Endocrinology Vol. 140, No. 9 4046-4055
Copyright © 1999 by The Endocrine Society
Evidence That the Inhibition of Luteinizing Hormone Secretion Exerted by Central Administration of Neuropeptide Y (NPY) in the Rat Is Predominantly Mediated by the NPY-Y5 Receptor Subtype1
Paula D. Raposinho,
Pierre Broqua,
Dominique D. Pierroz,
Amanda Hayward,
Yvan Dumont,
Remi Quirion,
Jean-Louis Junien and
Michel L. Aubert
Division of Biology of Growth and Reproduction, Department of
Pediatrics, University of Geneva School of Medicine (P.D.R., P.B.,
D.D.P., M.L.A.), 1211 Geneva 14, Switzerland; Ferring Research Ltd.
(P.D.R., P.B., D.D.P., A.H., J.-L.J.), Chilworth, United Kingdom SO17
7NP; Instituto Tecnológico e Nuclear (P.D.R.), 2685 Sacavem,
Portugal; and Douglass Hospital Research Center, Department of
Psychiatry, McGill University (Y.D., R.Q.), Verdun, Québec,
Canada H4H 1R3
1
Address all correspondence and requests for reprints to: Dr. M. L. Aubert, Hopital des Enfants, Hôpitaux Universitaires de Genève, 6 rue Willy-Donzé, 1211 Geneva 14, Switzerland. E-mail: aubert{at}cmu unige.ch.
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Abstract
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A number of studies have indicated that neuropeptide Y (NPY) is a
central regulator of the gonadotropic axis, and the Y1 receptor was
initially suggested to be implicated. As at least five different NPY
receptor subtypes have now been characterized, the aim of the present
study was to reinvestigate the pharmacological profile of the
receptor(s) mediating the inhibitory action of NPY on LH secretion by
using a panel of NPY analogs with different selectivity toward the five
NPY receptor subtypes. When given intracerebroventricularly (icv) to
castrated rats, a bolus injection of native NPY (0.72.3 nmol)
dose-dependently decreased plasma LH. Peptide YY (PYY; 2.3 nmol) was as
potent as NPY, suggesting that the Y3 receptor is not implicated.
Confirming previous data, the mixed Y1, Y4, and Y5 agonist
[Leu31,Pro34]NPY (0.72.3 nmol) inhibited LH
release with potency and efficacy equal to those of NPY. Neither the
selective Y2 agonist C2-NPY (2.3 nmol) nor the selective Y4 agonist rat
pancreatic polypeptide affected plasma LH, excluding Y2 and Y4
subtypes for the action of NPY on LH secretion. The mixed Y4-Y5 agonist
human pancreatic polypeptide (0.77 nmol) as well as the mixed Y2-Y5
agonist PYY336 (0.77 nmol) that displayed very low
affinity for the Y1 receptor, thus practically representing selective
Y5 agonists in this system, decreased plasma LH with potency and
efficacy similar to those of NPY, indicating that the Y5 receptor is
mainly involved in this inhibitory action of NPY on LH secretion.
[D-Trp32]NPY, a selective, but weak, Y5
agonist, also inhibited plasma LH at a dose of 7 nmol. Furthermore, the
inhibitory action of NPY (0.7 nmol) on LH secretion could be fully
prevented, in a dose-dependent manner (6100 µg, icv), by a
nonpeptidic Y5 receptor antagonist. This antagonist (60 µg, icv) also
inhibited the stimulatory action of NPY (0.7 nmol) on food intake. The
selectivity of PYY336, human PP,
[D-Trp32]NPY, and the Y5 antagonist for the
Y5 receptor subtype was further confirmed by their ability to inhibit
the specific
[125I][Leu31,Pro34]PYY binding
to rat brain membrane homogenates in the presence of the Y1 receptor
antagonist BIBP3226, a binding assay system that was described as being
highly specific for Y5-like receptors. With the exception of
[D-Trp32]NPY, all analogs able to inhibit LH
secretion were also able to stimulate food intake. Taken together,
these results indicate that the Y5 receptor is involved in the negative
control by NPY of the gonadotropic axis.
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Introduction
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NEUROPEPTIDE Y (NPY) is a 36-amino acid
peptide belonging to the pancreatic peptide (PP) family. In mammals,
this family consists of PP, peptide YY (PYY) of gastrointestinal
origin, and NPY (1). Although PP and PYY are primarily located in the
periphery, NPY is predominantly located within neurons of the central
and sympathetic nervous systems (2, 3, 4). In the rat, five NPY receptor
subtypes (Y1Y5) have been characterized on the basis of different
pharmacological profiles and/or cloning (5, 6). A sixth subtype (Y6)
has been cloned from mice (7, 8), but the existence of this subtype in
rats remains to be proven. The existence of additional NPY receptor
subtypes has been postulated. NPY receptor subtypes are members of the
seven-transmembrane domain, G protein-coupled receptors associated with
inhibition of adenylate cyclase.
