Receptor subtypes Y₁ and Y₅ mediate neuropeptide Y induced feeding in the guinea-pig

Abstract

1. Neuropeptide Y (NPY) is one of the most potent stimulants of food intake. It has been debated which receptor subtype mediates this response. Initially Y₁ was proposed, but later Y₅ was announced as a ‘feeding' receptor in rats and mice. Very little is known regarding other mammals. The present study attempts to characterize the role of NPY in feeding behaviour in the distantly related guinea-pig. When infused intracerebroventricularly, NPY dose-dependently increased food intake.

2. PYY, (Leu³¹,Pro³⁴)NPY and NPY(2 – 36) stimulated feeding, whereas NPY(13 – 36) had no effect. These data suggest that either Y₁ or Y₅ receptors or both may mediate NPY induced food intake in guinea-pigs.

3. The Y₁ receptor antagonists, BIBO 3304 and H 409/22 displayed nanomolar affinity for the Y₁ receptor (K_i values 1.1±0.2 nM and 5.6±0.9 nM, respectively), but low affinity for the Y₂ or Y₅ receptors. When guinea-pigs were pretreated with BIBO 3304 and H 409/22, the response to NPY was inhibited.

4. The Y₅ antagonist, CGP 71683A had high affinity for the Y₅ receptor (K_i 1.3±0.05 nM) without having any significant activities at the Y₁ and Y₂ receptors. When CGP 71683A was infused into brain ventricles, the feeding response to NPY was attenuated.

5. The present study shows that NPY stimulates feeding in guinea-pigs through Y₁ and Y₅ receptors. As the guinea-pig is very distantly related to the rat and mouse, this suggests that both Y₁ and Y₅ receptors may mediate NPY-induced hyperphagia also in other orders of mammals.

Introduction

Neuropeptide Y (NPY), which is a member of the pancreatic polypeptide hormone family that also includes pancreatic polypeptide (PP) and peptide YY (PYY), is one of the most evolutionarily highly conserved peptides known (Larhammar, 1996). The peptide is widely distributed within the central and peripheral nervous systems in mammals. Regulation of several important physiological activities such as food intake, blood pressure and circadian rhythms has been attributed to NPY. Five distinct Y receptor subtypes that bind NPY, PYY and PP with different affinities have been cloned in mammals. The receptors belong to the rhodopsin-like G-protein-coupled receptor superfamily, and have been given designations Y₁, Y₂, Y₄, Y₅ and y₆ (Larhammar et al., 2001; Michel et al., 1998). The pharmacologically defined Y₃ receptor has not been cloned (Herzog et al., 1993; Jazin et al., 1993), and may not exist as a separate gene product (Michel et al., 1998).

The NPY effects that have received the greatest attention over the past decade are those on feeding and metabolism. When NPY is injected intracerebroventricularly (i.c.v.) or directly into the hypothalamus, feeding is markedly increased (Haynes et al., 1998; Stanley & Leibowitz, 1985). This stimulatory effect has been reported not only in rodents (Iyengar et al., 1999; Levine & Morley, 1984; Morley et al., 1987a, 1987b), but also in virtually all vertebrates that have been studied (Boswell et al., 1993; Kuenzel et al., 1987; Kulkosky et al., 1989; Larsen et al., 1999; Miner et al., 1989; Morris & Crews, 1990; Pau et al., 1988; Parrot et al., 1986; Richardson et al., 1995; Volkoff & Peter, 2001). The only exceptions are the dog, which did respond to PP (Inui et al., 1991), and the baboon (Sipols et al., 1996). The receptor subtype mediating the effect of NPY on food intake is not yet firmly established. Currently available pharmacological data from rats and mice (see Kalra et al., 1999, for a review ) suggest that Y₁ and/or Y₅ receptors mediate the stimulatory effect of NPY on feeding. However, gene ablation studies have provided conflicting results. Mice lacking the NPY gene were initially reported to show no changes in feeding and metabolism unless crossed with mice having a genetic defect in the leptin gene, but later studies did observe reduced food intake and body weight (Erickson et al., 1996). When each of the NPY receptor genes was disrupted, there was a late-onset increase in body weight, both for Y₁ and Y₅ as well as for Y₂ (Kanatani et al., 2000b; Kushi et al., 1998; Marsh et al., 1998; Naveilhan et al., 1999; Pedrazzini et al., 1998).

