D Patel, N Patel
metabolic disorders, neuropeptide y, npy y1 and y5 receptor, obesity
D Patel, N Patel. Review Of NPY And NPY Receptor For Obesity. The Internet Journal of Pharmacology. 2009 Volume 8 Number 2.
The prevalence of obesity continues to increase throughout the world and the burden of obesity and related co morbidities is large. However existing drug therapies for obesity are limited and agents with high efficacy, safety and tolerability are expected to meet patient needs and lead to more substantial commercial success. Contemporary consideration has focused on physiology of neuropeptides Y (NPY) and its role in the regulation of energy homeostasis. NPY stimulates food intake, inhibits energy expenditure, and increases body weight and increases anabolic hormone level by activating the NPY Y1 and Y5 receptors in the hypothalamus. Based on these findings, several NPY Y1 and Y5 receptor antagonists have been developed in last two decade as potential anti-obesity agents and transgenic mice model lacking NPY, the NPY Y1 receptor or the NPY Y5 receptor have been generated. The data obtained to date with these newly developed tools suggests that NPY receptor antagonists, particularly NPY Y1 and Y5 receptor antagonist, have potentiality to bless the obesity patients worldwide. However, the redundancy of the neurochemical systems regulating energy homeostasis may limit the effect of ablating a single pathway. In addition, patients in whom the starvation response is activated, such as formerly obese patients who have lost weight or patients with complete or partial leptin deficiency, may be the best candidates for treatment with a NPY receptor antagonist. New leads are under research by major pharmaceutical companies to limit the side effects and explore the area to meet clinical requirement.
Obesity is defined as excess of adiposity for a given body size and results from a chronic imbalance between energy intake and energy expenditure, become the catastrophic illness to increase metabolic complication throughout the world like increase risk of diabetes, hypertension, dyslipidemias, cardiovascular disease, gallstone, osteoarthritis, and certain forms of cancer and reduce the life expectancy. The incidence of obesity and overweight individual continues to increase dramatically . Although large pools of scientific force work for regulation of food intake, an understanding of the neurophysiology of feeding behavior and energy expenditure in human still limitedly explored. Contemporary approaches are focused on decreasing caloric intake, either by appetite suppression or decreased nutrient absorption. However decrease in caloric intake is reported to result in a compensatory decrease in metabolic rate, which makes it difficult for patients to maintain weight loss for long period . Therefore optimal therapeutic approach to the treatment of obesity is expected to involve both reducing caloric intake and increasing energy expenditure. Based on the aforementioned, much new class of antiobesity compounds have been investigated. Some of them are in the early stages of development, and the practically non-existence of efficient antiobesity drugs convert the search for pharmacological treatment into a target of notable interest.
Neuropeptide Y (NPY) is a neuropeptide made up of 36 amino acids with an amide in carboxy terminal position (pancreatic polypeptide family) originally discovered in extracts of porcine brain, [3, 4] found in abundance in the central and peripheral nervous system, whose alterations provoke eating disorders (obesity, bulimia and anorexia nervosa), emotional disorders (depression and anxiety), cardiovascular disorders (hypertension), diabetes and other diseases. It is assumed that NPY regulates a variety of physiological processes, including vasoconstriction, nasal congestion, blood pressure, intestinal motility, anxiety, depression, pain, feeding, reproductive endocrinology, neuronal excitability and memory retention. For this reason it is thought that NPY receptor-specific ligands may ultimately have value in several therapeutic areas including the treatment of obesity. NPY has seen implicated in a wide variety of physiological effects involved in the regulation of blood pressure, anxiety, reproduction, thermoregulation, circadian rhythms, control of food intake, and others; hence, NPY and its receptors have sparked a great deal of basic research and drug development interest.  Of these, one of the most remarkable effects of NPY, and the one which has stimulated the most interest in the scientific community, is the strong orexigenic effect induced by injections of this peptide into the hypothalamus or cerebral ventricles of animals and decrease the energy expenditure of the patient . Indeed, chronic administration of NPY into the brain induces hyperphagia and weight gain. NPY receptors are a family of seven transmembrane G-protein coupled receptors (7t-GPCR), designated collectively as Y receptors that are expressed throughout the central and peripheral nervous systems where they mediate a variety of responses ranging from regulation of metabolism and food intake to regulation of neurotransmitter release in the vasculature and viscera.  Relationship of NPY and its receptors with several pathological disorders includes depression, anxiety related disorders, seizures in epilepsy, and potentiation of vasoconstriction. [8-10] for many years, there has been a debate over which receptor is primarily responsible for antiobesity effect, with evidence pointing to a role for both the Y1 and the Y5 subtypes. [11-13], this review will focus on the role played by NPY and NPY Y1 and Y5 receptors antagonist in the regulation of energy homeostasis.
