Phase-dependent resetting of the adrenal clock by ACTH in vitro

Abstract

The adrenal cortex has a molecular clock that generates circadian rhythms in glucocorticoids, yet how the clock is synchronized to the external environment is unknown. Using mPER2::Luciferase (mPER2Luc) knockin mice, in which luciferase is rhythmically expressed under the control of the mouse Per2 clock gene, we hypothesized that ACTH transmits entrainment signals to the adrenal. Adrenal explants were administered ACTH at different phases of the mPER2Luc rhythm. Treatment with ACTH 1-39 produced a phase delay that was phase-dependent, with a maximum at circadian time (CT)18; ACTH did not alter the period or amplitude of the rhythm. Forskolin produced a parallel response, suggesting that the phase delay was cAMP-mediated. The response to ACTH was concentration-dependent and peptide-specific. Pulse administration (60 min) of ACTH 1-39 also produced phase delays restricted to late CTs. In contrast to ACTH 1-39, other ACTH fragments, including α-melanocyte-stimulating hormone, which do not activate the melanocortin 2 (MC2/ACTH) receptor, had no effect. The finding that ACTH in vitro phase delays the adrenal mPER2luc rhythm in a monophasic fashion argues for ACTH as a key resetter, but not the sole entrainer, of the adrenal clock.

Keywords: adrenal clock; adrenocorticotropic hormone; nonphotic entrainment.

Fig. 1.

Representative experiment showing the mPER2luc rhythm in multiple adrenal explants before (A and C) and after detrending (B and D) under pretreatment conditions and after posttreatment with vehicle (A and B) or ACTH (10.0 nM) (C and D) at CT18. Note the peak phase of the rhythm following vehicle (B, black arrowhead in inset) of ∼110 h precedes the peak phase following ACTH of ∼118 h (D, black arrowhead in inset), reflecting an ACTH-induced phase delay in the mPER2Luc rhythm.

Fig. 2.

Phase response curve showing phase shifts in the adrenal mPER2luc rhythm following treatment with vehicle, ACTH, or forskolin administered at multiple circadian times (CTs). *P < 0.05 vs. vehicle.

Fig. 3.

A: concentration-response curve showing phase shifts in the adrenal mPER2Luc rhythm following treatment with ACTH at circadian time (CT)18. *P < 0.05 vs. vehicle; ^P < 0.05 vs. 0.1 nM ACTH; +P < 0.05 vs. 1.0 nM ACTH. B: comparison of the phase response of the adrenal mPER2Luc rhythms to ACTH 1–39 (10.0 nM), VIP 1–28 (10.0 nM), and CRH 1–41 (10.0 nM) administered at CT18. *P < 0.05 vs. vehicle.

Fig. 4.

Phase-response curve showing phase shifts in the adrenal mPER2Luc rhythm following pulses (60 min) of ACTH (1.0 nM) compared with vehicle. *P < 0.05 vs. vehicle.

Fig. 5.

A: effect of ACTH 7–38 on phase shifts in the adrenal mPER2Luc rhythm induced by pulse (60 min) treatment with ACTH 1–39 at circadian time (CT)18. *P < 0.05 vs. vehicle; ^P < 0.05 vs. ACTH 7–38. B: effect of ACTH 11–24 on phase shifts in the adrenal mPER2Luc rhythm induced by pulse treatment with ACTH 1–39 at CT18. *P < 0.05 vs. vehicle; ^P < 0.05 vs. ACTH 11–24. C: effect of pulse treatment with α-melanocyte-stimulating hormone (α-MSH) compared with ACTH 1–39 (1.0 nM) on phase shifts in the adrenal mPER2Luc rhythm at CT18. *P < 0.05 vs. vehicle; ^P < 0.05 vs. α-MSH.