It is important to study how the NPY system works in other animal species than rats and mice to get a broader perspective on this system. Evolutionary studies based on DNA data have shown that the guinea-pig (Cavia porcellus) is almost equally distantly related to rodents and humans (D'erchia et al., 1996), and thereby this animal could be a useful complementary model for NPY studies. Recently all of the guinea-pig NPY receptors have been cloned in our laboratory. The structural and pharmacological characteristics of the guinea-pig Y₁ and Y₂ receptors are similar to the human and rat orthologues (Berglund et al., 1999; Sharma et al., 1998). The guinea-pig Y₅ receptor has highest amino acid identity to the human Y₅ receptor reported for any non-primate NPY receptor orthologue and, in contrast to rat Y₅, it displays a virtually identical pharmacological profile to the human Y₅ (Lundell et al., 2001). Similarly, the guinea-pig Y₄ receptor resembles better the human orthologue than does the rat Y₄ (Eriksson et al., 1998). It has been reported that y₆ is a functional gene in mice, but a pseudogene in primates and guinea-pigs, and absent in rats (Burkhoff et al., 1998; Matsumoto et al., 1996; Starbäck et al., 2000). The aim of the present study was to determine whether centrally administered NPY or the related peptides known to stimulate feeding behaviour in rodents would increase food intake in guinea-pigs. Furthermore, the receptor subtype modulating feeding behaviour was investigated by using selective Y₁ and Y₅ receptor antagonists.

Methods

Compounds

NPY (porcine), PYY (porcine), (Leu³¹,Pro³⁴)NPY (porcine), NPY(2-36) (porcine), NPY(13-36) (porcine) were purchased from Bachem (King of Prussia, PA, U.S.A.). The peptides were dissolved in 0.9% saline. The Y₁ receptor antagonist, BIBO 3304, (R)N-{[4-(aminocarbonylaminomethyl)phenyl]methyl}-N2-(diphenylacetyl)-argininamide trifluoroacetate, was a generous gift from Boehringer Ingelheim (Pharma, Germany). Another Y₁ receptor antagonist, H 409/22, {(2R)-5-([amino(imino)methyl]amino)-2-[(2,2-diphenylacetyl) amino]-N-[(1R)-1-(4-hydroxyphenyl)ethylpentanamide} was provided by AstraHässle AB (Mölndal, Sweden). Both Y₁ antagonists were dissolved in sterile water after brief sonication. The Y₅ antagonist, CGP 71683A, [(4-{[(4-aminoquinazolin-2-yl)amino]methyl}-cyclohexyl)methyl]-(naphthylsulphonyl)amine, was synthesized by the Institute of Organic Synthesis (Riga, Latvia), and its chemical purity tests have been described elsewhere (Kask et al., 2001). It was dissolved in 30% DMSO.

Binding assays

Cells lines transfected with plasmids encoding the guinea-pig receptors were used for studying the selectivity and the affinities of the ligands. The agonist data shown in Table 1 have been reported previously (Berglund et al., 1999; Lundell et al., 2001; Sharma et al., 1998). The antagonists BIBO 3304, H 409/22 and CGP 71683A were tested on membrane fractions prepared using the same protocol. For binding assays, thawed aliquots of receptor membranes were resuspended in 25 mM HEPES buffer (pH 7.4) containing 2.5 mM CaCl₂, 1 mM MgCl₂ and 2 g l⁻¹ Bacitracin (Sigma, St. Louis, MO, U.S.A.) and homogenized using an Ultra-Turrax homogenizer. Binding experiments were performed in a final volume of 100 μl with 2 – 10 μg protein and ¹²⁵I-PYY (porcine) (Amersham Pharmacia Biotech) for 2 h at room temperature. Non-specific binding was defined as the amount of radioactivity remaining bound to the cell homogenate after incubation in the presence of 100 nM unlabelled NPY. In competition studies, various concentrations of the non-peptide compounds BIBO 3304, H 409/22 or CGP 71683A were included in the incubation mixture, along with ¹²⁵I-PYY. The peptide NPY was used as a reference for each experiment. Incubations were terminated by rapid filtration through GF/C filters, which had been pre-soaked in 0.3% polyethyleneimine, using a TOMTEC (Orange, CT, U.S.A.) cell harvester. The filters were washed with 5 ml of 50 mM Tris (pH 7.4) at 4°C and dried at 60°C. The dried filters were treated with MeltiLex A (Wallac) melt-on scintillator sheets, and the radioactivity retained on the filters counted using the Wallac 1450 Betaplate counter. The results were analysed using the Prism software package (Graphpad, San Diego, CA, U.S.A.).