NPY is a neuropeptide made up of 36 amino acids with an amide (Fig.1) in carboxy terminal position (pancreatic polypeptide family) originally discovered in extracts of porcine brain and distributed throughout the body. In the peripheral nervous system, NPY is located in postganglionic sympathetic neurons, adrenal medulla, enteric neurons, cardiac nonsympathetic neurons, certain noradrenergic perivascular neurons and parasympathetic neurons. In sympathetic neurons and adrenal medulla, the peptide is colocalized with the classical sympathetic neurotransmitter noradrenaline. In the brain, NPY containing neuronal cell bodies are found primarily in the locus coeruleus, the nucleus of the solitary tract and the arcuate nucleus of the hypothalamus. In addition, these NPY containing neuronal cell bodies often contain other neurotransmitters, such as noradrenaline, and send projections throughout the brain; hence, NPY can be found in most brain regions, particularly in the cortex, hippocampus, thalamus, hypothalamus and brainstem.  The orexigenic effects of NPY are mediated through the hypothalamus, while the anxiolytic effects of NPY appear to be mediated through the amygdala. Intra amygdalar NPY administration in satiated rats did not change total caloric intake, but it did produce a dose-dependent decrease in intake of and preference for high fat diet relative to low fat diet over 24 h. This neuropeptide is located in the cerebral cortex, ventral and dorsal striatum, and various limbic structures, including the hypothalamus and amygdala [15, 16]. NPY gene expression and peptide levels are increased in the Para ventricular and the arcuate nucleus following exposure to a high carbohydrate diet as compared with a high fat diet . The amygdala receives dense NPY innervations from the nucleus of the solitary tract, arcuate nucleus, and lateral septum  and contains a moderate amount of NPY Y1 receptors, which are important for the anxiolytic effects of NPY . The amygdala is also involved in feeding behavior . Lesions of the amygdala, particularly the posterodorsal region, have been shown to produce obesity and increase preference for a high carbohydrate diet, even in rats that had previously preferred a high fat diet [20, 21], and a number of neuropeptides have been shown to alter food intake following administration into the amygdala. Intra-amygdalar NPY administration have significantly increased the carbohydrate diet and less fat intake when rats were given a dietary choice, as has been reported for intra-PVN administration of NPY.
Energy homeostasis is the process by which adipose tissue, stored energy, is kept constant over time. Obviously, feeding behavior plays a crucial role in energy homeostasis, because to maintain a constant energy balance, energy (food) intake should match energy expenditure . Feeding behavior is a tightly regulated phenomenon, involving several factors for signaling the body needs in energy to the CNS, particularly to the hypothalamus. The hypothalamus integrates neuronal, metabolic and endocrine signals and stimulates different effectors pathways for activation of behavioral responses and neuroendocrine axes. It is certainly overly simplified to reduce the regulation of food intake to a series of cross-talks between certain hypothalamic circuitries that would control body fat mass. However, a variety of studies indicate that body weight is indeed controlled by genetic factors. Along these lines, the identification of several genes that are responsible for a change in body weight in rodents suggests that obesity is not solely due to a lack of discipline . In fact, a large number of molecules have been implicated in the control of food intake. In this complex network of pathways, one can distinguish those that provide orexigenic signals from those emitting anorexigenic signals.
AC: adenylate cyclase; ATP: adenosine triphosphate; cAMP: 3`, 5`-cyclic adenosine monophosphate; PLC: phospholipase C; PIP2: phosphatidyl inositol 4,5-diphosphate; IP3: inositol 1,4,5-triphospate; DAG: diacylglycerol; ROCC: receptor-operated calcium channel
NPY receptors are seven-transmembrane domain receptors that belong to the G-protein-coupled receptor family. After agonist stimulation, the inactivated GDP-bound proteins exchange GDP for GTP on the G
NPY receptors are a family of seven transmembrane G-protein coupled receptors, designated collectively as Y receptors that are expressed throughout the central and peripheral nervous systems where they mediate a variety of responses ranging from regulation of metabolism and food intake to regulation of neurotransmitter release in the vasculature and viscera. . The nomenclature reflects the large number of tyrosine (Y in the single letter code) present in their endogenous ligands, NPY, and the hormonal peptides, PYY and pancreatic polypeptide . NPY, peptide YY and pancreatic polypeptide elicit their physiological effects by interacting with at least six distinct G protein-coupled receptors designated Y1, Y2, Y3, Y4, Y5 and Y6 . With the exception of the NPY Y3 receptor, genes and/or cDNAs encoding each of these NPY receptors have been cloned. In contrast to other families of G protein-coupled receptors, the NPY receptors share only modest primary sequence homology (30–50%). In fact, some NPY receptors are structurally more related to G protein-coupled receptors outside of the NPY receptor family. The structural differences among NPY receptors is beneficial to drug discovery efforts since compounds with high affinity for a particular NPY receptor are less likely to interact with other NPY receptors. In addition to having distinct amino acid sequences, each of the NPY receptors is characterized by a unique pharmacological profile and distinct tissue localization. The Y1 receptor was cloned and sequenced in 1992, the Y2 receptor and the Y4/PP1 receptor in 1995, and the Y5 receptor in 1996.  The term dNPY receptors have been retained even though NPY is not the preferred endogenous ligand for all Y receptors. . Y1, Y2 and Y5 bind preferentially NPY and PYY, whereas Y4 binds preferentially PP . Y1 and Y5 receptors exhibit similar high affinities for NPY, PYY and [Pro34]-substituted analogs of NPY or PYY (e.g., [Leu31, Pro34] NPY), but can be distinguished by the selective, nonpeptide Y1 antagonist, BIBP 3226 . Y2 receptors exhibit high affinity for NPY, PYY, and long C-terminal fragments of NPY or PYY (e.g., NPY13–36), and are selectively blocked by the non-peptide antagonist, BIIE 0246 . Y2 and Y4 receptors are present only in intestinal circular muscle cells of rabbit and human where they mediate pertussis toxin (PTx)-insensitive contraction. Y1 receptors are present in smooth muscle cells from both layers: the receptors are negatively coupled to adenylyl cyclase, but unlike other Gi-coupled receptors, do not mediate contraction. Thus the efforts have been focused on Y1 or Y5 receptor selective antagonists. They are implicated in several biological roles including vasoconstriction,  learning and memory and energy balance. . It is highly expressed in several regions of the brain and is released into the circulation from neuronal stores in times of stress. In the CNS, NPY has been implicated in feeding, anxiety and depression, endocrine function, and metabolism. . NPY is a powerful stimulant of food intake when administered directly into the hypothalamus.