The highest concentrations of NPY in the brain are found within several
hypothalamic nuclei that regulate a variety of neuroendocrine and
autonomic functions (9), and one of the first reported actions of NPY
actually involved studies of the endocrine and eating behavior
responses to human PP (hPP) and NPY (10, 11, 12, 13, 14, 15). In these original
studies, it was shown that central administration of NPY stimulated
food consumption (12, 13, 14, 15), inhibited sexual behavior (14), and
modulated the activity of the gonadotropic axis (10, 11, 15). In
contrast to the straightforward action of NPY on feeding behavior, the
regulation of the gonadotropic axis by this neuropeptide is rather
complex. It was shown that NPY can stimulate LH release in sex
steroid-primed ovariectomized rats after central administration (10, 15). In contrast, in castrated animals, central administration of NPY
produced a striking inhibition of LH release, as seen in rats (10, 16, 17), rabbits (18), and nonhuman primates (19). Furthermore, in both
male and female intact rats, chronic intracerebroventricular (icv)
infusion of NPY led to a profound inhibition of the gonadotropic axis
(20, 21) and prolongation of sexual immaturity (22). Based on the
observation that this dual action of NPY on the gonadotropic axis could
be reproduced by the Y1 agonist
[Leu31,Pro34]NPY, but not by the Y2 agonist
NPY1336, both the stimulatory and inhibitory actions of
NPY were postulated to be mediated by the NPY Y1 receptor subtype (21, 23). However, after the cloning and pharmacological characterization of
the Y4 and Y5 receptors, it clearly appeared that
[Leu31,Pro34]NPY is a nonselective Y1, Y4,
and Y5 agonist (24, 25, 26). This observation led us to reevaluate the
pharmacological profile of the NPY receptor subtype involved in the
inhibitory action of NPY on the gonadotropic axis in the castrated rat.
For this purpose, we evaluated the effects of NPY analogs displaying
different selectivities toward the five NPY receptor subtypes on LH
secretion in the castrated rat:
[Leu31,Pro34]NPY, a nonselective Y1, Y4, and
Y5 agonist; C2-NPY, a selective Y2 agonist; PYY, that has very low
affinity for the Y3 receptor; rat PP (rPP), a selective Y4 agonist;
hPP, a nonselective Y4 and Y5 agonist; PYY336, a
nonselective Y2 and Y5 agonist; and finally,
[D-Trp32]NPY, a weak, but selective, Y5
ligand (25, 27, 28, 29). We also examined the effect of a selective Y5
antagonist (30). Finally, because several reports have led to the
proposal that feeding behavior and activity of the gonadotropic axis
are linked, and that NPY may be pivotal in this relationship (21, 31),
we also evaluated the effects of these analogs and the Y5 antagonist on
food intake. Together with literature data and binding studies
performed in the rat brain, these in vivo results have
indicated that both inhibition of the gonadotropic axis and stimulation
of food intake by NPY are predominantly mediated by the Y5 receptor
subtype.
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Materials and Methods
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Animals
Male Sprague Dawley rats (200220 g) obtained from Iffa Credo
(LArbresle, France) were fed standard laboratory chow ad
libitum and kept on a 12-h light, 12-h dark (lights on, 07001900
h) schedule in a temperature- and humidity-controlled room. Animal care
was performed according to protocols reviewed by the University of
Geneva School of Medicine ethical committee for animal experimentation
and approved by the State of Geneva Veterinary Office.
Materials
Porcine (p) NPY, hPYY336, rPP, hPP,
p[Leu31,Pro34]NPY, C2NPY, and pPYY were
purchased from Neosystem (Strasbourg, France).
[D-Trp32]NPY was obtained from Bachem California, Inc. (Torrance, CA).
[Leu31,Pro34]PYY was synthesized in Douglas
Hospital Research Center (Verdun, Canada). The Y5 antagonist,
trans-naphthalene-1-sulfonic
acid-{4-{[4-(3-dimethylamino-propylamino)-quinazolin-2-ylamino]-methyl}-cyclohexylmethyl}-amide
(30), was synthesized in the Medicinal Chemistry Department of Ferring
Research Ltd. (Chilworth, UK). This compound has a structure very
similar to that of CGP71683A, a Y5 antagonist described by the same
investigators and presented at the Fourth International Neuropeptide Y
Conference (London, UK) in October 1997. BIBP3226 was provided by Karl
Thomae GmbH (Biberach an der Riss, Germany).