Table 1.

Binding affinities of NPY, its analogues and Y₁ and Y₅ antagonists for the guinea-pig Y₁, Y₂ and Y₅ receptor subtypes

Animals and surgical procedures

The study was approved by the local ethical committee (C310/99 and C121/00). Juvenile male Dunkin – Hartley guinea-pigs (Bio Jet Service, Uppsala, Sweden) weighing 300 – 500 g were maintained in a 12-h light – dark cycle (lights on from 06.00 h to 18.00 h) in a temperature-controlled room (20 – 21°C). Two animals were housed in a polypropylene cage (60×80×25 cm) and kept separated by dividing each cage into two equal parts by a wall. Throughout the experiment, guinea-pigs were fed with powdered food (K5, Lactamin AB, Vadstena, Sweden) and hay was also freely available, except during the tests. Tap water was supplemented with approximately 0.5 mg ml⁻¹ of L-ascorbic acid and was freely available. The feeding experiments were performed between 09.30 h and 18.00 h.

Guinea-pigs were anaesthetized by intraperitoneal (i.p.) injection of a 1 : 3 (v v⁻¹) mixture of xylazine (Rompun vet. 20 mg ml⁻¹, Bayer, Gothenberg, Sweden) and ketamine (Ketalar 50 mg ml⁻¹, Parke Davis, Solna, Sweden). The animals were fixed into a stereotaxic frame (David Kopf Instruments, Tujunga, U.S.A.). The skull was exposed and a permanent stainless cannula (22 gauge) was implanted in the midline 6 mm below the bregma, according to the brain atlas of Luparello (1967). The guide cannula was fixed to the skull with screws and dental acrylic cement. The cannula was closed with a stainless steel stylet. After the surgical operation, animals were allowed to recover at least for 7 days. The animals were handled and weighed daily to habituate them to a partial restraint experience during i.c.v. infusions.

Experimental procedures

One hour before the drug administration, animals were moved into clean cages and food jars were removed. Different doses of NPY (0.9 – 10.8 nmol per animal) or saline were infused i.c.v. at a rate of 5 μl per min using a Hamilton infusion pump and syringes. The infusion volume was 10 μl. After the drug administration, the infusion cannula was left in place for an additional 1 min to avoid back diffusion along the cannula. The animals were returned to their home cage and food consumption was measured 1, 2, 3 and 4 h postinfusion. Food spillage was collected and subtracted from the intake. A video camera placed above the cage recorded the entire 4-h experiment. Afterwards, different eating parameters including the time spent on eating, number of meals and meal duration were analysed from the video tapes. Meals were divided into three categories based on duration: (1) short meals that lasted less than 1 min; (2) meals that lasted 1 – 5 min; (3) long meals that lasted over 5 min. Each animal received 2 – 4 different treatments in a randomized order with a 5 – 6 days recovery period between tests. At the end of the experiments, dye was infused and the staining of the third ventricle was examined.

Different doses of PYY (0.9 – 7.2 nmol per animal), (Leu³¹,Pro³⁴)NPY (0.9 – 10.8 nmol per animal), NPY(2 – 36) (0.9 – 10.8 nmol per animal), NPY(13 – 36) (3.6 – 25 nmol per animal) were infused i.c.v. to guinea-pigs. Saline and NPY (3.6 nmol per animal) were used as reference treatments. Food consumption and different eating parameters were measured using the same protocol as above.

