Introduction

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Neuropeptide Y (NPY) is a 36-amino acid polypeptide that is widely distributed within the peripheral and central nervous system (Tatemoto, 1982a,b; Gray and Morley, 1986). NPY has been shown to be involved in the regulation of several neuroendocrine functions, including feeding and drinking, central autonomic functions, learning, stress responses, and sexual and motor behaviors (Gray and Morley, 1986). When NPY is administered centrally, it stimulates food intake in rats and mice (Clark et al., 1984; Gray and Morley, 1986; Morley et al., 1987; Stanley et al., 1992). Central administration of NPY (either i.c.v. or direct injection into the paraventricular nucleus) stimulates feeding episodes by increasing meal size, duration, and frequency (Bivens et al., 1998). In fact, NPY is one of the most potent neuropeptides known to induce feeding in animals (Leibowitz and Alexander, 1991; Lynch et al., 1994; Nakajima et al., 1994) and has been suggested to be a physiological signal for food intake (Dube et al., 1994; Sahu et al., 1997). In addition, chronic administration of NPY into the lateral ventricle or direct injection into the paraventricular nucleus of the rat increases food intake and leads to obesity (Stanley et al., 1986; Vettor et al., 1994). The obese Zucker rat is known to possess a hyperactive hypothalamic NPY system (McCarthy et al., 1991; Dryden et al. 1995; Widdowson, 1997) and genetically obese ob/ob mice express higher levels of NPY mRNA in the hypothalamus (Wilding et al.,1993). Erickson et al. (1996) have noted an attenuation of the obesity syndrome of ob/ob mice with the loss of NPY. These observations imply a significant role for NPY in hyperphagia and obesity.

NPY appears to exert its actions via multiple receptor subtypes. Recent advances in cloning suggest that there are at least six different NPY receptors, Y1-Y6 (reviewed by Blomqvist and Herzog, 1997). The NPY family of peptides, which includes endogenous peptides Peptide YY (PYY) and pancreatic polypeptide (PP), as well as several N-terminal truncated and synthetic peptide agonists ([Leu³¹Pro³⁴]NPY (LP-NPY), NPY_2-36, NPY_3-36, NPY_13-36, PYY_3-36, Pro³⁴PYY, D-Trp³²NPY), have differential in vitro affinity to Y1-Y6 receptor subtypes (Corp, 1996; Blomqvist and Herzog, 1997). NPY has moderate to high affinity for all of the receptor subtypes. Endogenous NPY occurs as NPY_1-36 in the brain (Stenfors et al., 1997).

In vivo, the receptor subtype mediating the orexigenic effect of NPY is not yet clear, although it has been suggested to be the Y1 receptor or a subtype of the Y1 receptor (Stanley et al., 1992; Kanatani et al., 1996). More recently, the Y5 receptor has also been implicated in feeding (Gerald et al., 1996). Y5 receptors are shown to be down-regulated in the brain of obese Zucker rats (Widdowson, 1997). However, other studies have questioned such suggestions (Corp, 1996; O'Shea et al., 1997; Roche et al., 1997). Both Y1 and Y5 receptor-deficient mice respond to exogenously administered NPY, although at higher concentrations, the effect of NPY is blunted in both Y1- and Y5-knockout mice, suggesting that perhaps both receptors may contribute to stimulation of feeding by NPY (Marsh et al., 1998; Pedrazzini et al., 1998). However, such a contention is complicated by the fact that both types of mice develop higher body weights and increased body fat and that Y5-deficient mice are also hyperphagic. The purpose of these studies was to re-examine the actions of NPY peptide receptor agonists and prototypic Y1 receptor antagonists on feeding in normal CD-1 mice. A better understanding of the putative receptor subtypes involved in the orexigenic actions of NPY in the brain would also have potential implications for the development of subtype-selective agonists or antagonists for the treatment of eating disorders and obesity.

	Materials and Methods

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Animals. CD-1 mice (male, 20-25 g) were obtained from Charles River Breeding Laboratories (Portage, MI). Animals were maintained on a 12-h day/night cycle under controlled environmental conditions. Food and water were supplied ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at Eli Lilly and Company.

