Ghrelin, an Endogenous Growth Hormone Secretagogue, Is a Novel Orexigenic Peptide That Antagonizes Leptin Action Through the Activation of Hypothalamic Neuropeptide Y/Y1 Receptor Pathway
Mitsuyo Shintani,
Yoshihiro Ogawa,
Ken Ebihara,
Megumi Aizawa-Abe,
Fumiko Miyanaga,
Kazuhiko Takaya,
Tatsuya Hayashi,
Gen Inoue,
Kiminori Hosoda,
Masayasu Kojima,
Kenji Kangawa, and
Kazuwa Nakao
From the Department of Medicine and Clinical Science (M.S., Y.O., K.E.,
M.A.-A., F.M., K.T., T.H., G.I., K.H., K.N.), Kyoto University Graduate School
of Medicine, Kyoto; and the Department of Biochemistry (M.K., K.K.), National
Cardiovascular Center Research Institute, Osaka, Japan.
Address correspondence and reprint requests to Yoshihiro Ogawa, Department of
Medicine and Clinical Science, Kyoto University Graduate School of Medicine,
54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507 Japan. E-mail:
ogawa{at}kuhp.kyoto-u.ac.jp
.
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ABSTRACT
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Ghrelin, an endogenous ligand for growth hormone secretagogue (GHS)
receptor originally isolated from the stomach, occurs in the hypothalamic
arcuate nucleus and may play a role in energy homeostasis. Synthetic GHSs have
activated the hypothalamic arcuate neurons containing neuropeptide Y (NPY),
suggesting the involvement of NPY in some of ghrelin actions. This study was
designed to elucidate the role of ghrelin in the regulation of food intake. A
single intracerebroventricular (ICV) injection of ghrelin (5-5,000 ng/rat)
caused a significant and dose-related increase in cumulative food intake in
rats. Ghrelin (500 ng/rat) was also effective in growth hormone-deficient
spontaneous dwarf rats. Hypothalamic NPY mRNA expression was increased in rats
that received a single ICV injection of ghrelin (500 ng/rat) (
160% of
that in vehicle-treated groups, P < 0.05). The ghrelin's
orexigenic effect was abolished dose-dependently by ICV co-injection of NPY Y1
receptor antagonist (10-30 µg/rat). The leptin-induced inhibition of food
intake was reversed by ICV co-injection of ghrelin in a dose-dependent manner
(5-500 ng/rat). Leptin reduced hypothalamic NPY mRNA expression by 35%
(P < 0.05), which was abolished by ICV co-injection of ghrelin
(500 ng/rat). This study provides evidence that ghrelin is an orexigenic
peptide that antagonizes leptin action through the activation of hypothalamic
NPY/Y1 receptor pathway.
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INTRODUCTION
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Growth hormone secretagogues (GHSs) are synthetic compounds that cause the
release of growth hormone (GH) from the pituitary
(1). They act through the GHS
receptor (GHS-R) (2), a
previously orphaned G-protein-coupled receptor that is expressed in the
hypothalamus, pituitary, pancreas, etc.
(2,3,4).
GHSs stimulate GH release by a direct pituitary action
(5,6),
but several lines of evidence have suggested that it does so via a
hypothalamic mechanism as well
(7,8).
Clinically, GHSs alone or in combination with growth hormone-releasing hormone
(GHRH) have been used for diagnosis and treatment of various forms of GH
deficiency
(1,9,10).
Using cells expressing GHS-R as assay systems, Kojima et al.
(11) have recently isolated
from the rat stomach a novel GH-releasing acylated peptide of 28 amino acids.
Termed ghrelin, it can specifically stimulate GH release in vivo and in vitro.
Previous studies have demonstrated that central as well as peripheral
administration of GHS increases food intake and body weight in rats
(12,13,14,15,16).
Furthermore, GHS administration induces c-fos mRNA expression in the
hypothalamic arcuate neurons containing neuropeptide Y (NPY), a potent
stimulator of food intake
(17). Indeed, GHS-R is
colocalized with NPY in the rat hypothalamic arcuate nucleus
(18). Expressed in the
hypothalamic arcuate nucleus
(11), it is likely that
ghrelin acts also as an orexigenic peptide that regulates hypothalamic NPY
production. However, the effect of ghrelin on feeding behavior has not been
tested so far.
