Regulatory Peptides 125 (2005) 201 – 208

www.elsevier.com/locate/regpep

Inhibitory effect of ghrelin on food intake is mediated by the

corticotropin-releasing factor system in neonatal chicks
Ei-Suke Saitoa, Hiroyuki Kaiyab,*, Tetsuya Tachibanaa, Shozo Tomonagaa,
D. Michel Denbowc, Kenji Kangawab, Mitsuhiro Furusea
a
Laboratory of Advanced Animal and Marine Bioresources, Graduate School of Bioresource and Bioenvironmental Sciences,
Kyushu University, Fukuoka 812-8581, Japan
b
Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan
c
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA

Received 27 May 2004; received in revised form 25 August 2004; accepted 8 September 2004
Available online 18 October 2004

Abstract

It is known that, in rats, central and peripheral ghrelin increases food intake mainly through activation of neuropeptide Y (NPY) neurons.
In contrast, intracerebroventricular (ICV) injection of ghrelin inhibits food intake in neonatal chicks. We examined the mechanism governing
this inhibitory effect in chicks. The ICV injection of ghrelin or corticotropin-releasing factor (CRF), which also inhibits feeding and causes
hyperactivity in chicks. Thus, we examined the interaction of ghrelin with CRF and the hypothalamo-pituitary–adrenal (HPA) axis. The ICV
injection of ghrelin increased plasma corticosterone levels in a dose-dependent or a time-dependent manner. Co-injection of a CRF receptor
antagonist, astressin, attenuated ghrelin-induced plasma corticosterone increase and anorexia. In addition, we also investigated the effect of
ghrelin on NPY-induced food intake and on expression of hypothalamic NPY mRNA. Co-injection of ghrelin with NPY inhibited NPY-
induced increase in food intake, and the ICV injection of ghrelin did not change NPY mRNA expression. These results indicate that central
ghrelin does not interact with NPY as seen in rodents, but instead inhibits food intake by interacting with the endogenous CRF and its
receptor.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Ghrelin; Chick; Food intake; Anorexia; Corticosterone; CRF; NPY

