Stimulation of feeding in lean but not in obese Zucker rats by a selective neuropeptide Y Y5 receptor agonist

Obese Zucker rats are characterized by a reduced hypothalamic NPY receptor density. We tested the effects of intracerebroventricular injections of human NPY (hNPY) and [D-Trp32]NPY, a weak but selective NPY Y5 receptor agonist, on food intake in lean and obese Zucker rats. The effect of a maximal dose of hNPY (10 microg) on feeding was more pronounced in lean than in obese rats. [D-Trp32]NPY (10 microg) stimulated feeding in lean but not in obese Zucker rats. It did not affect the feeding response to hNPY, excluding the activation of an inhibitory receptor. These results are in favor of a down-regulation of the NPY 'feeding' receptor in the obese rat, which is suggested to be the Y5 subtype.     

CD5 labeling of thymic carcinomas and other nonlymphoid neoplasms

Neuropeptide Y (NPY) is a 36-amino-acid neurotransmitter which is widely distributed throughout the central and peripheral nervous system. NPY involvement has been suggested in various physiological responses including cardiovascular homeostasis and the hypothalamic control of food intake. At least six subtypes of NPY receptors have been described. Because of the lack of selective antagonists, the specific role of each receptor subtype has been difficult to establish. Here we describe mice deficient for the expression of the Y1 receptor subtype. Homozygous mutant mice demonstrate a complete absence of blood pressure response to NPY, whereas they retain normal response to other vasoconstrictors. Daily food intake, as well as NPY-stimulated feeding, are only slightly diminished, whereas fast-induced refeeding is markedly reduced. Adult mice lacking the NPY Y1 receptor are characterized by increased body fat with no change in protein content. The higher energetic efficiency of mutant mice might result, in part, from the lower metabolic rate measured during the active period, associated with reduced locomotor activity. These results demonstrate the importance of NPY Y1 receptors in NPY-mediated cardiovascular response and in the regulation of body weight through central control of energy expenditure. In addition, these data are also indicative of a role for the Y1 receptor in the control of food intake.     

Alternative splicing of Pax6 in bovine eye and evolutionary conservation of intron sequences

There are now six recognized neuropeptide Y (NPY) receptor subtypes (Y1-Y4 and two recently cloned distinct receptors labeled Y5), of which Y1 and one of the Y5's have been suggested could mediate the effect of NPY on feeding. The fragments NPY(2-36) and NPY(3-36), which bind Y1 only poorly, were injected intracerebroventricularly (icv) and found to have similar dose-response relationships to NPY in the stimulation of feeding. However NPY (13-36), which stimulates both Y2 and Y5, caused no increase in food intake, even at high doses. Maximal stimulation with the classical Y1 agonist [Pro34]-NPY produced only 50% of the maximum effect of NPY itself despite fully inhibiting adenylyl cyclase activity in vitro in a Y1 system. The novel fragment [Pro34]-NPY(3-36) is as effective at stimulating food intake as the classical Y1 analogue [Pro34]-NPY but bound to the Y1 receptor with only 1/20th of the affinity of NPY and failed to inhibit adenylyl cyclase through this receptor. [Pro34]-NPY(3-36) is therefore a relatively appetite-selective ligand. Coadministration of high dose NPY(13-36) and [Pro34]NPY did not enhance feeding compared with [Pro34]-NPY alone. In addition, the NPY Y1 receptor antagonist BIBP-3226, which does not bind Y2, Y4, or Y5 receptors, significantly reduced NPY induced feeding. These results indicate that the feeding effect of icv NPY involves a novel receptor and that it is functionally distinct from the recognized receptor subtypes.     

Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus

Leptin, the protein product of the adipose tissue-specific ob (obese) gene (1), reduces the body weight, adiposity and food intake of obese ob/ob mice on peripheral or central injection (2, 3, 4). [125I]leptin binding has been detected in mouse choroid plexus (5), from which a leptin receptor gene was expression cloned (5). The gene has at least 6 splice variants (6, 7). Leptin receptor mRNA was localized in the hypothalamus by in situ hybridization being particularly abundantly expressed in the arcuate nucleus (8). There is evidence linking the physiological effects of injected leptin with hypothalamic neuropeptide Y (9, 10) (NPY), which has potent central effects on food intake and energy balance (11), and is also expressed in the arcuate nucleus. Here we report dual in situ hybridization studies for leptin receptor and NPY gene expression in the mouse arcuate nucleus, where the majority of cells examined expressed both genes. This provides the first direct evidence that leptin acts on cells that express NPY mRNA.     