Leptin is an adipocyte-derived blood-born satiety factor that acts directly
on the hypothalamus, where it regulates a large number of molecules implicated
in energy homeostasis
(19,20,21).
We and others have demonstrated that the satiety effect of leptin is mediated
through the activation of the hypothalamic melanocortin system
(22,23,24).
Several lines of evidence have suggested that hypothalamic NPY also mediates
some aspects of leptin actions
(25,26,27,28).
Indeed, it has been reported that the leptin receptor is expressed in the
majority of the arcuate NPY neurons
(29). Currently, there is
evidence for the existence of at least six functional NPY receptor subtypes
(Y1-Y6)
(30,31).
NPY Y1 receptor antagonists suppress endogenous and exogenous NPY-induced
feeding, suggesting that Y1 receptor is a major NPY receptor subtype for its
orexigenic action
(32,33,34).
Furthermore, fasting-induced refeeding is severely affected in Y1-deficient
mice (35), suggesting that the
hypothalamic NPY/Y1 receptor pathway is activated in response to fasting when
plasma leptin concentrations are reduced
(36,37,38).
It has been reported that leptin can antagonize the action of exogenously
administered NPY (39). Given
that leptin reduces the otherwise increased hypothalamic arcuate NPY mRNA
expression in fasted rats
(28,40)
and that fasting-induced refeeding is inhibited by leptin treatment
(28,40,41),
it is conceivable that the satiety effect of leptin is mediated at least
partly through the inhibition of the hypothalamic NPY/Y1 receptor pathway.
The aim of this study is to elucidate the role of ghrelin in the regulation
of food intake. Here we provide evidence that ghrelin is an orexigenic peptide
that antagonizes leptin action through the activation of the hypothalamic
NPY/Y1 receptor pathway.
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RESEARCH DESIGN AND METHODS
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Seven-week-old male Sprague-Dawley rats (200-220 g) and 9- to 11-week-old
spontaneous dwarf rats (SDRs) (100-110 g)
(42,43)
were purchased from Japan SLC (Hamamatsu, Japan). They were housed in a
temperature-, humidity-, and light-controlled room (12-h light/12-h dark
cycle) and allowed free access to standard rat food (CE-2,352 kcal/100 g;
Japan CLEA, Tokyo), unless otherwise indicated. All experimental procedures
were approved by the Kyoto University Graduate School of Medicine Committee on
Animal Research.
Intracerebroventricular injection experiments. A stainless steel
intracerebroventricular (ICV) cannula (outer diameter 1.09 mm) (Becton
Dickinson, Sparks, MD) was implanted under anesthesia in the skull of rats 5-7
days before the injection experiments, using coordinates (6.5 mm anterior to
the lambdoidal suture; ± 1.4 mm lateral to the midline; 4.5 mm from the
dural surface)
(24,44).
The ICV cannula placement was confirmed in all rats by introducing Evans blue
after the experiments. Only the animals that showed the correct ICV cannula
placement were included in this study.
ICV injection of ghrelin in Sprague-Dawley rats and SDRs.
For the ICV injection of human ghrelin alone (5-5,000 ng/10 µl/rat)
(11), cumulative food intake
was measured in Sprague-Dawley rats and SDRs during 4 h at the early light
phase (10:00-14:00) after the ICV injection. It was reported that a single ICV
injection of ghrelin at doses of 10-200 pmol/rat (
33-660 ng/rat)
stimulates pituitary GH release dose-dependently
(45).
ICV co-injection of ghrelin and NPY Y1 receptor antagonist in
Sprague-Dawley rats. To examine the involvement of NPY/Y1 pathway in
ghrelin's orexigenic effect, we used an NPY Y1 receptor antagonist (J-115814)
(Banyu Pharmaceuticals, Ibaraki, Japan). In vitro binding analysis revealed
that J-115814 displaces [125I]peptide YY binding to the cloned
human and rat Y1 receptors with inhibitory constant (Ki)
values of 1.4 and 1.8 nmol/l, respectively, whereas it shows low affinities
for human Y2 (K>10,000 nmol/l), Y4 (Ki>620
nmol/l), Y5 receptors (K >6,000 nmol/l) (M. Hata, S. Mashiko, A.