The Neuropeptide YY1 Receptor
The neuropeptide YY1 receptor was first characterized during the study of vascular muscle contraction effects of NPY.  The NPY Y1 receptor was pharmacologically defined by its high affinity for NPY and [Pro34] - peptide YY and its low affinity for N-terminally truncated NPY analogues (Table 1). The mRNA and receptor binding sites of NPY Y1 receptor is abundantly expressed in many rat and human brain regions, including hypothalamic centers which control energy homeostasis. In contrast, levels of NPY Y1 receptor binding sites are very low throughout most of the human brain, perhaps due to instability of the receptor during the post-mortem interval . In the periphery, NPY Y1 receptor mRNA is expressed primarily in rodent kidney, heart, spleen, skeletal muscle, lung, gastro-intestinal tract and vascular smooth muscle  and in human kidney, heart, lung, colon, testis, adrenal gland, placenta, bone marrow and vascular smooth muscle 
The Neuropeptide YY2 Receptor
The neuropeptide YY2 receptor was also identified in early pharmacological studies as the NPY receptor responsible for regulation of noradrenalin release from sympathetic nerve terminals  The NPY Y2 receptor is pharmacologically unique in that it has high affinity for N-terminally truncated NPY analogues and low affinity for [Pro34] peptide YY (Table 1). The NPY Y2 receptor is localized in several rat and human brain regions, including hypothalamic nuclei regulating energy homeostasis  The NPY Y2 receptor is localized on NPY containing neurons in the brain, suggesting that this receptor is an auto receptor  In the periphery, the NPY Y2 receptor can be pharmacologically detected on the terminals of rat sympathetic, parasympathetic and sensory neurons, again functioning as an auto receptor or heteroreceptor . Negligible levels of NPY Y2 receptor mRNA are generally found in peripheral tissues , although low expression of NPY Y2 receptor mRNA has been reported in human and rat gastro-intestinal tract [49, 50].
The Neuropeptide YY3 Receptor
The neuropeptide YY3 receptor was formerly characterized pharmacologically in bovine adrenal chromaffin cells and has subsequently been found in rat heart, brainstem, hippocampus, colon and lung . The distinguishing pharmacological feature of the Y3 receptor is its much higher affinity for NPY than for peptide YY (Table 1). Although pharmacological evidence supports the existence of the NPY Y3 receptor in rats and cows, there are no reports of this receptor in humans and the receptor has not yet been cloned. Cloning of this receptor will be required to unequivocally validate its existence and determine its physiological role.
The Neuropeptide YY4 Receptor
The neuropeptide YY4 receptor was initially identified by molecular cloning. This receptor is pharmacologically characterized by its high affinity for pancreatic polypeptide and its low affinity for NPY (Table 1) . Therefore, the NPY Y4 receptor is probably a pancreatic polypeptide receptor rather than a NPY receptor. NPY Y4 receptor mRNA is sparsely expressed in brain; expression is seen primarily in the brainstem, but also at low levels in other brain regions such as the hypothalamus A different pattern of peripheral expression is seen in humans, with expression primarily detected in colon, small intestine, prostate and pancreas [56, 57].
The Neuropeptide YY5 Receptor
The neuropeptide YY5 receptor was also initially identified by molecular cloning  The NPY Y5 receptor is pharmacologically distinguished by its high affinity for both N-terminally truncated analogues of NPY and [Pro34] peptide YY (Table 1). In addition, the NPY Y5 receptor has high affinity for human pancreatic polypeptide, but much lower affinity for rat pancreatic polypeptide (Table 1). NPY Y5 receptor mRNA is discretely localized in rat and human brain, primarily in piriform cortex, olfactory tubercle and hypothalamus . NPY Y5 receptor binding sites have also been detected in these regions, although some groups fail to detect NPY Y5 receptor binding in hypothalamus  Interestingly, NPY Y5 receptor mRNA is almost always localized in neurons that also express NPY Y1 receptor mRNA . In the periphery, NPY Y5 receptor mRNA has been detected in rodent testis, spleen, pancreas, gastro-intestinal tract, vascular smooth muscle cells and cardiomyocytes [52, 59]. NPY has also been reported to alter diuresis, natriuresis and plasma glucose levels via NPY Y5 receptor activation in rats . However, when the Y5 receptor was cloned, comparison of the feeding effects of a wider range of agonists suggested that the Y5 rather than the Y1 receptor might be a better candidate 
The Neuropeptide YY6 Receptor
The neuropeptide YY6 receptor was also initially identified by molecular cloning . A functional NPY Y6 receptor gene is found in mice and rabbits, but a NPY Y6 receptor gene has not been detected in rats. The NPY Y6 receptor gene is a non-functional pseudogene in rabbits and primates . The NPY Y6 receptor has a pharmacological profile that is similar to that of the NPY Y1 receptor, but is somewhat more tolerant of N-terminal truncation (Table 1). In mice, NPY Y6 receptor mRNA has been detected in kidney, testis and brain, particularly in the hypothalamus [64, 65]. Although the NPY Y6 receptor clearly does not contribute to the physiological effects of NPY in humans, this receptor must be taken into account when considering physiological effects of NPY in mice.
