Download Potent Neuropeptide Y Y, Receptor Antagonist, 1229U91: Blockade

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Endocrinology
Copyright
0 1996 by The Endocrme
Vol
Prmfed
Potent Neuropeptide
Y Y, Receptor
Blockade
of Neuropeptide
Y-Induced
Food Intake
AKIO KANATANI,
SATOSHI
OZAKI,
Tsukuba
Research
137, No. 8
zn U S.A.
Society
AKANE
AND
Institute,
MASAKI
ISHIHARA,
IHAFtA
Banyu
Pharmaceutical
SHUICHI
ASAHI,
Co., Okubo
ABSTRACT
Neuropeptide
Y (NPY) is thought
to increase
food intake
through
the action of Y, (-like) receptors
in the hypothalamus.
To confirm
the
involvement
of Y, receptors
in feeding behavior,
selective
and potent
antagonists
for Y, receptors
are required.
In the present
study, we
showed that a peptide,
1229U91
[(Ile,Glu,Pro,Dpr,Tyr,Arg,Leu,Arg,
Tyr-NH,),
cyclic (2,4’),(2’,4)-diamide],
is a potent
and selective
antagonist
for Y, receptors.
1229U91
displaced
[?]peptide
YY (PYY)
binding
to membranes
of human
neuroblastoma-derived
SK-N-MC
cells that predominantly
express Y, receptors
with a K, value of 0.10
nM and inhibited
the NPY-induced
increase
in intracellular
calcium
levels (IC,, = 0.27 nM). In contrast,
the I(, values for [‘2511PYY binding
to Y, receptors
in membranes
of human
neuroblastoma-derived
SKN-BE2 cells and rat hypothalamus
were 700 nM and more than 1 PM,
Antagonist,
1229U91:
and Physiological
TAKESHI
3, Tsukuba
30033,
TANAKA,
Japan
respectively.
Although
[ ““I]PYY
could not detect Y, receptors
in the
rat hypothalamic
membranes,
[ ‘251]1229U91
revealed
binding
sites
with a high affinity
(&
= 18 PM), indicating
the presence
of Y,
receptors
in the hypothalamus.
Intracerebroventricular
injection
of
1229U91(30
pg) into male Sprague-Dawley
rats completely
inhibited
NPY (5 pg)-induced
food intake without
any other behavioral
change.
Furthermore,
intracerebroventricular
injection
of 1229U91
significantly suppressed
physiological
feeding behavior
after overnight
fasting. These results indicate
that Y, receptors
in the rat hypothalamus
mediate
NPY-induced
food intake,
and that physiological
feeding
behavior
after overnight
fasting may be largely
regulated
by NPY via
Y, receptors.
1229U91
may be useful
for further
elucidating
the
pathophysiological
roles of NPY in feeding behavior.
(Endocrinology
137: 3177-3182,
1996)
N
EUROPEPTIDE
Y (NPY) is a 36-amino
acid polypeptide in the pancreatic
polypeptide
family, which consists of NPY, peptide YY (PYY), and pancreatic
polypeptide
(1, 2). NPY is highly concentrated
within the hypothalamus
(3,4), and induces potent stimulation
of feeding behavior via
NPY receptors in the hypothalamus
of rats as well as other
species (5-9). Chronic
administration
of NPY to the brain
results in hyperphagia
and body weight gain, reduces energy
expenditure,
and increases lipogenic
activity in the liver and
adipose tissue (10, 11). It has also been reported
that concentrations
of NPY and its messenger RNA in the hypothalamus are markedly
increased during food deprivation
and
in some forms of genetic obesity accompanying
diabetes
(12-14). In addition,
antibodies
and antisense oligonucleotides of NPY reduce food intake significantly
(38,39). These
facts indicate that NPY may be one of the major regulators
of physiological
feeding behavior.
