Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists

Abstract

Synchronizing rhythms of behaviour and metabolic processes is important for cardiovascular health and preventing metabolic diseases. The nuclear receptors REV-ERB-α and REV-ERB-β have an integral role in regulating the expression of core clock proteins driving rhythms in activity and metabolism. Here we describe the identification of potent synthetic REV-ERB agonists with in vivo activity. Administration of synthetic REV-ERB ligands alters circadian behaviour and the circadian pattern of core clock gene expression in the hypothalami of mice. The circadian pattern of expression of an array of metabolic genes in the liver, skeletal muscle and adipose tissue was also altered, resulting in increased energy expenditure. Treatment of diet-induced obese mice with a REV-ERB agonist decreased obesity by reducing fat mass and markedly improving dyslipidaemia and hyperglycaemia. These results indicate that synthetic REV-ERB ligands that pharmacologically target the circadian rhythm may be beneficial in the treatment of sleep disorders as well as metabolic diseases.

Figure 1. SR9011 and SR9009 are synthetic REV-ERB agonists with activity in vivo

a, Chemical structures of SR9011 and SR9009. b, GAL4-REV-ERBα and GAL4-REVERBβ cotransfection assays in HEK293 cells illustrating the activity of SR9011 and 9009 and comparing the activity to GSK4112. c, Cotransfection assay in HEK293 cells with full-length REV-ERBα and a luciferase reporter driven by the Bmal1 promoter. d, Bioluminescence record from a Per2^LUC SCN treated with 5 μM SR9011 as indicated by the bar. The right panels display the period and amplitude of the oscillations prior to, during, and after treatment with SR9011. e, Expression of REV-ERB responsive genes after treatment with various doses of SR9011 or 100 mg/kg of SR9009 (i.p., b.i.d.) for 6-days. * indicates p<0.05. ** indicates p<0.05 vs. before SR9011 and during SR9011 treatment. Error bars indicate mean ± s.e.m. and n=6.

Figure 2. Synthetic REV-ERB ligands alter circadian behavior and the pattern of expression of core clock genes

a, Actograms illustrating the effect of single injections of vehicle, SR9011 (100 mg/kg, i.p.) or SR9011 (100 mg/kg, i.p.) on circadian behavior. C57Bl6 mice were initially maintained on a 12h:12h L:D cycle and altered to constant darkness (D:D) after 7-days. After 12 days on D:D the animals were injected with vehicle or compound at CT6. b, Analysis of wheel running activity during the subject dark period following injection of SR9011 i.p. at CT6 in mice kept under constant darkness. c, Normalized expression levels of several core clock genes following administration of SR9011 or vehicle under constant dark conditions. C57Bl6 mice were administered SR9011 (100 mg/kg, i.p.) at CT0 on a day of constant darkness. Gene expression was determined and normalized to cyclophilin. Data were double plotted. d, Actograms illustrating the effect of single injections of vehicle, SR9011, or SR9009 in mice maintained under 12:12 L:D conditions. e, Normalized expression levels of several core clock genes following administration of SR9011 or vehicle under L:D (12:12) conditions. Methods for e and e were otherwise identical to a and c. * indicates p<0.05. Error bars indicate mean ± s.e.m. and n=6–10 mice.

Figure 3. Activation of REV-ERB by SR9011 in vivo results in an increase in energy expenditure and weight loss

a, Treatment of mice (Balb/c) with SR9011 results in weight loss and fat mass loss. Animals were dosed with SR9011 (100mg/kg, i.p., b.i.d.) for 12 days. b, Oxygen consumption (VO₂) is increased in mice treated with SR9011. Results were obtained in using CLAMS and C57Bl6 mice were dosed as described in a except that the duration of treatment was 10 days. c, Oxygen consumption (VO₂) is increased during both the diurnal and nocturnal phases of C57Bl6 mice when they are treated with SR9011. Data obtained from the experiment described in b was analyzed for time of day differences. d, Mice treated with SR9011 are less active in the CLAMS as detected by the number of x-axis beam breaks. e, Total daily, diurnal and nocturnal food intake from the animals in the CLAMS study. f, The rate of food intake is not altered by SR9011 treatment. g, Respiratory exchange ratio (RER) is not altered by SR9011 treatment. h, After completion of the 10-day CLAMS experiment animals fat mass was assessed by DEXA. i, Results from a CLAMS experiment illustrating the diurnal increase in oxygen consumption prior to and immediately after administration of SR9011. Note the ~3h delay in the diurnal peak in VO₂ following administration of SR9011. For all b.i.d. dosing animals were dosed at CT0 and CT12. * indicates p<0.05. Error bars indicate mean ± s.e.m. and n=6–10 mice

Figure 4. REV-ERB ligands alter the pattern of circadian expression of metabolic genes in the liver, skeletal muscle and adipose tissue

C57Bl6 mice were administered a single dose of SR9011 (100 mg/kg, i.p.) at CT0 and groups of animals (n=6) were sacrificed and gene expression assessed by QPCR. Graphs were double plotted. a, Expression of core clock genes from the liver of vehicle treated vs. SR9011 treated mice. b, Expression of metabolic genes from the liver of vehicle treated vs. SR9011 treated mice. c, Expression of metabolic genes from the skeletal muscle of vehicle treated vs. SR9011 treated mice. d, Expression of metabolic genes from the white adipose tissue (WAT) of vehicle treated vs. SR9011 treated mice. * indicates p<0.05. Error bars indicate mean ± s.e.m. and n=6–10 mice

Figure 5. SR9009 treatment results in a decrease in fat mass and in plasma lipids in diet-induced obese mice

a, Diet-induced obese mice on SR9009 treatment lose weight vs. vehicle treated mice. C57Bl6 mice on a high fat diet were administered SR9009 (100mg/kg, i.p., b.i.d, at CT0 and CT12) for 30 days. b, Diet-induced obese mice on SR9009 treatment exhibit lower fat mass vs. vehicle treated mice. c, Fasting plasma triglycerides (TG), cholesterol (Chol), non-esterified fatty acids (NEFA) and glucose are decreased in SR9009 treated DIO mice. d, Plasma leptin and IL-6 levels from DIO mice e, Fasting plasma TG and Chol in lean C57Bl6 mice. Normal mice were administered 100 mg/kg, i.p., b.i.d. (at CT0 and CT12) SR9009 for 10 days. f, Fasting plasma TG and Chol are decreased by SR9011 treatment in lean C57Bl6 mice. g, Expression of metabolic genes in liver, WAT and skeletal muscle of DIO mice treated with SR9009 as described in a. Gene expression was measured by QPCR and normalized to Cyclophilin b expression. h, SR9009 treatment reduces weigh gain in ob/ob mice. Body weight and body fat content data are shown from ob/ob mice administered SR9009 for 12-days (100 mg/kg, i.p., b.i.d.).* indicates p<0.05. Error bars indicate mean ± s.e.m. and n=6–10 mice