The Small Molecule Nobiletin Targets the Molecular Oscillator to Enhance Circadian Rhythms and Protect against Metabolic Syndrome

Abstract

Dysregulation of circadian rhythms is associated with metabolic dysfunction, yet it is unclear whether enhancing clock function can ameliorate metabolic disorders. In an unbiased chemical screen using fibroblasts expressing PER2::Luc, we identified Nobiletin (NOB), a natural polymethoxylated flavone, as a clock amplitude-enhancing small molecule. When administered to diet-induced obese (DIO) mice, NOB strongly counteracted metabolic syndrome and augmented energy expenditure and locomotor activity in a Clock gene-dependent manner. In db/db mutant mice, the clock is also required for the mitigating effects of NOB on metabolic disorders. In DIO mouse liver, NOB enhanced clock protein levels and elicited pronounced gene expression remodeling. We identified retinoid acid receptor-related orphan receptors as direct targets of NOB, revealing a pharmacological intervention that enhances circadian rhythms to combat metabolic disease via the circadian gene network.

Keywords: Nobiletin; circadian clock; clock amplitude-enhancing small molecule; metabolic syndrome; natural flavonoid; retinoid acid receptor-related orphan receptors (RORs).

Figure 1. Nobiletin (NOB) enhances amplitude of circadian rhythms

(A) Nobiletin (NOB) chemical structure. (B) Primary screening of the NIH Clinical Collection (left) and Microsource Spectrum Collection (right) showing enhancement of the PER2::Luc Clock^Δ19/+ reporter rhythm by NOB. (C) Left: Dose-dependent effects of NOB on reporter rhythms from PER2::LucSV cells. Right: Quantification of amplitude response to NOB doses. (D) NOB was not able to rescue reporter rhythms in PER2::Luc Clock^Δ19/^Δ19 fibroblast cells. (E) Clock-enhancing effects of NOB in pituitary explants from PER2::Luc WT (left) and PER2::Luc Clock^Δ19/+ (right) reporter mice. (F) Left: Actograms illustrating the effect of vehicle or NOB on circadian behavior in RC-fed WT mice (n=5). Middle: Average wave plots summarizing wheel-running activity during 12–14 days of LD (12hr light, 12hr dark) or DD (Constant darkness) for indicated genotypes (n=5). Right: Daily total wheel-running activity during L:D or D:D conditions for the indicated genotypes (n=5). Data are represented as mean ± SEM.

Figure 2. NOB modulated energy homeostasis and behavior in diet-induced obesity (DIO) mice in a clock-dependent manner

(A) Average body weight of WT or Clock^Δ19/Δ19 mutant mice fed with high-fat diet and treated with either vehicle or NOB (WT.HF.Veh, WT.HF.NOB, Clk.HF.Veh and Clk.HF.NOB) for 10 weeks (n=8–15). (B) Daily food intake for the above 4 groups of mice (n=8–15). (C) Body mass composition as analyzed by NMR (n=3). (D) Histological analysis of white adipose fat (WAT) after 10-week treatment. WAT was subjected to H&E staining. (E) The diurnal rhythms of VO₂ (volume of oxygen consumed) in the 4 groups of mice (n=8). (F) Left: Actograms illustrating the effect of vehicle or NOB on circadian behavior in HFD-fed WT mice (n=7). Middle: Average wave plots summarizing wheel-running activity during 12–14 days of LD (12hr light, 12hr dark) or DD (Constant darkness) for indicated genotypes (n=7). Right: Daily total wheel-running activity during L:D or D:D conditions for the indicated genotypes (n=7). (G) Left: Actograms illustrating the effect of vehicle or NOB on circadian behavior in HFD-fed Clock^Δ19/Δ19 mutant mice (n=3). Middle: Average wave plots summarizing wheel-running activity during 10–12 days of LD (12hr light, 12hr dark) or DD (Constant darkness) for indicated genotypes (n=3). Right: Daily total wheel-running activity during L:D or D:D conditions for the indicated genotypes (n=3). Data are represented as mean ± SEM. ***p < 0.001. WT.HF.NOB vs. WT.HF.Veh.

Figure 3. NOB improved glucose and lipid homeostasis in diet-induced obesity (DIO) mice in a clock-dependent manner

(A) Fasting blood glucose levels in HFD-fed WT (left) and Clock^Δ19/Δ19 mutant mice (right) with Vehicle or NOB treatment at two opposite time points (n=8–15). (B) Effect of NOB on glucose tolerance in HFD-fed WT and Clock^Δ19/Δ19 mutant mice as measured by glucose tolerance test (GTT) (n=8–15). Right panel: Area under curve (AUC). (C) Effect of NOB on insulin tolerance in HFD-fed WT and Clock^Δ19/Δ19 mutant mice as measured by insulin tolerance test (ITT) (n=8–15). Right panel: Area under curve (AUC). (D) Blood insulin levels in HFD-fed WT mice and Clock^Δ19/Δ19 mutant mice with Vehicle or NOB treatment (n=8–15). (E) Total triglyceride (TG) levels and cholesterol (TC) levels in blood after 10-week treatment (n=8–15). (F) Total triglyceride (TG) levels and cholesterol (TC) levels in liver after 10-week treatment (n=8–15). (G) H&E staining of whole livers from HFD-fed WT and Clock^Δ19/Δ19 mutant mice after 10-week treatment. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. WT.HF.NOB vs. WT.HF.Veh.