Drugs. All peptide agonists including NPY (human), PYY (human), LP-NPY (porcine/human), PP (rat), PP (human), D-Trp³²NPY(human/rat), NPY_2-36 (porcine), NPY_3-36 (porcine), NPY_13-36 (porcine), PYY_3-36 (human), and Pro³⁴PYY(human) were obtained from Peninsula Laboratories (Belmont, CA). The NPY Y1 antagonists GR231118 [lle, Glu,Pro,Dpr,Tyr,Arg,Leu,Arg,Tyr-NH2)₂ cyclic (2,4'), (2'4)-diamide]. BIBP3226 [(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]arginiamide]; acetate salt) and its opposite enantiomer, BIBP3435 [(S)-N2-diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-arginiamide] (acetate salt), were synthesized at Lilly Research Laboratories for research purposes. All peptides were administered in 0.9% saline and BIBP3226 and BIBP3435 were administered in distilled water.

Intracerbroventricular Injection. All peptides and drugs were administered by i.c.v. injection into the lateral ventricle according to an adaptation of a procedure by Laursen and Belknap (1986). A 50-µl Hamilton syringe was fitted with PE-20 tubing so that the tip of the needle was 3.7 mm from the end of the tubing. The bregma was located with the tip of the needle by feeling for a slight depression in the middle of the skull and the needle was moved 2 mm lateral to the bregma for actual injection. Verification of the injection site was made histologically by staining the needle tract with cresyl violet for Nissl substances (Fig. 1) and also by injecting a tetrazolium dye into the site through the needle.

View larger version (108K): [in this window] [in a new window]   Fig. 1.   Verification of i.c.v. injection. Coronal sections of mouse forebrain illustrate the needle tract and injection site into the lateral ventricle (Nissl stain, cresyl violet). Further verification was performed by injecting a tetrazolium dye into the site through the needle. Scale bar, 20× (top), 40× (bottom).

Food Intake Measurements. Mice were individually housed and allowed to acclimate to 28 × 12 × 17 cm plastic cages for 30 to 40 min before the i.c.v. injection. Each mouse was weighed and provided with a preweighed amount of food (no water). Food weights were recorded 30, 60, and 90 min (or longer when necessary) after i.c.v. dosing with a Sartorius balance (Model LP22005; Artorius, Edgewood, NY) equipped with automatic printout of weights.

Neuromuscular Function. CD-1 mice were trained to perform on the horizontal screen test before the food intake experiment. Ninety minutes after the i.c.v. administration of drug, mice were tested on the horizontal screen test to determine any motor dysfunction due to the drug. The horizontal screen test was designed according to the method of Coughenour et al., (1977). The equipment used in this test consisted of six square (13 cm × 13 cm) wire screens (no. 4 mesh) mounted horizontally on a single metal rod. The mice were individually placed on top of each screen. The rod was rotated 180° so that the mice were at the bottom of the screens. A score of 2 was given to mice that climbed to the top of the screen in less than 60 s, a score of 1 was given to mice that could not climb over the screen in less than 60 s, and a score of 0 was given to mice that fell off the screen.

Data Analysis. Data are presented as mean ± S.E. Statistical analysis was performed with JMP version 3.2.2 (IBM platform; SAS Institute; Cary, NC). Data were analyzed by a repeated-measures ANOVA (Morrison, 1976), followed by post hoc analysis by Dunnett's test for multiple comparisons (Dunnett, 1964) as well as the Tukey-Kramer comparison test (Steel and Torrie, 1980). A p value of <.05 was considered statistically significant between groups unless indicated otherwise in the figure legend.

	Results

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Characterization of Effects of NPY (i.c.v.) on Food Consumption in Mice. NPY (i.c.v.) stimulated total as well as cumulative food consumption in CD-1 mice over a 90-min period in a dose-dependent manner (Fig. 2A). Various strains of mice (CD-1, CF-1, and Swiss-Webster) were similarly sensitive to the orexigenic effects of NPY at lower doses (23-230 pmol), although CD-1 mice were found to be consistently more sensitive to NPY at higher doses (data not shown). Hence, all characterization of NPY-induced feeding was carried out in this strain of mice.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   A, characterization of effects of NPY (i.c.v.) on food consumption in CD-1 mice over a 90-min period (mean ± S.E.). *p < .05 significantly different from vehicle. B, effect of PYY and NPY on food consumption. Total food consumption in 90 min (mean ± S.E.): PYY, ED50 = 38 ± 13 pmol (LCL = 20, UCL = 83 pmol); NPY, ED50 = 298 ± 143 pmol (LCL = 159, UCL = 1739 pmol). *p < .05, significantly different from vehicle; #p < .05, significant difference between two peptides.

