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Division of Endocrinology and Metabolic Medicine, Department of Medicine, Hammersmith Hospital, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, United Kingdom
Address all correspondence and requests for reprints to: Professor S. R. Bloom, Division of Endocrinology and Metabolic Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom. E-mail: sbloom{at}rpms.ac.uk
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Introduction |
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Neuropeptide Y (NPY) is the most potent physiological stimulant of feeding yet described. Intracerebroventricular (icv) or intrahypothalamic administration of NPY induces a powerful and prolonged drive to feed in most species tested, and repeated administration in the rat leads to obesity (5, 6). Increased expression and release of hypothalamic NPY occurs in animal models of diabetes and in the genetically obese Zucker rat and obøb mouse (7, 8, 9, 10). Both icv and intraparaventricular nuclear (PVN) administration of antibody to NPY cause a dose-dependent reduction in the feeding that follows a fast (11, 12). However, NPY also plays a crucial role in the control of many other physiological systems. Within the hypothalamus, it is clearly implicated in the regulation of growth (13), sexual function (13), and the stress response (14). It is not known if there is an NPY receptor that affects feeding alone, but, given the complex nature of NPY’s actions in the hypothalamus, the identification of such a receptor would have important implications for the therapeutic management of obesity.
NPY, peptide YY (PYY), and pancreatic polypeptide (PP) form a family of peptides with similar structures. There are currently five recognized receptor subtypes for these peptides (Y1-Y5) (15, 16, 17, 18, 19), but it is not proven if any of these mediate the feeding effect of NPY. The different subtypes of receptor for this family have been identified and characterized by their ability to bind NPY, PYY, and PP fragments and analogues (20, 21). The Y1 receptor binds with high affinity only full length NPY or full length analogues such as [Pro34]NPY and has much reduced affinity for C-terminal fragments such as NPY(13–36). This receptor has been cloned from human (15) and rat CNS (22) and is the sole NPY receptor expressed by the human neuroblastoma cell line SK-N-MC(15). It is also the predominant NPY receptor in rat cortex (23). The Y2 receptor, in contrast to Y1, has a much higher affinity for C-terminal fragments than for substituted analogues such as [Pro34]-NPY. This receptor has been cloned from human hippocampus (24), and from the human neuroblastoma cell line SMS-KAN(16). As in the human, this receptor is the predominant NPY subtype receptor in rat hippocampus (23). The human neuroblastoma cell lines are de facto standards for the characterization of ligands for Y1 and Y2 receptors.
A group of receptors with disparate pharmacological profiles have been labeled Y3 (25, 26, 27). These receptors all share a common low affinity for PYY, which is still capable of stimulating feeding (25). The Y4 receptor (17, 28) is characterized by its high affinity for rat PP, which does not stimulate feeding (29). Hence neither Y3 nor Y4 are directly involved in control of food intake. The role of NPY in the stimulation of feeding has been thought to be mediated by a Y1 receptor, with [Pro34]NPY reported to give a robust feeding response (11). The ability of the C-terminal fragment NPY(2–36) or NPY (3–36) to fully stimulate feeding at low concentrations, despite a much reduced Y1 affinity, opposes a crucial role for Y1 (30). The possibility that additional Y2 receptor activation is essential for the full feeding response to NPY has not been fully studied.
Recently, two new NPY receptors have been cloned, which have both been labeled Y5. Both of these receptors are expressed in the hypothalamus, one from rat (19) and one from mouse (31). These receptors share only approximately 30% amino acid homology and so can be considered entirely different receptors. In this paper, we shall refer to the rat receptor as Y5Nat and the mouse receptor as Y5JBC after the journals in which they were reported. The Y5Nat but not the Y5JBC receptor has been proposed as the receptor subtype mediating the feeding effect of NPY (19, 31). BIBP-3226 was developed as an NPY Y1 receptor antagonist, which is known not to have any effect at the Y2, Y4, and Y5Nat receptors (19, 32, 33). We have therefore also studied the effect of BIBP-3226 on NPY induced feeding. Here we report that a receptor distinct from any of those currently cloned is likely to mediate NPY induced feeding.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Norepinepherine bitartrate was purchased from Sigma (Poole, Dorset, UK). BIBP-3226 was supplied by Thomae GmbH (Biberach, Germany).
