Appetite suppression based on selective inhibition of NPY receptors

Abstract

AIM: The aim of this review is to critically assess available evidence that blockade of the actions of NPY at one of the five NPY receptor subtypes represents an attractive new drug discovery target for the development of an appetite suppressant drug.

RESULTS: Blockade of the central actions of NPY using anti-NPY antibodies, antisense oligodeoxynucleotides against NPY and NPY receptor antagonists results in a decrease in food intake in energy-deprived animals. These results appear to show that endogenous NPY plays a role in the control of appetite. The fact that NPY receptors exist as at least five different subtypes raises the possibility that the actions of endogenous NPY on food intake can be adequately dissociated from other effects of the peptide. Current drug discovery has produced a number of highly selective NPY receptor antagonists which have been used to establish the NPY Y1 receptor subtype as the most critical in regulating short-term food intake. However, additional studies are now needed to more clearly define the relative contribution of NPY acting through the NPY Y2 and NPY Y5 receptors in the complex sequence of physiological and behavioral events that underlie the long-term control of appetite.

CONCLUSIONS: Blockade of the NPY receptor may produce appetite-suppressing drugs. However, it is too early to state with certainty whether a single subtype selective drug used alone or a combination of NPY receptor selective antagonists used in combination will be necessary to adequately influence appetite regulation.

Introduction

Neuropeptide Y (NPY) exerts a powerful stimulatory effect on food intake after injection either into the cerebral ventricles or into the hypothalamus of satiated rats.1,2,3 Perhaps more importantly, infusion of NPY into the brain promotes continuing hyperphagia and an increase in body weight, which is accompanied by hyperinsulinemia and insulin resistance. These observations suggest that heightened brain NPY levels may be a contributing factor to the initiation and maintenance of the obese/diabetic state.4,5

Evidence for a physiological role of NPY in the control of food intake and body weight has been obtained from several studies and the following examples are instructive. Firstly, a close relationship exists between food intake and NPY expression levels in the hypothalamus. For example, hypothalamic NPY levels are increased in the dark phase when rats consume the majority of their daily food intake.6,7 Secondly, hypothalamic NPY levels are increased in certain models of hyperphagia, such as after food deprivation and food restriction as well as in spontaneous and experimentally induced diabetes.8,9,10,11 Hypothalamic NPY levels are also increased in monogenetic models of obesity such as the ob/ob mouse and Zucker fa/fa rat, which are hyperphagic and obese.12,13 Finally, inhibition of the synthesis or blockade of the actions of NPY with antibodies, antisense oligodeoxynucleotides (ODN), non-selective receptor antagonists, or other approaches leads to lower food intake in both freely feeding and energy-deprived animals.14,15,16,17,18,19 Together, these observations have been taken to suggest that NPY plays a physiologically important role in the control of food intake. Moreover, blockade of the central actions of NPY also leads to a decrease in food intake and body weight in the genetically obese Zucker fa/fa rat, which suggests a role for endogenous NPY in the pathogenesis of this obesity syndrome.20,21

The above findings have encouraged the pharmaceutical industry to develop NPY receptor antagonists as potential appetite suppressant and anti-obesity drugs. However, since both NPY and NPY receptors are widely distributed within the brain, it is not surprising that NPY has also been shown to play a role in the expression of behaviors other than food intake (Figure 1). The recent finding that the actions of NPY are mediated through at least five different receptors raises the possibility that the effects of NPY on appetite may be mediated through a specific ‘feeding’ receptor.22 In this case, selective blockade of this receptor would reduce the risk of inhibiting other actions of NPY which could lead to undesirable side-effects.

Figure 1
figure1

The actions of NPY.

Since drug discovery in this area relies upon NPY playing a physiologically important role in the control of food intake, the first part of this review will critically assess the available evidence supporting this basic assumption. We shall then evaluate the possibility that selective appetite suppressants with few side-effects can be produced by targeting individual NPY receptor subtypes, and finally discuss some pitfalls that seem likely to complicate the development of NPY antagonists.

Neuropeptide Y and food intake

Mechanisms of food intake

Food intake is a deceptively easy parameter to measure, but this simplicity masks a behavior with very complex motivation. Two schools of thought have emerged concerning the control of food intake—the physiological and the behavioral.23 The physiological school proposes that food intake is controlled by a combination of chemical (eg plasma glucose), hormonal (eg leptin) and neural (eg vagal afferents from the liver and viscera) inputs that converge on the brain.24,25,26

Feeding is undoubtedly influenced by these physiological signals, but it can also be initiated in the absence of energy depletion by learned associative cues. This is an aspect of food intake that is often overlooked. These cues can be linked both to palatability and the rewarding properties of the food eaten.27 Environmental cues such as stress, the time of day, place and an assessment of future food supply are also important regulators of food intake.28,29 Thus, it appears that experience and learning as well as an anticipation of future energy needs continuously interact with peripheral signals to control food intake. Consequently, many interconnected brain areas are used by the animal to initiate and then control the complex multifaceted behavior that results in a change in food intake.30

Food intake is highly variable from meal to meal, leading to well-tolerated variations in daily energy balance.31 This rather loose control of daily food intake contrasts with the stable body composition that is usually maintained for long periods of time, suggesting that over the long-term food intake must be very tightly controlled.23,31 Understanding the relative importance of the individual physiological and behavioral phenomena involved in the control of both short-term and long-term food intake is important, both for identifying new drug discovery candidates, and for utilizing them in treatment regimes that will maximize their appetite suppressant properties.

