Phase-Dependent Shifting of the Adrenal Clock by Acute Stress-Induced ACTH

Abstract

The adrenal cortex has a molecular clock that generates circadian rhythms in glucocorticoid production, yet it is unclear how the clock responds to acute stress. We hypothesized that stress-induced ACTH provides a signal that phase shifts the adrenal clock. To assess whether acute stress phase shifts the adrenal clock in vivo in a phase-dependent manner, mPER2:LUC mice on a 12:12-h light:dark cycle underwent restraint stress for 15 min or no stress at zeitgeber time (ZT) 2 (early subjective day) or at ZT16 (early subjective night). Adrenal explants from mice stressed at ZT2 showed mPER2:LUC rhythms that were phase-advanced by ~2 h, whereas adrenals from mice stressed at ZT16 showed rhythms that were phase-delayed by ~2 h. The biphasic response was also observed in mice injected subcutaneously either with saline or with ACTH at ZT2 or ZT16. Blockade of the ACTH response with the glucocorticoid, dexamethasone, prevented restraint stress-induced phase shifts in the mPER2:LUC rhythm both at ZT2 and at ZT16. The finding that acute stress results in a phase-dependent shift in the adrenal mPER2:LUC rhythm that can be blocked by dexamethasone indicates that stress-induced effectors, including ACTH, act to phase shift the adrenal clock rhythm.

Keywords: ACTH; acute stress; adrenal clock; circadian; dexamethasone; mPER2:LUC; restraint stress.

Figure 1

Schematic showing the time-line for each experiment relative to zeitgeber time (ZT). Mice were on a 12:12-h LD cycle (0600:1800 hours) with lights on at ZT0 and lights off at ZT12. Experimental manipulations were done in early subjective day (denoted by blue font) or early subjective night (denoted by red font). Dex, dexamethasone; TC, time of tissue collection.

Figure 2

Representative experiment showing the mPER2:LUC rhythm after detrending and baseline subtraction; adrenal explants were collected from control mice (black line) and mice exposed to acute (15 min) restraint stress at ZT2 (blue line) (A) or at ZT16 (red line) (B). The peak in adrenal PER2 bioluminescence is phase-advanced by ~2 h following restraint stress at ZT2 (A) and phase-delayed by ~2 h following restraint stress at ZT16 (B). The vertical dashed lines identify the time of the peak phase in the mPER2:LUC rhythm. Adrenal bioluminescence is reported as counts per second (cps).

Figure 3

Effect of acute (15 min) restraint stress at ZT2 (A) and at ZT16 (B) on the peak phase of the in vitro adrenal mPER2:LUC rhythm. Values represent means ± SEM (n = 6–7/group). Based on unpaired Student’s t-test, *p < 0.05 vs. Control at ZT2 and ZT16.

Figure 4

Effect of injection of ACTH or saline or no treatment (Control) at ZT2 (A) and at ZT16 (B) on the peak phase of the in vitro adrenal mPER2:LUC rhythm. Values represent means ± SEM (n = 5–6/group). Based on one-way ANOVA (using Dunnett’s correction for post hoc analysis), *p < 0.05 vs. Control at ZT2 and ZT16.

Figure 5

Effect of dexamethasone blockade on restraint stress-induced plasma ACTH (A) and corticosterone (B) at ZT16 in mPER2:LUC mice. Values represent means ± SEM (n = 4–5/group). Based on two-way ANOVA (using Bonferroni’s correction for post hoc analysis), *p < 0.05 vs. No Dex-No Stress and ^∧p < 0.05 vs. No Dex-Stress.

Figure 6

Effect of dexamethasone treatment before restraint stress at ZT3 (A) or at ZT16 (B) on the peak phase of the in vitro adrenal mPER2:LUC rhythm. Values represent means ± SEM (n = 4–5/group). Based on two-way ANOVA (using Bonferroni’s correction for post hoc analysis), *p < 0.05 vs. No Dex-No Stress and ^∧p < 0.05 vs. No Dex-Stress at ZT3 and ZT16.

Figure 7

Effect of dexamethasone treatment before restraint stress at ZT3 (A) or at ZT16 (B) on the period of the in vitro adrenal mPER2:LUC rhythm. Values represent means ± SEM (n = 4–5/group). Based on two-way ANOVA (using Bonferroni’s correction for post hoc analysis), *p < 0.05 vs. Dex-No Stress and ^∧p < 0.05 vs. No Dex-Stress at ZT3 and ZT16.