Cell culture
SK-N-MC and SMS-KAN cells were kindly donated by Dr. S. Legon
(Royal Postgraduate Medical School, London, UK) and Professor T.
Schwartz (Rigshospitalet, Copenhagen, Denmark) respectively. All cell
culture resources were supplied by GIBCO BRL (Life Technologies Ltd.,
Paisley, UK). Both cell lines were routinely maintained in 50%
modified Eagle’s medium/50% HAMS F12, supplemented with 10% FCS, 2
mM glutamate, 1 x nonessential amino acids, 100
µg/ml streptomycin, and 100 U/ml penicillin. Medium was changed every
48 h, and cells passaged when they reached 70% confluence
(approximately 7 days).
Peptide iodination
Porcine PYY was iodinated using the iodogen method. Peptide (5
nmol) was dissolved in 10 µl 0.2 M phosphate buffer (pH
7.2) and added to 10 µg
1,3,4,6-tetrachloro-3
,6-diphenylglycoluril (iodogen, Pierce,
Rockford, IL), plus 37 MBq Na125I (Amersham International,
Amersham, Buckinghamshire, UK). This was incubated on ice for 5 min and
products separated by reversed phase C18 HPLC (Waters
Novapak column, Millipore, Milford, MA), developed with a 15–45%
acetonitrile/water/0.05% trifluoroacetic acid gradient. Fractions (1.5
ml) were collected, and radioactive peaks assayed for receptor binding
activity. Active fractions were aliquoted, freeze-dried, and stored at
-20 C. The specific activity of the radioligand was 27 Bq/fmol.
Membrane preparation
Cell membranes were prepared by osmotic lysis and differential
centrifugation. Cells grown to confluence in 175 cm2 flasks
were scraped into PBS and centrifuged at 700 x g for 5
min at 4 C. The resulting pellet was suspended in 2 ml PBS. This was
added dropwise to 100 mls of stirring 1 mM HEPES buffer (pH
7.4) containing protease inhibitors [benzamidine (100 µg/ml),
bacitracin (100 µg/ml), aprotinin (30 µg/ml), soya bean trypsin
inhibitor (10 µg/ml), pepstatin (0.5 µg/ml), leupeptin (0.5
µg/ml), and antipain (0.5 µg/ml)] at 4 C. After 5-min stirring,
the cell suspension was centrifuged at 4 C for 15 min at 3000 x
g. The supernatant was then discarded and the pellet
disrupted in the same buffer containing 50 mM HEPES (pH
7.4) using an Ultra Turrax homogenizer (IKA Labortechnik, Staufen,
Germany). This was then centrifuged again at 4 C for 15 min at
3,000 x g. The supernatant was then centrifuged at 4 C
for 60 min at 48,000 x g.
For hippocampal and cortical membranes, animals were killed by CO2 asphyxiation, and brain regions rapidly dissected, frozen in liquid nitrogen and stored at -80 C until used. Membranes were prepared by homogenisation and differential centrifugation. The tissues were homogenized in ice cold 50 mM HEPES (pH 7.4) buffer containing 250 mM sucrose and protease inhibitors as above, using an Ultra-Turrax homogenizer. The homogenate was centrifuged for 20 min at 1500 x g and the supernatant then centrifuged for 1 h at 100,000 x g and 4 C. The pellet was resuspended in the same buffer without sucrose, and centrifuged for 1 h at 100,000 x g and 4 C.
The final pellets were resuspended in 50 mM HEPES buffer with protease inhibitors to a final protein concentration of 2–10 mg/ml. The membranes were then aliquoted and stored at -80 C.