Effects of Peptide NPY Receptor Agonists on Food Consumption in CD-1 Mice. The endogenous peptide PYY was much more potent than NPY in eliciting food consumption (Fig. 2B). N-terminal truncated peptides NPY_2-36 and NPY_3-36 also stimulated food consumption, with NPY_2-36 being as potent as NPY, whereas NPY_3-36 was less efficaceous; PYY_3-36 was found to be as effective as NPY, whereas Pro³⁴PYY was less efficaceous than NPY (Fig. 3A).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   A, effect of N-terminal truncated peptides NPY2-36, NPY3-36, PYY3-36, and Pro34PYY. *p < .05, significantly different from vehicle. B, effect of prototypic Y1, Y2, Y4, and Y5 agonists on food consumption. Y1 agonist, porcine/human LP-NPY; Y2 agonist, NPY13-36; Y4 agonist, rPP; Y4 and Y5 agonist, hPP; and Y5 agonist, D-Trp32NPY; *p < .05 significantly different from vehicle. (All data are expressed as total amount of food consumed in 90 min, mean ± S.E.)

Effects of Prototypic Y1, Y2, Y4, and Y5 Agonists on Food Consumption. Human and porcine LP-NPY, prototypic Y1 agonists, stimulated food consumption but were less efficaceous than NPY (Fig. 3B). The prototypic Y2 agonist NPY_13-36, prototypic Y4 agonists rat PP (rPP) and human PP (hPP), and the selective Y5 receptor agonist D-Trp³²NPY did not increase food consumption (Fig. 3B).

Effect of Coadministration of NPY with Y2-, Y4-, or Y5-Preferring Agonists. The possibility that the Y2-, Y4-, or Y5-preferring peptides NPY_13-36, rPP, hPP, and D-Trp³²NPY could either potentiate or antagonize NPY-induced food consumption was tested by coadministering these peptides with NPY. NPY_13-36, rPP, hPP and D-Trp³²NPY did not potentiate or block food consumption elicited by 230 pmol of NPY at the doses tested (Fig. 4).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Effect of NPY13-36- (A), rPP- (B), hPP-(C), or D-Trp32NPY-, Y2-, Y4-, Y4/Y5-, and Y5-preferring (D) peptides, respectively, on NPY-induced food consumption when coadministered with 230 pmol of NPY (data expressed as total amount of food consumed in 90 min, mean ± S.E.). There was no additive or antagonistic effect when any of the peptides were combined with NPY i.c.v. at the doses tested. #p < .05, significantly different from vehicle.

Effect of Peptide Y1 Antagonist GR231118 on NPY-Induced Food Consumption. Various doses of the peptide Y1 antagonist GR231118 were coinjected i.c.v. with 230 pmol of NPY. GR231118 did not block NPY-induced food consumption at any of the doses tested (Fig. 5). On the contrary, GR231118 tended to cause a slight increase in baseline food consumption. Doses higher than 8 nmol i.c.v. were not tested because the performance of mice in the horizontal screen test showed impairments with increasing doses (Fig. 7).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effect of vehicle, NPY (230 pmol) alone, peptide Y1 antagonist GR231118 (3.98 nmol) alone, and coadministration of various doses of GR231118 with NPY (230 pmol), on food consumption. Data expressed as total amount of food consumed in 90 min, mean ± S.E.; vehicle, NPY and GR231118 were administered i.c.v. #p < .05 significantly different from vehicle.