NPY activates two major types of NPY receptors, called Y,
and Y, receptors. These receptors are identified
on the basis
of their affinities to various NPY analogs (15-21). Although
intracerebroventricular
(icv) injection of the Y,-selective
agonist NPY-(13-36)
does not stimulate
food intake (22), icv
injection
of the Y,-selective
agonist [Leu31,Pro34]Nl?Y
induces food intake with a potency similar to that of NPY,
indicating
the involvement
of Y, receptors in NPY-induced
feeding behavior (6,22). NPY-(2-36)
is less potent than intact
NPY for most functions of Y, receptors (l&19, 23), whereas
it is more effective than NPY for stimulating
feeding behavior (6,22,40). Furthermore,
repeated injection of the antisense
oligonucleotides
of Y, receptors into the cerebroventricle
of
rats produces
behavioral
signs of anxiety without
anorexigenie activity (24). These data suggest that Y,-like receptors,
distinguishable
from Y, receptors,
may exist in the hypothalamus
to regulate food intake. However,
because of the
lack of selectivity and potency of the NPY antagonists
currently available,
it is unclear whether
Y, receptors are involved in regulating
food intake (25-28).
Recently, Daniels et al. (29, 30) reported
that 1229U91
[(Ile,Glu,Pro,Dpr,Tyr,Arg,Leu,Arg,Tyr-NH,),
cyclic (2,4’),
(2’,4)-diamide]
is a potent peptide NPY antagonist.
In their
report, 1229U91 had high affinities for Y, and Y, receptors
and inhibited
the NPY-induced
increase in perfusion
pressure in the isolated rat kidney (30). In contrast to the previous
report, we showed
in the present study that 1229U91 is a
potent and selective antagonist
for Y, receptors. Using this
potent Y,-selective
antagonist,
we investigated
the role of Y,
receptors in feeding behavior.
Materials
and Methods
Reagents
Human
NPY, PYY, [Leui’,Pro”‘]NPY,
NPY-(2-36)
and NPY-(13-36)
were synthesized
in our laboratories
using an automated
Applied
Biosystems model 431A peptide synthesizer
(Foster City, CA). 1229U91 was
synthesized
as described
previously
(29). [“‘I]PYY
was purchased
from
New England Nuclear-DuPont
(Boston, MA). 1229U91 was radiolabeled
by the Bolton-Hunter
method (31) with some modifications
and purified
by reverse
phase HPLC;
the reaction
mixture
was applied
to a C,,
column
(4.6 X 150 mm) equilibrated
with 28% acetonitrile
containing
Received
December
11, 1995.
Address
all correspondence
and requests
for reprints
to: Dr. Akio
Kanatani,
Tsukuba
Research
Institute,
Banyu
Pharmaceutical
Co.,
Okubo
3, Tsukuba
300-33,
Japan.
3177
3178
INVOLVEMENT
OF Y, RECEPTORS
0.1% trifluoroacetic
acid and eluted with an isocratic
solvent system at
a flow rate of 0.5 ml/min.
The elution of peptides
was monitored
by
measuring
absorbance
at 220 nm and determining
radioactivity.
The
culture reagents and BSA were obtained
from Life Technologies
(Grand
Island, NY). Bacitracin,
phenylmethylsulfonylfluoride,
and polyethylenimine
were obtained
from Sigma Chemical
Co. (St. Louis, MO). All
other chemicals
were of analytical
grades.
Human
neuroblastoma-derived
SK-N-MC
cells were obtained
from
the American
Type Culture
Collection
(Rockville,
MD). SK-N-BE2
cells
derived
from human neuroblastoma
were kindly
provided
by Dr. Robert A. Ross (Department
of Biological
Sciences, Fordham
University,
New York, NY). SK-N-MC
and SK-N-BE2
cells, planted
in a 175~cm*
culture flask, were grown in DMEM
supplemented
with 10% FBS, penicillin G (100 III/ml),
and streptomycin
(100 wg/ml)
in a 95% air-5% CO,
humidified
atmosphere
at 37 C.
experiments
The cells and tissues were washed with 50 mM HEPES buffer (pH 7.4)
containing
20% sucrose, then homogenized
and centrifuged
at 1,000 X
g for 15 min. The supernatant
was centrifuged
at 100,000 X g for 45 min.