Exogenous NPY and food intake

NPY enhances motivation and reward

The increase in food intake produced by administration of NPY into the brain could be due either to an increase in perceived hunger or to an enhancement of the motivating/rewarding properties of the food being eaten. Progress in this area is hampered by the difficulty in distinguishing a hungry animal from an animal that may be eating for some other reason. However, evidence has accumulated in recent years suggesting that both the motivation to eat and the subsequent rewarding properties of the food eaten may both play an important role in the action of exogenous NPY on food intake. Evidence supporting this hypothesis has come from a series of studies demonstrating that central administration of NPY directly enhances reward. For example, injection of NPY into the hypothalamus enhances the motivation to respond to rewarding stimuli.32,33 Furthermore, when presented with both palatable sweet food and regular chow, animals centrally injected with NPY choose the palatable food and will even tolerate an intense foot shock or other aversive stimuli to get it.34,35 The underlying increase in sweet intake in these experiments may be due to an associative learning effect of NPY-receptor stimulation to strengthen a gustatory preference for these foods.

Does exogenous NPY-stimulated food intake represent a true hunger state?

Whether NPY-induced eating represents a true hunger state has also been the focus of attention. For example, neither NPY nor peptide YY (PYY); (a close analog of NPY which acts on NPY receptors) when injected into the brain induces the same discriminative stimulus effects as food deprivation.36,37 That is, rats can readily perceive the distinction between stimulation of NPY receptors in the brain and food deprivation. Furthermore, injection of NPY into the brain produces generalized behavioral activation that enhances the appetitive phase (the period immediately before eating when the animal is actively looking for food) but does not change or may even decrease the consummatory phase (the period of biting, chewing and swallowing) of food intake.38,39,40 These are aspects of feeding different from deprivation-induced food intake and give reason to believe that exogenous NPY acts primarily to increase the motivation of animals to eat by mechanisms that are not the same as either food deprivation or other types of metabolic challenge.41 Indeed it has been suggested that the actions of exogenously administered NPY are more in keeping with pathophysiological states such as binge eating rather than normal food intake.42,43 Care should, however, be exercised in the interpretation of many of these studies, since NPY was injected intracerebroventricularly and the same behavioral changes may not be observed if NPY is injected in small doses directly into discrete areas of the hypothalamus. Examples of the major differences observed between food intake stimulated by exogenous NPY and food intake induced by food restriction are summarized in Table 1.

Table 1 Examples of differences between the effects of NPY and food deprivation on food intake

NPY and NPY receptor density in different brain areas in response to changes in energy balance

Evidence is accumulating to show that the content of NPY and NPY receptor density are changed in many brain areas in response to changes in food restriction and obesity. For example, energy deprivation produced either by fasting or by food deprivation has been shown to increase the NPY content of the hypothalamus (paraventricular nucleus, arcuate nucleus/median eminence), hippocampus and cortex and to decrease NPY levels in the striatum.44,45,46 In contrast, NPY levels are not altered in either the caudate nucleus or the nucleus accumbens.46 The increased NPY levels produced by energy deprivation are associated with decreased NPY Y5 and/or NPY Y2 receptor density in the hypothalamus (lateral (perifornical)), dorsal, ventromedial, hippocampus (CA3 region) thalamus (paraventricular and reuniens nuclei) and amygdala (centromedial nucleus).47,48 In ob/ob mice that have high brain NPY levels, similarly decreased receptor density has been shown in the hypothalamus, thalamus (midline group), cortex (cingulate, retrosplenal and granular) and hippocampus.49 In contrast, animals made obese by feeding a high-fat diet, in which hypothalamic NPY levels are low, increased NPY Y5 and/or NPY Y2 receptor density has been shown in the hypothalamus, amygdala and thalamus.47 Many of these brain areas are also influenced by other regulators of food intake such as GLP-1, MC-4 receptor agonists, AGRP, leptin, CART (cocaine and amphetamine related transcript) and the orexins.50,51,52,53 Together, these studies strongly suggest that food intake in response to changes in energy balance may involve the coordinated activity of NPY containing neurons and receptors in many brain regions, and not just those in the hypothalamus.

NPY injected into many brain areas alters food intake

NPY is commonly perceived to increase food intake only when injected into the hypothalamus, but it also exerts this effect at several extra-hypothalamic sites, including the frontal cortex, the hind brain and the hippocampus (Figure 2A).54,55,56,57,58 In the hippocampus, NPY increases the activity of acetylcholine-containing nerve terminals believed to be important in modulating reward and arousal,56 suggesting that stimulation of NPY receptors in this region may increase food intake by enhancing the perception of the rewarding properties of the food eaten.56

Figure 2
figure2

Sites of action of NPY on the brain to effect food intake. (A) NPY has been shown to stimulate food intake after injection into the cortex, hippocampus, hind brain as well as into the hypothalamus. (B) Injection of NPY into the ventricular system (black areas) ensures a wide distribution of the peptide within the brain (represented by the thick arrow). Injection of NPY into the hypothalamus may activate only certain other brain areas (represented by the thin arrows). The background figures are reprinted from Brain maps—structure of the rat brain, by LW Swanson (Elsevier: Amsterdam, 1998, 2nd edn), with permission from Elsevier Science.

Does NPY-induced hyperphagia mimic the actions of the endogenous peptide?

The above findings suggest that endogenous NPY may control food intake through coordinated activity in many parts of the brain; accordingly injection of NPY into the cerebroventricular system (or even directly into the hypothalamus) may not exactly mirror the effects of the endogenous peptide on food intake (Figure 2B). Some evidence exists to support these possibilities. First, the failure of NPY to increase the consummatory phase of food ingestion after intracerebroventricular (i.c.v.) injection appears to differ from that observed after direct hypothalamic injection where meal size (an index of the consummatory phase) is increased.38,40 Evidently, feeding after i.c.v. vs hypothalamic NPY may not necessary be based on the same underlying mechanisms. Second, both food intake and paradoxical taste aversions are produced after injection of NPY into the ventricular system of the brain, suggesting that sites additional to those involved in food intake are activated.40 This finding is supported by the observation that c-fos expression in the paraventricular nucleus (PVN) of the hypothalamus is increased after i.c.v. injection of NPY, but not after fasting when endogenous NPY levels at this site are increased.59,60

Endogenous NPY and food intake

Changes in food intake after blockade of the actions of endogenous NPY in the brain

Clearly, to adequately answer the question posed by the previous section it is necessary to compare feeding behavior after central injection of NPY with the effects of blocking the actions of endogenous NPY in the brain. The following is a discussion of the approaches that have been used to investigate the role of endogenous NPY in the control of food intake.