Ishihara, O. Okamoto, J. Haga, T. Ohe, T. Kanno, N. Murai, Y. Ishii, T.
Fukuroda, T. Fukami, M. Ihara, unpublished observations). Feeding induced by
NPY (5 µg/rat) was inhibited dose-dependently by ICV co-injection of
J-115814 (10-100 µg/rat) (data not shown).
For the ICV co-injection of ghrelin and J-115814, cumulative food intake
was measured in Sprague-Dawley rats for 4 h at the early light phase
(10:00-14:00) after a single ICV co-injection of ghrelin and J-115814 (500 ng
+ 10-30 µg/10 µl/rat) or vehicle (10 µl).
ICV co-injection of leptin and ghrelin in Sprague-Dawley
rats. For the ICV co-injection of leptin and ghrelin, cumulative food
intake was measured in Sprague-Dawley rats for a period of 4 h (19:00-23:00)
at the onset of the dark phase after a single ICV co-injection of leptin and
ghrelin (2 µg + 5-500 ng/10 µl/rat) or vehicle (10 µl).
Total RNA
extraction and RNA analysis. Total RNA was extracted from the whole
hypothalami (24) obtained from
Sprague-Dawley rats 4 h after a single ICV injection of ghrelin and/or leptin
or vehicle. A 232-bp rat NPY cDNA fragment was prepared by reverse
transcriptase-polymerase chain reaction using two oligonucleotide primers
(sense 5'-CTGTGTGGACTGACCCTCGC-3' and antisense
5'-CATTTTCTGTGCTTTCTCTC-3'). Northern blot analysis of NPY mRNA
was performed (24) using rat
NPY cDNA as a probe. A human ß-actin genomic probe (Wako Pure Chemical,
Osaka, Japan) was used to monitor the amount of RNA in each sample. We
confirmed that the intensity of hybridization signals shows a linear relation
to the amount of total RNA used in Northern blot analysis (data not shown).
The hybridization signal intensity was quantitated using an image analyzer
BAS-2500 (Fuji Photo Film, Tokyo) and normalized for the ß-actin signal
intensity.
Statistical analysis. All values were expressed as means ±
SE. Statistical significance of difference in mean values was assessed by
Duncan's multiplerange test following one-way analysis of variance.
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RESULTS
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ICV injection of ghrelin in Sprague-Dawley rats. A single ICV
injection of ghrelin (5-5,000 ng/rat) resulted in a significant and
dose-related increase in cumulative food intake in 8-week-old Sprague Dawley
rats compared with vehicle-treated groups during 4 h at the early light phase
(Fig. 1A). Treatment
with 5,000 ng ghrelin increased cumulative food intake approximately fourfold
relative to vehicle-treated groups (5.32 ± 1.28 vs. 1.25 ± 0.32
g, n = 10 each, P < 0.01). In this study, no significant
difference in cumulative food intake was noted between ghrelin- and
vehicle-treated animals 24 h after the injection (data not shown).

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FIG. 1 A: Effect of a single ICV injection of ghrelin (5-5,000 ng/rat)
on cumulative food intake in 8-week-old Sprague-Dawley rats. B:
Effect of a single ICV infection of ghrelin (500 ng/rat) on cumulative food
intake in 9- to 11-week-old SDRs. **P < 0.01 vs.
vehicle-treated groups.
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ICV injection of ghrelin in SDRs. To explore whether ghrelin's
orexigenic effect is mediated by increased release of GH, we examined the
effect of a single ICV injection of ghrelin on food intake in GH-deficient
SDRs. A single ICV injection of ghrelin at a dose of 500 ng/rat caused a
significant increase in cumulative food intake in SDRs compared with
vehicle-treated groups during 4 h after the injection (1.66 ± 0.08 vs.
0.70 ± 0.19 g, n = 8-10 each, P < 0.01)
(Fig. 1B).
