Figure 2

Fig.2. Main signaling pathways activated by NPY

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 a subunit, which then dissociates from the G bg dimer. Both the active GTP-bound G a subunit and the G bg complex can individually regulate the activity of specific downstream effectors. Initial studies demonstrated that NPY mediated some of its physiological effects through the inhibition of the adenylyl cyclase [24, 25]. Therefore, NPY was shown to decrease forskolin stimulated cAMP accumulation in a variety of cell lines and in tissues. However, NPY had no significant effect on basal cAMP levels. Since this response was pertussis toxin sensitive, it indicated that the receptors were coupled through inhibitory heterotrimeric Gi/o proteins. In addition, NPY appeared also to induce calcium mobilization, and is certainly linked to calcium signaling pathways through the receptor-operated calcium channel (ROCC) [26]. Both intracellular pathways are consistent with the vasoconstrictive properties of NPY and with its capacity to potentiate the actions of other vasoconstrictors. In smooth muscle cells, stimulation of calcium transients did not seem to depend on inositol 1, 4, 5-trisphosphate (IP3) production [27]. Interestingly, although NPY had no effect on IP3 levels alone, it could potentiate the action of angiotensin II on IP3 production. In addition, NPY had no detectable effect on the activation of protein kinase C (PKC). It is thus unlikely that NPY receptors couple to phospholipase C in smooth muscle cells. However, this view has been challenged, since treatment of smooth muscle cells with a specific phospholipase C inhibitor seemed to block not only NPY induced IP3 synthesis but also calcium mobilization [27]. In smooth muscle cells, NPY also induced rapid phosphorylation of myosin light chain, which represents a mandatory step for the initiation of contraction [28]. Using a series of NPY analogs, inhibition of adenylyl cyclase as well as stimulation of intracellular calcium release was shown to be mediated by NPYY1 receptors. Transfection experiments using NPYY1 cDNA clones confirm the coupling of this receptor subtype to inhibition of stimulated cAMP accumulation and to an increase in intracellular calcium [29]. In addition, it appeared that NPY could stimulate the mitogen-activated protein kinas (MAPK) pathways. This effect appeared sensitive to pertussis toxin and could be dependent on phosphatidylinositol 3 (PI-3)-kinase [30]. The PI-3 kinase induction were confirmed using human erythroleukemia (HEL) cells, in which NPY induced phosphorylation of ERK through NPYY1 receptors. In addition, NPY was shown to not activate the c-jun N-terminal kinase (JNK), the p38 kinase or PKC in HEL cells [31]. The intracellular signaling pathways associated with Y2 receptors have particularly been studied in rat dorsal root ganglion (DRG) neurons. In these cells, NPY appeared to inhibit calcium influx via the modulation of voltage-dependent calcium channels [32]. However, an NPY-induced increase in intracellular calcium secondary to IP3 production was also described in these cells. Coupling of NPYY2 to an inhibitory Gi protein was suggested by the sensitivity of the response to pertussis toxin [33]. In the human SMS-KAN neuroblastoma cell line, NPY was shown to inhibit w-conotoxin sensitive calcium influx (N-type channel), as well as angiotensin II-stimulated calcium release from intracellular stores [34]. However, contrary observation from DRG neurons, NPY was not able to trigger intracellular calcium release in SMS-KAN cells. Interestingly, in these cells NPY also inhibits forskolin-induced cAMP accumulation. The human glioblastoma cell line LN319 also proved useful in defining Y2-mediated pathways, since these cells exclusively express this receptor subtype. In these cells, NPY inhibits forskolin-induced cAMP accumulation and stimulates an increase in intracellular calcium via pertussis toxin-sensitive pathways [35]. The signaling pathways associated with the other NPY receptor subtypes have mainly been studied in transfected cells. Although the importance of these results is not questioned, functional data obtained in physiological situations are limited. Stably transfected HEC-1B with a human Y5 receptor cDNA showed no elevation of intracellular calcium upon NPY stimulation [36]. In contrast, NPY dose dependently increases calcium concentrations in LMtk– cells expressing a recombinant human Y5 receptor. This effect was blocked by addition of a selective Y5 antagonist. Finally, other pathways have been reported to be associated with activation of NPYY5 receptors. In primary mouse cardiomyocytes, NPY induces rapid phosphorylation of ERK, JNK and p38 MAPK as well as activation of PKC [37]. Moreover, NPY potentiate phenylephrine stimulated MAPK and PKC activation. These stimulatory effects appear to be pertussis toxin sensitive and mediated by NPYY5 receptors, since Y5 selective-agonists can mimic NPY actions and NPY potentiation of MAPK activation is abolished in NPYY5-deficient cells. However, these findings were not reproduced in Y5-transfected HEC-1B cells, in which NPY activated neither the MAPK nor the PKC pathways [35]. This observation might reflect distinct NPY intracellular couplings in different cell types. Besides the direct response elicited by NPY in various cell types, it also has capacity to amplify responses induced by other substances. In particular, NPY exerts a synergistic effect on Gq mediated nor epinephrine (NE) induced responses. This raises a question about the molecular mechanisms involved in the potentiating actions of NPY [38]. Gi-coupled NPY receptors could facilitate the effects of receptors associated to a Gq protein by changing the activity of PLC. The production of inositoltriphosphate (IP3) and the subsequent induction of calcium release are important in a number of cellular mechanisms such as contraction or calmodulin mediated activation of phosphorylation cascades. Moreover, PKC activation could also potentiate some of the effects produced by Gq coupled receptors. For instance, arachidonic acid generation by phospholipase A is dependent on PKC activity and intracellular calcium release [39]. PLC isoforms can be differently stimulated depending on the types of G protein that are activated in response to agonist stimulation. Indeed, Gq protein stimulates preferentially PLC b1 via the G a subunit, whereas Gi protein stimulates mainly PLC b2 through the G bg dimer. Interestingly, PLC b2 is downregulated by PKA. NPY could therefore facilitate the stimulation of PLC b2 activity via direct activation by the G bg heterocomplex and indirectly through its inhibitory actions on cAMP production and PKA activation.