Peptide YY-Preferring Receptor
A putative NPY receptor known as the peptide YY-preferring receptor has been characterized in several tissues. This receptor has approximately 5- to 10-fold higher affinity for peptide YY than for NPY and was initially found in crypt cells in the epithelium of the rat small intestine . Analysis of ligand affinities for cloned Y receptors expressed in various cell lines indicates that PP is a preferential agonist of Y4 receptors, whereas C-terminal fragments of NPY/PYY are preferential agonists of Y2 receptors, and [Pro34]-substituted analogs of NPY are preferential agonists of Y1 and Y5 receptors (or Y1 receptors in the absence of expressed Y5 receptors) . Recently,  provided convincing evidence that the peptide YY-preferring receptor in rat small intestine is in fact the NPY Y2 receptor. Thus, the peptide YY-preferring receptor can now be equated with the NPYY2 receptor. Inhibition of neurotransmitter release is a prototypical response mediated by Y2 or Y1 receptors in different tissues. Expression of Y receptors in non-neural tissues has rarely been mapped with precision and is usually surmised from complex pharmacological responses, which are usually a compound of nerve-mediated and direct effects 
Peptide YY (PYY) belongs to a family of structurally related peptides, including NPY and pancreatic polypeptide. PYY is a 36-amino acid peptide that is released from the gastrointest¬inal tract postprandially in proportion to the calories ingested, and is particularly released in response to dietary fat. The highest concentrations are found in the distal gut. PYY is released into the circulation, probably under neural control, before nutrients have reached this portion of the gastrointestinal tract. Further release occurs when nutrients do reach the distal gut, and PYY has therefore been proposed as an ‘ileal brake’ on food intake PYY3-36 acts via the presynaptic Y2 receptor in the ARC . It decreases NPY release from static hypothalamic explants and thus acts to decrease food intake. Its effects on the melanocortin system are a matter of ongoing debate. Although PYY increases α -MSH release from hypothalamic explants , other groups have reported that the anorectic effects of PYY are preserved in mice lacking the MC4 receptor . Developmental compensation in the knockout mice might provide an explanation for this apparent discrepancy. Recent work suggests that the integrity of the vagus nerve is also important . PYY, An important observation is that obesity results in impaired release of PYY postprandially. Importantly, how¬ever, the anorectic effects of PYY3-36 are preserved in obese individuals  and PYY3-36 therefore offers a potentially fruitful therapeutic target for the treatment of obesity. Stra¬tegies would include either the administration of exogenous 3-36 or another Y2 receptor agonist, or the augmentation of endogenous PYY release in overweight humans. One obstacle to the development of a PYY-based therapy is the difficulty some researchers have had in reproducing the effects on food intake of peripherally administered PYY3-36 in rodents . The reasons for this are unclear, although stress, such as that induced by handling and injecting, also power¬fully suppresses food intake, and in unacclimatised ani¬mals, the effects of PYY3-36 might therefore be masked. Stephen Bloom’s research group (Imperial College, London, UK) has shown that peptide YY (PYY), an agonist of the NPY2 receptor, is released from the gut after a meal in direct proportion to the calorie content of the meal; intraperitoneal injection of PYY in rats inhibits food intake and reduces weight. “This effect seems to be mediated through the arcuate nucleus: intra-arcuate injection of PYY inhibits food intake in rats, and also inhibits electrical activity of NPY nerve terminals, thus activating adjacent pro-opiomelanocortin neurons that release melanocortin, which decreases appetite” Infusion of normal postprandial concentrations of PYY decreases appetite and reduces food intake by 33% after 24 h .
Obese animals with lesions of the leptin system (ob/ob, db/db mice and fa/ fa rats) have increased prepro-NPY mRNA levels in the arcuate nucleus, increased NPY levels in various hypothalamic sites, and increased NPY release in the paraventricular nucleus. Similarly, a wealth of evidence links NPY with regions of the brain and with other peptides known to play a role in the regulation of feeding. For example, direct administration of NPY into various parts of the hypothalamus is more effective than i.c.v. administration in stimulating feeding . Low levels of leptin in response to fasting activate a subpopulation of NPY neurons that express mRNA for the long form of the leptin receptor . It has been reported that leptin lowers prepro-NPY mRNA levels in the hypothalamus  and reduces the expression of c-fos in NPY-expressing neurons of the arcuate nucleus . However, it appears difficult to detect a reduction in prepro-NPY mRNA expression in response to leptin if expression is already low . There is some evidence that NPY release is low in diet-induced obesity in animals , and if the same occurs in humans, this might limit the utility of drugs that antagonize NPY. Antisense studies have provided some support for a role for Y5 receptors in feeding , but administration of antisense to the Y1 receptor, if anything, tends to increase food intake.
Knockout of Y1 or Y5 receptors did little to enhance the status of either receptor as the NPY feeding receptor: both knockouts tend to obesity rather than leanness and co-expression of the Y5 knockout with the ob gene had no effect on the obesity phenotype. However, on the positive side, the Y1 knockout did display a marked reduction in fast-induced feeding, and both knock-outs caused some reduction in the feeding response to NPY [42; 80].
Thus, the ideal NPY receptor ligand might be one that not only antagonizes both Y1 and Y5 receptors, but also stimulates Y2 receptors, a rather extreme demand for the medicinal chemist. Studies with antagonists also support a role for both Y1 and Y5 receptors. However, a more fundamental question about the importance of the NPY system was prompted by the observations that the NPY peptide and Y1 and Y5 receptor knockout mice are perfectly viable and do not display any profound (body weight-related) phenotype. . In addition, in obese Zucker rats, which are characterized by reduced hypothalamic NPY receptor density, intracerebroventricular injections of a weak but selective NPY5 receptor agonist ([D-Trp32] NPY) did not stimulate feeding whereas it did in lean rats.  When transgenic mice lacking NPY were mated with
Strategy for therapy
First, the peptide should alter food intake without disrupting the normal behavioral satiety sequence of feeding in the rat . It is especially impressive if repeated or continuous administration of the peptide affects body weight. Secondly, the expression of the peptide or its mRNA should change in situations of altered fuel availability or energy balance, including animal models of obesity. Thirdly, histochemical, feeding, or electrophysiological studies should demonstrate interactions of the novel peptide with endocrine or neural pathways, or with peptides that are already believed to regulate energy balance. Fourthly, knockout or over expression of the gene for the peptide or one of its receptors would be expected to affect energy balance. Finally, blockade of the peptide's action using antisense, neutralizing antibodies or receptor antagonists should show that the endogenous peptide plays a role in feeding. More emphasis is given to those peptides that stimulate food intake (orexigenic peptides) than to those that decrease intake (anorectic peptides), since it is easier to identify antagonists of peptide receptors than agonists.