125I for binding was obtained from ICN Pharmaceuticals
Canada Ltd. (Montreal, Canada), and bacitracin was purchased from
Sigma Chemical Co. (St. Louis, MO). Schleicher and Schuell
no. 32 glass filters were obtained from Xymotech (Montreal, Canada).
125I was incorporated into the tyrosine residue of
[Leu31,Pro34]PYY using the chloramine-T
method as previously described (32), and the specific activity was
assumed to be a theoretical value (2000 Ci/mmol).
Surgical procedure
Castration and implantation of intracerebroventricular
cannula. Rats were weighed and anesthetized with ketamine/xylazine
(3 and 7 mg/kg, ip, respectively). An incision of the skin was made in
the middle of the scrotum, and the testicles and epididymis were
exposed. The testicles were separated from the epididymis and cut-off
after ligation of the artery. Cannulas, aimed at the right lateral
ventricle, were placed 1 mm posterior and 2 mm lateral to bregma and
extended 2 mm below the outer surface of the skull. Rats were allowed
to recover for 7 days, as described previously (20).
Implantation of jugular catheter. Under the same anesthetic,
the ventral side of the throat was shaved, and an incision was made
down the center of the throat. The right jugular vein was exposed and
cannulated using polythene tubing (OD, 1.0 mm) connected to a medical
grade silicone tubing (OD, 0.94 mm; silicone side in the jugular vein).
The tubing was secured, and the polythene side was externalized through
an incision made on the dorsal side of the neck. The catheter was
rinsed with 300 µl Ringers solution containing 0.1% heparin. Rats
were allowed to recover for at least 24 h in individual cages,
with food and water available ad libitum.
Experimental procedure
All experiments were carried out between 1418 h. Before the
start of the experiments, rats were weighed and placed in individual
cages with a preweighed amount of food. The different analogs,
dissolved in sterile distilled water, were injected (icv) in a volume
of 5 µl. The Y5 antagonist, dissolved in sterile distilled water
slightly acidified by acetic acid 2% (15% of the total volume, pH
6.5), was injected (icv) 15 min before NPY. Blood samples (200250
µl) were removed immediately before and 15, 30, 60, 90, and 120 min
after central injections. Each blood sample was replaced with an
equivalent volume of Ringers solution containing 0.1% heparin.
Plasma was extracted and stored at -20 C until determination of LH by
RIA. Food intake was measured after 2 h. At the end of the study,
the rats were anesthetized, and an icv injection of 5 µl methylene
blue dye was made. Animals were then killed by decapitation, and the
brain was inspected for uniform and complete spread of the dye in the
lateral ventricle. Data from any subject with inadequate spread of the
marker were discarded.
Determination of plasma LH levels
LH was determined by RIA using reagents prepared by Dr. A.
F. Parlow and provided by the NIDDK (Bethesda, MD), with the exception
of the second antiserum. NIDDK antirat LH S11 serum was used. Values
were expressed in terms of the RP-1 reference standard. For each
experiment, all plasma samples (vehicle control and tested analog) were
measured in the same RIA.
Ligand binding assays
Membranes were prepared as previously described (32). Briefly,
rats were killed by decapitation, and their brains rapidly removed and
homogenized in Krebs-Ringer phosphate (KRP) buffer at pH 7.4 of the
following composition: NaCl (120 mM), KCl (4.7
mM), CaCl2 (2.2 mM),
KH2PO4 (1.2 mM), MgSO4
(1.2 mM), dextrose (5.5 mM), and
NaHCO3 (25 mM) using a Brinkmann Instruments, Inc., Polytron (Westbury, NY; at setting 6 for
1520 sec). Homogenates were centrifuged at 49,000 x
g for 20 min, supernatants were discarded, and pellets were
washed, resuspended, and recentrifuged twice.
All binding assays were initiated by adding 100 µl of membrane
preparations in a final volume of 500 µl KRP containing 0.1%
(wt/vol) BSA, 0.05% (wt/vol) bacitracin, 125I-labeled
[Leu31,Pro34]PYY (2535 pM), and
various competitors (pNPY, hPYY336, hPP, rPP,
[Leu31,Pro34]pNPY, C2-NPY,
[D-Trp32]NPY, and the Y5 antagonist) at
concentrations ranging from 10-1210-6
M. All binding assays were performed in the absence or
presence of 1 µM BIBP3226 to block the Y1 receptor
subtype. Nonspecific binding was determined in the presence of 1
µM pNPY. After 2 h, the binding reaction was
terminated by rapid filtration through Schleicher and Schuell no. 32
glass filters (previously soaked in 1.0% polyethyleneimine) using a
cell harvester filtering apparatus (Brandel Instruments, Gaithersburg,
MD). Filters were rinsed three times with 3 ml cold KRP, and the
radioactivity remaining on filters was quantified using a
-counter
with 85% efficiency (Packard Instruments, Downers Grove, IL).