BIBO 3304 (30 nmol per animal), H 409/22 (100 – 200 nmol per animal) or saline were infused i.c.v. in a volume of 5 – 7 μl given 15 min before NPY (3.6 nmol in 5 μl) or saline. When the effect of CGP 71683A (60 nmol per animal) was tested, 30% dimethylsulphoxide (DMSO) was used as the vehicle. CGP 71683A or DMSO were infused in a volume of 10 μl 15 min before NPY (3.6 nmol in 5 μl) or saline. Food consumption (4 h response) and eating parameters were measured as above.

Calculations and statistical analysis

The mean and standard error of the mean (s.e.mean) were calculated. The statistical differences between groups were determined with one-way analysis of variance followed by the post hoc comparisons with the Dunnett test. When the presumptions of the one-way of analysis of variance were not fulfilled, the non-parametric tests (Kruskal – Wallis or Mann – Whitney) were used.

Results

NPY dose-dependently increased food intake in guinea-pigs. The NPY doses 3.6 nmol or higher caused statistically significant changes in food consumption (Figure 1). The treatment increased also time spent on eating and number of short and intermediate meals (⩽5 min) (Table 2). The number of long meals (>5 min) remained unchanged. NPY treatment had no effect on the eating rate, but at high doses it reduced the meal size (Table 2). It generally doubled the water consumption, but due to the large variation the changes were not statistically significant (Table 2). The effects of the reference treatments (saline and NPY 3.6 nmol) on food intake were similar in all parts of the study, confirming the reliability of the test conditions (Tables 2,3,4).

Figure 1.

Cumulative food intake induced by different doses of NPY following i.c.v. administration to conscious guinea-pigs. Mean±s.e.mean, n=8 in each group. Asterisks indicate values which are statistically different from the control values: ^*P<0.05; ^**P<0.01.

Table 2.

Eating parameters and water consumption after i.c.v. infusion of saline or different NPY doses in conscious guinea-pigs

Table 3.

Eating parameters and water consumption after i.c.v. infusion of NPY analogues or saline in conscious guinea-pigs

Table 4.

Eating parameters and water consumption in BIBO 3304 or H 409/22 pretreated guinea-pigs after i.c.v. infusion of NPY

Peptides PYY, (Leu³¹,Pro³⁴)NPY and NPY(2 – 36) were able to increase the 4-h feeding response in guinea-pigs (Figure 2). In this respect, these three peptides were almost equipotent with NPY. PYY potently increased the time spent on eating and the meal frequency. The highest PYY dose reduced the average meal size. (Leu³¹,Pro³⁴)NPY and NPY(2 – 36) also increased the time spent on eating and the meal frequency, but their dose-response curves were flatter compared to NPY and PYY (Figure 2). All peptides tended to increase the water intake (Table 3). On the other hand, NPY(13 – 36) caused no marked changes in food (Figure 2) or water consumption (Table 3).

Figure 2.

Food intake (left panel), eating time (middle panel) and number of meals (right panel) after i.c.v. administration of NPY, PYY, (Leu³¹,Pro³⁴)NPY, NPY(2 – 36) and NPY(13 – 36) to conscious guinea-pigs. Means, n=8. Original data are shown in Tables 2 and 3.

The Y₁ antagonists, BIBO 3304 and H 409/22, as expected had high affinity for membranes from cells transfected with the guinea-pig Y₁ receptor subtype, but not for membranes with either Y₂ or Y₅ receptors as determined by competition with ¹²⁵I-PYY (Table 1). The Y₅ antagonist, CGP 71683A had high affinity for membranes from cells with the guinea-pig Y₅ receptor, but was almost inactive on cell membranes with either guinea-pig Y₁ or Y₂ (Table 1).

BIBO 3304 at the dose of 30 nmol and H 409/22 at the dose of 200 nmol were able to inhibit the feeding response to NPY, while the lower H 409/22 dose was ineffective (Figure 3). Both Y₁ antagonists tended to reduce the time spent on eating but caused no change in the number of meals (Table 4). When H 409/22 was given alone, it had a modest stimulatory effect on feeding (Figure 3), but we did not observe any other notable changes in guinea-pigs' behaviour. As shown in Figure 4, the Y₅ antagonist, CGP 71683A suppressed NPY induced food intake. The compound tended to decrease the time spent on eating and the meal frequency in NPY treated animals, but the changes were not statistically significant (Table 5).

Figure 3.

