Endogenous ghrelin is an orexigenic peptide acting in the arcuate nucleus in response to fasting

Abstract

Ghrelin, a circulating growth-hormone releasing peptide derived from stomach, stimulates food intake through neuropeptide Y (NPY) neurons of the arcuate nucleus in the hypothalamus (ARC). We examined the effect of ghrelin microinjected into the ARC and the influence of intracerebroventricular (i.c.v.) pretreatment with a GHRH or NPY receptor antagonist on ghrelin-induced food intake in free-feeding male rats. Ghrelin (0.1–1 μg) stimulated food intake in a dose-dependent manner, and this effect was reduced by 55–60% by the Y₅ NPY receptor antagonist (10 μg i.c.v.), but not by the GHRH receptor antagonist MZ-4-71 (10 μg i.c.v.). We also evaluated the effects of passive ghrelin immunoneutralization by the microinjection of anti-ghrelin immunoglobulins (IgGs) intracerebroventricularly or directly into the ARC on food intake in free-feeding and fasted male rats. I.c.v. administration of anti-ghrelin IgGs decreased cumulative food intake over 24 h, whereas microinfusion of anti-ghrelin IgGs into the ARC induced only a short-lived (2 and 6 h) effect. Collectively, these data would indicate that centrally derived ghrelin has a major role in the control of food intake in rats and, in this context, blood-born ghrelin would be effective only in relation to its ability to reach the ARC, which is devoid of blood-brain barrier.

Introduction

Ghrelin, an acylated 28-amino acid peptide recently isolated from mammalian stomach, has been identified as the endogenous ligand for pituitary growth hormone secretagogue (GHS) receptors [1]. Ghrelin administration stimulates the pulsatile release of GH in experimental animals as well as in humans [2], [3], [4], [5]. Apart from endocrine activities, it has been shown that both the central and peripheral administration of ghrelin increases food intake in rodents and induces hunger in humans [3], [6], [7], [8], in agreement with the previously reported orexigenic action of synthetic GH secretagogues [9], [10]. The concentration of circulating ghrelin, principally derived from the stomach, is influenced by acute or chronic changes in nutritional state; in particular, ghrelin serum levels are increased in response to fasting and reduced by glucose intake [6], [11], [12].

Beyond the gastrointestinal tract, ghrelin immunoreactivity has also been found in the arcuate nucleus of the hypothalamus (ARC), especially in its ventral portion, and in the mediobasal hypothalamus [1], [13], [14]. The ARC is an integration site for peripheral and central feeding-regulatory signals and plays an important role in the control of food intake [14], [15]. The nucleus is located at the base of the hypothalamus on both sides of the third ventricle and, owing to the weak blood-brain barrier in this region of the brain, it is exposed to peripheral signals, such as circulating adrenal and gonadal steroids, and large peptides such as leptin and insulin [16], [17]. The ARC also expresses GHS receptors like other hypothalamic nuclei, including the paraventricular (PVN) and the ventromedial (VMH) nuclei [18], [19], [20]. Within the ARC, GHS receptors are expressed in 94% of neuropeptide Y (NPY)-containing neurons [21], and several experimental data indicate that both central and systemic administration of ghrelin leads to activation of the orexigenic pathway of ARC NPY/Agouti-related peptide neurons projecting to the PVN and lateral hypothalamic area [22], [23], [24].

To achieve further proof that the orexigenic activity of centrally administered ghrelin is mediated by a direct interaction of the peptide with ARC neurons, we examined the feeding response to ghrelin injected stereotaxically into the ARC nucleus of free-feeding rats treated or not with a NPY or GHRH receptor antagonist into the lateral ventricles. We also studied the effect of passive ghrelin immunoneutralization on food intake in free-feeding and fasted male rats, with the aim to provide further evidence for a physiological role of endogenous ghrelin in the ARC nucleus in the control of feeding. Passive immunoneutralization was done by microinjecting anti-ghrelin immunoglobulins (IgGs) directly into the ARC nucleus or into the lateral ventricles. We also compared the effects of central immunoneutralization of ghrelin with those of the peripheral administration of anti-ghrelin IgGs on food intake in free-feeding and in fasted male rats, to determine whether circulating ghrelin is really a physiological orexigenic signal from the gastrointestinal tract involved in the regulation of food consumption.