All binding experiments were repeated three times, each in triplicate,
and results are expressed as a percentage of the specific binding,
representing the mean ± SEM. IC50 values
(i.e. concentration of unlabeled peptide required to compete
for 50% of specific binding of the radioligand) of the various
peptides and Y5 antagonist were calculated from the competition binding
assays data using GraphPad software (GraphPad Software, Inc., San Diego, CA).
Statistical analysis
The effect of each NPY analog on plasma concentrations of LH was
analyzed by one-way ANOVA followed by Dunnetts t test to
examine the differences between each postinjection time point and the
preinjection basal value. The effects of NPY analogs on food intake
were analyzed by a one-way ANOVA followed by Dunnetts t
test. For the study with the Y5 antagonist, one-way ANOVA followed by
Students-Newman-Keuls test were performed.
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Results
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NPY-induced inhibition of LH release in the castrated rat:
dose-response study
An icv bolus injection of NPY (0.077 nmol) produced a
dose-related inhibition of LH secretion, as seen from plasma LH levels
measured during the 120-min period after NPY injection (Fig. 1
). The lowest NPY dose tested (0.07
nmol) was ineffective. For the other four doses, a significant decrease
(P < 0.05) was first observed after 30 min. Minimum LH
values were reached after 30 min for the 0.23-nmol dose and at later
times with increasing doses. With the highest dose (7 nmol), plasma LH
levels were still decreasing after 120 min.

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Figure 1. Effects on plasma LH levels of an icv bolus
injection of NPY at different doses (0.077.0 nmol) in the castrated
rat. Each point represents the mean ±
SEM for plasma LH in five to seven rats.
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Effects of NPY analogs on plasma LH levels in the castrated rat
Various NPY analogs with varying specificities for the different
NPY receptor subtypes were tested icv at a single dose of 2.3 nmol,
except for [D-Trp32]NPY, for which 7 nmol
were injected. Plasma LH levels were followed for 120 min after
injection. A highly significant inhibition of LH secretion was observed
for five of the NPY analogs tested:
[Leu31,Pro34]NPY, hPP, PYY,
PYY336, and [D-Trp32]NPY [F =
5.7 to 27; P < 0.001; Fig. 2
]. Two analogs, C2-NPY and rPP, did not
affect plasma LH levels (Fig. 2
).

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Figure 2. Effects on plasma LH levels of an icv bolus
injection of various NPY analogs in the castrated rat.
[Leu31,Pro34]NPY, hPP, PYY336,
C2-NPY, rPP, and PYY were administered as a bolus injection of 2.3
nmol, and [D-Trp32]NPY was given as a bolus
injection of 7 nmol. Each point represents the mean
± SEM for plasma LH. The number of rats per group is
indicated in parentheses in the lower left
corner. *, Significantly lower than the pretreatment LH levels,
as determined by Dunnetts test (P < 0.05).
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Dose-response curves for NPY (0.077 nmol) and the four active NPY
analogs [Leu31,Pro34]NPY (0.232.3 nmol),
PYY336 (0.237 nmol), hPP (0.237 nmol), and
[D-Trp32]NPY (0.77 nmol), plotted at the
time of maximal LH inhibition, are shown in Fig. 3
. All compounds produced a highly
significant, dose-related decrease in plasma LH levels: NPY, F(5, 44) = 25.17 and P < 0.001;
[Leu31,Pro34]NPY, F(3, 38) = 15.7 and
P < 0.001; PYY336, F(4, 41) = 23.51
and P < 0.001; hPP, F(4, 46) = 25.71 and
P < 0.001; and [D-Trp32]NPY,
F(3, 23) = 8.05 and P < 0.001. At the 2.3-nmol
dose, NPY, PYY336,
[Leu31,Pro34]NPY, and hPP generated similar
highly significant (P < 0.001) levels of LH inhibition
(76.7 ± 3.0%, 71.8 ± 3.0%, 67.3 ± 8.2%, and
64.0 ± 4.5%, respectively). With
[D-Trp32]NPY, 55.0 ± 6.5% of
inhibition was achieved at a dose of 7 nmol only.