Receptor binding
Membranes (100 µg protein) were incubated with 40
pM (500 Bq) 125I-PYY in the presence or absence
of unlabelled peptides as indicated. Binding was carried out in a final
volume of 500 µl assay buffer (20 mM HEPES pH7.4, 5
mM CaCl2, 1 mM MgCl2,
1% (wt/vol) BSA) for 90 min at 30 C. Bound and free label were then
separated at 4 C by centrifugation for 2 min at 15,000 x
g. The pellet was washed in 1 ml assay buffer and
recentrifuged. Bound 125I-PYY was then quantified in a
-counter. Total specific binding was defined as the difference in
counts between assays in the presence (nonspecific) and absence (total)
of 200 nM NPY. Analysis of equilibrium competition data was
carried out using ReceptorFit programs (Lundon Software, Inc.,
Cleveland, OH) to give KD values for each ligand. Membranes
prepared from rat brain tissues were found to contain more than one
binding site for 125I-PYY. In this situation, two-site and
one-site curves were calculated for each set of data and compared by
F-test. Two site curves were considered a significantly better fit when
P < 0.05. Sites with high affinity for
[Pro34]-NPY were taken to be Y1 receptors, and those with
low affinity for this peptide, Y2.
To ensure that BIBP-3226 was not acting via the galanin receptor, we also investigated the ability of BIBP-3226 to compete for the membrane binding of 125I-Galanin as previously described (34).
Adenylyl cyclase studies
Adenylyl cyclase activity was studied in SK-N-MC cell lysates,
prepared according to Gordon et al. (35). Confluent cultures
of SK-N-MC cells grown in 75-cm2 flasks were washed with
ice-cold PBS and then with ice-cold 1 mM Tris-HCl (pH 7.4)
containing 2 mM EDTA, pepstatin (0.5 µg/ml), leupeptin
(0.5 µg/ml), and antipain (0.5 µg/ml). The cells were allowed to
lyse in 3 ml of the same buffer for 15 min at 4 C and then disrupted in
a glass Teflon homogenizer. Aliquots (20 µl) of the lysate were
incubated for 10 min at 25 C in a total volume of 100 µl reaction
mixture containing 25 mM Tris/HCl (pH 7.4), 2
mM MgCl2, 1 mM EDTA, 100
µM GTP, 100 µM ATP, 1 mM ATP,
20 mM creatine phosphate, 2 mM isobutyl
methylxanthine, 20 µg creatine kinase, 20 µg myokinase, 1 µCi [
-32P] ATP and 33 nCl 3H cAMP and, where
indicated, various experimental agents. The reaction was stopped by the
addition of 10 µl 62.5% trichloroacetic acid, and insoluble matter
separated by centrifugation (15000 x g, 3 min). The
32P-cAMP was measured by the method of Salomon et
al. (36). To measure the inhibition of adenylyl cyclase, lysates
were incubated with 10 µM isoproterenol and 1
µM NPY or analogue. This concentration of isoproterenol
was shown in preliminary experiments to give a robust stimulation of
adenylyl cyclase activity, without being maximal. Results are given as
percentage of isoproterenol stimulated adenylyl cyclase activity in the
absence of exogenous inhibitor.