The pellets were resuspended
in 5 mM HEPES buffer
(pH 7.4) and
centrifuged
again. The membrane
fraction
was resuspended
by a homogenizer
in the same buffer and used for this study.
Binding
of [‘*“1]1229U91
and [“s I]PYY to membrane
preparations
was performed
in 0.2 ml 25 rnM Tris buffer (pH 7.4) containing
10 mM
MgCI,,
1 mM phenylmethylsulfonylfluoride,
0.1% bacitracin,
and 0.5%
BSA. The membranes
(100-300
pg/ ml) were incubated
at 25 C for 60 and
120 min with [‘251]1229U91
(30 PM) and [ ‘*“I]PYY
(25 PM), respectively.
Bound and free peptides were separated
by filtration
using a GF / C glass
filter (Whatman,
Maidstone,
UK) presoaked
with 0.3% polyethylenimine. Specific binding
of [ ‘ZsI]1229U91
and [‘251]PYY was defined as the
difference
between total binding and nonspecific
binding
in the presence
of I PM 1229U91 and PYY, respectively.
Measurement
([Ca’ ‘13
of intracellular
calcium
ion concentrations
[Ca”],
was measured
fluorometrically
using a Ca”-sensitive
fluorescent dye, fura-2. SK-N-MC
cells were harvested
using 0.25% trypsin
and 0.02% EDTA. The cells (1.0 X lo7 cells) were washed
once with
DMEM
containing
20 mM HEPES and 0.3% BSA (pH 7.4; DMEMI
HEPES/ BSA), suspended
in I ml DMEM
/ HEPESI BSA, and incubated
with 2 FM furaacetoxymethylester
at 37 C for 30 min. The suspensions
were diluted with a IO-fold volume
of DMEM/HEPES/BSA
and again
incubated
at 37 C for 20 min. The fura-2-loaded
cells were washed with
DMEM/HEI’ES/BSA
and resuspended
in an equal volume
of KrebsHenseleit-HEPES
buffer containing
0.1% BSA (pH 7.4). In a cuvette, 0.5
ml of the resultant
suspension
was stirred continuously
at 37 C during
the measurement.
Test compound
or vehicle was added 5 min before the
addition
of 10 nM NPY, and fluorescent
intensity
at an emission
wavelength of 500 nm and excitation
wavelengths
of 340 and 380 nm was
monitored
with a CAF-110
intracellular
ion analyzer
(JASCO, Tokyo,
Japan). [Ca’+], values were calculated
according
to the previously
reported method
with some modifications
(32).
protocols
Adult male Sprague-Dawley
rats (7 weeks old, 280-350
g; Charles
River Japan, Yokohama,
Japan) were maintained
in individual
cages
under controlled
conditions
of temperature
(23 2 2 C) and light-dark
cycle (lights on from 0700-1900
h). Water and pellet food (CE-2, CLEA
Japan, Tokyo, Japan) were available
ad libitum. Rats were anesthetized
with sodium
pentobarbital
(50 mg/ kg, ip; Dainabot,
Tokyo, Japan). A
permanent
21-gauge
stainless
steel cannula
was stereotaxicaily
implanted into the right lateral ventricle.
The stereotaxic
coordinates
used
were as follows:
0.9 mm posterior
to the bregma,
1.2 mm bilateral to the
midsagittal
sinus, and 1.5 mm ventral
to the brain surface. After 1 week
of recovery,
rats were used in experiments
while fully satiated and fasted
overnight.
Groups of 10 animals received icv injections
of NPY, 1229U91,
a mixture
of these two compounds,
or vehicle (10 mM PBS containing
10 ~10.05% BSA), and their food intake was monitored.