1. Introduction release of GH [1–3,9–11] and energy homeostasis [12,13].

Ghrelin, a natural ligand for the GH secretagogue (GHS)
receptor (GHS-R), has been identified in mammalian and
non-mammalian species [1–5]. This peptide has a unique
octanoylation at Ser3 residue, and this modification is
necessary for the hormonal activity [1]. Ghrelin is mainly
produced in the stomach [1,6,7], and has also been detected
by immunohistochemistry in the hypothalamic arcuate
nuclei (ARC) of colchicine-treated rats and mice [1,8].
Central or peripheral ghrelin plays important roles in the
* Corresponding author. Tel.: +81 6 6833 5012x2479; fax: +81 6 6835
5402.
E-mail address: kaiya@ri.ncvc.go.jp (H. Kaiya).
0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.regpep.2004.09.003
In rats, either intracerebroventricular (ICV) or peripheral
(intraperitoneal or intravenous) administration of ghrelin
increases food intake [14–16]. The orexigenic effect of
ghrelin is mediated by activating some neuropeptides such
as neuropeptide Y (NPY), agouti-related protein (AGRP)
and orexin [16,17]. In agreement with the feeding effect,
plasma ghrelin levels are increased by fasting and decreased
by re-feeding [15,18].
In the chicken, ghrelin and its cDNA were identified in
the proventriculus [2]. Ghrelin increases plasma GH levels
after intravenous injection in the chick [2] and 4- to 5-week-
old young chickens [19], as well as in rodents and humans
[13]. However, the effect on feeding is different between
mammals and chickens. The ICV injection of ghrelin and
another GHS-R agonist, GH-releasing peptide (GHRP)-2,
202 E.-S. Saito et al. / Regulatory Peptides 125 (2005) 201–208
potently inhibits food intake of neonatal chicks [20,21]. In
the present study, we examined the mechanism whereby
ghrelin inhibits food intake in chicks. Although mono-
amines decrease food intake in mammals [22–24] and
chickens [25–29], ICV injection of ghrelin does not affect
hypothalamic monoamine levels in chicks [30]. Intravenous
injection of ghrelin increases plasma corticosterone levels,
although direct or indirect action on adrenal glands has been
unclear yet [2]. Furthermore, ICV-injected ghrelin induces
hyperactivity as measured by stepping and vocalizations
[30].
Similar phenomena have been demonstrated by cortico-
tropin-releasing factor (CRF). CRF plays a major role in
behavioral responses to stressors and in activation of the
hypothalamo-pituitary–adrenal (HPA) axis [31]. In mam-
mals, ICV injection of CRF inhibits food intake, elicits
anxiety behavior, and induces glucocorticoid release from
the adrenal glands [32–34]. In chicks, ICV injection of CRF
also induces anxiety behavior (hyperactivity) and anorexia
[35,36]. In rodents, it is reported that ghrelin stimulates CRF
and AVP release from hypothalamic explants [37]. Fur-
thermore, ICV-injected ghrelin increases plasma ACTH and
corticosterone concentrations [37,38]. These facts suggest
that central ghrelin may interact with CRF and, subse-
quently, the HPA axis.
In the present study, we examined whether inhibitory
effect of ghrelin is mediated by central CRF and its
receptor. These studies used a non-selective CRF receptor
(CRF-R) blocker, astressin, in combination with ghrelin.
Furthermore, it is known that ghrelin-induced hyperphagia
in rats results in activation of hypothalamic NPY [16].
Therefore, we examined whether ICV injection of ghrelin
affects expression of hypothalamic NPY mRNA in neonatal
chicks.
2. Material and methods
2.1. Chicks
One-day-old male layer chicks were purchased from a
local hatchery (Murata Hatchery, Fukuoka, Japan). The
birds were kept in a windowless temperature-controlled
room at 28 8C under continuous light, and had free access
to water and a commercial starter diet (Toyohashi Feed and
Science, Aichi, Japan) prior to the experiments. Birds were
placed in individual cages 1 day before the experiments.
Before each experiment, birds were weighed and divided
into experimental groups based on their body weight so
that the average weight between groups was as uniform as
possible. The body weight is reported in the figure
legends. Experimental procedures followed the guidance