The effect of NPY (3.6 nmol i.c.v.) on 4 h food intake (upper panel) and number of meals (lower panel) in conscious guinea-pigs pretreated with the Y₁ receptor antagonists, BIBO 3304 and H 409/22. Mean±s.e.mean, n=8 – 9. Asterisks indicate values which are statistically different from the controls or the group otherwise indicated: ^*P<0.05; ^**P<0.01; ^***P<0.001.

Figure 4.

The effect of NPY (3.6 nmol i.c.v.) on 4 h food intake (upper panel) and number of meals (lower panel) in conscious guinea-pigs pretreated with the Y5 receptor antagonist, CGP 71683A. Mean±s.e.mean, n=7. Asterisks indicate values which are statistically different from the controls or the group otherwise indicated: ^*P<0.05; ^**P<0.01; ^***P<0.001.

Table 5.

Eating parameters and water consumption in DMSO or CGP 71683A pretreated guinea-pigs after i.c.v. infusion of NPY or saline

Discussion

After infusion into the third brain ventricle, NPY increased food intake in guinea-pigs in a dose-related manner. This feeding-stimulating effect of NPY has been reported in most vertebrates that have been studied, including mouse (Iyengar et al., 1999), rat (Levine & Morley, 1984; Morley et al., 1987a, 1987b), golden hamster (Kulkosky et al., 1989), ground squirrel (Boswell et al., 1993), rabbit (Pau et al., 1988), pig (Parrot et al., 1986), sheep (Miner et al., 1989), rhesus monkey (Larsen et al., 1999), bird (Kuenzel et al., 1987; Richardson et al., 1995), snake (Morris & Crews, 1990) and fish (Volkoff & Peter, 2001). In the dog, feeding was stimulated by PP, not by NPY or PYY (Inui et al., 1991). The single exception is a study with male baboons that was unable to demonstrate any effect of central NPY administration on food intake (Sipols et al., 1996). Here, the effective i.c.v. dose range for guinea-pigs was comparable to that reported for rats and mice (Levine & Morley, 1984; Morley et al., 1987a).

The herbivorous guinea-pig has a stomach of small volume that necessitates a frequent intake of small meals (Hirsch, 1973). The animal does not show diurnal fluctuation in its feeding pattern, and thus it eats as much during the light as the dark period (Hirsch, 1973; Horton et al., 1975), whereas the rat as a nocturnal animal eats most of its daily intake during the dark period. In the present study, the control animals spent 11 – 26 min during the 4 h follow-up on eating. Their meal frequency was between 13 – 17 per 4 h, and the meals usually lasted less than 5 min. In agreement with this, Jilge (1985) has reported that guinea-pigs in laboratory conditions eat approximately 99 times per 24 h and the average meal duration is 67 s. Longer meals (6 – 10 min) have been observed by Hirsch (1973), but the number of meals in his study was also 4 – 5 times smaller than in our study. According to Hirsch (1973), meal frequency is a parameter independent of age, lighting conditions and water availability. As juvenile animals grow, they start to eat larger meals at a faster rate. Therefore, it was important that the average animal weights in experimental groups were adjusted to be as close to each other as possible (390±15 g).

When NPY was infused i.c.v. to guinea-pigs, not only the amount of food eaten increased but also other components of feeding behaviour such as the time spent on eating and the number of meals were elevated (Table 2). In a manner similar to NPY, food intake was stimulated by PYY, the analogue (Leu³¹,Pro³⁴)NPY and the N-terminally truncated fragment, NPY(2 – 36). The peptides were all equally potent (Figure 2). The orexigenic activity of the peptide analogues did not directly correlate with affinity for one subtype of NPY receptors. As shown in Table 1, NPY and PYY bind with high affinity to the guinea-pig receptors Y₁, Y₂ and Y₅, whereas NPY(2 – 36) is potent at Y₂ and Y₅. On the other hand, (Leu³¹,Pro³⁴)NPY binds with high affinity to the Y₁ receptor, but its affinity for the guinea-pig Y₅ receptor is only moderate. Among the peptides tested, those activating both Y₁ and Y₅ receptors increased eating time and number of meals more potently than those stimulating either receptor subtype alone. Since food intake was not stimulated by NPY(13 – 36), a peptide which exhibits low affinity for Y₁ and therefore could be expected to stimulate mainly Y₂ receptors, the Y₂ receptor activation seems not to elicit feeding. Nor did this Y₂ agonism inhibit food intake. The combined peptide data are interpreted to mean that ligands activating either Y₁ or Y₅ receptors are capable of stimulating food intake in guinea-pigs.

