Retinoic Acid-Related Orphan Receptor γ (RORγ): A Novel Participant in the Diurnal Regulation of Hepatic Gluconeogenesis and Insulin Sensitivity
Figure 6
Transcriptional regulation of glucose metabolic genes by RORγ.
(A) Sequence and activation of the RORγ binding region of the G6pase(−500/+58) proximal promoter. The ROREs and PPRE are indicated in bold. Activation of the G6pase promoter by RORγ was examined by transfecting Huh-7 cells as indicated with pCMV-β-Gal, pCMV10-3xFlag-RORγ, -RORα or -PPARα (with 10 µM Wy14,643) expression vectors and a pGL4.10 reporter driven by G6Pase(−500/+58) or the promoter in which the RORE and PPRE were mutated. Luciferase activities were normalized to the control transfected with the empty expression vector. (B) Inhibition of the activation of the G6pase(−500/+58) promoter by RORγ-selective antagonist “A”. (C) Activation of the Pparδ regulatory region by RORγ. Sequence of the RORγ binding region in intron 2 of Pparδ. The three potential ROREs are indicated in bold. Huh-7 cells were co-transfected with pCMV-β-Gal, pCMV10-3xFlag-RORγ or -RORα expression vector, and the pGL4.27 reporter plasmid containing the Pparδ (intron 2) or the intron in which the ROREs are mutated. (D) Inhibition of the activation of the Pparδ (intron 2) by the RORγ-selective antagonist. Data represent mean ±SEM, * P<0.05 by ANOVA. (E) Loss of RORα does not affect the circadian expression of G6pase and Pparδ in liver of WT and RORαsg/sg mice (n = 4). (F) Comparison of G6pase and Pparδ expression in liver collected from WT, RORαsg/sg, RORγ−/−, and RORαsg/sgRORγ−/−DKO mice at ZT8 and ZT20. Data represent mean ±SD, * P<0.05, ** P<0.01, *** P<0.001 by ANOVA. (G) Huh-7 cells were co-transfected with pGL4.27 in which the reporter was under the control of Gck (intron 1) or Gck (intron 1) containing a mutated RORE or truncated Gck (intron 1) without the RORE. (H) Huh-7 cells were co-transfected with pGL4.10 plasmid containing the mouse Gckr promoter (−685/+42) or the promoter containing mutated ROREs. Data represent mean ±SEM, * P<0.05 by ANOVA.