Knockout mice

Knockout of the gene encoding for NPY produces a mouse with few metabolic perturbations.61 For example, NPY-deficient mice have a normal hormonal profile, grow and eat normally and have a normal response to both diet and chemically-induced obesity (monosodium glutamate or gold thioglucose).61,62,63 Most studies conducted with these animals show a normal refeeding response to short-term fasting, although one recent study has challenged this conclusion;61,64 the difference between these studies appears due to whether wild-type or heterozygous animals were used as controls. NPY knockout animals do however, express an anxiogenic-like phenotype are hypoalgesic with an increased susceptibility to epilepsy.64,65

Since these findings argue against an important physiological role for NPY in the control of food intake, how can they be interpreted? Two main explanations have been suggested. First, NPY may not be a critical feeding regulator; in its absence, food intake is maintained by the action of other neuropeptides. For example, AGRP expression is increased in the hypothalamic neurons of the NPY knockout mouse.66 Alternatively, during development, other neural pathways may compensate for the lack of the NPY. This is a distinct possibility, since neural pathways in the brain that control food intake are highly redundant and inhibition of one generally leads over time to the return of normal feeding. This point could be addressed by the use of inducible knockout animals, in which the expression of NPY would be prevented at the time of experiment.

Anti-NPY antibodies

Specific antibodies against NPY have been used acutely to block the actions of endogenous NPY on food intake. Injection of anti-NPY antibodies into the brain of rats attenuates spontaneous feeding and that induced by 2-deoxyglucose, ventromedial hypothalamic (VMH) lesions and fasting.14,67,68,69,70,71,72 Spontaneous food intake in the Zucker fa/fa rat is also reduced by administration of NPY antibodies into the brain.21 These studies suggest that endogenous NPY does in fact play a physiologically important role in modulating the activity of brain feeding circuits—at least in the short-term. However, only total food intake was measured in response to antibody administration in most of these studies. Therefore, it cannot be assumed that the observed reduction in food intake was behaviorally specific rather than an aversive effect of the antibodies. Indeed, injection of anti-NPY antibodies directly into the VMH produced a large increase in amphetamine-like motor activity and decreased resting behavior, which is thought to explain the concurrent decrease in food intake observed in that study.73

Antisense oligonucleotides (ODNs)

The results obtained with this approach have been mixed. Initial use of this technology demonstrated that injection of ODNs directly into the arcuate nucleus of the hypothalamus reduced food intake.74 Other studies have also shown central administration of NPY antisense ODNs to decrease food intake, meal size and meal duration without inducing sickness or taste aversion.75 However, side effects and toxicity associated with the use of NPY ODNs are often prominent.75,76,77 For example, a recent comprehensive study of the effect of several different types of ODNs on food intake has shown either no effect on feeding or significant side effects associated with inhibiting the production of NPY.77 The use of different types of ODNs could explain the discrepant results described above,74,75,76,77 but these studies do raise concerns about the specificity and toxicity of antisense molecules within the central nervous system. This remains a particular problem when studying behaviors such as food intake, which can be strongly influenced by both toxic and non-selective interactions.

Miscellaneous approaches

Ventricular injection of the non-selective peptide NPY receptor antagonist 1229U91 has been shown to inhibit NPY-induced food intake, the refeeding response to short-term fasting as well as spontaneous food intake in Zucker fa/fa rats.78,79 By contrast, 1229U91 did not inhibit either spontaneous food intake in normal rats or after the induction of dietary-induced obesity—both cases in which brain NPY levels are low.79 The product 1229U91 has high affinity for NPY Y1 and Y4 receptors and acts as an antagonist of the former and as an agonist at the latter.80 Furthermore, 1229U91 has also been shown to interact with high affinity to the NPFF(2) receptor, a neural substrate related to NPY that may also be involved in feeding.81 These findings should be considered when interpreting the outcome of studies using this molecule.

Other non-selective NPY antagonists such as GI 264879A and WRY amide have also been shown to decrease food intake.18,19 However, since the existence of possible non-NPY actions of these peptides have not been systematically evaluated the results of these studies also need to be interpreted with caution.

An anti-NPY monoclonal antibody has been prepared with cytotoxic molecules attached (ricin A chain and monensin) with the intention of destroying NPY neurons. This modified antibody has been shown to reduce both fasting-induced food intake and spontaneous food intake for 10 days after direct injection into the arcuate nucleus.15

Does endogenous NPY play an important role in the control of food intake?

In view of the discussion above what can be said regarding the role of endogenous NPY in the control of food intake? Firstly, following energy depletion and repletion there is an appropriate change in brain NPY levels and NPY receptors in many of the brain areas activated by exogenous NPY. In addition, a good correlation exists between changes in NPY expression and food intake. These positive observations are supplemented by the ability of anti-NPY antibodies, NPY antisense, immunotoxins and non-selective NPY antagonists to reduce food intake in animal models where brain NPY levels are high. On the other hand, energy balance in NPY knockout mice appears essentially normal, and there are questions regarding the selectivity of all of the approaches used to inhibit the action of NPY. Furthermore, it remains unknown whether blockade of endogenous NPY affects food intake through the same behavioral mechanisms as those produced by exogenous NPY. Certainly, confidence that endogenous NPY is involved in the control of food intake would be considerably enhanced by the use of specific and selective manipulations that also sought to define the behavioral mechanism(s) by which food intake was changed.

NPY receptor subtypes

How many NPY receptor subtypes?