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Figure 3. Dose-response relationship for the inhibition of
LH secretion achieved by a bolus icv injection of either native NPY
(dose range, 0.077 nmol) or different NPY analogs:
[Leu31,Pro34]NPY (0.232.3 nmol), hPP
(0.237 nmol), PYY336 (0.237 nmol), and
[D-Trp32]-NPY (0.77 nmol). Each
point represents the mean ± SEM for
the percent decrease in plasma LH relative to the pretreatment values
at the time of maximal effect (60 min for hPP and
[D-Trp32]NPY and 90 min for the other
analogs) in 511 rats.
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Effects of a Y5 receptor antagonist (Y5-ant) on NPY-induced
inhibition of plasma LH levels
The Y5 antagonist was tested in the presence of native NPY in the
castrated rat. When given as a bolus icv injection (60 µg) 15 min
before NPY (0.7 nmol), Y5-ant fully inhibited the lowering effect of
NPY on plasma LH levels without affecting basal plasma LH levels (Fig. 4
). The antagonistic action of Y5-ant was
dose related; a significant inhibition was reached with the dose equal
to or greater than 60 µg, and full reversion was achieved at a dosage
of 100 µg (Fig. 5
).

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Figure 4. Effect of a nonpeptidic Y5 receptor antagonist
(Y5-ant) on the inhibition of plasma LH induced by a bolus icv
injection of NPY. Y5-ant (60 µg) or its vehicle was administered icv
15 min before NPY (0.7 nmol) or the vehicle of NPY. Each
point represents the mean ± SEM for
plasma LH in six to nine rats. *, Significant difference between NPY
alone and NPY associated with Y5-ant for each time point (comparing
open symbols), as determined by Student-Newman-Keuls
test (P < 0.05).
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Figure 5. Dose-dependent antagonism by the Y5
antagonist of NPY-induced inhibition of LH. Y5-ant (6- to 100-µg
bolus icv injection) was injected 15 min before NPY (0.7 nmol, icv).
Each bar represents the mean ± SEM for
plasma LH at 60 min postinjection of NPY in eight or nine rats. ,
Significantly different from vehicle-injected rats; *, significantly
different from NPY-injected rats, as determined by Student-Newman-Keuls
test (P < 0.05).
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Binding of NPY, NPY analogs, and the Y5 receptor antagonist to rat
brain membrane homogenates
To evaluate the affinities at the Y1 and Y5 receptors of the NPY
analogs and the Y5 antagonist, competition of the binding of
125I-labeled [Leu31,Pro34]PYY to
rat brain membrane homogenates was assessed in the presence or absence
of 1 mM BIBP3226. Under such binding assay conditions,
[Leu31,Pro34]NPY was active for both Y1-like
and Y5-like activities (Table 1
). Both
hPP (known as a mixed Y4-Y5 specific analog) and PYY336
(known as a mixed Y2-Y5 specific analog) had a very weak affinity for
Y1-like activity and a strong binding capacity for the Y5-like type of
binding (Table 1
). The Y2-selective NPY analog C2-NPY and the
Y4-selective peptide rPP displayed very weak activity for both types of
binding. Finally, the nonpeptidic Y5 receptor antagonist described by
Novartis clearly induced strong inhibition of the Y5-like type of
binding and no inhibition of the Y1-like type of activity in this
competitive binding system (Table 1
). In addition, this compound was
found to have no affinity for Y1 receptors in intact human
neuroblastoma SK-N-MC cells (IC50, >1000
nM).
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Table 1. Comparative binding parameters of NPY, PYY, rPP,
hPP, [Leu31, Pro34]-NPY, C2-NPY,
PYY336, [D-Trp32]NPY, and the
Y5 antagonist against [125I][Leu31,
Pro34]NPY binding sites in the presence of 1
µM BIBP3226 in the rat brain membrane
homogenates
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Effects of NPY and NPY analogs on food intake in the castrated
rat
Food intake induced by bolus injection of NPY or NPY analogs
was assessed during 2 h in the castrated rats used for the study
on LH secretion. Significant stimulation of food intake was observed
for all NPY analogs able to decrease LH secretion, except for
[D-Trp32]NPY:
[Leu31,Pro34]NPY [F(3, 38) = 3.5;
P = 0.025], PYY336 [F(4, 41) = 5.2;
P = 0.002], hPP [F(4, 46) = 17.4;
P < 0.001], and NPY [F(5, 44) = 20.7;
P < 0.001; Fig. 6
). The
minimal effective dose was 0.7 nmol for NPY, hPP, and
PYY336 and 2.3 nmol for
[Leu31,Pro34]NPY. At the dose of 2.3 nmol,
PYY336 and NPY stimulated food intake to a comparable
extent (PYY336, 7.0 ± 0.9 g; NPY, 5.7 ±
1.1 g), whereas [Leu31,Pro34]NPY and hPP
were less efficient ([Leu31,Pro34]NPY,
3.1 ± 1.2 g; hPP, 1.9 ± 0.6 g).
