Fig. 3.

High glucose regulates LXR transactivation of the ChREBPα promoter via O-GlcNAc. A: Huh7 cells maintained in 25 mM glucose were transfected with ChREBPα-Firefly luciferase reporter (ChREBPα-prom), Renilla luciferase control plasmid (pRL-CMV) and RXRα expression vector and/or LXRα or LXRβ expression vectors or empty vector (pcDNA3.1) (n = 3). Dual luciferase assay was performed and lysates were immunoblotted with antibodies against FLAG, RXRα, and β-actin as loading control. A schematic figure of the ChREBPα reporter construct (ChREBPα-prom) including the two LXREs is shown. B: Huh7 cells maintained in 5 mM glucose were transfected with ChREBPα-prom or pGL3-Basic with empty expression vector or RXRα and LXRα expression vectors. All transfections contained pRL-CMV. After 6 h, the cells were treated with 1 mM or 25 mM glucose for 24 h (n = 3). Dual luciferase assay was performed and lysates were subjected to SDS-PAGE and immunoblotted with an antibody detecting O-GlcNAc epitopes on proteins (CTD 110.6) with β-actin as loading control. One representative Western blot is shown (n = 3). C: Huh7 cells maintained in 5 mM glucose (upper panel) and 25 mM glucose (lower panel) were transfected with 40 nM siRNA, nontargeting pool (siSrc), or human OGT (siOGT). After 24 h (25 mM glucose) and 48 h (5 mM glucose) the cells were further transfected with ChREBPα-prom, pRL-CMV with empty vector, or RXRα and LXRα expression vectors. Dual luciferase assay was performed after another 24 h. Lysates were subjected to SDS-PAGE and immunoblotted with antibodies against OGT, O-GlcNAc (CTD 110.6), LXRα, and RXRα and β-actin as loading control (n = 3). All values are given as mean ± SEM. Statistical differences are shown as *P < 0.05 or ***P < 0.001 between respective controls or as ^###P < 0.001 between indicated groups.