for Animal Experiments in Faculty of Agriculture and in
the Graduate Course of Kyushu University and the Law
(No. 105) and Notification (No. 6) of the Japanese
Government.
2.2. Peptides and ICV injection
Chicken ghrelin-26-C8 was synthesized at Daiichi
Suntory Pharma, Institute for Medicinal Research and
Development (Gunma, Japan) [2]. Ovine CRF (oCRF) and
porcine/bovine NPY (NPY) were purchased from Peptide
Institute (Osaka, Japan). Astressin, a CRFR antagonist of a
human/rat CRF analog, was purchased from Sigma (St.
Louis, MO, USA). Peptides were dissolved in a 5%
mannitol solution containing 0.1% Evans Blue for prevent-
ing absorption of ghrelin, and injected ICV in a volume of
10 Al using a microsyringe as described by Davis et al. [39].
The control group was given a 5% mannitol solution
containing Evans Blue alone while the intact group was
not given any administration.
2.3. Experimental designs
Except for Experiment 1 described below, the remaining
experiments involved the measurement of food intake and/
or corticosterone levels. In the feeding experiment, 4-day-
old chicks were deprived of food for 3 h prior to injection in
order to stimulate and synchronize feeding.
In experiments measuring plasma corticosterone levels,
4- or 6-day-old chicks were used. Food and water were
available for ad libitum consumption until ICV injections in
order to minimize stress. Following the ICV injection, birds
were deprived of food only. Blood was collected by heart
puncture. After centrifugation at 9000g for 5 min at 4 8C,
plasma was separated and stored at 80 8C until use for the
corticosterone measurement. Plasma corticosterone concen-
trations were measured using a commercial corticosterone
enzyme immunoassay kit (Correlate-EIA, Assay Designs,
Ann Arbor, MI) according to the manufacturer’s protocol.
2.3.1. Experiment 1. Detection of ghrelin and GHS-R in
chick brain by quantitative RT-PCR
Ghrelin exists in the hypothalamic ARC of rodents, as
well as the stomach [1,6]. Little is known about the
production of ghrelin and its receptor, GHS-R in chick brain
regions. We conducted quantitative real-time PCR (Q-PCR).
The corpus striatum, cerebellum, optic lobes and brainstem
including hypothalamus were dissected from three chicks (4-
day-old, approximately 50 g body weight). Total RNA was
extracted using Trizol reagent (Invitrogen, Carlsbad, CA).
First-strand cDNA was synthesized using 3 Ag of DNase I
(Invitrogen)-treated total RNA and using SuperScript II
reverse transcriptase (Invitrogen) and random primers at 25
8C for 10 min followed by 42 8C for 50 min. Synthesized
cDNA was washed with phenol–chloroform followed by
ethanol precipitation. The precipitated cDNA was reconsti-
tuted in 30 Al of sterilized water, and 2 Al of the cDNA
solution was used as a template. Q-PCR analysis was
performed using the LightCycler system (Roche Applied
Science, Indianapolis, IN) with the FastStart DNA Master
SYBR Green I kit (Roche Applied Science). A chicken
 E.-S. Saito et al. / Regulatory Peptides 125 (2005) 201–208
ghrelin fragment (445 bp) was amplified with a sense primer
(5V-ATACAAGAAAACCAACAGCAAGAT-3V) and an
antisense primer (5V-ACTAAGGAAGGAAATAAAA-
TAAGC-3V). The amplification reaction was conducted with
95 8C for 10 min, followed by 35 cycles at 95 8C for 15 s, 59
8C for 10 s, and 72 8C for 15 s. The primers spanned the third
and fourth introns of the chicken ghrelin gene [2] and did not
produce any non-desired products. A chicken GHS-R
fragment (232 bp) was amplified with a sense primer (5V-
GGGCCGTCTCCTTCATTAGTG-3V) and an antisense
primer (5V-TTCCTCTTCCTCCTCCACAGC-3V). The
amplification conditions were identical to that of ghrelin.
Although these primers did not span the intron of the chicken
GHS-R gene, we did not find any amplified products except
the desired product. Furthermore, we confirmed that there
was no contamination of the genomic DNA using non-
reverse-transcription reaction samples. Data from this
quantitative PCR were normalized by total RNA according
to Bustin SA [40].
2.3.2. Experiment 2. Effect of ICV ghrelin on corticosterone
release
Although intravenous injection of ghrelin increases
