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

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FIG. 3.
FIG. 3.

OGT overexpression increases ChREBP protein content and activity in the liver of C57BL/6J mice. OGT (5 × 109 pfu/mouse) was overexpressed in the liver of C57BL/6J mice through penis vein injection. An equivalent dose of GFP adenovirus was used as control. Mice were fasted for 24 h or refed a regular diet following the fasting period (n = 6–10 mice per group). A: Liver extracts were immunoblotted with O-GlcNAc, ChREBP, and OGT antibodies. GFP was used as loading control. Representative Western blots (Wb) are shown. B: Quantitative RT-PCR analysis of OGT and ChREBP. Data are means ± SEM. n = 6 per group. *P < 0.01, OGT vs. GFP mice; #P < 0.01, refed vs. fasted. C: ChREBPOG were obtained by WGA binding experiments. Specificity of the binding was confirmed by GlcNAc (0.5 mmol/L) competition. β-Actin was used as a loading control. Lanes were run on the same gel but were noncontiguous. D: ChREBP levels (cytosolic and nuclear) and nuclear ChREBPOG were analyzed by Western blot. Lamin A/C was used as a control. Representative Western blots are shown. Lanes were run on the same gel but were noncontiguous. E: ChREBP at the ChoRE containing region of the L-PK promoter were measured by ChIP analysis. Immunoprecipitated L-PK promoter sequence was analyzed by quantitative PCR. Data are means ± SEM. n = 3 per group. *P < 0.01 compared with fasted GFP mice; #P < 0.01 compared with refed GFP mice. F: Quantitative RT-PCR analysis of L-PK. Data are means ± SEM. n = 6 per group. *P < 0.01 compared with fasted GFP mice; ##P < 0.005 compared with refed GFP mice. (A high-quality color representation of this figure is available in the online issue.)

This Article

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