Genes encoding five NPY receptor subtypes have been identified—NPY Y1, NPY Y2, NPY Y4, NPY Y5 and NPY Y6.22,82,83,84 The NPY Y3 receptor has not yet been cloned but has been identified pharmacologically on the basis of a unique in vivo binding profile.85 The NPY Y6 receptor is a pseudogene in primates86,87 and the encoding gene has not been detected in the rat.88 However, in both the mouse and rabbit the NPY Y6 receptor gene encodes for a functional NPY/PYY receptor.86,89 Comparison of the amino acid sequences of the human (h) NPY hY1, NPY hY4 and NPY hy6 receptors shows 41–48% identity (Table 2). By contrast, the deduced amino acid sequences of the NPY hY2 and NPY hY5 receptors reveal significant divergence from the other NPY subtypes (Table 2). However, despite these structural variations all receptor subtypes bind NPY with high affinity.

Table 2 NPY receptor amino acid identity

Evidence for the existence of other NPY receptor subtypes

Several other NPY receptors have been cloned in sub-mammalian species. For example, NPY zYa, NPY zYb and NPY zYc receptors from the zebra fish,90,91 the NPY Yb receptor from Atlantic cod92 and unclassified NPY receptors from Lymnaea stagnalis93 and Drosophila melanogaster.94 The NPY cod, NPY Yb/zYb and NPY zYc receptors display NPY Y1-like pharmacology while the NPY zYa subtype seems to have a pharmacological profile closer to the NPY Y5 receptor.90 However, amino-acid sequence comparisons and phylogenetic analyses have shown that NPY zYb, zYc and zYa receptors are not simple orthologues of existing mammalian NPY receptors.90,91 In addition, neither the NPY receptors from Lymneae stagnalis nor Drosophila have counterparts in mammals. Therefore, it is possible that additional mammalian NPY receptors remain to be discovered. Recently, an orphan receptor named GPR74 has been identified that binds PYY with high affinity and has 25% identity with the NPY Y1 receptor.95,96 This receptor has been provisionally designated as the ‘NPY Y7 receptor’ subtype, a classification that awaits confirmation from other groups.

Gene structure of human neuropeptide Y receptor subtypes

The NPY Y1 and NPY Y5 receptor genes are both localized on human chromosome 4.97,98,99 The 5-untranslated regions of both the NPY Y1 and NPY Y5 receptor genes are encoded by several alternative 5 exons.100 These exons are transcribed under the control of different promoters that allow for the tissue-specific expression of the receptors.100,101 An important observation is that the human NPY Y1 and NPY Y5 receptor genes are transcribed in opposite directions from a common promoter region.98 Therefore, co-regulatory mechanisms controlling both NPY Y1 and Y5 gene transcription may exist. In favor of this hypothesis is the observation that, in rat brain, NPY Y5 receptor mRNA always coincides with the presence of the receptor, although the converse is not necessarily the case. Consequently, pharmacological treatments that modify NPY Y1 receptor expression could also affect that of the NPY Y5 receptor and vice versa.

Evidence for obesity linkage to NPY receptor subtypes.

Polymorphisms in existing NPY receptor subtypes have been evaluated for linkage to the obese state. In most studies, no linkage between receptor mutations and obesity have been found.102,103,104 For example, neither the polymorphisms Pstl (NPY Y1/Y5)101 and Glu-4-Ala (NPY Y5)104 nor the silent polymorphism Gly-426-Gly (NPY Y5)103 have associations with extremes of body weight. Conversely in a recent study, three novel single nucleotide polymorphisms (P1, P2 and P3) located in the NPY Y5 receptor gene non-coding region had an association with extreme obesity in Pima Indians.105 Although these studies suggest that the NPY Y5 receptor may be linked to some forms of obesity, it remains to be determined whether these mutations have functional consequences, are markers for another obesity locus, or have relevance to human obesity in other populations.

Distribution of NPY receptor subtypes in the brain

NPY receptors are distributed widely throughout the brain. Autoradiographic studies using the non-selective ligand 125I-PYY have revealed the presence of NPY receptors in over 100 different regions from all levels of the brain and spinal cord.106 The fact that these receptors exist as five functional subtypes raises the possibility that one exists only to mediate the effect of NPY on food intake. In this ideal but highly unlikely situation, a clear separation of the actions of NPY on food intake from all other central effects of the peptide could then be obtained (Figure 1). The possibility of making a ‘clean’ drug is an important consideration since regulatory authorities are unlikely to accept major behavioral side effects in an anti-obesity drug.107

One approach to understanding the role of a particular NPY receptor subtype in the control of food intake would be to study its distribution within the brain. The receptor subtype whose distribution best matched the brain areas acted on by NPY to control of food intake (see above) would then be an ideal candidate for drug discovery. One obvious limitation to this approach is the fact that the brain areas utilized by NPY in the control of food intake are not well understood. Furthermore, mRNA's encoding for the NPY Y1, NPY Y2, NPY Y4 and NPY Y5 receptor subtypes all appear to be very widely distributed in rat brain—both in areas known to be involved in the control of food intake and in others that are not (Table 3).108,109,110,111,112,113,114,115 Furthermore, there is uncertainty whether the encoding mRNA accurately represents either the location or degree of expression of the receptor protein, for the following reasons. Firstly, although the receptor protein and the encoding mRNA mostly overlap, they do not coincide perfectly.109,111 Therefore, the level of expression of the mRNA and the protein in a given location could well be markedly different.111 In rat arcuate nucleus, for example, mRNA encoding the NPY Y1 receptor is strongly expressed, while the NPY Y1 protein, revealed either with radiolabelled agonists and antagonists or by immunohistochemistry, is present only at low concentrations.112,113,114 Secondly, the regional distribution and expression of each NPY receptor subtype appears to be species-dependent.110,111,115 Finally, specific tools, such as radiolabeled non-peptide ligands, are available at present only for the NPY Y1 receptor subtype.112 The distribution of this receptor can therefore be mapped with reasonable accuracy, but accurate mapping of other NPY receptor subtypes awaits the use of highly selective ligands.