Obesity and mild hyperinsulinemia found in neuropeptide Y-Y1 receptor-deficient mice

To elucidate the role of neuropeptide Y (NPY)-Y1 receptor (Y1-R) in food intake, energy expenditure, and other possible functions, we have generated Y1-R-deficient mice (Y1-R-/-) by gene targeting. Contrary to our hypothesis that the lack of NPY signaling via Y1-R would result in impaired feeding and weight loss, Y1-R-/- mice showed a moderate obesity and mild hyperinsulinemia without hyperphagia. Although there was some variation between males and females, typical characteristics of Y1-R-/- mice include: greater body weight (females more than males), an increase in the weight of white adipose tissue (WAT) (approximately 4-fold in females), an elevated basal level of plasma insulin (approximately 2-fold), impaired insulin secretion in response to glucose administration, and a significant changes in mitochondrial uncoupling protein (UCP) gene expression (up-regulation of UCP1 in brown adipose tissue and down-regulation of UCP2 in WAT). These results suggest either that the Y1-R in the hypothalamus is not a key molecule in the leptin/NPY pathway, which controls feeding behavior, or that its deficiency is compensated by other receptors, such as NPY-Y5 receptor. We believe that the mild obesity found in Y1-R-/- mice (especially females) was caused by the impaired control of insulin secretion and/or low energy expenditure, including the lowered expression of UCP2 in WAT. This model will be useful for studying the mechanism of mild obesity and abnormal insulin metabolism in noninsulin-dependent diabetes mellitus.     

Immunocytochemical localization of the NPY/PYY Y1 receptor in enteric neurons, endothelial cells, and endocrine-like cells of the rat intestinal tract

Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides that are considered to mediate inhibitory actions on gastrointestinal motility, secretion, and blood flow. Several receptor subtypes for these peptides have been identified and the Y1, Y2, Y4/PP1, Y5, and Y5/PP2/Y2b receptors have been cloned. In this article we report the immunocytochemical localization of the Y1 receptor to myenteric and submucosal nerve cell bodies, endothelial cells, and scattered endocrine-like cells of rat intestinal tract. Moreover, double immunofluorescence demonstrates that subpopulations of the Y1 receptor-positive nerve cell bodies are immunopositive for NPY, vasoactive intestinal polypeptide, and nitric oxide synthase. In part, such co-localizations were made possible by use of peroxidase-mediated deposition of tyramide, which permitted use of antisera derived from the same species. Our observations suggest the existence of multiple neuronal, endothelial, and endocrine target sites for NPY and PYY and that some of the actions of these regulatory peptides can be mediated by vasoactive intestinal peptide and nitric oxide synthase.     

Zinc deficiency increases hypothalamic neuropeptide Y and neuropeptide Y mRNA levels and does not block neuropeptide Y-induced feeding in rats

Zinc deficiency reduces intake and produces an unusual approximately 3.5-d cycle of intake in rats. The mechanism underlying the anorexia and cycling has not yet been defined; current hypotheses suggest that alterations in amino acid metabolism and neurotransmitter concentrations may be a part of this anorexia. Recent reports indicate that appetite-stimulating neuropeptide Y (NPY) may be elevated during zinc deficiency. This suggests that a resistance to NPY may exist during zinc deficiency because NPY levels are high, yet appetite is low. The purpose of this study was to measure NPY peptide and mRNA concentrations during zinc deficiency in specific nuclei of the hypothalamus in which peptide and mRNA for NPY are known to be associated with appetite, and also to determine whether zinc-deficient rats are responsive to central infusions of NPY. Both NPY peptide levels in the paraventricular nucleus and NPY mRNA levels in the arcuate nucleus were higher (P < 0.05) in zinc-deficient rats than in zinc-adequate rats. When rats were administered exogenous NPY to the paraventricular nucleus, both zinc-deficient and zinc-adequate rats responded similarly by increasing food intake. These results suggest that NPY is elevated during zinc deficiency in an attempt to restore normal food intake levels, rather than being reduced and thereby contributing to the anorexia associated with zinc deficiency. During zinc deficiency, NPY receptors are able to bind NPY and initiate an orexigenic response.     

Expression of tenascin-C in intraductal carcinoma of human breast: relationship to invasion

A growing body of evidence indicates that a number of peptides expressed in the mammalian hypothalamus are involved in the regulation of food intake and energy balance. Among these, melanin-concentrating hormone (MCH) and neuropeptide Y (NPY) are potent appetite stimulants, whereas alpha-melanocyte-stimulating hormone (alpha-MSH), neurotensin, and glucagon-like peptide (GLP)-1(7-36) amide have appetite-suppressing properties. However, the functional interactions between pathways involving these neuropeptides remain incompletely understood. In the current study, we describe the functional interactions between orexigenic (appetite-stimulating: MCH and NPY) and anorectic (appetite-suppressing: alpha-MSH, neurotensin, and GLP-1) peptides after intracerebroventricular (i.c.v.) administration in the rat. The i.c.v. administration of GLP-1 completely prevents the orexigenic effects of both MCH and NPY. However, i.c.v. administration of alpha-MSH prevents only the orexigenic effect of MCH, as we have previously shown, but does not prevent the effect of NPY on food intake. Similarly, i.c.v. administration of neurotensin prevents only the orexigenic effect of MCH, but does not prevent the appetite-stimulating effect of NPY. Thus, our study suggests that the functional interactions between these neuropeptides are specific, although the underlying mechanisms are as yet unexplored.     

