Evidence Supporting a Role for Constitutive Ghrelin Receptor Signaling in Fasting-Induced Hyperphagia in Male Mice

Abstract

Ghrelin is a potent orexigenic peptide hormone that acts through the growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor highly expressed in the hypothalamus. In vitro studies have shown that GHSR displays a high constitutive activity, whose physiological relevance is uncertain. As GHSR gene expression in the hypothalamus is known to increase in fasting conditions, we tested the hypothesis that constitutive GHSR activity at the hypothalamic level drives the fasting-induced hyperphagia. We found that refed wild-type (WT) mice displayed a robust hyperphagia that continued for 5 days after refeeding and changed their food intake daily pattern. Fasted WT mice showed an increase in plasma ghrelin levels, as well as in GHSR expression levels and ghrelin binding sites in the hypothalamic arcuate nucleus. When fasting-refeeding responses were evaluated in ghrelin- or GHSR-deficient mice, only the latter displayed an ∼15% smaller hyperphagia, compared with WT mice. Finally, fasting-induced hyperphagia of WT mice was significantly smaller in mice centrally treated with the GHSR inverse agonist K-(D-1-Nal)-FwLL-NH2, compared with mice treated with vehicle, whereas it was unaffected in mice centrally treated with the GHSR antagonists D-Lys3-growth hormone-releasing peptide 6 or JMV2959. Taken together, genetic models and pharmacological results support the notion that constitutive GHSR activity modulates the magnitude of the compensatory hyperphagia triggered by fasting. Thus, the hypothalamic GHSR signaling system could affect the set point of daily food intake, independently of plasma ghrelin levels, in situations of negative energy balance.

Growth hormone secretagogue receptor signalling affects high‐fat intake independently of plasma levels of ghrelin andLEAP2, in a 4‐day binge eating model

“…Plasma ghrelin levels were assessed using an enzymatic immunoassay from Bertin Pharma (Montigny le Bretonneux, France; catalogue no. A05118), as reported previously . This commercial assay specifically detects the bioactive acylated form of the peptide and does not cross‐react with des‐acylghrelin.…”

“…SLBN1014V). Mice were i.c.v.‐treated with 2 nmol/mouse of [D‐Lys3]‐GHRP‐6, this dose was chosen based on previous work showing that it blocks the orexigenic effects of i.c.v.‐injected ghrelin . JMV2959 was synthesised as described previously .…”

“…JMV2959 was synthesised as described previously . Mice were i.c.v.‐treated with 3 nmol/mouse of JMV2959, with this dose being chosen based on our previous experience showing that it blocks the orexigenic effects of i.c.v.‐injected ghrelin . K‐(D‐1‐Nal)‐FwLL‐NH 2 was synthetised by automated solid‐phase peptide synthesis, as described previously .…”

“…K‐(D‐1‐Nal)‐FwLL‐NH 2 was synthetised by automated solid‐phase peptide synthesis, as described previously . Mice were i.c.v.‐treated with 1 nmol/mouse of K‐(D‐1‐Nal)‐FwLL‐NH 2, with this dose being chosen based on our previous experience showing that it blocks the orexigenic effects of i.c.v.‐injected ghrelin and reduces the fasting‐induced compensatory hyperphagia . Importantly, mice i.c.v.‐injected with GHSR ligands did not show any sickness‐like behaviour, such as a spiky coat, hunched posture, altered breathing rate, laboured movements, reduced activity and/or subdued behaviour.…”

“…In wild‐type (WT) mice, the relevance of ghrelin‐independent GHSR activity on food intake regulation can be uncovered after a prolonged fast. In particular, plasma ghrelin levels and GHSR gene expression in the hypothalamic arcuate nucleus (ARC) increase in 48‐hour fasted mice, and they decline to basal levels soon after refeeding . However, GHSR protein remains elevated in ARC neurones during the first days of refeeding and its action stimulates the biosynthesis of orexigenic peptides that, in turn, enhance the compensatory hyperphagia several days after the fast .…”