plasma corticosterone levels [2], it has not been known
whether ICV injection of ghrelin stimulates corticosterone
release in chicks. We examined the dose–response effect and
time-course changes in plasma corticosterone levels after
ghrelin injection. Chicks (4 days old) were used in these
experiments. For the dose–response experiment, chicks were
injected with 20 pmol or 2 nmol of ghrelin, and blood was
collected 10 min after injection. For the time-course experi-
ment, chicks were injected with 20 pmol of ghrelin, and
blood was collected 10 or 30 min after injection.
2.3.3. Experiment 3. Effects of astressin on ghrelin-induced
anorexia and corticosterone release
Chicks (4 days old) were injected with 20 pmol of
ghrelin alone or 20 pmol of ghrelin in combination with 6
nmol of astressin. Food intake was measured at 30, 60 and
120 min after injection. Injected doses of astressin were
determined based on those of Baram et al. [41] and Jones et
al. [42]. In a separate experiment, 4-day-old chicks were
given the same injections described above, and blood was
collected 30 min after injection.
2.3.4. Experiment 4. Effects of ghrelin on hypothalamic NPY
mRNA levels and NPY-induced feeding
It is known that, in rats, ghrelin-induced hyperphagia is
mediated through hypothalamic NPY. Nothing is known
about the influence of ghrelin on hypothalamic NPY in
chicks, therefore, this experiment examined whether ICV
injection of ghrelin affects hypothalamic NPY mRNA
expression. Chicks (4 days old) were provided feed for ad
libitum consumption, and were injected with 50 pmol of
ghrelin or vehicle. Thirty minutes after injection, they were
sacrificed with an intraperitoneal overdose of sodium
203
pentobarbital, and the brainstem including hypothalamus
removed from five individuals. The samples were immedi-
ately treated with RNAlater (Sigma) and stored at 80 8C
until extraction of RNA. The first-strand cDNA synthesized
using 2 Ag of DNase I (Invitrogen)-treated total RNA was
reconstituted in 50 Al of sterilized water, and 2 Al of the cDNA
solution was used as a template. NPY mRNA was measured
by Q-PCR analysis using the LightCycler system. NPY
fragment (194 bp) was amplified with a sense primer (5V-
GGCACTACATCAACCTCATC-3V) and an antisense primer
(5V-CTGTGCTTTCCCTCAACAAG-3V). The amplification
reaction was conducted at 95 8C for 10 min, followed by 35
cycles at 95 8C for 15 s, 56 8C for 10 s and 72 8C for 8 s.
In addition, we examined the effect of ghrelin on NPY-
induced hyperphagia. Fasted chicks (4 days old) were
injected with 10 pmol of ghrelin alone, 50 pmol NPY alone,
or with 10 or 100 pmol ghrelin in combination with 50 pmol
NPY. Food intake was measured at 30, 60 and 120 min after
the injection. Only data from those birds that were found to
have dye in the lateral ventricle following sacrifice with an
intraperitoneal overdose of pentobarbital sodium were
included in later analysis.
2.4. Data analysis
Results are expressed as the meansFS.E.M. The data
were analyzed using a commercially available package,
StatView (Version 5, SAS Institute, Cary, USA, 1998). For
analysis of feeding data, comparisons between means were
made using Fisher’s PLSD test. For analysis of cortico-
sterone data, a one-way ANOVA followed by Duncan’s
multiple range test was employed. For comparisons between
means of NPY mRNA levels, a Mann–Whiney U-test was
applied. Differences were considered to be significant when
P is less than 0.05.
3. Results
3.1. Experiment 1. Detection of ghrelin and GHSR in chick
brain by quantitative RT-PCR
Ghrelin and GHS-R mRNA were detected in all parts of
brain examined (Fig. 1). Ghrelin mRNA was ranging from
40 to 200 molecules/Ag total RNA, but the number was
much smaller compared with that of the proventriculus
(16 003.5F2836.2 molecules/Ag total RNA, n=10). On the
other hand, the number of GHS-R mRNA was more
abundant and was ranging from 2000 to 6000 molecules/
Ag total RNA.
3.2. Experiment 2. Effects of ICV ghrelin on plasma
corticosterone levels
Plasma corticosterone levels significantly increased 30
min after ICV injection of ghrelin in a dose-dependent
204 E.-S. Saito et al. / Regulatory Peptides 125 (2005) 201–208

