Activation of growth hormone secretagogue receptor induces time-dependent clock phase delay in mice

Early studies have reported a phase-shifting effect of growth hormone secretagogues (GHSs). This study aimed to determine the mechanism of action of GHSs. We examined the response of the hypothalamic suprachiasmatic nuclei (SCN) to growth hormone releasing peptide-6 (GHRP-6) by assessing effects on the phase of locomotor activity rhythms, SCN neuronal discharges, and the potential signaling pathways involved in the drug action on circadian rhythms. The results showed that bolus administration of GHRP-6 (100 μg/kg ip) at the beginning of subjective night (CT12) induced a phase delay of the free-running rhythms in male C57BL/6J mice under constant darkness, but did not elicit phase shift at other checked circadian time (CT) points. The phase-delay effect of GHRP-6 was abolished by d-(+)-Lys-GHRP-6 (GHS receptor antagonist), KN-93 [calcium/calmodulin-dependent protein kinase II (CaMK) II inhibitor], or anti-phosphorylated (p)-cAMP response element-binding protein (CREB) antibody. Further analyses demonstrated that GHRP-6 at CT12 induced higher calcium mobilization and neuronal discharge in the SCN compared with that at CT6, decreased the levels of glutamate and γ-aminobutyric acid, increased the levels of p-CaMKII, p-CREB, and period 1, and delayed the circadian expressions of circadian locomotor output cycles kaput, Bmal1, and prokineticin 2 in the SCN; these signaling changes resulted in behavioral phase delay. Collectively, GHRP-6 induces a CT-dependent phase delay via activating GHS receptor and the downstream signaling, which is partially similar to the signaling cascade of light-induced phase delay at early night. These novel observations may help to better understand the role of GHSs in circadian physiology.