[D-Trp32]NPY at the doses tested (0.77
nmol) was unable to stimulate food intake.

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Figure 6. Dose-response relationship for the stimulation of
food intake induced by a bolus icv injection of either native NPY (dose
range, 0.077 nmol), or different NPY analogs:
[Leu31,Pro34]NPY (0.232.3 nmol), hPP
(0.237 nmol), PYY336 (0.237 nmol), and
[D-Trp32]-NPY (0.77 nmol). Food intake
was evaluated over 2 h starting in the early afternoon. Each
point represents the mean ± SEM for
the quantity of food ingested in 710 rats.
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Effects of the Y5 receptor antagonist (Y5-ant) on NPY-induced
stimulation of food intake in the castrated rat
The Y5 antagonist was able, when given as an icv bolus injection
of 60 µg, to fully inhibit the stimulatory effect of NPY (0.7 nmol)
on 2-h food consumption. Y5-ant alone (60 µg) had no effect on food
intake (Table 2
).
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Table 2. Effect of administration of a nonpeptidic Y5
receptor antagonist on NPY-induced stimulation of food intake in the
castrated rat
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Discussion
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Early studies of NPY action demonstrated that a bolus injection
into the lateral ventricle of rats (recognized as central, or icv,
injection) could induce a robust feeding response in satiated rats
(12, 13, 14, 15), a reduction in sexual behavior (14), and a dual effect on LH
secretion (15). Indeed, bolus injection of NPY elicited a modest
increase in LH secretion in intact males and gonadectomized, sex
steroid-primed females (15), whereas such a bolus injection induced a
striking, dose-dependent decrease in plasma LH in castrated rats (10, 17). The physiological significance of the stimulatory effect of NPY in
sex steroid-intact animals has been reviewed extensively, indicating
that in several situations, NPY clearly provides an excitatory signal
for LH release (31). The significance of the inhibitory effect of bolus
NPY injection on LH and FSH secretion was greatly highlighted by the
demonstration that chronic, 1-week central infusion of NPY into the
lateral ventricle fully inhibited the gonadotropic axis in intact
female rats, with a collapse of estrous cyclicity (20) and delayed
sexual maturation in female rats (22, 33), and a spectacular inhibition
of testicular function in male rats (21). Interestingly, chronic,
central infusion of NPY produced at least three different effects: 1)
unabated increase in food intake leading to obesity, 2) suppression of
gonadotropin secretion (21, 34), and 3) suppression of GH secretion,
with low insulin-like growth factor I secretion as a consequence (21).
Even if these different actions of NPY appear to be coordinated, it is
not clear whether they are mediated by the same NPY receptor
subtype(s). To reinvestigate NPY receptor selectivity for the action on
LH secretion, the most simple model was chosen: the inhibitory action
of central, bolus NPY injections at different doses on LH secretion in
castrated male rats that yields most precise answers in terms of
pharmacological action.
The panel of NPY agonists used in this study was chosen because their
specificities toward the different Y receptors are well documented by
numerous studies published over several years (for review, see Refs. 6, 7). Recently, their specificities were further established by
functional studies of the specific Y receptor, expressed in stable cell
lines such as, for example, the analysis proposed by Gerald et
al. (25). Also, the specificities of several NPY agonists or
antagonists for the Y1 and Y5 receptor subtypes in rat brain were
reevaluated recently by two of us, using a paradigm of binding
competition of
[125I][Leu31,Pro34]PYY to rat
brain membrane homogenates in the absence or presence of 1
mM BIBP3226, a highly specific Y1 receptor antagonist (35),
that allows clear discrimination between Y1 and Y5 receptor binding
specificities (29).
In the present study we confirmed the clear dose-dependent inhibitory
action of centrally administered NPY on LH secretion in the male
castrated rat, first described by Kalra and Crowley in the eighties
(15). The involvement of the Y2 receptor subtype for this NPY action on
LH secretion was eliminated in another study published by Kalra
et al. in 1992 (23) and by us, as chronic administration of
NPY1336 had no effect on the pituitary-testicular axis of
male rats (21). Consistent with this, C2-NPY, a specific Y2 analog, had
no effect on LH secretion in the castrated rat in the present study.