Effects of Nonpeptide Y1 Antagonist BIBP3226 and Its Opposite Enantiomer, BIBP3435, on NPY-Induced Food Consumption. Various doses of the nonpeptide Y1 antagonist BIBP3226 and its opposite enantiomer, BIBP3435, were coinjected i.c.v. with 230 pmol of NPY. BIBP3226 significantly attenuated NPY-induced feeding. However, BIBP3435, which has no affinity for Y1 receptors, also attenuated NPY-induced feeding at doses comparable to those of BIBP3226 (Fig. 6A) in vitro. Both compounds had no effect on baseline feeding when administered alone (in the absence of exogenous NPY). BIBP3226 and BIBP3435 also attenuated LP-NPY-induced feeding (Fig. 6B) as well as PYY-induced feeding (Fig. 6C) with no effect on baseline feeding.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   A, effects of coadministration of A, NPY (230 pmol i.c.v.) with various doses (i.c.v.) of BIBP3226 or BIBP3435, its opposite enantiomer, on food consumption. The effects of vehicle, NPY (230 pmol) alone, and drug (26 nmol) alone are also shown. Drug alone did not significantly affect food consumption compared to vehicle. When coadministered, BIBP3226 attenuates NPY-induced feeding in a dose-dependent manner, but this effect is not stereoselective. B, effects of coadministration of LP-NPY (230 pmol i.c.v.) with various doses (i.c.v.) of BIBP3226 or BIBP3435 on food consumption. The effects of vehicle, LP-NPY (230 pmol) alone, and drug (26.5 nmol) alone are also shown. Drug alone did not significantly affect food consumption compared to vehicle. When coadministered, BIBP3226 attenuates LP-NPY-induced feeding, but this effect also is not stereoselective. C, effects of coadministration of PYY (230 pmol i.c.v.) with various doses (i.c.v.) of BIBP3226 or BIBP3435 on food consumption. The effects of vehicle, PYY (230 pmol) alone, and drug (26 nmol) alone are also shown. Drug alone did not significantly affect food consumption compared to vehicle. When coadministered, BIBP3226 attenuates PYY-induced feeding in a dose-dependent manner, but this effect is not stereoselective. All data are expressed as a total food consumed in 90 min; mean + S.E. *p < .05 significantly different from peptide (NPY, LP-NPY, or PYY); #p < .05, significantly different from vehicle.

Effects of GR231118, BIBP3226, and BIBP3435 on Neuromuscular Function. The behavioral specificity of the effects of the Y1 antagonists on feeding was evaluated by the horizontal screen test. BIBP3226, BIBP3435, and GR231118 did not show significant neuromuscular deficits at doses tested for food consumption (Fig. 7). However, at higher doses, all three drugs caused impairment in performance on the horizontal screen. The effects of BIBP3226 and BIBP3435 at 37.5 and 60 nmol are shown in Fig. 7. The effect of higher doses of GR231118 is not shown in the figure.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effect of Y1 antagonists BIBP3226, BIBP3435, and GR231118 on sedation and ataxia as measured by the horizontal screen test. Doses of drug that blocked stimulated food consumption occurred in the absence of performance deficits in this test. However, at doses higher than those tested for feeding effects, BIBP3226, BIBP3435, and GR231118 showed significant behavioral impairment in this test (data for GR231118 not shown). Performance in the horizontal screen test is scored as: 2 = climbs over screen in 60 s; 1 = does not climb over screen in 60 s but does not fall off the screen; 0= falls off the screen. *p < .05, significantly different than vehicle.

	Discussion

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The effects of NPY and related analogs on food consumption were characterized in CD-1 mice. The relative ability of various peptide analogs to elicit food intake after i.c.v. administration into the lateral ventricle was found to be PYY > NPY_2-36 = NPY = PYY_3-36 > Pro³⁴ PYY > NPY_3-36 > LP-NPY > NPY_13-36 = D-Trp³²NPY = PP. The potent, dose-dependent effects elicited by NPY were consistent with previously reported studies in rats in which NPY was injected into specific hypothalamic nuclei or into the fourth ventricle (Clark et al., 1984, 1985; Kalra et al., 1991; Stanley et al., 1992; Corp, 1996). The endogenous peptide PYY was the most potent peptide to stimulate feeding. The N-terminal truncated analog of PYY, PYY_3-36, the substituted analog of PYY, Pro³⁴PYY, and the N-terminal truncated NPY analogs NPY_2-36 and NPY_3-36 also potently stimulated food consumption. LP-NPY stimulated feeding but was less efficaceous than NPY or PYY. NPY_13-36 and D-Trp³²-NPY had no stimulatory effect on feeding even at fairly high doses. Both hPP and rPP (another endogenous peptide from the NPY family) did not stimulate feeding in CD-1 mice at the doses tested. The relative ability in vivo of the various peptides to stimulate feeding was somewhat different from the relative selectivity of these peptides to bind in vitro to any one of the currently reported cloned NPY Y1-Y6 receptors (Table 1).