Effects of ghrelin on hypothalamic NPY mRNA expression. Northern
blot analysis revealed that NPY mRNA expression is increased significantly in
the hypothalamus obtained from rats that received a single ICV injection of
500 ng ghrelin compared with vehicle-treated rats 4 h after the injection
(
1.6-fold, n = 12 each, P < 0.05) (Fig.
2A and
B).

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FIG. 2A : Northern blot analysis of hypothalamic NPY mRNA expression in
8-week-old Sprague-Dawley rats 4 h after the ICV injection of ghrelin (500
ng/rat). Representative blots are shown.
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FIG. 2B : Northern blot analysis of hypothalamic NPY mRNA expression in
8-week-old Sprague-Dawley rats 4 h after the ICV injection of ghrelin (500
ng/rat). Graph shows the quantification of NPY mRNA levels relative to
ß-actin mRNA levels. Data are presented as relative ratios ± SE
(n = 12 each). *P < 0.05 vs. vehicle-treated
groups.
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ICV co-injection of ghrelin and Y1 receptor antagonist. To assess
whether the orexigenic effect of ghrelin is mediated by NPY and, if so, to
assess whether Y1 receptor is involved, we examined the effect of ICV
co-injection of ghrelin and Y1 receptor antagonist in rats. After a single ICV
injection of 500 ng ghrelin, cumulative food intake was increased by
400%
relative to vehicle-treated groups (5.12 ± 0.38 vs. 1.25 ± 0.32
g, n = 10 each, P < 0.01)
(Fig. 3). By co-injection of
J-115814 at doses of 10 and 30 µg/rat, increased food intake by ghrelin was
reversed in a dose-dependent manner. Treatment with 30 µg J-115814 reversed
completely the ghrelin-induced increase in cumulative food intake relative to
vehicle-treated groups (1.22 ± 0.71 vs. 1.25 ± 0.32 g,
n = 10 each, P > 0.1). In this study, treatment with 30
µg J-115814 alone did not affect food intake in rats
(Fig. 3).

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FIG. 3 Effect of a single ICV co-injection of ghrelin and NPY Y1 receptor
antagonist on cumulative food intake in 8-week-old Sprague-Dawley rats.
**P < 0.01 vs. vehicle-treated groups.
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ICV co-injection of leptin and ghrelin. A single ICV injection of 2
µg leptin resulted in a significant reduction of cumulative food intake in
rats relative to vehicle-treated groups (1.03 ± 0.21 vs. 3.11 ±
0.24 g, n = 12 each, P < 0.01)
(Fig. 4). This is consistent
with our previous report (24).
Co-injection of ghrelin (5-500 ng/rat) reversed dose-dependently the
inhibition of food intake by leptin. Treatment with 500 ng ghrelin reversed
the leptin-induced inhibition of food intake (2.19 ± 0.23 vs. 1.03
± 0.21 g, n = 12 each, P < 0.01). Co-injection of
500 ng ghrelin and 2 µg leptin decreased significantly food intake relative
to that in vehicle-treated groups (2.19 ± 0.23 vs. 3.11 ± 0.24
g, n = 12 each, P < 0.05). In this study, a single ICV
injection of 500 ng ghrelin increased cumulative food intake (5.07 ±
0.37 vs. 3.11 ± 0.24 g, n = 12 each, P < 0.01)
during 4 h at the onset of the dark phase.

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FIG. 4 Effect of a single ICV co-injection of leptin and ghrelin on cumulative
food intake in 8-week-old Sprague-Dawley rats. *P <
0.05 and **P < 0.01 vs. vehicle-treated groups;
#P < 0.01 vs. leptin-treated groups.
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Effects of ICV co-injection of leptin and ghrelin on hypothalamic NPY
mRNA expression. Northern blot analysis revealed a significant reduction
of hypothalamic NPY mRNA expression in rats that received a single ICV
injection of leptin (2 µg/rat) relative to vehicle-treated groups 4 h after
the injection (
65%, n = 12 each, P < 0.05) (Fig.
5A and
B). The leptin-induced
decrease in NPY mRNA expression was reversed by ICV co-injection of ghrelin at
a dose of 500 ng/rat (n = 12 each, P < 0.01) (Fig.