The experiments
were performed
between
0900-1130
h. Results are given as the mean 5
SE. Statistical
significance
of the differences
between
groups was calculated using ANOVA
followed
by Bonferroni’s
test.
Results
on human YI and Yz receptors
cell lines
Effects of 1229U91
neuroblastoma-derived
Fw. 1. Inhibition
of J’“sI’PYY
specific
binding
to human
neuroblastoma-derived cell membranes
by NPY, NPY analogs, and 1229U91.
The membranes
were incubated
with 25 PM [‘asI]PYY
for
2 h at 25 C. Data are expressed
as a
percentage
of total
specific
binding.
Each point represents
the mean 2 SE of
three
independent
experiments
performed
in duplicate.
A, SK-N-MC
cell
membranes
as human
Y, receptors.
B,
SK-N-BE2
cell membranes
as human
Y, receptors.
5
0
120
in
of human
The specific binding
of [ ‘251]PYY to membranes
SK-N-MC
cells was inhibited
by NPY and related peptides
in the following
studies. The rank order affinities [NPY =
[Leu3’,Pro34]NPY
> NPY-(2-36)
> NPY-(13-36)]
revealed
the presence of Y, receptors
in SK-N-MC
cells (Fig. 1A).
[‘251]PYY specific binding
to Y, receptors in the membranes
was also inhibited
by 1229U91 with a high affinity (Ki =
0.10 nM).
In human
SK-N-BE2
cell membranes,
the rank
order affinities
[NPY = NPY-(2-36)
> NPY-(13-36)
>>
[Leu3’,I+034]NPY]
indicated
the presence of Y, receptors.
1229U91 displaced
[‘251]PYY specific binding
in SK-N-BE2
cell membranes
with a low potency (K, = 700 nM; Fig. 1B).
Therefore,
the affinity of 1229U91 was 7000 times higher to
Y, receptors than to Y, receptors in human neuroblastomaderived cell membranes
(Table 1).
It has been reported that Y, receptors in human SK-N-MC
8)
A)
a
b
Endo . 1996
Vol 137 . No 8
BEHAVIOR
In vivo experimental
Cell culture
Binding
IN FEEDING
120
1
100
loo
e
60
60
F
p
3
60
60
40
40
20
20
-a-o-#-
1
NPY
[Lau31,Pro34]NPY
NPY(Z-36)
‘i;
.o
%
B
ul
E
B
r
c-4
c
0
74
-14
Log[Ligand,
M]
:
O-14
-12
-10
Log[Ligand,
-8
M]
-6
-4
INVOLVEMENT
OF Y, RECEPTORS
in rat hypothalamic
membranes
In rat hypothalamic
membranes,
the specific binding
of
[‘251]PYY was displaced by NPY and the Y, agonist NPY-(1336), but not by the Y,-selective ligands [Leu3’,l’ro34]NI’Y
and
1229U91 (Fig. 3). This indicated
that [‘251]PYY bound to Y,
receptors preferentially
and failed to detect Y, receptors in the
rat hypothalamic
membranes. To detect Y, receptors in the rat
hypothalamic
region, we synthesized
[‘251]1229U91 iodinated
with [‘251]Bolton-Hunter
reagent. The saturation binding analyses revealed that [‘251]1229U91 bound to the membranes with
a high affinity (Ka = 18 PM) and showed a single class of binding
sites (Fig. 4). The receptor subtype recognized
by [‘251]1229U91
was the typical Y, receptor deduced according to the rank order
affinities: 1229U91 > NPY = [Leu31,Pro34]NPY
> NPY-(2-36)
> NPY-(13-36)
(Fig. 5).