Fig. 1. Quantitative PCR of ghrelin mRNA and GHS-R mRNA in the brain, and its PCR products. Four parts of the brain from three chicks were examined.
After PCR amplification, the amplicons were electrophoresed and visualized.

manner ( Pb0.05 or Pb0.01 vs. each intact or control group,

respectively) (Fig. 2a). In a time-course experiment, plasma
corticosterone levels significantly increased 10 min after 20
pmol ghrelin injection, and the increased levels were still
significantly higher even 30 min after injection compared
with the time controls ( Pb0.05) (Fig. 2b).

3.3. Experiment 3. Effect of astressin on ghrelin-induced

anorexia and corticosterone release

The ICV injection of ghrelin significantly inhibited food

intake for 120 min post-injection ( Pb0.0003 vs. control
group at each time point) (Fig. 3a). Co-injection of 6 nmol
astressin with ghrelin attenuated ghrelin-induced anorexia
60 and 120 min after the injection ( Pb0.05 and Pb0.001 vs.
ghrelin-treated group, respectively). By 120 min post-
injection, food intake of the ghrelin plus astressin group
had recovered to control levels. Astressin alone had no
effect on food intake during the experimental period.
Fig. 3b shows plasma corticosterone levels after ICV
injection of ghrelin, astressin, ghrelin plus astressin.
Simultaneous treatment with astressin and ghrelin attenu-
ated the ghrelin-induced increase in plasma corticosterone
levels 30 min after injection ( Pb0.001 vs. ghrelin-treated
group and P=0.8619 vs. control group). Astressin alone had
no effect on plasma corticosterone levels.

