibody, with anti-IgG antisera serving as a control. Pol II was subsequently immunoprecipitated from the resulting eluate using a Pol II antibody, again anti-IgG was used as a control. Values are means ± SEM from four individual experiments. *P < 0.01 vs. 20 mM glucose, ‡ P < 0.05 vs 20 mM glucose.

A, B. 832/13 cells were cultured in 2 or 20 mM glucose in the presence or absence of 10 µM forskolin for 6 h. C, D. Cells were transduced with adenoviruses expressing GFP, wild-type ChREBP or dominant-negative ChREBP for 24 h, followed by culture in 2 or 20 mM glucose for an additional 6 h. E, F. Cells were transfected with an siScramble or siCBP duplex for 48 h, then cultured for 6 h in 2 or 20 mM glucose. Relative promoter and coding region occupancy were determined by chromatin immunoprecipitation assay, using antibodies against either acetylated histones H3 (A, C, E) or H4 (B, D, F), and normalized to control IgG. Data represent means ± SE from three to five independent experiments. **P < 0.05 vs. 2 mM glucose control samples, *P < 0.05 vs. 20 mM glucose control samples, ‡ P < 0.05 vs 20 mM glucose.