In vivo feeding studies
Adult male Wistar rats (250–300 g) were maintained in
individual cages under controlled temperature (21–23 C) and light
(11-h light, 13-h dark), with ad libitum access to food (RM1
diet, SDS United Kingdom, Ltd.) and water. Rats were anaesthetized by
ip injection of a mixture of Ketalar (ketamine HCl 60 mg/kg,
Parke-Davis, Pontypool, UK) and Rompun (xylazine 12 mg/kg, Bayer UK,
Ltd., Bury St. Edmunds, UK). Permanent 22-gauge stainless steel
cannulae were implanted 0.8 mm posterior to bregma on the midline and
6.5 mm below the outer surface of the skull using a Kopf stereotactic
frame with the incisor bar set at 3 mm below the interaural line. After
surgery, a wire plug was inserted into each cannula to prevent
blockage. All animals were allowed a period of 7 days to recover before
being used in the study. During this period, an icv injection of
angiotensin II (150 ng/animal) was given and animals not demonstrating
a prompt and sustained drinking response were excluded. The animals
were handled daily for 5 days before the study to minimize nonspecific
stress. The placement of the cannulae was verified at the end of the
study by the injection of 10 µl ink, removal of the brain, and
examination of coronal brain slices. Substances were administered by a
stainless steel injector, projecting 0.5 mm below the tip of the
cannulae. The injector was connected by polyethylene tubing (id, 0.5
mm; od, 1 mm) to a Hamilton syringe (Reno, NV) in a syringe pump set to
dispense 10 µl solution/min. All compounds were dissolved in 0.9%
saline and each study involved an injection of 10 µl of peptide or
saline. Immediately after the icv injections, rats were placed into
their home cage with a preweighed amount of chow and free access to
water. After a 2-h period, the remaining food was reweighed. The food
intake for each rat was calculated as the stimulated intake in grams
over saline infused controls. All treatments were given at least
48 h apart, between 0800 h and 1100 h, and experiments
were of cross-over design unless otherwise stated.
Study 1. Five separate groups of rats (n = 14 to 18 per group) were studied simultaneously. Each group was studied at one dose for NPY, NPY(2–36), NPY(3–36), NPY(13–36), [Pro34]NPY, and [Pro34]NPY(3–36). The doses used were 0.24, 0.72, 2.4, 7.2, and 24 nmol. In a group, each rat received all six peptides at the same dose. A dose of 50 nmol was also used for NPY(13–36).
Study 2. To investigate the possibility that Y2, in addition
to Y1 activation, is needed for the full feeding effect of NPY, we
coadministered equivalent doses (based on the binding data in Table 1
) of the Y1 agonist [Pro 34]NPY and the
Y2 agonist NPY(13–36) and compared the feeding response to that of NPY
alone. There were seven groups of rats (n = 8 to 10 per group).
Each received two injections 2 min apart and was studied on one
occasion only. Doses of 7.2 nmol (NPY and [Pro34]-NPY)
and 14.4 nmol (NPY 13–36) were used.
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Statistical analysis
All results are given as mean ± SEM.
Comparison between groups of data were made using analysis of variance.
Post-hoc comparisons were made using Tukey’s test. Statistical
significance was taken as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The classical Y1 agonist
[Pro34]-NPY showed the greatest degree of specificity,
with a greater than 1000-fold difference between its affinity for the
Y1 and Y2 receptors. NPY (3–36) and NPY (13–36) showed a lower degree
of specificity, with respectively, 20-fold and 4-fold higher affinities
for the Y2 receptor. [Pro34]-NPY(3–36) showed similar
binding to the corresponding unsubstituted C-terminal fragment in
SK-N-MC cells but was unable to compete for binding to SMS-KAN cell
membranes, presumably due to the [Pro34] substitution.
These results were mirrored closely by those obtained in competition
curves using rat cortex (predominantly Y1) and hippocampus
(predominantly Y2). BIBP-3226 was found to bind specifically to the Y1 receptor, showing no competition for 125I-PYY binding to the Y2 receptor at concentrations exceeding 1 µM. An inhibition constant of 31 nM was established for BIBP-3226 binding to the Y1 receptor, an affinity approximately 60 times lower than that of NPY. To test the ability of BIBP-3226 to bind to galanin receptors, membranes were incubated with 40 pM 125I-galanin in the presence or absence of 1 µM BIBP-3226. No significant reduction in 125I-galanin binding was seen in the presence of 1 µM BIBP-3226 (8152 ± 191.75 vs. 8937.75 ± 186.5 counts per min). In the presence of 200 nM galanin; however, binding was reduced by 92% (701.25 ± 15.5 counts per min). This suggests that BIBP-3226 is not interacting directly with the galanin receptor to prevent activation.