Physiological role of NPY in energy homeostasis
Effect of exogenous NPY on energy intake and energy expenditure
NPY is potent orexigenic peptide identified to date have a variety of behavioral, physiological and endocrine systems that are critical in the modulation of energy homeostasis. Exogenous NPY directly injected to the specific part of brains such as perifornical hypothalamus of rats causes a tremendous increase in food consumption, even under conditions of satiation [75, 85]. Central administration of NPY to rats also decreases energy expenditure by decreasing sympathetic nervous system activity; as a result, thermogenic activity in brown adipose tissue, a key regulator of energy expenditure in rodents, is diminished . By increasing food consumption and decreasing energy expenditure, central administration of NPY results in a state of positive energy balance that will promote weight gain if chronically maintained. Hence, NPY containing neuronal pathways in the hypothalamus are most critical in the regulation of energy homeostasis. Central administration of NPY also induces hyperinsulinemia, hyperglucagonemia, increased plasma non-esterified fatty acids and insulin resistance independent to the hyperphagic effect of NPY . Chronic central administration of NPY to normal rats results in many of the physiological abnormalities observed in the obese state, including hyperphagia, accelerated weight gain, increased adiposity, hypertriglyceridemia, hyperinsulinemia and hypercorticosteronemia . As observed with other obesity models, NPY induced obesity is glucocorticoid dependent, as adrenalectomy prior to chronic central NPY administration prevents this obesity syndrome from developing , and dexamethasone treatment of adrenalectomized rats restores the response to chronic NPY administration. Hence, chronic central administration of NPY results in obesity that is strikingly reminiscent of spontaneous, genetic and diet-induced obesity in rodents and man. Transgenic mice modestly over expressing NPY have significantly increased body weight, plasma glucose and plasma insulin compared to wild type mice when maintained on a palatable high sucrose diet, although not when maintained on a low fat diet . This effect could be partially explained by significant but transient hyperphagia immediately following introduction of the high sucrose diet.
Role of endogenous NPY in the modulation of energy homeostasis
A large body of evidence also suggests that endogenous NPY plays a central role in energy homeostasis. Immunoneutralization of hypothalamic NPY decreases feeding, even in rats deprived of food for 24 h  Administration of NPY antisense oligodeoxynucleotides to the brains of rats leads to the expected decrease in NPY levels in the arcuate nucleus and also significantly reduces natural feeding behavior [81, 92]. Furthermore, administration of certain potent NPY receptor antagonists to rodents decreases food intake and body weight. Consistent with a role in the tonic modulation of energy homeostasis, expression of NPY mRNA and the synthesis of NPY are altered with changes in nutritional state and metabolic need, as well as in a number of genetic and diet-induced models of obesity. Depriving lean rats of food for 48 h leads to a significant increase in NPY mRNA expression in the arcuate nucleus, and an increase in NPY itself in the arcuate nucleus and the paraventricular nucleus . Thus, increased NPY synthesis and release may mediate the hyperphagic response observed after fasting. Streptozotocin induced diabetic rats, a model of insulin dependent diabetes with high metabolic demand, have increased NPY mRNA expression in the arcuate nucleus and increased NPY levels and NPY release in the paraventricular nucleus, which may in part be responsible for the hyperphagia exhibited by these animals . The obese ob/ob transgenic mouse, which lacks the hormone leptin, and the obese db/db mouse and Zucker rat, which do not have functional leptin receptors, both exhibit increased levels of hypothalamic NPY mRNA and peptide . Thus, increased NPY transmission may be partially responsible for the hyperphagia and obesity that are characteristic of these mice. Suffice it to note that there appears to be a dysregulation of the NPY system in animals that are prone to develop obesity under the appropriate dietary conditions . Considering the wealth of data indicating that the NPY system is important in energy homeostasis, results from transgenic mice in which the NPY system has been manipulated have been somewhat paradoxical. Mice that are deficient in NPY have normal food intake and body weight, and display the same hyperphagia as their wild type counterparts after food deprivation . Further studies indicated that plasma corticosterone, insulin, and glucose levels were normal in NPY deficient mice . A lack of NPY also did not affect the development of obesity induced by diet or chemical means . Studies by a separate group, however, demonstrated that NPY deficient mice have decreased food intake after a 24–48 h fast . The conflicting data on the effect of NPY deficiency on energy homeostasis in mice is puzzling in view of the wealth of data supporting a role for the peptide in energy homeostasis. However, the results may suggest that mice readily compensate during development for the absence of a key neurotransmitter. Indeed, NPY deficient mice are more sensitive to the hyperphagic effect of agouti-related protein, a melanocortin receptor antagonist that is co-expressed with NPY in arcuate neurons . Furthermore, many neurotransmitter and neuropeptide knockout mice do not display overt phenotypes; phenotypes are frequently seen only under specific physiological conditions or when the organism is stressed in some way. In this regard, it is interesting to note that the obese phenotype of ob/ob mice is attenuated when NPY is absent, suggesting that NPY is partially responsible for this obesity syndrome . These data also implicate NPY as a downstream mediator of leptin action in the brain (see below). In contrast to the ob/ob mouse, the obesity characteristic of the genetically obese UCP-DTA or Ay mice was not affected when these mice were crossed with NPY deficient mice .
