| Background Kojima et al. (1999) reported the purification of a 28-amino acid peptide acting as a growth hormone secretagogue receptor (GHS-R) agonist and dubbed it ghrelin. They reported that the molecule contains an octanoyl group at a serine residue, and this feature was associated with the biological activity of ghrelin. This peptide, which is produced primarily by endocrine cell of the stomach, is released into the circulation. Ghrelin is also produced by neurons of the hypothalamus and third ventricle. In addition to stimulation of growth hormone secretion by binding to GHS-R, ghrelin also stimulates food intake, weight gain, and participates in the long-term regulation of body weight and energy homeostasis. Many studies suggest a physiological role of ghrelin in the initiation of feeding. There is considerable interest in the mechanism by which ghrelin exerts its appetite-stimulating effects, and, in particular, the action sites of ghrelin in the central nervous system (CNS). In tne present study, many observations support the hypothesis that the neuropeptide Y (NPY) and agouti-related peptide (AgRP) (NPY/AgRP) neurons within the arcuate nucleus (ARC) are suggested to be the primary location for the orexigenic activity of ghrelin. Pharmacological evidence indicates that ghrelin’s effects on food intake are mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the central nervous system. This is forebrain mechanism of ghrelin stamulating food intake. Although there is strong evidence supporting a hypothalamic mode of action, there is also evidence that ghrelin may work via hindbrain. A present study by Zigman using in situ hybridization demonstrated the presence of GHS-R in all three components (solitary nucleus, area postrema and dorsal motor nucleus of vagus nerve) of the dorsal vagal complex (DVC). With the new evidence provided by Faulconbridge et al, ghrelin (150 p mol) delivered to the third and fourth ventricles significantly and comparably increased cumulative food intake, and ghrelin (10 pmol) microinjected unilaterally into the DVC significantly increased food intake 3 h after treatment. Thereby, we conclude that ghrelin may be effected by brain stem. So what is the mechanism by which DVC ghrelin administration induces a hyperphagic response?Objective The aim of this study was to determine whether the ARC NPY/AgRP neural pathway is required for hyperphagic responses stimulated by ghrelin delivery within the DVC and the underlying mechanism.Methods1. The measurement of food intake Healthy male Sprague-Dawley rats (n=32) were divided into two groups at random, ghrelin group (n= 16) received injection of 20 pmol ghrelin, while saline group(n=16) received injection of 0.5μl saline. Each rats received a 22G guide cannula on DVC, and the experiments begin after at least 7 days recovery from surgery. On at least three occasuions before experimental testing, rats were given saline injections to habituate them to the injection procedure. Cumulative food intake was measured at 1h,2h,3h and 4h, after DVC ghrelin injection by feeding and activity analyser (Feeding and Activity Analyser 47552-002, UGO BASILE, ITALY)2. NPY and AgRP gene expression:Healthy male SD rats(n=72) were divided into two groups at random, ghrelin group (n=36) received injection of 20 pmol ghrelin, while saline group (n=36) received injection of 0.5μl saline. Each group included three sub-groups(n=12) according to the detection time after ghrelin (or saline) injection. Each rats received a 22G guide cannula on DVC, and the experiments begin after at least 7 days recovery from surgery. On at least three occasuions before experimental testing, rats were given saline injections to habituate them to the injection procedure. Hypothalamus ARC mRNA was extracted at 1h,2h, 3h after microinjection of ghrelin or saline into the DVC and the expression of NPY/AgRP mRNA was detected by using Real-Time PCR.3. Hypothalamic NPY neurons immunohistochemistry Healthy male SD rats (n=16) were divided into two groups at random, ghrelin group(n=8) received injection of 20 pmol ghrelin, while saline group (n=8) received injection of 0.5μl saline. Each rats received a 22G guide cannula on DVC, and the experiments begin after at least 7 days recovery from surgery. On at least three occasuions before experimental testing, rats were given saline injections to habituate them to the injection procedure. At 1h,2h and 3 h after ghrelin injection, the rats were killed, and then the brains were sectioned in the coronal plane on a freezing microtome. In each section, an area within the ARC nucleus was selected for counting the NPY-positive neurons and analyzing optical density. The amount and mean optical density of NPY positive neurons were obtained by microscope and analyzed with the image analysis software.Result1. Effects of ghrelin on food intake Cumulative food intakes in ghrelin group were significantly increased over the 4 h test period compared with those in control group [1 h:(0.92±.99) g vs (0.61±0.13) g, P<0.05; 2 h:(2.03±0.10) g vs (1.21±0.17) g, P<0.01；3 h:(2.38±0.11) g vs (1.62±0.19) g, P<0.05]. The promoting effect of ghrelin was the greatest at 2 h after injection, and the effects continued at 3 h after injection. No significant difference was observed between control and ghrelin groups at 4 h after injection.2. Effect of ghrelin on hypothalamic NPY, AgRP mRN A expressionHypothalamic NPY and AgRP mRN A expression levels were significantly higher in ghrelin group than those in control group at 1,2 and 3 h after injection. Ghrelin administration increased NPY mRNA levels by 238.09%,648.84% and 156.82% over those in control group at 1,2 and 3 h after injection, respectively. On the other hand, ghrelin administration increased AgRP mRNA levels by 161.45%,576.82% and 121.30% over those in control group at 1,2 and 3 h, respectively. NPY/AgRP mRNA expressions at 2 h after ghrelin injection exhibited higher levels than those at 1 and 3 h after injection. No significant differences were found between ghrelin groups at 1 and 3 h after injection.3. Effects of ghrelin on NPY expression in the ARC The numbers of NPY-immunopositive neurons in the ARC nucleus was significantly increased at 1,2 and 3 h after ghrelin injection compared with that in control group, respectively (1h,3 h, P<0.05; 2 h, P<0.01). Ghrelin’s promoting effect was most pronounced at 2 h after peptide injection. There was no significant difference between the numbers of NPY-immunopositive neurons at 1 and 3 h in ghrelin group. Quantitative analysis revealed significant increases of mean optical density at 1,2 and 3 h after ghrelin administration compared with that in control group (1,3 h, P<0.05; 2 h,P<0.01). In ghrelin group, the mean optical density of NPY-positive neurons at 2 h after injection was significantly higher than those at 1 and 3 h after injection. There was no significant difference between the mean optical densities of NPY-positive neurons at 1 and 3 h after injection in ghrelin group.Conclusion1. Ghrelin may be acte on GHS-R in the DVC, and then cause the increase in the expression of ARC NPY/AgRP Mrna by upper fibers.2. The activation of NPY/AgRP neurons in the ARC is involved in the mediation of the hyperphagic response to brainstem ghrelin administration in neurologically intact rats. |
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