Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats

Abstract

Studies showed that the metabolic unlike the neuroendocrine effects of ghrelin could be abrogated by co-administered unacylated ghrelin. The aim was to investigate the interaction between ghrelin and desacyl ghrelin administered intraperitoneally on food intake and neuronal activity (c-Fos) in the arcuate nucleus in non-fasted rats. Ghrelin (13 μg/kg) significantly increased food intake within the first 30 min post-injection. Desacyl ghrelin at 64 and 127 μg/kg injected simultaneously with ghrelin abolished the stimulatory effect of ghrelin on food intake. Desacyl ghrelin alone at both doses did not alter food intake. Both doses of desacyl ghrelin injected separately in the light phase had no effects on food intake when rats were fasted for 12 h. Ghrelin and desacyl ghrelin (64 μg/kg) injected alone increased the number of Fos positive neurons in the arcuate nucleus compared to vehicle. The effect on neuronal activity induced by ghrelin was significantly reduced when injected simultaneously with desacyl ghrelin. Double labeling revealed that nesfatin-1 immunoreactive neurons in the arcuate nucleus are activated by simultaneous injection of ghrelin and desacyl ghrelin. These results suggest that desacyl ghrelin suppresses ghrelin-induced food intake by curbing ghrelin-induced increased neuronal activity in the arcuate nucleus and recruiting nesfatin-1 immunopositive neurons.

Introduction

Ghrelin is a 28-amino acid peptide hormone with an n-octanylated serine residue that is predominantly produced by the X/A-like endocrine cells of the gastric oxyntic mucosa in rodents and humans [19]. Ghrelin has been shown to act on the brain–gut axis as the only peripherally produced and centrally active peptide that stimulates food intake in rats [25], [34], [38], mice [2], [36] and humans [37]. Ghrelin exerts its orexigenic action through activation of the functionally active, signal-transducing form of the growth hormone secretagogue receptor type 1a (GHS-R1a), renamed GRLN [9] located in the hypothalamus–pituitary axis [24].

Ghrelin circulates in two major forms: n-octanyl-ghrelin (ghrelin), and des-n-octanyl ghrelin (desacyl ghrelin), which lacks the hydrophobic octanyl group, esterified to Ser³. Acylation is essential for the binding of ghrelin to the GHS-R1a, and although desacyl ghrelin does not bind to the GHS-R1a, it may be biologically active [12], [14], [24]. One group of investigators reported that desacyl ghrelin exerts an inhibitory effect on food intake after intraperitoneal (ip) or central (intracisternal or intracerebroventricular, icv) administration in rodents [8]. Most likely, the effect is mediated by neurons in the arcuate nucleus (ARC) and paraventricular nucleus (PVN) of the hypothalamus [8]. Transgenic mice over-expressing desacyl ghrelin displayed a decrease in food intake and body weight [1], [7]. However, subsequent studies showed that centrally administered desacyl ghrelin stimulates food intake independently from the GHS-R1a while peripheral administration of the peptide had no effects in rodents and humans [32]. There is also evidence suggesting that desacyl ghrelin may counteract ghrelin’s role in the control of energy balance and glucose metabolism [1], [11], [12].

Recent reports indicate that the effects of both ghrelin and desacyl ghrelin on food intake are mediated at least in part by distinct brain nuclei, as demonstrated by differential brain Fos expression in response to peripheral administration of each peptide [8], [13]. However, both peptides injected ip induce Fos expression in the ARC of the hypothalamus in rodents [8], [13], and double labeling showed that peripheral injection of ghrelin activates arcuate neurons containing the orexigenic peptides neuropeptide Y (NPY) and Agouti-related protein [36]. However, the neuropeptidergic phenotypes of activated neurons in the ARC induced by peripheral desacyl ghrelin are unknown. Recently, it could be demonstrated that a new peptide called nesfatin-1, derived from the nucleobindin-2 protein (NUCB2) modulates food intake and body weight in rats [27]. In addition, immunohistochemical mapping of the hypothalamus shows that nesfatin-1 immunopositive neurons are – among other areas – localized in the ARC [6], [27]. Therefore, nesfatin-1 was chosen as a candidate for the neurohistochemical analysis performed in this study in response to peripheral simultaneous administration of ghrelin and desacyl ghrelin.

We recently reported that simultaneous administration of peripheral cholecystokinin (CCK) or bombesin together with ghrelin blocks the orexigenic effect of ghrelin and modulates Fos expression in hypothalamic brain nuclei in rats [15], [17]. In contrast, amylin was not able to modulate ghrelin-induced food intake [15]. To further explore the role of desacyl ghrelin in the regulation of food intake and its interaction with ghrelin we investigated whether desacyl ghrelin injected intraperitoneally (ip) influences ghrelin-induced food intake under ad libitum feeding conditions in rats. Additionally, we explore the role of desacyl ghrelin injected alone on food intake in 12-h fasted rats. Furthermore, we assessed changes in neuronal activity in response to peripheral ghrelin, desacyl ghrelin and simultaneous injection of both peptides by mapping the Fos expression in the ARC in rats.