Integration of NPY with other brain pathways regulating energy homeostasis
In addition to NPY, many other neurotransmitter and neuropeptide systems in the brain have been shown to influence energy homeostasis . Consistent with its key role in the regulation of energy homeostasis, NPY has been shown to interact with numerous neurotransmitter and neuropeptide systems that are thought to play a role in this energy homeostasis process. Leptin, a hormone secreted by adipose tissue that is critical in the regulation of body weight, exerts many of its physiological effects on body weight regulation by acting on target neurons in the brain . It is likely that leptin is a key messenger that communicates information about adipose tissue energy stores to the central nervous system and has been shown to decrease NPY mRNA levels in vivo and to decrease NPY release from hypothalamic slices in vitro. Thus, NPY is probably one of the downstream mediators of leptin action in the brain, which is supported by the observation that the obese phenotype of the leptin-deficient ob/ob mouse is partially ameliorated by genetic deletion of NPY . Activation of the melanocortin MC3 and MC4 receptors by melanocortin peptides such as α-melanocyte stimulating hormone (α -MSH) decreases food intake, increases energy expenditure and decreases body weight . Conversely, blockade of these receptors by an endogenous antagonist known as agouti-related peptide (AGRP) has the opposite effect . Furthermore, genetic ablation of the melanocortin MC3 or MC4 receptor has been shown to cause obesity in both mice and, in the case of the melanocortin MC4 receptor, in humans . Interestingly, AGRP is expressed exclusively in NPY containing neurons originating in the arcuate nucleus . Thus, activation of arcuate NPY neurons releases NPY and AGRP, both of which increase food intake and body weight via activation of two distinct pathways. The obvious implication of this finding is that both arcuate NPY /AGRP-containing neurons and arcuate melanocortin containing neurons project to one or more common target neurons that express both NPY receptors (Y1 and/or Y5) and melanocortin receptors (MC3 and/or MC4). Since most arcuate melanocortin-containing neurons also express cocaine and amphetamine-regulated transcript (CART), another peptide that has been implicated in the central regulation of energy homeostasis , these target neurons probably also express CART receptors. Identification of the neurotransmitters engaged by these target neurons may unveil a ‘‘final common pathway’‘ in the regulation of energy homeostasis and would thus suggest very interesting additional basic research and drug development approaches in the field of obesity. It has been suggested and necessitates confirming that these target neurons contain γ-aminobutyric acid (GABA), CART, thyrotropin releasing hormone (TRH), and/or melanin concentrating hormone (MCH) . NPY has also been shown to interact with a number of other hormones, neurotransmitters and neuropeptides that are thought to play a role in the regulation of body weight (e.g., insulin, galanin, GABA, corticotrophin releasing hormone, melanin concentrating hormone, orexin, ghrelin, biogenic amines, opiate peptides) [103, 108]. NPY may also be involved in metabolic control of energy homeostasis by inhibition of fatty acid synthase as evidenced by the decrease in NPY levels . Taken together, these data strongly support a role for NPY in the complex and highly integrated brain neurochemical system that regulates energy homeostasis. Markedly, these manifold systems must interact with one another and the outputs of each of these systems must be highly integrated to achieve tight control of energy homeostasis.
Role of NPY receptor subtypes in the regulation of energy homeostasis
To design potential anti-obesity drugs, it is necessary to identify the NPY receptor subtypes that mediate the ability of NPY to promote positive energy balance and increased body weight. Several lines of experimental evidence now point to a primary role of the NPY Y1 and Y5 receptors in mediating the effects of NPY on energy homeostasis. Central administration of selective NPY Y1 or Y5 receptor peptide agonists to rodents increases food intake, suggesting that activation of either NPY receptor subtype results in an orexigenic response . On the contrary, administration of selective NPY Y1 and Y5 receptor antagonists partially decreases spontaneous, NPY induced and/or fasting-induced food intake. Antisense oligonucleotides of NPY Y5 receptor also decrease in food intake . Furthermore, the pharmacology of the NPY receptor mediating feeding and decreases in body temperature and energy expenditure in rats strongly resembles that of the NPY Y5 receptor . Both the NPY Y1 and Y5 receptors are also regulated by changes in nutritional status . The NPY Y1 and Y5 receptors have also been proposed to play a role in the central and peripheral effects of NPY on plasma glucose and insulin levels . Studies of mice lacking NPY Y1 receptors also support a role for this receptor in mediating the effects of NPY on energy homeostasis. NPY Y1 receptor-deficient mice exhibit slightly diminished natural and NPY stimulated feeding. In addition, feeding following a 24-h fast was significantly diminished by about 45% . Paradoxically, NPY Y1 receptor-deficient mice develop mild late onset obesity and moderate hyperinsulinemia. These mice exhibit reduced locomotor activity during the nocturnal period, which could be one mechanism by which the late onset obesity occurs. Studies of mice lacking NPY Y5 receptors have failed to provide strong support for a role of this receptor in mediating the effects of NPY on energy homeostasis . NPY Y5 receptor deficient mice have normal growth and feeding when young. Core body temperature in NPY Y5 receptor deficient mice is normal, indicating a lack of a gross effect on metabolic rate. Feeding induced by low doses of NPY was unchanged, while feeding induced by higher doses of NPY was reduced in NPY Y5 receptor deficient mice. Interestingly, NPY induced feeding was completely abolished in NPY Y5 receptor-deficient mice in which NPY Y1 receptors were also blocked by a NPY Y1 receptor antagonist. This confirms that activation of both NPY Y1 and Y5 receptors are responsible for NPY induced feeding. NPY Y5- deficient ob/ob mice were equally as obese as ob/ob mice, indicating that the attenuation of obesity in the ob/ob mouse lacking NPY is not mediated through the Y5 receptor . Like the NPY Y1 receptor-deficient mice, NPY Y5 receptor-deficient mice unexpectedly develop mild late onset obesity; however, this is more likely due to an increase in food intake. The data from the NPY Y1 and Y5 receptor deficient mice confirm that both receptors mediate NPY induced food intake and that the NPY Y1 receptor may also play a minor role in spontaneous food intake and a more significant role in food intake after deprivation. As discussed above, it is possible that these data underestimate the role of the NPY Y1 and Y5 receptors in normal energy homeostasis if mice can readily compensate for their absence. For example, an increased involvement of the NPY Y6 receptor in energy homeostasis may occur in the absence of the NPY Y1 or Y5 receptor in mice. It is also possible that the NPY Y1 and Y5 receptors are only involved in energy homeostasis under specific conditions (e.g., situations of negative energy balance such as starvation, obese patients who have lost substantial weight, etc.) It would also be interesting to examine energy homeostasis in mice lacking both the NPY Y1 and Y5 receptors. Unfortunately, the close proximity of the genes for the NPY Y1 and Y5 receptors precludes the generation of mice lacking both receptors via simple cross breeding of the individual knockout mice.
