Retinoic acid-related orphan receptor γ (RORγ): a novel participant in the diurnal regulation of hepatic gluconeogenesis and insulin sensitivity

Abstract

The hepatic circadian clock plays a key role in the daily regulation of glucose metabolism, but the precise molecular mechanisms that coordinate these two biological processes are not fully understood. In this study, we identify a novel connection between the regulation of RORγ by the clock machinery and the diurnal regulation of glucose metabolic networks. We demonstrate that particularly at daytime, mice deficient in RORγ exhibit improved insulin sensitivity and glucose tolerance due to reduced hepatic gluconeogenesis. This is associated with a reduced peak expression of several glucose metabolic genes critical in the control of gluconeogenesis and glycolysis. Genome-wide cistromic profiling, promoter and mutation analysis support the concept that RORγ regulates the transcription of several glucose metabolic genes directly by binding ROREs in their promoter regulatory region. Similar observations were made in liver-specific RORγ-deficient mice suggesting that the changes in glucose homeostasis were directly related to the loss of hepatic RORγ expression. Altogether, our study shows that RORγ regulates several glucose metabolic genes downstream of the hepatic clock and identifies a novel metabolic function for RORγ in the diurnal regulation of hepatic gluconeogenesis and insulin sensitivity. The inhibition of the activation of several metabolic gene promoters by an RORγ antagonist suggests that antagonists may provide a novel strategy in the management of metabolic diseases, including type 2 diabetes.

Figure 1. Loss of RORγ improves insulin and glucose tolerance in a ZT-dependent manner.

ITT (A) and GTT (C) were examined at ZT4–6 and ZT18–20 in WT and RORγ^−/− mice fed a HFD for 6 weeks (n = 7–12). Data represent mean ±SEM, * P<0.05, ** P<0.01, *** P<0.001 by ANOVA. (B, D) Comparison of AUC for ITT and GTT by one way ANOVA. AUC was also calculated by 2-way ANOVA; for ITT: Time period P = 0.080 and Genotype P = 0.0002; for GTT: Time period P = 0.073 and Genotype P = 0.013 (not shown). (E) Loss of RORγ did not affect Akt activation. Total and phosphorylated of Akt were examined by Western blot analysis in liver, BAT, WAT, and skeletal muscle (SM) isolated from WT(HFD) and RORγ^−/−(HFD) mice 30 min after intraperitoneal injection of either 0.75 U/kg insulin or PBS. (F) Representative Western blot analysis (n = 2) of total and phosphorylated Akt in primary mouse hepatocytes isolated from WT and RORγ^−/− mice. Cells were treated with 20 nM insulin or PBS for 10 min before proteins were isolated.

Figure 2. Loss of RORγ leads to reduced hepatic gluconeogenesis at daytime.

(A) The hyperinsulinemic-euglycemic clamp test was performed during daytime (ZT2–9) in WT(HFD) and RORγ^−/−(HFD) mice and the glucose infusion rate (GIR), whole-body glucose disappearance (Rd), basal endogenous hepatic glucose production (Basal HGP), and endogenous hepatic glucose production during the clamp (Clamp HGP) were determined. (B) Suppression rate of hepatic glucose production by insulin in WT(HFD) and RORγ^−/−(HFD) mice. (C) PTT was examined at ZT4–6 and ZT18–20 in WT(HFD) and RORγ^−/−(HFD) mice (n = 8) as indicated. (D) Comparison of AUC for PTT by one way ANOVA. AUC for PTT was also evaluated by 2-way ANOVA: Time period, P = 0.0001; Genotype, P = 0.0009 (not shown). (E) PTT was examined at ZT4–6 in RORγ^−/−(HFD) mice injected with either empty or RORγ-containing adenovirus injection (n = 6). Data represent mean ±SEM, * P<0.05, ** P<0.01, *** P<0.001 by ANOVA.

Figure 3. Blood insulin and hepatic glycogen levels are reduced in RORγ^−/− mice.

(A) Comparison of food consumption between WT(HFD) and RORγ^−/−(HFD) mice (n = 8) during day- and nighttime. (B) Serum glucose and insulin levels were analyzed in WT(HFD) and RORγ^−/−(HFD) mice (n = 5) every 4 h over a period of 24 h. (C) Comparison of glucose-stimulated insulin secretion (GSIS) in WT and RORγ^−/− mice. Mice were fed either a HFD (n = 5–6) or ND (n = 2–3) and GSIS was analyzed as described in Materials and Methods. (D) Analysis of insulin content in pancreas of WT(HFD) and RORγ^−/−(HFD) mice (n = 10–14) collected at ZT16. (E) Comparison of glycogen accumulation in livers of WT(HFD) and RORγ^−/−(HFD) mice (n = 5) collected every 4 h over a period of 24 h. (F) Analysis of glycogen accumulation in livers from WT(HFD) and RORγ^−/−(HFD) mice (n = 7) collected at ZT4 after 16 h fasting. (G) Liver glycogen accumulation was enhanced in liver of RORγ^−/− mice (n = 6) injected with RORγ-expressing adenovirus. (H, I) Oxygen consumption (VO₂), CO₂ production (VCO₂), respiratory exchange ratio (RER), and heat production were measured during 3 successive days using metabolic cages and the average in each ZT was plotted as a ratio between RORγ^−/−(ND) and WT(ND) mice (n = 8). Data represent mean ±SEM, * P<0.05, ** P<0.01, *** P<0.001 by ANOVA.