Figure 4. NOB efficacy against metabolic syndrome in Type 2 diabetic db/db mice requires a functional circadian clock

(A) Left panel: Average body weight of db/db or db/db Clock^Δ19/Δ19 double mutant mice fed with regular chow and treated with either vehicle or NOB (Db.Veh, Db.NOB, Db.Clk.Veh and Db.Clk.NOB) for 10 weeks (n=6–8). The mice were 6–8 weeks old at the beginning of the treatment. Right panel: Average body weight gain for these 4 groups of mice. (B) Fasting blood glucose levels in db/db (left) and db/db Clock^Δ19/Δ19 double mutant mice (right) at two opposite time points (n=6–8). (C) Effect of NOB on glucose tolerance in db/db and db/db Clock^Δ19/Δ19 double mutant mice as measured by glucose tolerance test (GTT) (n=6–8). Right panel: Area under curve (AUC). (D) Effect of NOB on insulin tolerance in db/db and db/db Clock^Δ19/Δ19 double mutant mice as measured by insulin tolerance test (ITT) (n=6–8). Right panel: Area under curve (AUC). (E and F) Blood total triglyceride (TG) levels and cholesterol (TC) levels in db/db and db/db Clock^Δ19/Δ19 mice (n=6–8). (G) Effects of NOB on circulating insulin levels in both db/db and db/db Clock^Δ19/Δ19 mice (n=6–8). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. NOB vs. Veh. #p < 0.05 and ###p < 0.001. Db.Veh vs. Db.Clk.Veh.

Figure 5. NOB restored circadian oscillation of clock and metabolic output genes in the liver of HFD-fed WT mice

(A and B) Liver samples were collected from HFD-fed WT mice with vehicle or NOB treatment (WT.HF.Veh and WT.HF.NOB). WT mice fed with regular chow (WT.RC.Veh) were used as controls for comparison (N=3–4). Western blotting (A) and Real-time qPCR (B) analyses were performed to determine protein and mRNA expression of clock genes. (C) Heat map of microarray gene expression data indicating that the expression patterns of 56 genes were altered by HFD, and NOB reversed, to varying degrees, their expression to approximate RC levels in WT mouse liver at both ZT2 and ZT14 time points. Color scale indicates median normalized signal intensity in relative values. (D) Functional classification of 56 genes in (C) by the Gene Ontology (GO) program. Percentages of genes sharing GO biological processes are shown. (E) Real-time qPCR analysis of mRNA expression of clock-controlled metabolic output genes in the livers from treated mice as above. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. WT.HF.NOB vs. WT.HF.Veh.

Figure 6. NOB enhanced RORα/γ transcriptional activity via direct binding to RORα/γ

(A and B) Saturation curves and Scatchard plots from filter binding assays for RORα-LBD and RORγ-LBD. (A) Saturation curves for 25-[3H]-OHC binding were generated with 100ng of RORα-LBD (top) and 200ng of RORγ-LBD (bottom) (n=3). Dissociation constant values are shown. (B) Saturation curve results in Fig. 5E were subjected to Scatchard analysis and Scatchard plots for 25-[3H]-OHC are shown for RORα-LBD (top) and RORγ-LBD (bottom) corresponding to (n=3). This analysis gave a dissociation constant (Kd) of 6.10 nM and a total number of binding sites (Bmax) of 100 fmol/mg of protein for RORα, and 6.67 nM and 410 fmol/mg of protein for RORγ. (C and D) In vitro competitive radio-ligand binding assay indicating the direct binding of NOB (C), but not NAR (C) or Naringenin (D), to RORα-LBD and RORγ-LBD within the indicated dose range. Inhibitory constant values are shown. (E and F) Mammalian one-hybrid assays showing ligand interaction with ROR-LBD. HEK293T cells were cotransfected with a GAL4 reporter construct with expression vectors for GAL4 DBD-RORα LBD or GAL4 DBD-RORγ LBD. Cells were treated with varying concentrations of NOB (E) and its non-methoxylated analog NAR (F). SR1001 served as a positive control in (F). (G) NOB dose-dependently increased Bmal1 promoter-driven luciferase reporter expression with wild-type, but not mutant, RORE in the presence of RORα or RORγ in Hepa1-6 cells. (H) Knockdown of RORα/γ expression by siRNAs abrogated NOB induction of Bmal1 promoter-driven luciferase reporter expression in both Hepa1-6 and U2OS cells. (I) Real-time qPCR analysis of RORα/γ target genes from the same mouse liver samples as in Figure 5A. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. WT.HF.NOB vs. WT.HF.Veh.