Table 3 NPY receptor subtype mRNA distribution in rat brain

Additional effort is clearly needed in this area to define more precisely the anatomical distribution of each NPY receptor subtype within the brain. Currently, our knowledge can point mainly to potential side effects. For example, the presence of NPY Y1 receptors in the amygdala which may be linked to anxiety and NPY Y5 receptors in the hippocampus which are implicated in epilepsy.116,117

NPY receptor subtypes in the control of food intake

The NPY Y5 receptor will be considered first, since this subtype has been designated the ‘feeding’ receptor. However, is there any solid evidence to support this assumption?

The NPY Y5 receptor

Amino-acid substitutions within the NPY molecule produce analogs which bind to the various receptor subtypes with different affinities. When the affinity of these modified peptides for the rat NPY Y5 receptor is compared with their ability to stimulate food intake, a significant positive correlation emerges, with those peptides having the highest affinity for the NPY Y5 receptor appearing to produce greater stimulation of food intake (Figure 3).118 The activity of four NPY analogs [K4, RYYSA19-23]PP2-36; [Ala31, Aib32]pNPY; [DTrp34]hNPY and [cPP1-7, NPY19-23, Ala31, Aib32, Gln34]hPP are of considerable interest since they have been shown to have both high affinity and selectivity for the rat NPY Y5 receptor (Table 4) and all potently stimulate food intake.119,120,121

Figure 3
figure3

Correlation between in vivo potency (ED50 in nmol) and in vitro affinity (IC50 in nmol) for the NPY Y1, Y2, Y3, Y5 receptor subtypes. Reprinted from Wyss et al.118 Regulatory Peptides 1998; 75–76: 363–371, with permission from Elsevier Science.

Table 4 Affinity of NPY peptide analogs for NPY receptor subtypes

Taken together, the above studies show that single injections of NPY Y5 selective agonists can stimulate food intake for times up to 24 h. However, it is a characteristic of G-protein coupled receptors that they down regulate with long-term stimulation. The results in Figure 4 show that the continuous infusion of hPP (a NPY Y5 and NPY Y4 agonist) into the brain of free-feeding rats results in a continuous hyperphagia which is associated with an increase in body weight. It is unlikely that the effects of hPP in these experiments are due to NPY Y4 receptor stimulation since this receptor subtype does not appear to be correlated with food intake (Figure 3).118 These new findings indicate that, at least over the period of study, stimulation of the NPY Y5 receptor subtype leads to maintained food intake without evidence of tolerance.

Figure 4
figure4

Effect of chronic infusion of hPP on food intake and body weight. In these studies, normal Sprague–Dawley rats were prepared with a chronic indwelling catheter implanted into the right lateral cerebroventricle. The cannula was connected to an Alzet osmotic minipump located beneath the skin of the back which dispensed hPP (11 µg/24 h) for 14 days. The cannula was implanted on day 0. After a 7 day recovery period, the infusion of hPP was started and continued until day 14.

The available evidence strongly suggests that stimulation of the NPY Y5 receptor with exogenous peptides increases both short- and long-term food intake. This then raises the question as to whether food intake changes when the action of endogenous NPY on the NPY Y5 receptor is prevented. This answer is crucial for drug discovery, since selective inactivation of the NPY Y5 receptor still leaves all other NPY receptor subtypes implicated in the control of food intake open to the peptide (Figure 5). The following section of the review considers the evidence both for and against a physiologically important role for NPY acting through the NPY Y5 receptor in the control of food intake.

Figure 5
figure5

Possible effects of NPY receptor stimulation on food intake. In the hypothalamus, release of NPY from the arcuate nucleus leads to stimulation of NPY receptors in the paraventricular nucleus. The NPY Y1, Y2 and Y5 receptors have both pre- and postsynaptic locations. Blockade of the NPY Y5 receptor, for example, leaves the NPY Y1 and the NPY Y2 receptors open to stimulation. NPY may also activate NPY receptors in other areas of the brain involved in the control of food intake, for example the cortex, hippocampus and hind brain. NPY receptor antagonists may (but should not) have significant interaction with non-NPY receptors (×).

Knockout of the NPY Y5 receptor

Knockout of the NPY Y5 receptor subtype produces a mouse of essentially normal phenotype when young; it grows and feeds normally and has normal feeding responses to both fasting and to centrally administered leptin.122 In these respects it resembles the NPY knockout mouse described above. Interestingly, with age the NPY Y5 receptor knockout mouse becomes hyperphagic and mildly obese. Since neither of these findings are in agreement with a role for the NPY Y5 receptor in mediating NPYs' stimulation of feeding, how can they be reconciled? Two main explanations have been suggested. The initially normal phenotype may be because the NPY Y5 receptor subtype is not a critical feeding receptor; in its absence, food intake is maintained by the action of NPY on other receptor subtypes such as the NPY Y1 receptor for instance (Figure 5). Alternatively, during development the lack of the NPY acting through the NPY Y5 receptor may be compensated for completely by other neural pathways as described above. The late developing obesity has been explained by postulating a presynaptic location for the NPY Y5 receptor. In this position the NPY Y5 subtype could act as an autoreceptor negatively inhibiting the release of NPY. Its absence in the NPY Y5 knockout would then leave the release of NPY unopposed—leading to overproduction of the peptide and to increased food intake (Figure 5). Indeed, there is some evidence for the presence of a presynaptically located NPY Y5 receptor, at least in the subiculum of the rat brain.123

Inhibition of the production of the NPY Y5 receptor with antisense oligodeoxynucleotides (ODNs)

The physiological role of endogenous NPY acting through the NPY Y5 receptor has also been investigated using the antisense approach. Repeated injections of high doses of antisense ODNs to the NPY Y5 receptor over a 2 day period has been shown to reduce NPY-induced food intake, spontaneous food intake and to inhibit the refeeding response to an overnight fast in rats.124,125,126 These data obtained using antisense ODNs in general contradict the conclusion of the knockout mouse that the NPY Y5 receptor has no involvement in the control of fasting-induced and spontaneous food intake. Interestingly, in one of the above studies antisense ODNs directed against the NPY Y5 receptor did not affect either the microstructure of food intake or the food intake response to galanin, indicating some specificity of this approach.124 In general, however, concerns about selectivity and toxicity have not been rigorously addressed. Another criticism leveled at the above studies is their failure to determine whether the antisense molecules actually entered their target cells to reduce NPY Y5 receptor density. In addition, the effect of the antisense molecules on food intake was in some cases faster than the 60–96 h turnover expected for G-protein-coupled receptors, further adding to the problems in data interpretation.