Figure 4. Genome-wide mapping of RORγ and RORα binding sites in mouse liver.

(A) Summary of ChIP-Seq analysis using an anti-RORγ antibody and mouse hepatic chromatin. The RORγ binding regions were identified by SISSRs, P<0.001. (B) Genomic position of RORγ-binding regions on the mouse genome relative to the nearest gene. The promoter is defined as the region up to 5 kb upstream from TSS. (C) Distance from the center of each peak identified as a RORγ-binding site to transcriptional start site (TSS) of the nearest gene. (D) Motif analysis. De novo consensus motif analysis was performed within the RORγ binding sites using MEME program. This analysis identified a classic RORE motif, a DR1-like motif, and a RORE variant motif. (E) Venn diagram representing the overlap of the 3 consensus motifs within the RORγ binding regions. (F) Summary of ChIP-Seq analysis using an anti-RORα antibody and mouse hepatic chromatin. The RORα binding peaks were identified by SISSRs, P<0.001. (G) Venn diagram representing the overlap between the RORγ and RORα binding sites. Examples of genes containing common RORγ and RORα, binding sites and genes containing binding regions unique to RORγ are indicated.

Figure 5. RORγ regulates the circadian expression of genes involved in gluconeogenesis and glycolysis pathways.

(A) Circadian expression pattern of G6pase, Pepck, Glut2, Pklr, Gck, Gckr, Gys2, Pparδ, and Dlat in liver of WT(ND) and RORγ^−/−(ND) mice (n = 4). RNA was isolated every 4 h over a period of 24 h. (B) Pklr protein levels at ZT4 and ZT16 in whole liver lysates prepared from WT and RORγ^−/− mice fed either a ND or HFD (n = 2–3). Pklr was examined by Western blot analysis. (C) Differential expression of several metabolic genes in liver of WT(HFD) and RORγ^−/−(HFD) mice collected at ZT0 and ZT12 (n = 5). (D) Differential expression of Pcx and Klf15 in WT and RORγ^−/− livers collected at ZT12. (E) Adenovirus mediated over-expressing of RORγ in RORγ^−/− liver enhanced the expression of several glucose metabolic genes. (F) G6pase, Pepck, Gck, Glut2, and Gys2 expression in primary hepatocytes isolated from RORγ^−/− mice (n = 3) infected with either empty or RORγ lentivirus. Data represent mean ±SD, * P<0.05, ** P<0.01, *** P<0.001 by ANOVA.

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 RORα^sg/sg mice (n = 4). (F) Comparison of G6pase and Pparδ expression in liver collected from WT, RORα^sg/sg, RORγ^−/−, and RORα^sg/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.

Figure 7. Liver-specific RORγ deficient mice exhibit improved insulin sensitivity and reduced gluconeogenesis.

(A) RORγ expression in liver and kidney collected from RORγ^fx/fxAlb-Cre⁺ and -Alb-Cre⁻ mice at ZT8 and ZT20 (n = 4–5). GTT (B), ITT (C), and PTT (D) were examined during ZT4–6 in RORγ^fx/fxAlb-Cre⁺ and -Alb-Cre⁻ mice fed with a HFD (n = 7–11). (E) Serum insulin levels were measured at ZT4 (n = 8) and ZT16 (n = 15–16) in RORγ^fx/fxAlb-Cre⁺ and -Alb-Cre⁻ mice on a HFD. Hepatic glycogen was measured at ZT0. Data represent mean ±SEM, * P<0.05, ** P<0.01 by ANOVA. (F) The expression of a series of glucose metabolic genes was analyzed in the liver collected at ZT8 or ZT20 (n = 4–5). Data represent mean ±SD, * P<0.05, ** P<0.01, *** P<0.001 by ANOVA. (G) RORγ coordinates the regulation of circadian rhythm, hepatic glucose metabolism, and insulin sensitivity. Genome-wide cistromic profiling and promoter analysis revealed that RORγ is targeting and regulating a number of metabolic genes critical in the control of glycolysis, gluconeogenesis and glycogenesis pathways. The loss of RORγ in hepatocytes reduces the expression of these genes and hepatic gluconeogenesis in a diurnal time-dependent manner that results in improved insulin sensitivity. Due to reduced hepatic glucose production, RORγ^−/− mice may require less insulin than WT mice to maintain blood glucose levels. A decrease in glucose uptake due to lower insulin levels as well as reduced Gys2 expression may in part be responsible for the reduced accumulation of liver glycogen. Our study supports the model that the circadian regulation of several glucose metabolic genes by RORγ in liver is linked to its circadian control of gluconeogenesis, insulin sensitivity, and glucose tolerance and is consistent with the idea that RORγ functions as a positive regulator of gluconeogenesis and is positively linked to increased risk for type 2 diabetes.