3.4. Experiment 4. Effects of ghrelin on NPY-induced

feeding and hypothalamic NPY mRNA levels
Fig. 4 shows the effects of ghrelin and NPY on food
intake. A low dose (10 pmol) of ghrelin alone significantly
inhibited food intake, and 50 pmol of NPY alone stimulated
food intake. Co-injection of 10 pmol of ghrelin with 50
pmol of NPY had no effect on NPY-induced food intake, but
100 pmol of ghrelin with 50 pmol of NPY clearly inhibited
the NPY-induced food intake.
Fig. 2. Dose-dependent (a) and time-dependent (b) increases in plasma
corticosterone levels after ICV ghrelin injection. The number of chicks used
for the dose-dependency experiment and their body weights were as
follows: intact, 56.3F1.1 g (n=6); control, 56.0F1.4 g (n=6); 20 pmol
ghrelin, 55.5F1.1 g (n=6); 2 nmol ghrelin, 54.8F0.9 g (n=6), respectively.
The number of chicks used for the time-course experiment and their body
weights are as follows: intact, 50.4F1.5 g (n=5); 10 min control, 52.1F1.3
g (n=7); 10 min ghrelin, 50.6F1.0 g (n=5); 30 min control, 50.2F1.3 g
(n=6); 30 min ghrelin, 52.0F1.0 g (n=7), respectively.
 E.-S. Saito et al. / Regulatory Peptides 125 (2005) 201–208
Fig. 3. Effects of ICV injections of 20 pmol ghrelin, 6 nmol astressin and
their combination on (a) food intake and (b) plasma corticosterone
concentration. The number of chicks used and their body weights in the
feeding experiment were as follows: control, 48.8F0.8 g (n=9); ghrelin,
48.7F0.9 g (n=9); ghrelin+astressin, 48.8F0.9 g (n=8); astressin, 48.6F0.8
g (n=10), respectively. The number of chicks used and their body weight in
the corticosterone measurement were as follows: intact, 57.4F1.0 g (n=5);
control, 59.0F0.9 g (n=5); astressin, 57.6F0.7 g (n=5); ghrelin, 56.2F0.9
g (n=5); ghrelin and astressin, 57.6F0.6 g (n=7), respectively. Values
represent the meansFS.E.M. Groups with different letters are significantly
different ( Pb0.05).
The ICV injection of ghrelin (50 pmol) did not change
hypothalamic NPY mRNA 30 min after injection
(1299.9F70.6 molecules/ng total RNA for control versus
1253.9F156.1 molecules/ng total RNA for ghrelin, n=5
each).
4. Discussion
The results of the present study show that (i) the ghrelin
gene is expressed in various regions of the chick brain; (ii)
exogenous ICV-injected ghrelin activates the HPA axis
resulting in an increase in plasma corticosterone; and (iii)
205
the ghrelin-induced anorexia and plasma corticosterone
increase is attenuated by a CRF antagonist, astressin.
Ghrelin is known as an orexigenic peptide in rodents and
human, and its effect is mediated via NPY, AGRP and
orexin. In the chick, however, it is likely that ghrelin does
not work via NPY neurons, but rather with the CRF and its
receptor system. In the chicken, the effect of ghrelin on
corticosterone release is more pronounced compared with
that in other animals including rats and humans [2]. There is
a possibility that corticosterone release stimulated by ghrelin
may play an important role in energy regulation in birds.
Ghrelin is predominantly produced in the gastric fundus
in rats and humans [1,6,7,43,44]. In addition to the stomach,
a few ghrelin-producing cells are located in the hypothala-
mic ARC [1] and are probably involved in the regulation of
GH release from the pituitary gland [11] and feeding
behavior in the rat [16,45]. In the chicken, ghrelin mRNA
expression is also detected in several parts of brain [2]
suggesting a central action(s) of ghrelin. Although it was
reported that ICV injection of ghrelin inhibits food intake in
chicks [20,21] to show the presence of the GHS-R mRNA in
the brain was necessary to demonstrate the effects of ghrelin
on a central function. Recently, the sequences of chicken
GHS-R cDNA have been reported from two laboratories
[3,46,47]. The GHS-R mRNA is widely distributed in
several parts of the brain such as the hypothalamus,
telencephalon, cerebellum, optic lobe and brainstem [46].
In the present study, we quantified, for the first time, ghrelin
mRNA in addition to the GHS-R mRNA from four parts of
Fig. 4. Effects of ICV injections of ghrelin, NPY and their combinations on
food intake. Ghrelin was injected at doses of 10 or 100 pmol. NPY was
injected at a dose of 50 pmol. The number of chicks used and their body
weights are as follows: control, 50.7F1.5 g (n=6); ghrelin, 50.5F1.8 g
(n=4); NPY, 51.0F1.5 g (n=5); 10 pmol ghrelin and NPY, 50.8F0.9 g
(n=6); 100 pmol ghrelin+NPY, 49.7F1.1 g (n=6), respectively. Values
represent the meansFS.E.M. Groups with different letters are significantly
different ( Pb0.05).
206 E.-S. Saito et al. / Regulatory Peptides 125 (2005) 201–208
chick brain using Q-PCR. Consequently, we detected
ghrelin mRNA in the brain and found that the number
was very low. The rank order was cerebellumNcorpus
striatumNoptic lobesNbrainstem. In the rat, ghrelin-produc-
ing cells are located in the hypothalamus. Why ghrelin is
produced in several brain area other than the hypothalamus
in chicks remains unclear. On the other hand, expression of
the GHS-R mRNA was more abundant than that of ghrelin
mRNA. The rank order (brainstemNcerebellumNoptic
lobesNcorpus striatum) was similar to the previous result
of Geelissen et al. [46]. This indicates the sites of actions for
central ghrelin. Although the presence of GHS-R mRNA in
several brain regions in rats has been demonstrated, little is
known about the functional relevances until now [48,49].
Further studies need to clarify this issue not only in the
chicken, but also in mammalian species.
ICV-injected ghrelin stimulated corticosterone release in
dose-dependent and time-dependent manners in this study.
Although similar responses have observed in the previous