Effects of 1229U91
on feeding
Discussion
NPY is thought to stimulate
food intake, probably
via Y,
receptors. However,
because of the lack of potent and selective antagonists
for Y, receptors, it has not been clarified
whether
Y, receptors regulate physiological
feeding behavior. Recently,
nonpeptide
Y, antagonists,
BIBP3226
and
SR120819A, have been reported
with K, values of 7.2 and 21
nM for Y, receptors, respectively
(41, 42). These antagonists
are highly selective for Y, receptors because their K, values
for Y, receptors
are more than 1 and more than 10 /AM,
respectively.
These compounds
are effective in viva, as they
inhibit the NPY-induced
increase in blood pressure. However, there are no reports that these Y,-selective
antagonists
show inhibitory
effects on feeding behavior. In the present
study, we showed that 1229U91 is to date the most potent and
selective antagonist
for Y, receptors, with a Ki value of 0.10
behavior
NPY injected into the cerebroventricle
induced
rapid and
dose-dependent
feeding behavior in rats (Fig. 6A). A significant difference between rats given saline vehicle and those
given NPY was observed at a dose of 5 pg; consequently,
we
attempted
to determine
the inhibitory
effect of 1229U91
against this dose of NPY given intracerebroventricularly.
1229U91 (5 and 30 pg alone) did not change the cumulative
TABLE
1. K, (nM)
Membrane
values
source:
for NPY,
its analogs,
Human
Radioligand:
NPY
[Leu31,Pro34]NPY
NPY-(2-36)
NPY-(13-36)
1229u91
and
1229U91
for radioligand
SK-N-MC
3179
BEHAVIOR
food intake compared
with the respective vehicles, indicating that it had no effect on food intake (Fig. 6, B and C, stippled
bay). When 1229U91 (5 and 30 pg) was coadministered
with
NPY (5 pg) icv, it dose dependently
inhibited
NPY-induced
food consumption
(Fig. 6, B and C, solid bay). The highest dose
of 1229U91(30
pg, icv) completely
inhibited
exogenous NPYinduced feeding behavior, whereas we did not observe any
remarkable
changes in other behaviors, including
sedation or
barrel-rolling,
at any of the doses tested.
In rats fasted overnight,
a bolus icv injection of 1229U91,
5 min before food presentation,
significantly
attenuated feeding behavior
(Fig. 7). 1229U91 dose-dependently
inhibited
spontaneous
food intake after both the 5- and 30-pg doses.
The mean cumulative
food consumption
with both doses of
1229U91 was significantly
lower than that observed with the
saline vehicle for 4 h after access to food.
cells are coupled with second messenger
systems, such as
stimulation
of [Ca2+li increase and inhibition
of CAMP accumulation
(20, 33). As shown in Fig. 2A, the NPY-induced
increase in [Ca2+li in SK-N-MC
cells was more potent than
that induced by N-terminal-truncated
peptides of NPY. Although 1229U91 did not induce an increase in [Ca2+li even
at 1 FM, 1229U91 inhibited
the NPY (10 nM)-induced
[Ca2+li
increase dose dependently,
with an IC,, of 0.27 nM (Fig. 2B).
NPY receptors
IN FEEDING
Human
[‘2”I]PYY
binding
to membrane
Rat
SK-N-BE2
[‘z511PYY
1.0
1.6
5.5
19
0.10
preparations
hypothalamus
[‘z”IJPYY
0.75
r’~“111229u91
0.30
740
0.60
1.3
>lOOO
>lOOO
2.1
4.2
700
24
21
100
460
0.18
4
120-O-+-
NPY
NPY(Z-36)
FIG. 2. A, Agonistic
effects
of NPY,
NPY analogs,
and 1229U91
on mobilization of [CactIi.
B, 1229U91
antagonism of the 10 nM NPY-induced
[Ca”l,
increase
in SK-N-MC
cells. Different
concentrations
of 1229U91
were added
5 min before the addition
of 10 nM NPY
at 37 C. The results are expressed
as a
percentage
of the maximal
effects of 1
PM or 10 nM effects. Each point shows
the mean t SE from three independent
experiments
performed
in duplicate.