5A and
B). Co-injection of
500 ng ghrelin and 2 µg leptin increased NPY mRNA expression relative to
vehicle-treated groups (
130%, n = 12 each, P <
0.01). Treatment with 500 ng ghrelin alone increased NPY mRNA expression by
160% relative to vehicle-treated groups (Fig.
5A and
B).

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FIG. 5A : Northern blot analysis of hypothalamic NPY mRNA expression in
8-week-old Sprague-Dawley rats 4 h after the ICV co-injection of leptin (2
µg/rat) and ghrelin (500 ng/rat). Representative blots are shown.
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FIG. 5B : Northern blot analysis of hypothalamic NPY mRNA expression in
8-week-old Sprague-Dawley rats 4 h after the ICV co-injection of leptin (2
µg/rat) and ghrelin (500 ng/rat). Graph shows the quantification of NPY
mRNA levels relative to ß-actin mRNA levels. Data are presented as
relative ratios ± SE (n = 12 each). *P
< 0.05 and **P < 0.01 vs. vehicle-treated groups;
#P < 0.01 vs. leptin-treated groups.
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DISCUSSION
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This study demonstrates that a single ICV injection of ghrelin, a newly
discovered naturally occurring GHS, increases food intake in rats. This is
consistent with previous reports that central administration of GHRP-6 and
KP-102, both of which are synthetic GHSs, increases food intake and body
weight in rats
(12,13).
Although originally isolated from the stomach, ghrelin also occurs in the
hypothalamic arcuate nucleus
(11). Furthermore, GHS-R is
expressed in several hypothalamic nuclei critical for energy homeostasis, such
as the arcuate nucleus, ventromedial hypothalamic nucleus, and paraventricular
nucleus
(3,18).
These findings, taken together, suggest that ghrelin acts centrally as an
orexigenic peptide. It has been reported that peripheral administration of
GHSs can increase food intake and body weight in rats
(15). Expression of GHS-R is
also detected in peripheral tissues, such as the pituitary, pancreas, and
renal pelvis
(2,3,4).
Currently, it is unknown that ghrelin, which is released from the stomach into
the circulation, can affect feeding behavior centrally, peripherally, or
both.
In this study, ghrelin can increase food intake in GH-deficient SDRs,
indicating that ghrelin does not require GH for its orexigenic effect. This is
consistent with the notion that the orexigenic effect of hexarelin analogs
when injected centrally and peripherally does not involve GH release
(15). It was reported that
systemic administration of GHS can induce c-fos mRNA expression in
the hypothalamic arcuate GHRH neurons
(14,17),
where it can stimulate the release of GHRH
(46). Given that GHRH is a
potent orexigenic peptide
(47), it is interesting to
speculate that ghrelin increases food intake via GHRH. However, the orexigenic
effect of KP-102 is not blocked by GHRH antagonist
(13). Thus, it is likely that
ghrelin stimulates feeding behavior via mechanisms different from those of
GHRH.
A previous study showed that GHS-R mRNA is expressed in a large population
of hypothalamic arcuate neurons containing NPY
(18). Indeed, systemic
administration of GHS can induce c-fos mRNA expression in some of
hypothalamic arcuate NPY neurons
(17). These findings suggest
that GHS can act through GHS-R that is expressed in hypothalamic arcuate
neurons containing NPY. In this study, we demonstrate that a single ICV
injection of ghrelin can increase hypothalamic NPY mRNA expression.
Furthermore, the orexigenic effect of ghrelin is abolished by ICV co-injection
of Y1 receptor antagonist. These observations suggest that ghrelin increases
food intake at least partly through the activation of the hypothalamic NPY/Y1
pathway. It is interesting to speculate that other actions of ghrelin might be
mediated by the hypothalamic NPY/Y1 pathway.
Evidence has accumulated indicating that NPY plays a role in leptin action
(25,26,27,28).
Hypothalamic NPY production is increased in leptin-deficient ob/ob
mice or db/db mice or fa/fa rats with leptin receptor
mutation
(25,48,49),
and Y1 antagonist partially reverses the spontaneous feeding of fa/fa
rats
(32,34).