study in which ghrelin was injected intravenously [2], the
fact that ICV ghrelin stimulates corticosterone release
demonstrated for the first time. However, little is known
whether ghrelin is acting directly on the adrenal gland or
indirectly through the hypothalamus by affecting the HPA
axis. Based on the presence of GHS-R mRNA in the
adrenal gland [46], this would suggest that a direct
stimulation of adrenal corticosterone release is possible.
On the other hand, the expression of GHS-R mRNA in the
hypothalamus and pituitary might suggest an indirect
stimulation of corticosterone release through the release of
hypothalamic CRF or arginin–vasotocin [50], or through a
direct affect on ACTH release from systemic circulation.
Some mammalian studies support the hypothesis that
ghrelin activates the HPA axis through CRF: GHRPs do
not release glucocorticoids from the adrenal glands directly,
but via stimulation of ACTH secretion [51], and; intra-
peritoneal injection of ghrelin at a dose of 3 nmol increases
hypothalamic CRF mRNA and serum corticosterone con-
centrations in mice [38]. Furthermore, ghrelin increases
plasma ACTH and corticosterone concentrations by ICV
injection in the rat [37]. The results of the present study
support the idea even in chicks. In addition, the effect of
ghrelin on plasma corticosterone was completely blocked
by simultaneous treatment of astressin, a CRF antagonist.
These results suggest that, in chicks as well, ICV-injected
ghrelin, and possibly peripherally injected ghrelin with
different pathways, stimulates the release of endogenous
CRF, which activates the HPA axis, resulting in cortico-
sterone release from the adrenal glands.
Exogenous CRF reduces food intake in chicks [35,36,
52,53], as well as in rats [54,55], mice [56] and marsupials
[57]. However, the mechanism governing the inhibition of
food intake by ghrelin is clearly different from those
following CRF injection; ghrelin-induced anorexia was
attenuated by co-administration of astressin, suggesting that
endogenous CRF released by ghrelin is involved in the
inhibition of food intake. Larsen et al. [58] reported an
inhibitory mechanism of food intake in rats by glucagon-
like peptide (GLP)-1: central administration of GLP-1
caused anorexia by activating the HPA axis, which caused
an increase in plasma corticosterone levels at the same time.
Furthermore, it was recently reported that central anorectic
actions of prolactin-releasing peptide in rats, and those of
pituitary adenylate cyclase-activating polypeptide (PACAP)
and vasoactive intestinal peptide (VIP) in chicks were
mediated by CRF-Rs [59,60]. These anorectic pathways are
similar to the present results.
It is noteworthy that, in chicks, ghrelin-induced anorexia
is mediated by the CRF system. In chicks, GLP-1-induced
anorexia is mediated by the noradrenergic system without
changes in plasma corticosterone levels [61]. In contrast,
ghrelin does not alter monoamines in the brain of chick [30].
These results suggest that central inhibition of food intake in
chicks is controlled by complex peptidergic and non-
peptidergic mechanisms.
It is known that, in rats, ghrelin-induced hyperphagia is
controlled by some orexigenic peptides such as orexin,
AGRP and NPY [16,17]. The interaction of ghrelin and
NPY is well documented: ICV or peripheral injection of
ghrelin activates c-fos in the ARC NPY neurons [16,62–64];
ICV injection of ghrelin increases NPY mRNA in the ARC
and food intake [65,66]; almost all of NPY neurons in the
ARC co-localize with GHS-R [67]; isolated single NPY
neuron directly responds to ghrelin with their intracellular
calcium increase [68], and ICV injection of NPY Y1
antagonists inhibit ghrelin-stimulated food intake [16,45,
64,69]. On the other hand, in chicks, ICV injection of NPY
stimulates food intake [70], but AGRP [71] and orexin [72]
have no effect, suggesting different mechanisms controlling
feeding behavior. In the present study, ghrelin did not affect
the expression of hypothalamic NPY mRNA, and co-
administration of ghrelin with NPY inhibited NPY-induced
food intake. These results indicate that central ghrelin does
not interact with NPY in chicks, unlike in rodents. This
would be one of the reasons why ghrelin inhibits food intake
in chicks.
In summary, we demonstrated here that the inhibitory
effect of central ghrelin on food intake is caused by
activating the endogenous CRF system. Furthermore, the
fact that ghrelin does not interact with hypothalamic NPY in
chicks would also be an additional reason of the anorexia.
However, it is unclear whether this pathway for ghrelin-
induced anorexia by the CRF system is a species-specific
effect or a developmental phenomenon in the chicken
because nothing is known about the effect of ghrelin on
feeding in neonatal animals. This needs to be examined
using adult chickens. The ICV injection of ghrelin induces
an orexigenic effect in mammals and goldfish, but an
opposite effect is observed in the neonatal chick. Today,
although it is widely known that ghrelin is a peptide
associated with energy regulation, these differences may be
associated with specific avian physiology.
 E.-S. Saito et al. / Regulatory Peptides 125 (2005) 201–208
Acknowledgements
This study was supported by a Grant-in-Aid for Scientific
Research from Japan Society for the Promotion of Science,
and in part by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and
Technology of Japan. We are grateful to Yasuo Kitajima,
Masaru Matsumoto, Yoshiharu Minamitake at the Daiichi
Suntory Pharma, Institute for Medicinal Research and
Development, Gunma, Japan for synthesizing chicken
ghrelin.
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