-10
-a
Log[Ligand,
-6
M]
-4
,
-2
02,
0 -14
-12
-10
Log[1229U91,
-a
M]
-6
,
-4
INVOLVEMENT
3180
OF Y, RECEPTORS
nM and about 7000-fold greater selectivity
for Y, receptors
than for Y, receptors. As a consequence,
it may be a useful
tool for investigating
the role of Y, receptors
in feeding
behavior.
We characterized
NPY receptors in the rat hypothalamus
that regulate
food intake. [‘251]PYY binding
as well as
[3H]NPY
binding
(data not shown)
to the hypothalamus
membranes
showed a low affinity for the Y,-selective agonist
[Leu3’,Pro34]NPY,
indicating
that Y, receptors are predominant in this region. This result is consistent with other reports (34-37). However, we did not detect Y, receptors in the
hypothalamus
in these experiments,
although
it has been
suggested
that NPY-induced
food intake is mediated
via
Y,-type receptors (6, 22). To detect Y, receptors, we synthesized
an iodinated
Y,-selective
ligand,
[‘251]1229U91.
[““1]1229IJ91
bound with a high affinity to a single class of
binding
sites in rat hypothalamic
membranes.
The binding
sites of [‘251]1229U91
showed
a high
affinity
for
[Leu3* Pro3”]NPY, but a low affinity for NPY-(13-36).
These
data indicate clearly that Y, receptors with high affinity for
[Leu31,Pro34]NPY
and 1229U91 are present in the rat hypothalamus. There were no differences in Y, receptors between
-.-++
-Cl+
IN FEEDING
Endo . 1996
Vol 137 . No 8
BEHAVIOR
human SK-N-MC
cells and rat hypothalamus
with respect to
rank order affinities
of NPY and its analogs, including
NPY-(2-36),
in the saturated
binding
experiments
with
[‘251]1229U91.
Consequently,
the NPY receptors recognized
by 1229U91
in the rat hypothalamus
were typical
Y,
receptors.
To investigate
the involvement
of rat hypothalamic
Y,
receptors
in feeding
behavior,
we studied
the effect of
1229U91 on NPY-induced
food intake. Concomitant
administration of 1229U91 with NPY completely
inhibited
the NPYinduced
response in a dose-dependent
manner. In several
previous
reports (25-28), NPY antagonists
failed to inhibit
NPY-induced
food intake because of insufficient
potency, so
it is remarkable
that the Y,-selective
antagonist
can completely inhibit the anorexigenic
activity of icv injected NPY.
These results suggest that Y, receptors in the rat hypothalamus must participate
in feeding behavior. However, it is not
clear whether other Y,-like receptor subtypes are present and
influence feeding behavior; therefore, the cloning and characterization
of all NPY receptors in the hypothalamus
will be
an important
step in this direction.
In contrast to the present finding,
Daniels et al. (30) dem-
NPY
[Leu3’,Pro34]NPY
NPV(2-36)
NPY(13-36)
1229u91
n
+
-O-
NPY
[~eu3l,~ro3~lNpY
-&
NPYi13-36)
60-
-i4
Log[Ligand,
M]
-io
Log[Ligand,
FIG. 3. Inhibition
of [ i2”IlPYY
specific binding
to rat hypothalamic
membranes
by NPY, NPY analogs,
and 1229U91.
The membranes
were incubated
with 25 pM [‘““IIPW
for 2 h at 25 C. Data are expressed as a percentage
of total specific binding
and represent
the
mean of two independent
experiments
performed
in duplicate.
F
5
FIG. 4. A,
Saturation
curve
of
1’“5111229U91
specific binding to rat hypothalamic
membranes.
B, Scatchard
plot of the same data. The membranes
were incubated
with [‘25111229U91
for
1 h at 25 C. Each point represents
duplicate
determinations
from two independent
experiments.