Thus, the profile of the various peptides in feeding studies did not correspond to the reported binding profile of these peptides to either Y2, Y4, or Y5 receptors (Gerald et al., 1995, 1996; Lundell et al., 1995; Bard et al., 1995; Gehlert et al., 1996; Gregor et al., 1996a; Hu et al., 1996, Chen et al., 1997). For example, the prototypic Y2 agonist NPY_13-36 was inactive in eliciting feeding. The prototypic Y4 receptor ligand rPP was also inactive in eliciting feeding in CD-1 mice. Likewise, hPP, a prototypic Y4 and Y5 agonist, and D-Trp³²NPY, a selective Y5 agonist (Gerald et al., 1996; Hu et al., 1996; Chen et al., 1997), did not stimulate feeding. In addition, NPY_13-36, rPP, hPP, and D-Trp³²NPY, when coadministered with NPY, also did not potentiate or block NPY-induced feeding at the doses tested.

These results with rPP, hPP, and D-Trp³²-NPY in CD-1 mice are somewhat different from those described in published reports in rats. Clark et al. (1985) have previously shown rPP to increase feeding in the rat (although it was much less potent than NPY). Likewise, hPP (at doses > 500 pmol; Clark et al., 1984; Gerald et al., 1996) and D-Trp³²NPY (at doses > 1000 pmol; Gerald et al., 1996; Wyss et al., 1998) previously have been shown to elicit small increases in feeding in the rat, although only single doses were tested in the latter two studies. Marsh et al. (1998) also have shown a small but significant increase in feeding with hPP in wild-type mice but not in Y5 receptor-deficient mice, at a 5-µg dose (>1000 pmol). These previously published effects are seen at significantly higher doses than those tested in the current studies where a dose-response with rPP, hPP, and D-Trp³²NPY revealed no hint of activity. These results do not appear to be related to species differences either because the mouse Y5 receptor is shown to be 97% homologous to the rat Y5 receptor (Nakamura et al., 1997). Moreover, NPY, PYY, and LP-NPY, as well as other peptides tested in this study, are reported to have similar relative potencies for binding to the rat and mouse Y5 receptors (Gerald et al., 1996; Chen et al., 1997; Nakamura et al., 1997). The current observations with peptide analogs in CD-1 mice do not agree with the relative selectivity of these peptides for the Y5 receptor, NPY_2-36 > NPY = PYY = LP-NPY > hPP > Pro³⁴ PYY > D-Trp³²NPY > NPY_13-36 (Gerald et al., 1996; Hu et al., 1996). Consistent with these observations, in a recent study, Marsh et al. (1998) suggested that the NPY-induced feeding response may not be mediated exclusively via Y5 receptors and that more than one NPY subtype may be involved. The authors showed that exogenously administered NPY (i.c.v.) stimulated feeding in Y5 receptor-deficient mice, although higher doses of NPY showed a blunted response compared with wild-type mice. Y5 receptor-deficient mice were also found to be hyperphagic. Overall, the present results with NPY and related peptide analogs do not suggest that NPY-induced feeding in mice is elicited via either Y2, Y4, or Y5 receptors alone.