Adenylyl cyclase studies
Figure 1 shows that NPY and
[Pro34]-NPY cause a similar activation of the Y1
receptor. Isoproterenol (10 µM) gave a robust stimulation
of adenylyl cyclase activity, increasing activity by approximately
5-fold over basal. Full length NPY and [Pro34]-NPY, both
at a concentration of 1 µM, inhibited isoproterenol
stimulated adenylyl cyclase activity by 52.1 ± 4.7% (n = 5,
P < 0.005) and 62.4 ± 8.1% (n = 6,
P < 0.005) respectively. The same concentration of
[Pro34]-NPY (3–36), or of NPY (13–36) had no
significant effect on adenylyl cyclase activity (n = 7).
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shows a dose dependent
stimulation of feeding by icv NPY reaching a maximum at 7.2 nmol.
NPY(2–36) and NPY(3–36) exhibited similar dose-response relationships
to NPY. [Pro34]NPY and [Pro34]NPY(3–36)
demonstrated similar maximum effects to each other, which were
significantly lower than that achieved with NPY (F(25,
315)=12.3:P < 0.001). NPY(13–36) failed to show an
increase in feeding at any of the doses studied up to 50 nmol (results
not shown).
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shows that NPY and
[Pro34]NPY stimulate feeding (P < 0.001)
as expected. NPY gave a greater feeding response than
[Pro34]NPY (P < 0.005). NPY(13–36)
(14.4 nmol) did not stimulate feeding, either alone or in combination
with maximal [Pro34]NPY, suggesting that Y2 activation
has no effect on Y1 induced feeding. As expected, neither of the
control groups combining [Pro34]NPY or NPY(13–36) with
NPY showed a difference from NPY alone.
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shows the effect of BIBP-3226
on feeding stimulated by NPY (P < 0.05), galanin
(P < 0.001) and norepinephrine (P <
0.001). All three stimulants significantly increased food intake
compared with ethanol/saline controls, and in all three cases this
effect was completely blocked by prior injection of 60 nmol BIBP-3226.
BIBP-3226 had no significant effect on basal food intake.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our in vivo studies have involved two cell lines which are
de facto standards for the characterization of Y1 and Y2
receptor ligands. However, these are both human cell lines and, in
comparing their NPY receptors to behavioral effects in the rat, the
possibility of species variation must be taken into account. For this
reason, we have compared the binding profiles of the receptors on these
cell lines to those of receptors in rat cortex and hippocampus
membranes, tissues containing predominantly Y1 and Y2 receptors
respectively. It can be seen from Table 1 that the affinities for Y1
and Y2 receptors of the various ligands studied here does not vary
between rat and human, except for NPY itself, which shows a very high
affinity for cortex membranes.
We also studied adenylyl cyclase activity using lysates made from these cells. Preliminary experiments (results not shown) compared adenylyl cyclase activity in cell membranes and cell lysates, and we found that the results achieved using cell lysates were more robust and less variable than those achieved with cell membranes. It is well recognized that lysed cells are not optimal for binding studies and, as expected, pilot studies showed the dose response to be shifted to the right in comparison with membrane binding studies (data not shown). We therefore used high doses of peptide for adenylyl cyclase studies. These assays show that [Pro34]-NPY gives the same maximal inhibition of adenylyl cyclase in SK-N-MC cell lysates as NPY. This is an important difference between this classical Y1 system and NPY’s stimulation of feeding, where [Pro34]-NPY is only able to give approximately half of the stimulation of NPY. Another important difference is seen with [Pro34]-NPY(3–36). This peptide is unable to inhibit adenylyl cyclase activity at the same concentration at which [Pro34]-NPY completely inhibited it, although the two were equipotent in the feeding studies. It is not possible to state whether this peptide is capable of inhibiting adenylyl cyclase activity through the Y1 receptor at higher doses without carrying out entire dose response curves. However, there does seem to be a significant difference between the two [Pro34]-substituted peptides at the Y1 receptor that is not present in the activation of feeding.