Screening Assay for NPY antagonist [114-120]:
In present scenario large number of NPY antagonist are developed due wide application of system in curing various type of disease. Genetic level study inspires the scientist to find specific cell based assay for evaluating the particular activity. Different cellular assay reported in literature were presented here used in analysis of NPY antagonist. The majority of assay used are cAMP assay, immuno reactive calcitonin gene-related peptide (iCGRP) radioimmuno assay, ligand binding assay, OB-R bioluminescence resonance energy transfer (BRET), amplified luminescent proximity homogeneous assay represented as below.
Challenges in development:
Antagonism of NPY remains a tantalizing target, but it appears difficult to find a developable compound with a suitable pharmacology, and it has yet to be demonstrated that such a compound can reduce obesity in animals, let alone humans. A particular challenge will be to demonstrate anti-obesity activity in situations where leptin levels are high and NPY release is low . Most of the evidence in support of the Y5 hypothesis has been generated in rodents, but recently, there have been reports suggesting that Y5 antagonists have little or no effect in rodent feeding models . The ability to interpret observations of this type is critically dependent on the quality of the compounds used in the studies and a good understanding of their properties. Numerous highly selective antagonists have been developed and their properties to modulate NPY induced feeding have been extensively investigated. Y1 receptors apparently modulate food intake since a wide range of selective antagonists produces unequivocal reduction of NPY or deprivation induced food intake. Y5 antagonist appears to reduce food intake and body weight. This apparent discrepancy could be explained by interplay between the y1 and y5 receptor. This is supported by a comparative study, examining the effects of NPY in Y1 and Y5 receptor deficient mice. NPY induced feeding was reduced in Y1 deficient mice while it was not significantly influenced in the Y5. Of special interest is that Y5 agonist induced feeding was reduced in the Y1 Knockout animals. The authors concluded that the Y1 receptor might be modulated by the action of the Y1 receptor. In line with a key role for the Y1 receptor is the observation that Y1 but not Y5 receptor gene expression was altered in association with hyperphagia in rats.
The strongest support for the study in rhesus monkeys showing that icv., administration of Y1 antagonist attenuates NPY mediated feeding..
Effects of NPY on other body chemical:
Results from animal studies provide strong support for the hypothesis that NPY modulates nociception at the level of the spinal cord  and higher level nociceptive processing centers . Although evidence exists for both inhibitory and stimulatory effects of NPY in electrophysiological and behavioral studies , the antinociceptive effects of NPY are likely due to activation of the Y1 receptor, and could be mediated, at least in part, by inhibition of exocytosis of neuropeptides from the spinal cord . In mice, injection of capsaicin evoked the release of peripheral immunoreactive substance P (iSP) from skin, and this effect was blocked in mice lacking the Y1 receptor . The controversial evidence which show disagreement, as Y1 receptor inhibits immuno reactive calcitonin gene related peptide (iCGRP) release from spinal cord, most likely by a presynaptic mechanism of action . Morphine suppresses a number of immune parameters, such as natural killer (NK) cell activity and lymphocyte proliferation, by acting through α-opioid receptors in the central nervous system. Studies have implicated the sympathetic nervous system in mediating the immunomodulatory effects of acute morphine treatment. Opioid involvement in feeding elicited by NPY was confirmed initially by the ability of the general opioid antagonist, naloxone to decrease the magnitude, but not the latency of NPY-induced feeding following systemic and ventricular administration  The selective NPY Y1 receptor antagonist BIBP3226 blocked morphine’s effect on splenic NK activity but did not attenuate the suppression splenocyte proliferative responses to Con- A or LPS. Furthermore, intravenous NPY administration produced a dose-dependent inhibition of splenic NK activity but did not suppress lymphocyte proliferation. 
The role of neuropeptide Y (NPY) in the regulation of luteinizing hormone (LH) release is clearly established in mammals . Some attempts have been made to characterize the nature of NPY receptor subtypes involved in the regulation of LH and growth hormone (GH) secretion. Y2 receptors have been suggested to be involved in the regulation of LH and GH secretion in male rats , reported those NPY Y1 receptors, but not the Y2 receptors, are involved in the regulation of GnRH and LH secretion in female rats.
Radiolabled NPY peptide analogue:
To better characterize NPY receptor subtypes, several novel selective analogues of the NPY sequence have been developed in the last few years. These analogues show high selectivity towards single NPY receptors and facilitate insights into the localization and physiological role of each receptor subtype in health and disease. Besides the important role of NPY in the regulation of food intake and other physiological functions, recently NPY receptors have been identified to play an important role in several types of cancer. To further enlighten all these numerous functions the radioactive labeling of peptides is still an indispensable and versatile tool. By using the appropriate radioisotope and labeling strategy several different radioisotopes can be incorporated into the sequence of subtype selective NPY analogues. Depending on the radioisotope the resulting radiolabel led NPY analogues can be used for diagnostic or therapeutic applications and enable us to better investigate single receptor subtypes in vitro and in vivo. Using this novel NPY new insights and new aspects of receptor localization and physiological function were obtained. In combination with different radiolabel ling strategies, these novel selective analogues will allow the study of NPY receptors in further applications like tumor diagnosis and therapy. 