Very recently, another NPY receptor subtype has been reported in mice (Gregor et al., 1996b; Weinberg et al., 1996) and was initially classified as the mouse Y5 subtype. More recent studies have shown that the human homolog of this receptor was not identical with the human Y5 receptor and that both in other primates and humans, the gene for this receptor contained a frame shift mutation that very likely rendered the receptor functionally inactive (Gregor et al., 1996b; Matsumoto et. al., 1996; Rose et al., 1997). This receptor has now been classified as the Y6 subtype. In a very recent study, Burkhoff et al. (1998) have confirmed that the Y6 receptor is present in the mouse, in several other species, and in several tissues in human, but is not present in the rat. Because the Y6 receptor is also localized in the hypothalamus in the mouse, it is important to consider the possibility that the feeding profile obtained in these studies may be mediated via the Y6 receptor. The reported profile for the peptides to bind to the Y6 receptor is NPY = PYY = LP-NPY > NPY_2-36. The feeding effects observed in the present studies do not show a similar profile of stimulation with these peptides. Moreover, the overall ability of these peptides to stimulate feeding in the current studies corresponds to results published with earlier studies in the rat (Stanley et al., 1992; Corp, 1996), a species that has been reported to lack Y6 receptors (Burkhoff et al., 1998). Thus, the feeding effects observed in the present study do not appear to be mediated via Y6 receptors.

Although Y3 receptors have not been cloned in humans to date, PYY does not bind to Y3 receptors (Blomqvist and Herzog, 1997). Because the current in vivo studies show PYY to be the most potent stimulator of feeding, Y3 receptors appear to be unlikely to have mediated the observed effects.

The role of the Y1 receptor in NPY-induced feeding in CD-1 mice was evaluated with receptor agonists as well as antagonists. Based on the results obtained in mice to the Y1 receptor alone, LP-NPY, a prototypic Y1 receptor ligand, was not as potent or efficaceous as NPY in eliciting feeding, although the two peptides are equally potent in binding to the Y1 receptor. Moreover, NPY_2-36, a peptide that is not very potent at binding to Y1 receptors, was very potent at stimulating feeding. In addition, PYY was found to be more potent than NPY in stimulating food consumption, although the two peptides are equipotent at binding to the Y1 receptor in vitro (Blomqvist and Herzog, 1997). These discrepancies are similar to those noted by Stanley et al. (1992) in studies in the rat.

The results obtained with prototypic Y1 antagonists do not unequivocally support mediation of NPY-induced feeding via the currently defined Y1 receptor, either. GR231118 (1229U91), a potent peptide NPY Y1 antagonist (Daniels et al., 1995), did not block NPY-induced food consumption at doses that did not show other behavioral impairments. Doses higher than 8 nmol caused performance deficits in the horizontal screen test and, hence, were not evaluated for effects on NPY-induced feeding. Given the high affinity of GR231118 to the Y1 receptor, however, one might have expected to see some hint of antagonism at the doses tested. In contrast to our results, Kanatani et al. (1996) have reported blockade of NPY-induced feeding in the rat with GR231118 [5 µg (1150 pmol) NPY versus 5 µg (2 nmol) and 30 µg (12.5 nmol) GR231118 i.c.v.]. Marsh et al. (1998) have also shown blockade of NPY-induced feeding in Y5-deficient mice as well as wild-type mice with a single dose of GR231118 [5 µg (1150 pmol) NPY versus 2.5 µg (2 nmol) GR231118]. However, no data were shown in these studies to rule out nonspecific effects of GR231118 on food consumption (such as neurological or motor deficits). Moreover, the selectivity profile of GR231118 for NPY receptor subtypes (Matthews et al., 1997; Parker et al., 1998; Schober et al., 1998) makes it difficult to interpret effects of this antagonist unequivocally in vivo. Matthews et al. (1997), for example, have shown that GR231118 binds with high affinity to both cloned human Y1 and Y4 receptors and much lower affinity to human Y2 receptors, whereas in the rat hypothalamus, GR231118 differentiates a high-affinity site corresponding to Y1 receptors as well as a low-affinity site corresponding to Y2 receptors. GR231118 has also been shown to be an agonist at Y2, Y4, and Y5 receptors and has high affinity for mouse Y6 receptors (Parker et al., 1998). In the present studies, GR231118 tended to potentiate NPY-induced food consumption, perhaps due to these reported multiple receptor affinities of the peptide.

