FIG. 5.

Glucose-dependent binding of Mlx to the Gck promoter. A: Effects of overexpression of ChREBP-WT on Pklr, G6pc, Gckr, and Gck mRNA expression. Hepatocytes were either untreated (open bars) or treated with vectors for expression of ChREBP-WT at two viral titres (twofold dilution). After 18-h preculture, they were incubated for 4 h with 5 mmol/L (5) or 25 mmol/L (25) glucose. mRNA levels are expressed relative to untreated at 5 mmol/L glucose. Means ± SEM, n = 4–10. *P < 0.05, **P < 0.001, effect of ChREBP-WT. B and C: Recruitment of Mlx and ChREBP to the Pklr promoter and the Gck promoter. Hepatocytes were incubated for 4 h with 5 mmol/L glucose or 25 mmol/L glucose + 2 μmol/L S4048 [(25)S]. Chromatin immunoprecipitation was performed as described in research design and methods using either control IgG, or antibody to Mlx or ChREBP. The promoter and coding regions of the Pklr (B) and Gck (C) genes were amplified by real-time RT-PCR and binding of Mlx and ChREBP is expressed relative to the IgG control at 5 mmol/L glucose. For Pklr, the carbohydrate response element–containing region (30) of the promoter was amplified. For Gck, three regions of the promoter (29) spanning the residues indicated (D) were amplified. Results are means ± SEM, n = 4. *P < 0.05 relative to 5 mmol/L glucose.