11.0 Summary and perspective:
Much of the data published on small molecule, selective NPY1 and Y5 antagonists to date has been compiled using compounds that suffer from a variety of pharmacokinetic issues such as poor brain penetration, or short in vivo half-lives which prevent definitive interpretation of the results . Since NPY was first isolated in 1982, a large number of studies have shown that NPY and its receptors have a pivotal role in the regulation of food intake and energy homeostasis. Findings, mainly in rodents, as well as in a few other animal species, suggest a therapeutic potential for antiobesity drugs by modulating NPY receptors. However, small molecules often cause non mechanism based feeding suppression and body weight reductions. Thus a validation of compound selectivity in some way would be essential. In addition, evidence to support the utility of NPY related ligand in human is still limited. Further evaluation of selective compounds in higher species than rodents may be necessary to predict efficacy in humans and to understand the roles of NPY signals in energy homeostasis more clearly. Furthermore, some pharmaceutical companies have describes the use of combination therapies for the treatment of obesity. Although a sustained weight loss of 5- 10 % of body weight has been shown to improve the co-morbidities associated with obesity. Existing weight reducing drugs have limited efficacy when used alone and have significant side effects. Hence, it may be difficult to maintain the reduced body weight for a long time with a single drug. The phenotype of the NPY receptor knockout mice has not always been consistent with the results obtained by pharmacological studies in vivo or inviter. These discrepancies may, at least in part, be due to compensatory changes in the expression of the remaining Y- receptor types and each NPY receptor might act in a complementary manner. Thus, manipulation of two or more NPY subtype could be an interesting and challenging method for the treatment of obesity.
Data from mice lacking NPY or the NPY Y1 receptor suggest that activation of the NPY Y1 receptor by NPY plays a role in maintaining normal food intake as well as food intake after deprivation. Studies with selective NPY Y1 receptor antagonists also suggest a role for NPY Y1 receptors in maintaining food intake under conditions of real or apparent deprivation (e.g., food-deprived animals and genetically obese animals in which leptin signaling to the hypothalamus has been disrupted). The role of the NPY Y5 receptor in energy homeostasis is less clear, however. Mice lacking the NPY Y5 receptor do not differ from wild type mice in paradigms that assess food intake and body weight under a variety of conditions. Furthermore, the reported effects of NPY Y5 receptor antagonists on food intake and body weight are conflicting. Although administration of some NPY Y5 receptor antagonists is associated with reduced food intake and body weight gain, evidence for the specificity of these effects is lacking. The modest effects of NPY deficiency, NPY Y1 receptor deficiency and NPY Y1 receptor antagonists and the lack of any consistent effect of NPY Y5 receptor deficiency and NPY Y5 receptor antagonists on energy homeostasis suggest that the role of NPY in the regulation of body weight is more complicated than previously envisioned. Overall, these results may indicate that the high level of redundancy in the regulation of body weight insures that mice can substantially compensate for the loss of a single neuropeptide or neuropeptide receptor under normal conditions. However, the data obtained to date suggests that NPY plays a critical role in energy homeostasis under very specific physiological conditions, particularly conditions of real or apparent deprivation. These data indicate that NPY receptor antagonists may be most useful in human conditions where appetite is increased and energy expenditure is decreased due to activation of the starvation response. Such conditions include obese patients who are dieting, formerly obese patients who have lost substantial weight, and patients with complete or partial leptin deficiency. Significant progress has been made in the identification of structurally diverse, orally bioavailable NPY Y1 and Y5 receptor antagonists that can cross the blood–brain barrier. However, there is a clear need for further studies with both NPY Y1 and Y5 receptor antagonists in order to clarify their potential as anti-obesity agents. Most studies have been performed in lean rodents or in genetically obese rodents that do not mimic common obesity in the human population. It would be desirable to evaluate the effects of compounds in diet-induced obese rodents and non-rodents as disease models that more closely mimic human obesity. Other critical issues in the development of NPY receptor antagonists as effective agents for obesity management are the need to overcome counter balancing effects of the multiple complementary mechanisms involved in energy homeostasis that tend to oppose any changes in body weight and the identification of patient subclasses most likely to benefit from treatment.
In summary, numerous investigations to date suggest that NPY is implicated in the pathophysiology of a number of diseases including feeding and metabolic disorders, anxiety, seizures, memory, circadian rhythm, drug addiction, pain, cardiovascular diseases, rhinitis, and endothelial cell dysfunctions. Thus, the design of selective antagonists or agonists of NPY receptors could be useful compounds for the treatment of all these diseases. However, one should keep in mind that the large tissue distribution of NPY receptors and their stimulation or blockade by insufficiently selective drugs could produce untoward effects.
Authors are highly thankful to collegue of medicinal chemistry department for commenting and reviewing in preparation of manuscript.
NPY : Neuropeptide Y
7t-GPCR : seven transmembrane G-protein coupled receptors
AC : adenylate cyclase
ATP : adenosine triphosphate
cAMP : 3`, 5`-cyclic adenosine monophosphate
PLC : phospholipase C
PIP2 : phosphatidyl inositol 4, 5-diphosphate
IP3 : inositol 1, 4, 5-triphospate
DAG : diacylglycerol
ROCC : receptor-operated calcium channel
GMP : Guanosine Monophosphate
GDP : Guanosine Diphosphate
GTP : Guanosine Triphosphate
PKC : Protein KInase C
MAPK : Mitogen-activated protein kinase
PI-3 Kinase : Phosphatidylinositol 3 kinase
HEL : Human erythroleukemia
JNK : c-jun N-terminal kinase
DRG : dorsal root ganglion
NE : Norepinephrine
PYY : Peptide YY
α -MSH : α-melanocyte-stimulating hormone
AGRP : Agouti-related peptide
CART : cocaine and amphetamine-regulated transcript
GABA : γ-aminobutyric acid
TRH : Thyrotropin- releasing hormone
MCH : Melanin concentrating hormone