In binding studies using cell lines stably expressing guinea-pig NPY receptors, BIBO 3304 and H 409/22, a novel Y₁ antagonist structurally related to BIBP 3226, the first Y₁ antagonist developed, were potent and selective ligands that could discriminate between Y₁ and Y₅ receptors. They displayed nanomolar affinity for the Y₁ receptor, BIBO 3304 being five times more potent than H 409/22 (Table 1). Both were devoid of affinity for the Y₅ receptor. The pretreatment with either 30 nmol of BIBO 3304 or by 200 nmol of H 409/22 was able to block the feeding response to exogenous NPY in guinea-pigs. The Y₁ antagonists suppressed the amount of food consumed and tended to reduce eating time after NPY, but caused no change in the number of meals. When H 409/22 was given alone, it had a modest orexigenic activity. A similar kind of elicitation of feeding has been observed after administration of 1229U91, another Y₁ receptor antagonist (Haynes et al., 1998). Since BIBO 3304 and H 409/22 do not block Y₅ receptors, their inhibitory actions on NPY induced food intake imply that Y₁ receptors are mediating this response. In agreement with the present results, it has been reported that BIBO 3304 inhibits NPY, (Leu³¹,Pro³⁴)NPY and NPY(2 – 36) induced as well as fasting induced food intake in rats (Polidori et al., 2000; Wieland et al., 1998). The compound also inhibits the hyperphagia after orexin-A administration (Yamanaka et al., 2000), but not that obtained after galanin or norepinephrine (Wieland et al., 1998).

At present, no other Y₅ antagonists than CGP 71683A has been publicly available, and therefore it has been used as a reference compound when studying the role of the Y₅ receptor. CGP 71683A has been shown to be a competitive antagonist of the Y₅ receptor in functional assays in cell lines and isolated organs (Criscione et al., 1998; Duhault et al., 2000; Dumont et al., 2000). In the present study, it had high affinity for the guinea-pig Y₅ receptor without showing any significant activities at the Y₁ and Y₂ subtypes (Table 1). When guinea-pigs were pretreated with CGP 71683A, the feeding response to NPY was markedly attenuated, suggesting that the compound is an effective antagonist of NPY-induced feeding mediated through Y₅ receptors. Previous rodent data have also shown the inhibitory effects of CGP 71683A on NPY-induced food intake in lean and obese animals (Della Zuana et al., 2001; Duhault et al., 2000; Polidori et al., 2000) and on spontaneous food intake in diabetic, 24 h fasted and free-feeding animals (Criscione et al., 1998; Kask et al., 2001).

It is interesting that the blockade of Y₁ and Y₅ receptors had different effects on the number of meals consumed by NPY treated animals. We had initially expected that the number of meals should correlate with the amount of food ingested, but both Y₁ antagonists suppressed food intake in NPY treated animals without reducing meal frequency (Figure 3), whereas the Y₅ antagonist reduced both food intake and meal frequency (Figure 4). Since NPY has a robust anxiolytic-like effect (Heilig et al., 1989), while the blockade of Y₁ receptors increases anxiety (Kask et al., 2001), it is possible that the effects of ligands on the anxiety level may also modulate feeding behaviour, assuming that the compounds can reach the centres for anxiety, primarily the amygdala, during the 4 h period of the experiment. On the other hand, high doses of NPY and PYY seemed to induce hyperactivity in guinea-pigs, and one reflection of this activation is the increased amount of small meals consumed (Tables 2 and 3). Indeed, injections of NPY into frontal cortex or lateral brain ventricle in rats have been shown to increase locomotion and exploration (Levine & Morley, 1984; Morley et al., 1987b; Smialowski et al., 1992), although not in all studies (Heilig et al., 1988). Our observation that increased locomotion might be connected to Y₅ receptor stimulation is in agreement with the previous finding showing that the Y₅ antagonist, CGP 71683A, decreases exploratory behaviour in rats (Kask et al., 2001). The suppression of food intake after CGP 71683A administration is not, however, related to the changes in locomotor activity (Kask et al., 2001).