-i2
-s
M]
FIG. 5. Inhibition
of [iz51]1229U91
specific binding
to rat hypothalamic membranes
by NPY, NPY analogs,
and 1229U91.
The membranes were incubated
with 30 pM [‘25111229U91
for 1 h at 25 C. Data
are expressed
as a percentage
of total specific binding
and represent
the mean of two independent
experiments
performed
in duplicate.
150
P
Log[Ligand,
M]
Bound
(tmol/mg
protein)
FIG. 6. A, Dose dependency of NPY-induced food intake. B and C, Effects of
two doses of 1229U91 on the response to
5 pg NPY. *, P < 0.05 compared with
vehicle (no drug) control. The graphs
show the cumulative food intake in
SpraguedDawley rats for 2 h after icv
injection of drug. Data are reported as
the mean i: SE. #, P < 0.05 compared
with rats injected with NPY alone. n =
9-11 rats/group.
0.0
vehicle
w9
NPY
SF9
a:
vehicle
15
s
-
m
: NPY alone
-I)+
+
vehicle
1229u91
1229u91
F‘:::
: 1229U9l
alone
NPY 5pQ
+ 1229u913&lg
m
: mlxfure
NPY
5w
NPY 5c(g
+ 1229119151.lg
of NPY and 1229U91
-40
3 kg
30 pg
P
0
1
7. Effects of 1229U91 on feeding
behavior in overnight-fasted rats. The
lines show the cumulative food intake in
Sprague-Dawley rats for 8 h after icv
injection of drug (left panel). Total
amounts of food intake during 24 and
24-48 h are illustrated by the points
(right panel).
Data are reported as the
mean +. SE. *, P < 0.05 compared with
vehicle (no drug) control. n = 4-8
rats/group,
FIG.
I3
i
f
-20
- 10
2b
Time (hr)
that 1229U91 was the nonselective antagonist for
Y, and Y, receptors. In particular, there was a large discrepancy between their data and our results regarding the affinities of 1229U91 for Y, receptors. In the previous report,
1229U91showed both a high affinity for Y2 receptors, with
an I& of 0.021 nM in rat brain membranes, and a high
affinity for Y1 receptors, with an I&-, of 0.20 nM in SK-N-MC
cells (30). However, we speculate that NPY receptor subtypes
in rat whole brain membranes might be mainly Y, receptors,
becausethese researchers reported that the Y,-selective agonist [Leu31,Pro34]NPY inhibited [3H]NPY binding to the
membranes with a high affinity (ICsO= 2.6 nM) (30). In this
and other reports, [Leu31,Pro34]NPYshowed a low affinity to
inhibit [‘251]PYY binding to Y, receptors (I&, = >lOO nM)
(16, 20). In addition, it is well known that rat frontal cortex
is rich in Y, receptors, whereas other sitesin the brain are rich
in Y, receptors (34-37). Therefore, 1229U91is a Y,-selective
antagonist.
In conclusion, 1229U91is a potent and selective Y, receptor
antagonist. Because 1229U91 inhibits both NPY-induced
food intake and the natural appetite induced by overnight
onstrated
-30
Time
24-48
(hr)
fasting in rats, NPY must regulate physiological feeding behavior at least in part via Y, receptors in the rat hypothalamus. It is well known that NPY inhibits energy expenditure
in brown adipose tissue and enhances energy deposition in
white adipose tissue (10, 11). Therefore, Y, antagonists are
expected to reduce body fat in addition to their effects on
food intake. It will be interesting to study the effects of
1229U91 on feeding behavior in obese and diabetic animal
models as a prelude to developing antiobesity drugs.
Acknowledgments
The authors are grateful for excellent technical support from Mr. Y,
Tsuchiya, Mr. M. Itoh, and Mr. T. Numazawa. We also express our
thanks to Ms. D. LeBlanc (Merck Co., Rahway, NJ) for her critical reading
of the manuscript.
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