O-GlcNAcylation Increases ChREBP Protein Content and Transcriptional Activity in the Liver

(Downloading may take up to 30 seconds. If the slide opens in your browser, select File -> Save As to save it.)

Click on image to view larger version.

FIG. 2.
FIG. 2.

Elevated O-GlcNAc levels by GlcNH2 administration in vivo increases ChREBP protein content. Four groups of C57BL/6J mice were studied: a 24-h fasted group, a fasted group refed a regular diet for 18 h, and the last groups of mice were force-fed with glucose (5 g/kg) or GlcNH2 (2.5 g/kg) after the fasting period. Mice were killed at the indicated time and livers were analyzed. A: Western blot (Wb) analysis of O-GlcNAc and ChREBP levels. β-Actin was used as a loading control. Lanes were run on the same gel but were noncontiguous. Representative Western blots are shown. Quantification of the ratio of total ChREBP compared with β-actin content is shown. Data are means ± SEM. n = 6–10 per group. **P < 0.005 compared with the fasting state. B: ChREBPOG was evaluated by purification of proteins with an O-GlcNAc–specific lectin, WGA. Specificity of the binding was confirmed by GlcNAc (0.5 M) competition. A representative Western blot is shown. β-Actin was used as a loading control. Quantification of the ratio of ChREBPOG compared with total ChREBP content is shown. Data are means ± SEM. n = 6–10 per group. **P < 0.005 compared with the fasting state. C: Immunofluorescence analysis of ChREBP in liver sections from 24-h fasted, refed, and force-fed mice with either glucose (5 g/kg) or GlcNH2 (2.5 g/kg) after the fasting period. No signal was obtained when liver sections were incubated with the secondary antibody only (data not shown). D: Detection of nuclear ChREBPOG by WGA binding in nuclear extracts from 24-h fasted, refed, and/or force-fed mice with either glucose (5 g/kg) or GlcNH2 (2.5 g/kg) for 8 h. A representative Western blot is shown. n = 6–10 per group. Lanes were run on the same gel but were noncontiguous. E: Quantitative RT-PCR analysis of ChREBP (blacks bars) and L-PK (white bars) in the livers of the four groups of mice. Data are means ± SEM. n = 6–10 per group. *P < 0.01, **P < 0.005 compared with fasted mice (within the same color). (A high-quality digital representation of this figure is available in the online issue.)

This Article

  1. Diabetes vol. 60 no. 5 1399-1413