We have demonstrated that [Pro34]-NPY gives approximately 50% of the stimulation of food intake seen with NPY despite being able to stimulate the cloned Y1 receptor to the same extent as NPY, as shown by our adenylyl cyclase experiments. A similar feeding effect was seen with [Pro34]-NPY(3–36). It is unlikely that there is any Y2 receptor activation by the doses of [Pro34] substituted peptides used in these experiments. In contrast, NPY(13–36) has very low affinity for the Y1 receptor, and therefore might be expected solely to stimulate Y2 receptors. Because this peptide produces no feeding effect alone, it seems that Y2 activation alone elicits no feeding response. However, we hypothesized that Y2 receptor activation might be necessary to see the full feeding effect of Y1 activation. This might explain why [Pro34] substituted NPY analogues give a lower maximal effect than NPY. To test this hypothesis, we compared the feeding effects elicited by [Pro34]-NPY and NPY(13–36), alone and in combination, to the maximal effect of NPY. If our hypothesis was correct, then [Pro34]-NPY administered together with NPY(13–36) would give an effect approaching that of NPY, even though the Y2 agonist alone caused no increase in feeding. In fact, this did not turn out to be the case, suggesting that our hypothesis was incorrect and providing evidence that a receptor other than the Y1 receptor is involved in feeding. This might be a single receptor, at which [Pro34]-NPY acts as a partial agonist or it might be that the Y1 receptor elicits a proportion of the effect with another receptor which does not recognize [Pro34]-NPY or NPY 13–36 responsible for the rest.
We have found that, as well as inhibiting the feeding response to NPY,
BIBP-3226 inhibits galanin and noradrenaline induced feeding. This is
an unexpected result because we (Table 1) and others (32, 33) have
shown BIBP-3226 to be a specific NPY Y1 receptor antagonist. We have
shown that BIBP-3226 is unable to bind to galanin receptors on RIN 5AH
cell membranes, suggesting that its effect on galanin induced feeding
at least, is not a direct effect. There are a number of possible
explanations for the unexpected actions of BIBP-3226 on feeding apart
from non specific toxicity:
It is possible that BIBP-3226 blocks the NPY receptors mediating feeding and that activation of these receptors is required for the stimulation of food intake by galanin and noradrenaline.
A second possibility is that is that BIBP-3226 may inhibit NPY receptors not directly involved in the control of feeding. This in turn might cause behavioral changes that reduce food intake. We have observed a brief period (10 min) of decreased grooming, rearing, and locomotor activity following the injection of 60 nmol BIBP-3226 (results not shown), followed by a return to apparently normal behaviour. It was for this reason that we allowed a 30-min period between injection of BIBP-3226 and the stimulant of feeding.
Others have reported that NPY (2–36) is either more (30, 37) or less (38) potent than NPY following icv administration, but these widely quoted studies either failed to cover a dose range appropriate to the claims made or were technically flawed. The study by Jolicoeur et al. reports that NPY(2–36) is more potent than NPY (37) but fails to show a significant difference in stimulated food intake following icv NPY and NPY(2–36). Feeding responses to both NPY and NPY (2–36) in their study were well below what we and others have demonstrated following equivalent doses of the same peptides (3, 30). The insertion of temperature recorders into the rats rectum, which was performed repeatedly throughout the period of monitoring of food intake, may well have accounted for the poor responses. Further, the highest dose of NPY or NPY (2–36) used by Kalra et al. (30) was 0.47 nmol, well below the dose that we found gave 50% of maximum stimulation of feeding. The comprehensive microinjection study by Stanley et al. (11) demonstrates that NPY(2–36) is more potent at lower doses than NPY but maximum response to either compound was not assessed. The proposed role for the Y1 receptor in mediating the feeding effect of NPY is based on studies in which only lower doses have been assessed(11, 30). Our study shows that at maximum stimulation the classical Y1 receptor agonist [Pro34]-NPY accounts for only 50% of the feeding effect of NPY, although at lower doses, this difference is not apparent. While McLaughlin et al. claim that NPY and NPY (2–36) stimulate feeding by 486% and 219% respectively, they did not demonstrate any significant difference between these two (38). The percentage difference is fully accounted for by a difference in the control groups in their study and the absolute amounts eaten are virtually identical. The 2-h food intake in animals given 5.0 nmol NPY was 12.6 g compared with 12.2 g in those given 5.0 nmol NPY (2–36) (38).