The neuropeptide YY1 receptor was first characterized during the study of vascular muscle contraction effects of NPY. [48] 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 [49]. 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 [50] and in human kidney, heart, lung, colon, testis, adrenal gland, placenta, bone marrow and vascular smooth muscle [51]

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 [48] 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 [52] The NPY Y2 receptor is localized on NPY containing neurons in the brain, suggesting that this receptor is an auto receptor [53] 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 [48]. Negligible levels of NPY Y2 receptor mRNA are generally found in peripheral tissues [54], although low expression of NPY Y2 receptor mRNA has been reported in human and rat gastro-intestinal tract [49, 50].

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 [41]. 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 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) [55]. 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 was also initially identified by molecular cloning [58] 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 [59]. NPY Y5 receptor binding sites have also been detected in these regions, although some groups fail to detect NPY Y5 receptor binding in hypothalamus [60] Interestingly, NPY Y5 receptor mRNA is almost always localized in neurons that also express NPY Y1 receptor mRNA [61]. 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 [62]. 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 [63]

The neuropeptide YY6 receptor was also initially identified by molecular cloning [64]. 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 [65]. 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.

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 [66]. 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) [42]. Recently, [67] 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 [68]

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 [69]. 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 [69], other groups have reported that the anorectic effects of PYY are preserved in mice lacking the MC4 receptor [70]. 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 [71]. 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 [72] 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 [73]. 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 [74].

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 [86]. 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 [87]. 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 [88]. 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 [89], 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 [90]. This effect could be partially explained by significant but transient hyperphagia immediately following introduction of the high sucrose diet.

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 [91] 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 [93]. 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 [94]. 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 [95]. 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 [96]. 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 [97]. Further studies indicated that plasma corticosterone, insulin, and glucose levels were normal in NPY deficient mice [98]. A lack of NPY also did not affect the development of obesity induced by diet or chemical means [99]. Studies by a separate group, however, demonstrated that NPY deficient mice have decreased food intake after a 24–48 h fast [100]. 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 [101]. 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 [102]. 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 [99].

In addition to NPY, many other neurotransmitter and neuropeptide systems in the brain have been shown to influence energy homeostasis [103]. 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 [103]. 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 [102]. 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 [104]. Conversely, blockade of these receptors by an endogenous antagonist known as agouti-related peptide (AGRP) has the opposite effect [105]. 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 [103]. Interestingly, AGRP is expressed exclusively in NPY containing neurons originating in the arcuate nucleus [106]. 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 [107], 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) [103]. 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 [109]. 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.

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 [58]. 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 [110]. 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 [111]. Both the NPY Y1 and Y5 receptors are also regulated by changes in nutritional status [112]. 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 [62]. 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% [113]. 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 [42]. 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 [42]. 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.