Receptor subtype Y4 could be eliminated as well, as the specific Y4
receptor agonist rPP was inactive. Furthermore, PYY, which binds only
poorly to the Y3 receptor subtype (28), was as active as NPY to inhibit
LH secretion, making it unlikely that the Y3 receptor subtype could be
involved. At this point, only receptor subtypes Y1 and Y5 were still
candidates for the mediation of the action of NPY on LH secretion.
Three analogs were particularly useful to demonstrate that NPY action
on LH secretion in the castrated rat is predominantly mediated by the
Y5 receptor subtype: PYY336, a mixed Y2 and Y5 agonist;
hPP, a mixed Y4 and Y5 agonist; and
[D-Trp32]NPY, a weak but selective Y5 ligand
(25, 27, 28, 29). Those three analogs were found to inhibit LH secretion
with potency and efficacy similar to those of NPY despite very weak
affinity for the Y1 receptor subtype. Taken together, the data obtained
with PYY336, hPP, and
[D-Trp32]NPY indicated that the Y5, rather
than the Y1, receptor subtype is involved in mediation of the
inhibitory action of NPY on LH secretion in the castrated rat. These
data make it very unlikely that the Y1 receptor subtype is still
involved, although this possibility cannot be completely eliminated at
this point. For such a demonstration, a pure Y1 receptor agonist or a
powerful, selective, and nontoxic Y1 receptor antagonist is still
needed. There are unfortunately no Y1 antagonists that could be used
for such a demonstration. It is known that the nonpeptidic BIBP3226
(35) and the more recently available BIBO3304 (36) are specific Y1
antagonists, but their use in in vivo models is limited due
to their neurotoxicity. In a preliminary experiment performed in our
laboratory, BIBO3304 (10 µg/rat), coinjected centrally (icv) with NPY
(3 µg/rat), was unable to inhibit the inhibitory effect of NPY on LH
secretion (data not shown). When tested at a larger concentration (30
µg/rat), BIBO3304 clearly induced neurotoxic manifestations, thus
preempting the use of this antagonist at meaningful concentrations.
1229U91 (GW1229) is another Y1 antagonist that could have been used in
our model (37). However, this peptidic NPY analog is known to display
high binding affinity in vitro for both Y1 and Y4 receptors
and weak affinity for Y2 and Y5 receptor subtypes (38). It is therefore
very difficult to make any meaningful demonstration of Y1 specificity
by using such a compound.
We synthesized a Y5 antagonist that was described as having an
IC50 of 2.3 nM for the cloned Y5 receptor (30).
In our hands, this nonpeptidic receptor antagonist was found to be
highly specific to displace the 125I-labeled
[Leu31,Pro34]PYY/BIBP3226-insensitive sites
in the rat brain and to display no binding to Y1 receptors of
neuroblastoma SK-N-MC cells, thus confirming its Y5 specificity. This
Y5 receptor antagonist, when administered icv, with no apparent
toxicity problem, fully reversed, in a dose-dependent manner, the
inhibitory action of NPY on LH secretion. This finding strongly
reinforces the concept that the Y5 receptor subtype is mainly, if not
exclusively, involved in the NPY inhibitory action on LH secretion.
The Y5 receptor subtype was originally described as a receptor
subtype mediating the orexigenic effect of NPY (25). Our data with Y5
agonists and a specific Y5 antagonist confirmed that the Y5 receptor
subtype is involved in the stimulation of food intake by exogenous NPY.
The dose-response studies indicated that the Y5 agonist
PYY336 was as potent as NPY, confirming the work of
Gerald et al. (25). Another Y5 agonist, hPP, was found to be
as potent as [Leu31,Pro34]NPY in stimulating
food intake. Finally, the Y5 antagonist that was used in the present
study clearly blocked NPY-induced stimulation of food intake in the
castrated rat. [D-Trp32]NPY, a weak but
specific Y5 ligand (25, 27, 29), that was found to be an agonist in our
LH assay was unable to significantly induce food intake at 7 nmol, the
highest dose used in the present study. In another study, central
administration of [D-Trp32]NPY at a dose of 2
nmol stimulated food intake in rats (25). On the other hand, several
groups have reported an antagonistic action of
[D-Trp32]NPY on NPY-induced foodintake (27, 39).