Because of these criticisms, the studies with antisense ODNs that point to a role of the NPY Y5 receptor in the physiological control of food intake need to be verified using another approach.

Inhibition of the NPY Y5 receptor with non-peptide antagonists

This is currently an area of intense interest to pharmaceutical companies, who would view a selective NPY Y5 antagonist that safely reduced food intake as a potential blockbuster anti-obesity drug. Since pharmaceutical companies closely guard the identity and activity of proprietary compounds, it is perhaps understandable why little of real relevance has been published in this area. At the time of writing, many different compounds from a variety of different chemical series had been described as NPY Y5 antagonists.127,128,129,130,131,132,133 The activity of only a few of these compounds has been described in detail, although more information is now starting to appear in the literature. The following examples illustrate the current problems facing drug discovery in this area.

CGP 71683A—an example of an active NPY Y5 receptor antagonist

Perhaps the most extensively studied of the known NPY Y5 receptor antagonists is CGP 71683A (Figure 6).134 This product has very high affinity for the NPY Y5 receptor and very low affinity for the NPY Y1, Y2 and Y4 receptor subtypes (Table 5). In LMTK mouse fibroblasts expressing the NPY Y5 receptor CGP 71683A attenuates NPY-induced intracellular calcium transients, thus attesting to its antagonistic properties. At first sight, the effects of this compound appear very promising since after intraperitoneal administration CGP 71683A strongly inhibits NPY-induced food intake. Furthermore, in both fasted and streptozotocin diabetic rats, animal models associated with elevated activity of hypothalamic NPY neurons, CGP 71683A significantly suppressed the attendant hyperphagia.

Figure 6
figure6

Molecular structure of some currently known non-peptide Y receptor antagonists.

Table 5 Specificity of selected NPY receptor antagonists

Non-selectivity and non-overt toxicity

To investigate its cross-reactivity, CGP 71683A has recently been studied in a series of both NPY and non-NPY receptor binding assays (Table 5).135 Surprisingly, CGP 71683A was found to have equally high affinity for the serotonin reuptake recognition site and for cholinergic muscarinic receptors in the rat brain as for the NPY Y5 receptor. Reasonably high affinity for α2-adrenergic receptors was also observed—a finding in agreement with the original published studies on this compound.134 Since increased serotonin as well as changes in muscarinic and α-adrenergic receptor activity can affect food intake, these observations call into doubt the conclusion that the hypophagia observed with CGP 71683A is due only to inhibition of NPY Y5 receptors.136,137,138 In the original description of this compound, CGP 71683A did not appear to be overtly toxic and did not change the microstructure of feeding behavior, induce significant taste aversion or produce anxiety.134,139 However, when injected into the brain, CGP 71683A induced a dose-dependent inflammatory response that appeared to correlate with the fall in food intake.135 Inflammatory mediators have been shown to negatively affect food intake and could be a further explanation for the decreased appetite observed with this compound.140 In accordance with the above studies, CGP 71683A has recently been shown to reduce food intake equally in both wild type and in NPY-knockout mice.64 This latter result confirms that the activity of CGP 71683A resides in a mechanism or mechanisms of action other than NPY Y5 receptor blockade.

Other examples of active NPY Y5 receptor antagonists

Both of the compounds Banyu X and Novartis 10 have recently appeared in the patent literature as NPY Y5 receptor antagonists (Figure 6).141,142 Both products decrease fasting-induced food intake after a single intraperitoneal injection (Table 5), consistent with the suggested role of the NPY Y5 receptor in the control of food intake. However, Banyu X (despite having a different chemical structure from CGP 71683A) also had significant cross-reactivity for the serotonin reuptake site, the norepinephrine uptake site and for muscarinic receptors (Table 5). In our hands, Novartis 10, produced a strong conditioned taste aversion when given at a dose that also lowered food intake. Thus, many of the products reported in the literature have been shown to affect food intake, but all have the potential to do so by mechanisms that are additional or even alternative to NPY Y5 receptor blockade.

L 152804—an example of a non-active NPY Y5 receptor antagonist

L 152804 is a potent and selective NPY Y5 receptor antagonist which, after oral administration, enters the hypothalamus in concentrations high enough to block the NPY Y5 receptor (Figure 6).143 L 152804 appears to bind specifically to the NPY Y5 receptor subtype since it has no significant affinity for over 120 different binding assays and seven enzyme assays. When injected into the brain or administered orally, L 152804 blocked the increase in food intake produced by the NPY Y5 selective agonist bPP but not the increase in food intake produced by NPY. Finally, when administered orally to Zucker fa/fa rats and to db/db mice, L 152804 did not affect spontaneous food intake.144 Recently, other potent and highly selective NPY Y5 antagonists have been described, which enter the brain in high concentrations.145,146 However, like L 152804, all of these compounds are ineffective at reducing food intake after acute oral administration to both spontaneously feeding and food deprived rodents as well as to ob/ob mice.