It has been shown that leptin treatment can reduce the otherwise increased
hypothalamic arcuate NPY mRNA expression during fasting
(28,40),
when it inhibits significantly fasting-induced refeeding
(28,40,41).
Furthermore, fasting-induced refeeding is severely affected in Y1-deficient
mice (35). Thus, NPY, when
induced in response to leptin signaling deficiency, may activate Y1 receptor,
thus leading to a marked induction of feeding. It is also conceivable that the
satiety effect of leptin is mediated at least partly through the inhibition of
hypothalamic NPY/Y1 pathway. In this study, we demonstrate that the satiety
effect of leptin is abolished by ICV co-injection of ghrelin, which indicates
the antagonism of the satiety effect of leptin by ghrelin. It has been
reported that the biologically active longest isoform of leptin receptor mRNA
is expressed in
50% of hypothalamic arcuate NPY neurons
(50), where GHS-R mRNA is
mostly expressed (>90% of hypothalamic arcuate NPY neurons)
(18). It is likely that
ghrelin and leptin act on the same NPY neurons to regulate hypothalamic NPY
production. Therefore, we postulate that ghrelin and leptin share the
hypothalamic NPY/Y1 pathway as one of the downstream mechanisms; the
antagonism of the satiety effect of leptin by ghrelin may be due to the
ghrelin-induced activation of the hypothalamic NPY/Y1 pathway. Evidence has
suggested that the Y5 receptor is also involved in the control of food intake
(51,52).
Further studies are needed to elucidate the role of the hypothalamic NPY/Y5
receptor pathway in orexigenic action of ghrelin.
In this study, although the leptin-induced decrease in hypothalamic NPY
mRNA expression is completely abolished by ICV co-injection of ghrelin, the
satiety effect of leptin is only partially reversed by ghrelin. These
observations suggest the involvement of other orexigenic or anorexigenic
systems in the antagonism of leptin action by ghrelin. Because Agoutirelated
protein (AGRP), an endogenous antagonist of the hypothalamic melanocortin
system (24), is expressed in
the arcuate NPY neurons (53),
it is interesting to see whether ghrelin can increase hypothalamic AGRP mRNA
expression. Indeed, we have observed that hypothalamic AGRP mRNA expression is
increased significantly in rats that received a single ICV injection of
ghrelin compared with vehicle-treated rats (M.S., Y.O., K.N, unpublished
data). These observations suggest that the orexigenic effect of ghrelin is
mediated at least partly by increased production of AGRP, which should
contribute to the inhibition of the hypothalamic melanocortin system. In this
regard, it is interesting to know the role of the hypothalamic
-MSH/MC4-R/AGRP pathway in the orexigenic action of ghrelin.
In conclusion, we demonstrate that ghrelin is a novel orexigenic peptide
that antagonizes leptin action through the activation of hypothalamic NPY/Y1
receptor pathway. This study suggests that ghrelin may play an important role
in the regulation of energy homeostasis.
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ACKNOWLEDGMENTS
|
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This work is supported in part by research grants from the Japanese
Ministry of Education, Science, Sports, and Culture; the Japanese Ministry of
Health and Welfare; the Yamanouchi Foundation for Research on Metabolic
Disorders; the Foundation for Total Health Promotion; the Kato Memorial Trust
for Nanbyo Research, the Inamori Foundation; the Naito Foundation; the Uehara
Memorial Foundation; and "Research for the Future" (RFTF) of the
Japanese Society for the Promotion of Science (JSPS-RFTF 96100204 and
98L00801).
We would like to thank S. Yura for discussions and M. Ihara and A. Kanatani
for providing J-115814. The authors also acknowledge M. Nagamoto for technical
assistance and Y. Isa and Y. Nakajima for secretarial assistance.
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FOOTNOTES
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Posted on the World Wide Web at
www.diabetes.org/diabetes
on <<insert date> 2001.
AGRP, agouti-related protein; GH, growth hormone; GHRH, growth
hormone-releasing hormone; GHS, growth hormone secretagogue; GHS-R, GHS
receptor; ICV, intracerebroventricular; NPY, neuropeptide Y.
Received for publication August 16, 2000
and accepted in revised form November 9, 2000
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