The results obtained with BIBP3226, a more selective nonpeptide antagonist, also do not suggest Y1 receptor mediation of NPY-induced feeding. BIBP3226, as well as its opposite enantiomer, BIBP3435, which is inactive at Y1 receptors, blocked feeding elicited by NPY, LP-NPY, and PYY at doses that did not cause other observable behavioral deficits. BIBP3226 is highly selective for Y1 receptors, having no significant affinity for several neurotransmitter receptors as well as human Y2 and Y4 receptors (Rudolf et al., 1994; Wieland et al., 1995; Matthews et al., 1997) and rat Y2, Y4, and Y5 receptors (Gerald et al., 1996). The affinity of the acetate salt of the antagonist (the form used in this study) for human Y1 receptors (DG Gehlert and DA Schober, unpublished observations) is consistent with reported affinities (K_i approximately 7 nM; Rudolf et al., 1994). It is, thus, a good tool to examine in vivo the effects of the Y1 receptor. O'Shea et al. (1997) and Kask et al., (1998) previously have shown BIBP3226 to block NPY-induced feeding in rats, although these studies did not compare the effects of BIBP3435 to determine whether the effects of BIBP3226 were stereoselectively elicited via the Y1 receptor.

The results with Y1 agonists and antagonists are in keeping with previously raised inconsistencies regarding the role of Y1 receptors in feeding, such as the ability of NPY_2-36 to elicit feeding more potently than NPY, in contrast to their respective binding affinities to the Y1 receptor (Stanley et al., 1992), as well as the inability of Y1 receptor antisense oligonucleotide probes to block NPY-induced feeding responses, although it resulted in the rats becoming anxiogenic (Wahlestedt et al., 1993; Lopez-Valpuesta et al., 1996). A recent study by Kask et al. (1996) with BIBP3226 further confirmed that Y1 receptors appear to mediate anxiogenic behavior. Unpublished observations from our laboratory, however, show that the opposite enantiomer, BIBP3435 does not cause anxiogenic behavior. Given that both enantiomers were active in blocking NPY-induced feeding, one could further surmise that: 1) the effect of the enantiomers on NPY-induced feeding must not be occurring via Y1 receptors; 2) blockade of feeding responses can occur independent of anxiogenic behavior; 3) the feeding effects were nonspecific; or 4) BIBP3226 may be less than optimal as a Y1-antagonist tool. Higher doses of both isomers caused sensorimotor dysfunction, although blockade of feeding by BIBP3226 and BIBP3435 occurred at doses that did not cause behavioral dysfunction. Recent evidence from studies on knockout mice (Pedrazzini et al., 1998) also question the role of the Y1 receptor in NPY-induced feeding. These studies showed that exogenous NPY elicited feeding in Y1 receptor-deficient mice, although at higher doses the feeding response to NPY was slightly blunted.

In summary, these studies, based on currently available agonist and antagonist tools, suggest that NPY-induced food consumption does not appear to be mediated via the Y1 receptor as it is currently defined. In addition, the ability of the peptide agonists to elicit feeding was not similar to the reported ability of these agonists to bind to known cloned NPY-receptor subtypes, Y1-Y6. Overall, the effects of the peptide agonists in CD-1 mice were not very different from those seen in previous studies performed in rats (Clark et al., 1984, 1985; Stanley et al., 1992; Corp, 1996) although there were some differences noted in CD-1 mice with effects of rPP, hPP, and D-Trp³²-NPY compared to published reports in rats. Based on the relative profile of food consumption elicited by selective prototypic agonists to the NPY family of receptors, it is difficult to attribute NPY-induced feeding to any one of the currently defined NPY Y1-Y6 receptors. Thus, it is possible that NPY-induced feeding may be mediated in vivo by a combination of more than one NPY receptor subtype as suggested by Corp (1996), Pedrazzini et al., (1998), and Marsh et al. (1998). The possibility that there may be subtypes of the Y1 receptor also needs to be considered, as has been previously suggested (Palea et al., 1995). Alternativly, food consumption may be mediated by an undiscovered NPY receptor subtype. Subtype-selective, structurally diverse, safe, nonpeptide NPY receptor antagonists are needed to unequivocally tease out the receptor subtype(s) mediating NPY-induced feeding and to further understand the role of NPY in central regulation of appetite and obesity.