During the preparation of this manuscript, Gerald et al. (19) have reported the cloning of a rat hypothalamic NPY receptor (Y5Nat), which they suggest mediates the increase in food intake seen with NPY. They have shown that a number of peptides that inhibit adenylyl cyclase activity through the Y5 receptor also increase food intake, with a similar order of potency in each case. We found that NPY(13–36) at doses of up to 50 nmol (70 times the minimal effective dose of NPY) fails to stimulate feeding. Although Gerald et al. (19) did not test NPY(13–36) on feeding, they did show it to activate the Y5Nat receptor with an EC50 of 20 nM (20 times less potent than NPY). This is evidence against Y5Nat being the feeding receptor. Further, they report that BIBP-3226 at a dose of 10 nmol had no effect on NPY (0.3 nmol) induced food intake (19). They also show that BIBP-3226 does not block NPY inhibition of adenylyl cyclase via the Y5Nat receptor even at concentrations up to 1 µM, consistent with the hypothesis that the Y5Nat receptor mediates feeding. However, we demonstrate inhibition of NPY (1.2 nmol) induced feeding by 60 nmol BIBP-3226. We have also carried out the experiment using a lower dose of BIBP-3226 (30 nmol) and found this dose did not decrease NPY induced feeding (results not shown). This suggests that Gerald et al. may not have used a sufficiently high dose of BIBP-3226 in their study to demonstrate blockade of NPY induced feeding. The failure of NPY(13–36) to stimulate feeding and the ability of BIBP-3226 to block NPY induced feeding raise the intriguing possibility that the Y5Nat receptor is not the sole mediator of NPY induced feeding.
In conclusion, these findings suggest that the feeding effect of NPY involves a hitherto undescribed NPY receptor. Current pharmacological strategies are aimed at developing antagonists to the Y1 and Y5Nat receptors (19, 32). However, specific therapeutic manipulation of the feeding effects of NPY will be easier to achieve if an antagonist to the appetite specific receptor is identified.
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Acknowledgments |
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Footnotes |
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Received June 21, 1996.
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N. Pronchuk, A. G. Beck-Sickinger, and W. F. Colmers Multiple NPY Receptors Inhibit GABAA Synaptic Responses of Rat Medial Parvocellular Effector Neurons in the Hypothalamic Paraventricular Nucleus Endocrinology, February 1, 2002; 143(2): 535 - 543. [Abstract] [Full Text] [PDF]
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C. L. Chaffer and M. J. Morris The Feeding Response to Melanin-Concentrating Hormone Is Attenuated by Antagonism of the NPY Y1-Receptor in the Rat Endocrinology, January 1, 2002; 143(1): 191 - 197. [Abstract] [Full Text] [PDF]
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S. H. Russell, C. J. Small, A. R. Kennedy, S. A. Stanley, A. Seth, K. G. Murphy, S. Taheri, M. A. Ghatei, and S. R. Bloom Orexin A Interactions in the Hypothalamo-Pituitary Gonadal Axis Endocrinology, December 1, 2001; 142(12): 5294 - 5302. [Abstract] [Full Text] [PDF]
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F. Zammaretti, G. Panzica, and C. Eva Fasting, Leptin Treatment, and Glucose Administration Differentially Regulate Y1 Receptor Gene Expression in the Hypothalamus of Transgenic Mice Endocrinology, September 1, 2001; 142(9): 3774 - 3782. [Abstract] [Full Text] [PDF]
