Glucocorticoid regulation of the circadian clock modulates glucose homeostasis

Abstract

Circadian clock genes are regulated by glucocorticoids; however, whether this regulation is a direct or secondary effect and the physiological consequences of this regulation were unknown. Here, we identified glucocorticoid response elements (GREs) at multiple clock genes and showed that 3 were directly regulated by the glucocorticoid receptor. We determined that a GRE within the core clock gene Per2 was continuously occupied during rhythmic expression and essential for glucocorticoid regulation of that gene in vivo. We further demonstrated that mice with a genomic deletion spanning this GRE expressed elevated leptin levels and were protected from glucose intolerance and insulin resistance on glucocorticoid treatment but not from muscle wasting. We conclude that Per2 is an integral component of a particular glucocorticoid regulatory pathway and that glucocorticoid regulation of the peripheral clock is selectively required for some actions of glucocorticoids.

Fig. 1.

Glucocorticoids stimulate transcriptional rhythmicity of specific circadian clock genes in primary mouse MSCs. The transcript levels at 4-h intervals were measured by quantitative PCR and normalized to time = 0 h. Graphs represent the relative transcriptional levels of genes averaged over at least 3 independent experiments with MSCs isolated from different mice (plotted in log₂ scale, and error bars represent ± SEM). (A) Per2, Rev-ERBβ, Npas2, and Per1 expression levels oscillate in response to glucocorticoids. (B) E4bp4 and Dec2 respond to glucocorticoids without oscillation. (C) Clock and GR expression levels do not respond to glucocorticoids. See Table S1 for primer sequences.

Fig. 2.

GR directly regulates transcription of circadian clock components in mouse and human primary MSCs. (A) Per2, E4bp4, Per1, and Timeless rapidly respond to glucocorticoid stimulation. Relative transcript levels of mouse primary MSCs treated with vehicle (DMSO) or 1 μM dex for 4 h were measured by quantitative PCR. The transcript levels are normalized to the vehicle-treated samples, and the data represent the average of 3 independent experiments (mean + SEM). (B–D) GR occupies genomic sites at Per2, E4bp4, and Per1. ChIP was performed with primary mouse MSCs treated with vehicle (DMSO) (gray) or 1 μM dex (black) for 4 h. Quantitative PCR was used to quantify enrichment of computationally identified putative GBSs surrounding 64 kb of the corresponding gene TSSs (Fig. S3). The samples were normalized to amplification of a genomic region near the Hsp70 gene (mean + SEM). (E) GR-occupied GBSs at Per1, Per2, and E4bp4 are conserved in sequence across species. Aligned rat, human, and canine sequences of GBSs were obtained from the UCSC Genome Browser (20). Conserved bases are highlighted in red. (F) GR occupancy of GBSs at Per1, Per2, and E4bp4 is conserved in human primary MSCs. ChIP experiments with primary human MSCs [treated with 1 μM dex for 1.5 h] were performed and analyzed as indicated previously (mean + SEM).

Fig. 3.

Endogenous GR-occupied GBS in Per2 is functional in vivo. (A) Diagram of Per2 is displayed (exons shown as solid black bars, and introns shown as lines) along with the GR-occupied GBS (Per2 GBS.6,7) located at the +22.8-kb position (red box). PCR genotyping using primers spanning Per2 GBS.6,7 demonstrates that the 2.1-kb region containing Per2 GBS.6,7 is deleted in mutant cells but not in WT cells. A GR-occupied site at Per1 (Per1 GBS.3) was used as a positive control for DNA amplification. (B) Deletion of the endogenous genomic region harboring the GR-occupied Per2 GBS.6,7 specifically abolishes the glucocorticoid transcriptional response of Per2 but not other targets occupied by GR (Sgk, Mt1). Cells were treated with vehicle (DMSO) (white and gray) or 1 μM dex (black and red) for 4 h. Transcript levels from 3 independent experiments were measured by quantitative PCR (mean ± SEM). (C) Deletion of Per2 (red) dampens oscillations of other clock components induced by glucocorticoids compared with the response in WT cells (black) (mean ± SEM.). (D) A 500-bp DNA fragment from the endogenous Per2 gene that spans Per2 GBS.6,7 is responsive to dex in a luciferase reporter assay. Site-directed mutagenesis of Per3 GBS6 results in ablation of the dex response (mean ± SD).

Fig. 4.

Continuous GR activity is required to initiate circadian rhythm and maintain a synchronized phase. (A) Simultaneous treatment with RU486 antagonized dex-stimulated transcriptional oscillation. Cells were treated with 100 nM dex alone (red) or 100 nM dex and 1 μM RU486 simultaneously (gray) at time = 0 h (arrows). The samples were quantified as described previously (average and range of 2 independent experiments). (B) Addition of RU486 after dex treatment disrupts glucocorticoid-stimulated circadian rhythm. One micromolar RU486 (black) or vehicle (DMSO) (red) was added to the culture medium after 22 h of treatment with 100 nM dex (arrows). Transcript levels from 3 independent experiments were measured by quantitative PCR (mean ± SEM). (C) GR remains bound at GR-occupied Per2 GBS.6,7 during cycling of expression. ChIP experiments were performed in a time course after treatment with 1 μM dex. The data were processed as described previously and normalized to ChIP samples treated with vehicle (DMSO) for 8 h (mean ± SEM).

Fig. 5.

Per2 is required and specific for alterations in glucose homeostasis induced by glucocorticoids in vivo. (A) Blood glucose levels were monitored in mice following an injection with 2 mg/kg glucose. Glucocorticoid-induced glucose intolerance depends on Per2. Per2^Brdm1 mice (n = 10) are protected from glucocorticoid-induced glucose intolerance (red) compared with WT mice (n = 6) (blue) (mean ± SEM; P = 0.0574 at 15 min, P = 0.002 at 30 min). Per2^Brdm1 (n = 7) and WT mice (n = 8) injected with PBS (gray and black) had similar glucose tolerance. (B) The decline in blood glucose from basal levels of mice was monitored following injections with 0.75 units/kg insulin. Per2^Brdm1 mice (red) were more sensitive to insulin than WT mice (blue) (mean ± SEM; P = 0.0104 at 15 min, P = 0.0048 at 30 min, P = 0.021 at 60 min). (C) DEXA scans of mice injected with glucocorticoids were compared with those of PBS-injected animals. Per2^Brdm1 mice treated with glucocorticoids had a similar decrease in lean mass (P = 0.0014) as WT animals. Probability values were calculated using unpaired Student's t tests.

Fig. 6.

Per2^Brdm1 mice have elevated leptin levels. A panel of candidate genes was screened for differential expression levels in adipose tissue with quantitative PCR. Adipose leptin expression was 2.7-fold higher in Per2^Brdm1 mice compared with WT mice. Five mice from each genotype were used, and the experiment was repeated 3 times (mean ± SD). The same genes (except for the adipokines) were also screened in muscle, with similar results.