It has been reported that CGP 71683A binds to the serotonin (5-HT) re-uptake recognition site and cholinergic muscarinic receptors with virtually identical affinity to its binding to Y₅ receptors (Della Zuana et al., 2001). Furthermore, chronic administration of CGP 71683A may produce local inflammatory changes near the site of injection (Della Zuana et al., 2001). Due to these non-specific actions and its poor solubility, CGP 71683A is not a good tool to use in vivo, and new selective Y₅ antagonists are needed to characterize the physiological importance of the Y₅ receptor. Recent reports on novel agents blocking Y₅ receptors do not entirely support the view that Y₅ receptors are crucial for NPY-induced feeding in rats (Kanatani et al., 2000a; Polidori et al., 2000). Therefore, our results with CGP 71683A in the guinea-pig should be regarded with caution until investigated with other Y₅ antagonists.

The role of NPY in the modulation of food intake is well established in rats and mice (see Kalra et al., 1999 for review). Here, we have shown that NPY stimulates feeding behaviour also in guinea-pigs. Even if there are species differences in the level of expression and distribution of the NPY receptor subtypes in the brain (Dumont et al., 1998; Gehlert & Gackenheimer, 1997), and in the binding characteristics of the cloned receptors (Lundell et al., 2001; Eriksson et al., 1998; Starbäck et al., 2000), NPY and its analogues, as well as the Y₁ and Y₅ antagonists, seemed to behave in a similar manner in guinea-pigs as reported earlier for rats (Haynes et al., 1998; Polidori et al., 2000; Wieland et al., 1998). Our results favour the hypothesis that both Y₁ and Y₅ receptors mediate NPY-induced food intake. Duhault et al. (2000) have recently shown that combined administration of Y₁ and Y₅ receptor antagonists is a more efficient intervention to inhibit food intake than the use of either antagonist alone. Although Y₁ or Y₅ knockout mice do not show overt disturbances of body weight and food intake, they develop late-onset obesity through different mechanisms. Y₁ deficient mice have lowered metabolic rate, while Y₅ deficient mice appear to be hyperphagic (Kanatani et al., 2000b; Kushi et al., 1998; Marsch et al., 1998; Pedrazzini et al., 1998). Furthermore, feeding responses to NPY are lowered in both Y₁ and Y₅ deficient mice compared to wild-type animals (Kanatani et al., 2001). These observations generally support the view that both receptor subtypes are involved in food intake regulation, but through different mechanisms. This issue could be addressed by developing mutant mice lacking both the Y₁ and Y₅ receptors. Another approach to study the contribution of Y₁ and Y₅ receptors to food intake regulation is to develop highly subtype selective agonists for the NPY receptors. Such compounds have been described for the Y₁ and Y₅ receptors (Cabrele et al., 2000; Söll et al., 2001) and their in vivo effects in guinea-pigs are under investigation in our laboratory.

In conclusion, the results presented here show that NPY through Y₁ and Y₅ receptors stimulates feeding behaviour in the guinea-pig as it does in the rat. As guinea-pigs and rats are very distantly related, this suggests that the dual involvement of Y₁ and Y₅ receptors may reflect the ancestral situation for mammals before the radiation of the present mammalian orders. Our results help provide a firmer basis for extrapolation to humans regarding the role of NPY and its receptors in the control of food intake.

Abbreviations

BIBO 3304

(R)N-{[4-(aminocarbonylaminomethyl)phenyl]methyl}-N2-(diphenylacetyl)-argininamide trifluoroacetate

CGP 71683A

[(4-{[(4-aminoquinazolin-2-yl)amino]methyl}-cyclohexyl)methyl]-(naphthylsulphonyl)amine

H 409/22

{(2R)-5-([amino(imino)methyl]amino)-2-(2,2-diphenylacetyl) amino]-N-[(1R)-1-(4-hydroxyphenyl)ethylpentanamide}

NPY

neuropeptide Y

PP

pancreatic polypeptide

PYY

peptide YY