The results presented in this section show definitively that stimulation of the NPY Y5 receptor, either acutely or chronically, can enhance food intake. It is also possible to block the effects of ligands acting at the NPY Y5 receptor with selective antagonists. However, it is less certain that the action of endogenous NPY acting through this receptor plays an indispensable role in the maintenance of food intake. Therefore the NPY Y5 receptor subtype may not be a target for drug discovery (ie may not be ‘drugable’). However, NPY acting through the NPY Y5 receptor may well play a less critical role in the control of food intake, but after blockade its role is immediately taken over by NPY acting through another NPY receptor. Furthermore, as pointed out several times in this review, appetite is a very complex motivated behavior and further investigation may reveal a function of the NPY Y5 receptor subtype in an aspect of acute and perhaps long-term food intake. Clearly extensive studies of NPY Y5 antagonists in a variety of different animal models, both alone and in combination with antagonists of other NPY receptors, are now needed to define the role of NPY acting through this receptor subtype in the control of food intake.

The NPY Y2 receptor

Although a significant positive correlation exists between the affinity of modified NPY peptides for the rat NPY Y5 receptor subtype and their ability to stimulate food intake, no such correlation exists for the NPY Y2 receptor (Figure 3).118 Indeed, an analog of NPY (NPY13-36) with some selectivity for the NPY Y2 receptor subtype (Table 4) has very little, if any, effect on food intake when injected into the brain.147 The recent discovery of cyclo S-S [Cys20, Cys24]pNPY—a highly selective ligand of the NPY Y2 receptor may allow further study of the role of this subtype in the control of food intake (Table 4).148

Knockout of the NPY Y2 receptor

Knockout of the NPY Y2 receptor produces an animal which is both hyperphagic and obese.149 The exact mechanism or mechanisms underlying the phenotype of these animals is unclear at the present time. However, evidence is accumulating to support the idea that the NPY Y2 subtype has a presynaptic location and may act to inhibit NPY release from hypothalamic neurons.150 Thus the simplest explanation of the observed phenotype is that chronically increased NPY release after knockout of the NPY Y2 receptor is the cause of the hyperphagia and obesity of these animals (Figure 5). Furthermore, since NPY acting through the NPY Y2 receptor also controls the presynaptic release of catecholamines, increased monoamine release may also contribute to the changes in food intake and energy expenditure observed in the NPY Y2 knockout model.

Acute injection of NPY into the cerebral ventricles has been shown to induce a similar increase in food intake in both wild-type and NPY Y2 receptor knockout mice. These observations confirm that the NPY Y2 receptor is not a critical component for the expression of NPY-induced food intake. Interestingly, NPY Y2 receptor knockout mice have a normal feeding response to acute starvation.149 This is further evidence supporting the suggestion that the NPY Y2 receptor subtype does not modify the acute feeding response—in this case to endogenous NPY.

Inhibition of the NPY Y2 receptor with non-peptide antagonists

Recently, a non-peptide NPY Y2 receptor antagonist, BIIE0246 has been described as a selective high affinity antagonist of the NPY Y2 receptor.151 This compound also has significant affinity for muscarinic receptors (Table 5). If confirmed, these findings may make BIIE0246 a questionable tool to investigate the role of the NPY Y2 receptor in the control of food intake.

The NPY Y4 receptor

As with the NPY Y2 receptor, no correlation exists between NPY Y4 receptor affinity and food intake for a wide range of NPY peptide analogs (Figure 3).118 Rat pancreatic polypeptide (PP) which has very high affinity and reasonably good selectivity for the NPY Y4 receptor has little or no effect on food intake in rats.118,147 In addition, the NPY Y4 receptor has the lowest level of expression in rat brain when compared to other NPY receptor subtypes.110

The NPY Y1 receptor

Stimulation of the NPY Y1 receptor

The NPY Y1 receptor is pharmacologically characterized by high affinity for NPY and PYY and progressively lower affinity for the N-terminally truncated analogs: NPY2-36, NPY3-36, NPY13-36 and PP.118 The first apparently selective NPY Y1 receptor agonist was created by replacing both Ile31 and Glu34 in porcine NPY with the corresponding residues from hPP. Subsequently, only the Pro34 substitution was found necessary for Y1 selectivity (Y1 vs Y2). Use of these tools—[Leu31, Pro34] NPY and [Pro34] NPY—in rodents has shown that both strongly stimulate food intake.118,147 Although both have recently been shown to have significant affinity for both the NPY Y4 and the Y5 receptor subtypes118 their in vitro profiles on cloned Y-receptors (Y1, Y2, Y4 and Y5) based on negative coupling to cAMP appears to show some functional NPY Y1 selectivity.118,152 However, recently described NPY ligands with improved selectivity for the NPY Y1 receptor, such as c[D-Cys29-L-Cys34]NPY and in particular [Phe7, Pro34]pNPY and [Arg6, Pro34]pNPY should prove very useful for further elucidating the effect of this receptor subtype in the control of food intake (Table 4).148,153

Interestingly, when the affinity of modified NPY peptides for the NPY Y1 receptor is compared with their ability to stimulate food intake after i.c.v. administration, a significant positive correlation emerges (Figure 3).118 However, this correlation is not as tight as for the NPY Y5 receptor and other approaches have been used to examine the role of the NPY Y1 receptor in controlling food intake.

Knockout of the NPY Y1 receptor

Compared with controls, the feeding response of the NPY Y1 receptor knockout mouse to acute i.c.v. bolus injections of hNPY is reduced, as are the responses to hPYY3-36 as well as to h, b and rPP.154 That a residual component to the feeding response of some of these peptides remains is not surprising, since none is totally selective for the mouse NPY Y1 receptor.154 Thus it is entirely possible that each of these peptides had sufficient interaction with the remaining NPY receptor subtypes to maintain a partial feeding response (Figure 5). However, the observation that after knockout the feeding response to NPY analogs is attenuated provides evidence that stimulation of the NPY Y1 receptor subtype may be involved in the control of food intake.