The comparison of dose-response curves for inhibition of LH secretion
and stimulation of food intake indicates that the rank ordering of
peptide activity for inhibition of LH secretion, NPY =
PYY336 = hPP =
[Leu31,Pro34]NPY >
[D-Trp32]NPY, paralleled the rank ordering of
peptide activity for stimulation of food intake, NPY =
PYY336 > hPP =
[Leu31,Pro34]NPY >>
[D-Trp32]NPY. Together with the antagonist
study, this observation suggests that the same subtype is mediating
both effects of NPY. However, the different sensitivity to
[D-Trp32]NPY we have evidenced also suggests
the possible existence of minor differences between the Y5 subtypes
mediating those two actions, for example, differences in receptor
density or different second messengers. The identity of the major NPY
receptor(s) mediating the stimulation of food intake is still
uncertain, and recent data have indicated that both Y5 and Y1 could be
involved (36, 36, 40, 41, 42, 43), or possibly that mediation is through the Y5
subtype with participation of the Y1 subtype. Wyss et al.
recently demonstrated that correlation between in vivo
ED50 for stimulation of food intake by NPY and in
vitro IC50 for the binding to receptors expressed on
cell lines is strong for the Y5, weak for the Y1, and nonsignificant
for the Y2 and Y4 subtypes (40). Repeated icv injections of Y5
antisense oligodeoxynucleotides prevented both NPY-induced and
fasting-induced food intake in rats (41). NPY Y1 receptor-deficient
mice have normal food intake and exhibit modest obesity and
hyperinsulinemia (43). Finally, Wieland et al. demonstrated
that the Y1 receptor subtype is still involved in the stimulation of
food intake by injecting the Y1 antagonist BIBO3304 (30 µg/rat)
locally at the level of the paraventricular nucleus that in these
conditions could inhibit both the feeding response induced by 1 µg
NPY as well as the hyperphagia induced by a 24-h fast (36). Obviously,
more work is necessary to delineate the Y receptor subtype(s) for the
action of NPY on feeding, but this was not the primary aim of the
current study.
Much work is also still needed to understand the role of NPY in the
modulation of gonadotropin secretion in physiological conditions, its
role as an excitatory signal to LH release (31), and its role to
clarify the involvement of NPY action on that axis in several
pathophysiological situations. The demonstration that null mutation of
the NPY gene produced transgenic mice that were almost normal, with
normal fertility, clearly cast some doubts on the relevance of the
described specific stimulatory and inhibitory actions of NPY, in
particular on reproduction (44, 45). It is quite likely, however, that
the important redundancy of peptides from the PP family and NPY
receptor subtypes made possible a rapid reprogramming of the actions
normally assigned to NPY, and then knockout mice appear normal at birth
and in adulthood. Recently, similar findings were published with gene
deletion of the Y1 receptor subtype (43, 46) and the Y5 receptor
subtype (47) with, as an outcome, fertile mice. One important
limitation of these studies is that NPY or specific NPY receptor are
lost from conception, allowing the development of compensatory systems
that could take the place of the missing NPY receptor. Development of
useful inducible promoters will be an important advance for these
transgene techniques.
The elegant studies by Kalra et al. have indicated that NPY
is involved in the modulation of LH release at the time of the
ovulatory peak, stressing the participation of NPY in the triggering of
a large secretion of LH (31). As important is the observation that NPY
can inhibit LH/FSH secretion, at least when administered exogenously
(21, 34). There are several presumptions that endogenously produced NPY
can also modulate gonadotropin secretion in pathological conditions
(33, 48). Gene expression for NPY and the synthesis and release of this
peptide are enhanced in several pathological situations associated with
hypogonadism, such as malnutrition (49), obesity (50), and diabetes
(51, 52). Increased NPY output in the hypothalamus, such as that seen
in diabetic rats that drives robust food intake (52), could also
fulfill an inhibitory action on LH secretion. The recent demonstration
that leptin administration to ob/ob mice reestablished LH
secretion and fertility with a concomitant decrease in gene expression
for NPY in the hypothalamus at least indicates that such a specific
inhibitory action of NPY on gonadotropin secretion is possible
(53).
In conclusion, our results indicate that the Y5 receptor subtype is
involved in the inhibitory control of the gonadotropic axis by NPY and
confirm other work that have considered this subtype as a mediator of
NPY action on food consumption.
 |
Acknowledgments
|
|---|
We acknowledge the excellent technical assistance of Jean-Pierre
Giliberto and Christiane Rey. The skillful technical assistance of
Jean-Jacques Goy and Ramon Junko in our animal quarters is gratefully
acknowledged. We thank Dr. Graeme Semple, Ferring Research Institute
Ltd. (Chilworth, UK), for synthesis of the Novartis nonpeptidic Y5
receptor antagonist. We thank Drs. H. N. Doods and K. Rudolf from
Karl Thomae GmbH (Germany) for their generous gift of the Y1
antagonist BIBP3226.
 |
Footnotes
|
|---|
1 This work was supported by a grant from the Swiss National Research
Science Foundation (3139729-93) and by Ferring Research Institute
Ltd. 
Received November 2, 1998.
 |
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