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 A. Kanatani, M. Hata, S. Mashiko, A. Ishihara, O. Okamoto, Y. Haga, T. Ohe, T. Kanno, N. Murai, Y. Ishii, et al. A Typical Y1 Receptor Regulates Feeding Behaviors: Effects of a Potent and Selective Y1 Antagonist, J-115814 Mol. Pharmacol., March 1, 2001; 59(3): 501 - 505. [Abstract] [Full Text]
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C. J. Small, M. S. Kim, S. A. Stanley, J. R.D. Mitchell, K. Murphy, D. G.A. Morgan, M. A. Ghatei, and S. R. Bloom Effects of Chronic Central Nervous System Administration of Agouti-Related Protein in Pair-Fed Animals Diabetes, February 1, 2001; 50(2): 248 - 254. [Abstract] [Full Text]
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A. Kanatani, S. Mashiko, N. Murai, N. Sugimoto, J. Ito, T. Fukuroda, T. Fukami, N. Morin, D. J. MacNeil, L. H. T. Van der Ploeg, et al. Role of the Y1 Receptor in the Regulation of Neuropeptide Y-Mediated Feeding: Comparison of Wild-Type, Y1 Receptor-Deficient, and Y5 Receptor-Deficient Mice Endocrinology, March 1, 2000; 141(3): 1011 - 1016. [Abstract] [Full Text] [PDF]
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 A. Wraith, A. Törnsten, P. Chardon, I. Harbitz, B. P. Chowdhary, L. Andersson, L.-G. Lundin, and D. Larhammar Evolution of the Neuropeptide Y Receptor Family: Gene and Chromosome Duplications Deduced from the Cloning and Mapping of the Five Receptor Subtype Genes in Pig Genome Res., March 1, 2000; 10(3): 302 - 310. [Abstract] [Full Text]
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 P. J. Larsen, M. Tang-Christensen, C. E. Stidsen, K. Madsen, M. S. Smith, and J. L. Cameron Activation of Central Neuropeptide Y Y1 Receptors Potently Stimulates Food Intake in Male Rhesus Monkeys J. Clin. Endocrinol. Metab., October 1, 1999; 84(10): 3781 - 3791. [Abstract] [Full Text] [PDF]
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 S. Iyengar, D. L. Li, and R. M. A. Simmons Characterization of Neuropeptide Y-Induced Feeding in Mice: Do Y1-Y6 Receptor Subtypes Mediate Feeding? J. Pharmacol. Exp. Ther., May 1, 1999; 289(2): 1031 - 1040. [Abstract] [Full Text]
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 L. Sun and R. J. Miller Multiple Neuropeptide Y Receptors Regulate K+ and Ca2+ Channels in Acutely Isolated Neurons From the Rat Arcuate Nucleus J Neurophysiol, March 1, 1999; 81(3): 1391 - 1403. [Abstract] [Full Text] [PDF]
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K. Meeran, D. O’Shea, C. M. B. Edwards, M. D. Turton, M. M. Heath, I. Gunn, S. Abusnana, M. Rossi, C. J. Small, A. P. Goldstone, et al. Repeated Intracerebroventricular Administration of Glucagon-Like Peptide-1-(7-36) Amide or Exendin-(9-39) Alters Body Weight in the Rat Endocrinology, January 1, 1999; 140(1): 244 - 250. [Abstract] [Full Text]
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 Y. Dumont, A. Fournier, and R. Quirion Expression and Characterization of the Neuropeptide Y Y5 Receptor Subtype in the Rat Brain J. Neurosci., August 1, 1998; 18(15): 5565 - 5574. [Abstract] [Full Text] [PDF]
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 J. Robidoux, L. Simoneau, S. St-Pierre, H. Ech-Chadli, and J. Lafond Human syncytiotrophoblast NPY receptors are located on BBM and activate PLC-to-PKC axis Am J Physiol Endocrinol Metab, March 1, 1998; 274(3): E502 - E509. [Abstract] [Full Text] [PDF]
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 C. J. Small, D. G. A. Morgan, K. Meeran, M. M. Heath, I. Gunn, C. M. B. Edwards, J. Gardiner, G. M. Taylor, J. D. Hurley, M. Rossi, et al. Peptide analogue studies of the hypothalamic neuropeptide Y receptor mediating pituitary adrenocorticotrophic hormone release PNAS, October 14, 1997; 94(21): 11686 - 11691. [Abstract] [Full Text] [PDF]
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