The experiments described above demonstrate that blockade of the NPY Y1 receptor can inhibit NPY-induced food intake. Seemingly in line with these conclusions is the observation that knockout of the NPY Y1 subtype produces an animal with slightly reduced food intake and a significantly reduced refeeding response to short-term fasting.155 Paradoxically, NPY Y1 receptor knockout animals also have increased body fat and are hyperinsulinemic.155,156 The increased fat mass is thought to be due to decreased activity-associated thermogenesis and/or the concurrent hyperinsulinemia.156 Hyperinsulinemia could also be the reason underlying the decreased food intake observed in these animals.157

Inhibition of NPY Y1 receptor production with antisense oligodeoxynucleotides (ODNs)

The data obtained from the use of this approach have been conflicting, with antisense ODNs directed against the NPY Y1 receptor either increasing, decreasing or producing no change in food intake.158,159,160,161 However, a paradoxical increase in food intake has been the consistent finding in the majority of studies utilizing this approach.158,159,160

Inhibition of the NPY Y1 receptor with non-peptide antagonists

Recently, several non-peptide antagonists of the NPY Y1 receptor have been synthesized. Of these, BIBP3226 was the first highly-potent, high affinity NPY Y1 antagonist that had low affinity for other NPY Y receptor subtypes.162,163 After administration into the brain, this compound either partially or completely reversed the increase in food intake produced by NPY, [Leu31, Pro34] NPY and hPYY3-36. BIBP3226 and also decreased the refeeding response to short-term fasting and the intake of highly palatable food.164 However, BIBP3226 has also been shown to produce an anxiety-like state—presumably linked to blockade of NPY Y1 receptors in the amygdala and induced other behaviors such as barrel rolling and catatonia.162,165 Thus, the conclusion that the changes in food intake produced by BIBP3226 were behavioral may not be correct. Complicating matters further, BIBP3226 has recently been shown to act as an antagonist of the NPFF(2) receptor, which is also implicated in feeding modulation.81 Thus, BIBP3226 may affect food intake by mechanisms both related and unrelated to NPY Y1 receptor inhibition.162

Recently, BIBO3304, a derivative of BIBP3226, has been synthesized (Figure 6, Table 5), and has high affinity for the NPY Y1 receptor and very low affinity for the NPY Y2, NPY Y4 and NPY Y5 receptors.166 To avoid causing an anxiety-like state by blockade of NPY Y1 receptors in the amygdala, BIBO3304 has been injected directly into the PVN, and there inhibited both the refeeding response to fasting and the increase in food intake produced by NPY, [Leu31, Pro34]NPY, NPY2-36 and NPY3-36.166 The fact that BIBO3304 diminished the feeding response to peptide agonists with affinity towards both the NPY Y1 and the NPY Y5 receptor subtype has been taken as in vivo evidence for interplay between these two receptor subtypes in the control of food intake.166 A similar decrease in the agonist response to supposedly NPY Y5 selective analogs such as hPP is also observed in the NPY Y1-knockout mouse.154 An indication of the improved specificity over BIBP3226 is provided by the observations that the increase in food intake produced either by galanin or by norepinephrine is unaffected by BIBO3304.166

Other NPY Y1 receptor antagonists have been synthesized with oral activity. For example, J-104870, a compound with high affinity for the hNPY Y1 receptor and very low affinity for the hNPY Y2, hNPY Y4 and hNPY Y5 receptors, has been reported (Figure 6).167 Relatively high doses of this product were shown to block the increase in food intake produced by concurrent injection of NPY into the lateral ventricle of satiated normal rats.167 Spontaneous food intake in Zucker fa/fa rats can be decreased by administering high doses of J-104870 into the lateral ventricle (200 µg) or after oral administration at 100 mg/kg.167

Another potent and selective NPY Y1 antagonist from the same group, J-115814, has recently been shown to decrease food intake in db/db and normal mice but not in NPY Y1-knockout mice.144,168 This latter observation is very important since in general a systematic evaluation of the cross-reactivity of each compound with non-NPY receptors has not usually been performed. The potential of individual compounds to produce changes in food intake by non-specific mechanisms is clearly illustrated by the example of AMG 68, an NPY Y1 receptor antagonist, which paradoxically lowers food intake in the NPY knockout mouse.64

In conclusion, evidence exists to suggest that NPY acting through the NPY Y1 receptor is important for modulating short-term food intake—for example, after fasting and in monogenetic models of obesity. Nonetheless, concerns must remain about the ability of NPY Y1 receptor antagonists to reduce food intake without eliciting side effects such as increased anxiety or changed blood pressure.

Conclusions

We have attempted to assess critically the available evidence that blockade of the actions of NPY represents an attractive new drug discovery target.

Current drug discovery efforts have produced a number of highly selective NPY receptor antagonists. These have been used to establish the NPY Y1 subtype as apparently the most critical in regulating short-term food intake. However, additional work is needed to clarify the role of the NPY Y2 and NPY Y5 receptors in mediating the actions of NPY on food intake and will need to be performed using antagonists possessing the properties outlined in Table 6. It may turn out that combinations of NPY receptor antagonists represent the best approach to the modulation of the complex sequence of physiological and behavioral events that underlie normal and disordered appetite.169 Clearly, care will also have to be exercised in the extrapolation of animal data using NPY antagonists to humans, where each NPY receptor subtype may have a different distribution and function. Therefore, the final answer as to whether any NPY receptor antagonist will be useful in the control of food intake, without producing unacceptable side effects, can only be definitively answered by clinical studies in man.

Table 6 Criteria for accepting changes in food intake as due to NPY Yx receptor blockade

Finally, will a drug based on blockade of NPY receptor subtypes eventually produce an attractive appetite suppressant? Only time can answer this question, but any new drug that has the potential both to decrease food intake and increase sexual appetite16,31 would seem to be worth the effort.

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Chamorro, S., Della-Zuana, O., Fauchère, J. et al. Appetite suppression based on selective inhibition of NPY receptors. Int J Obes 26, 281–298 (2002). https://doi.org/10.1038/sj.ijo.0801948

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Keywords

  • neuropeptide Y
  • food intake
  • reward; NPY receptor knockout
  • NPY receptor antagonists
  • NPY antisense oligodeoxynucleotides
  • NPY antibodies

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