125I]Leu31, Pro34-PYY is a high affinity radioligand for rat PP1/Y4 and Y1 receptors: evidence for heterogeneity in pancreatic polypeptide receptors

Cloned receptors for the PP-fold peptides are subdivided into Y1, Y2, PP1/Y4, Y5 and Y6. NPY and PYY have similar affinity for Y1, Y2, Y5 and Y6 receptors while PP has highest affinity for PP1. Pro34-substituted analogs of NPY and PYY have selectivity for Y1 and Y1-like receptors over Y2 receptors. In the present study, we found the putative Y1-selective radioligand, [125I]Leu31, Pro34-PYY, also binds with high affinity to the rat PP1 receptor in cell lines expressing the receptor. However, in rat brain sections, [125I]Leu31, Pro34-PYY does not appear to bind to the interpeduncular nucleus, a brain region containing a high density of [125I]-bPP binding sites. Therefore, it appears there is additional heterogeneity in receptors recognizing PP.     

Increased neuropeptide Y concentrations in specific hypothalamic nuclei of the rat following treatment with methysergide: evidence that NPY may mediate serotonin's effects on food intake

Neuropeptide Y (NPY) is a potent central appetite stimulant found in hypothalamic neurons that have close anatomical associations with neurons containing serotonin, a powerful anorectic agent. To determine whether the two neurotransmitters interact functionally, we have studied the effects on regional hypothalamic NPY concentrations of acute and chronic administration of methysergide, a 5-HT1BC/serotonin receptor antagonist. Chronic methysergide treatment (10 mg/kg/day) was given by subcutaneously implanted osmotic minipumps (n = 8). Acute effects of methysergide were determined 4 h after a single injection (10 mg/kg) in a separate group (n = 8). Controls (n = 8) had implanted minipumps delivering saline, and also received a saline injection 4 h before sacrifice. Food intake was significantly increased (p < 0.01) by both acute and chronic methysergide treatment. In the chronically treated rats, NPY levels were significantly increased over controls in the arcuate nucleus (ARC; by 41%, p = 0.02) and paraventricular nucleus (PVN; by 40%, p < 0.01). Acute methysergide treatment also increased NPY concentrations in the ARC (by 81%, p < 0.01) and PVN (by 30%, p < 0.01). Methysergide administration, which stimulated feeding, therefore raised NPY concentrations in the ARC, where NPY is synthesized, and in the PVN, a major site of NPY release where NPY injection induces hyperphagia. These findings suggest that NPYergic and serotoninergic innervations in the hypothalamus interact to regulate food intake, and raise the possibility that increased NPY release may mediate the hyperphagic effect of serotoninergic 5-HT1BC/receptor blockade.     

Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells

Neuropeptide Y (NPY) has been shown to inhibit insulin secretion from the islets of Langerhans. We show that insulin secretion in the insulinoma cell line RIN 5AH is inhibited by NPY. 125I-Peptide YY (PYY) saturation and competition-binding studies using NPY fragments and analogues on membranes prepared from this cell line show the presence of a single class of NPY receptor with a Y1 receptor subtype-like profile. Inhibition of insulin secretion in this cell line by NPY fragments and analogues also shows a Y1 receptor-like profile. Both receptor binding and inhibition of insulin secretion showed the same orders of potency with NPY > [Pro34]-NPY > NPY 3-36 > NPY 13-36. The Y1 receptor antagonist, BIBP 3226, blocks NPY inhibition of insulin secretion from, and inhibits 125I-PYY binding to, RIN 5AH cells. Northern blot analysis using a Y1-receptor specific probe shows that NPY Y1 receptors are expressed by RIN 5AH cells. Y5 receptors are not expressed in this cell line. Neuropeptide Y inhibition of insulin secretion is blocked by incubation with pertussis toxin, implying that the effect is via a G-protein (Gi or Go) coupled receptor. Neuropeptide Y inhibits the activation of adenylyl cyclase by isoprenaline in RIN 5AH cell lysates, and the stimulation of cAMP by glucagon-like peptide-1 (7-36) amide (GLP-1). It also blocks insulin secretion stimulated by GLP-1, but not by dibutyryl cyclic AMP. Hence, we suggest that NPY inhibits insulin secretion from RIN 5AH cells via a Y1 receptor linked through Gi to the inhibition of adenylyl cyclase.     

Expression cloning of a human brain neuropeptide Y Y2 receptor

The 36-amino acid peptide, neuropeptide Y (NPY), is a member of a peptide family that includes the endocrine peptides, peptide YY (PYY), and pancreatic polypeptide (PP). NPY receptors have been broadly subdivided into postsynaptic Y1 receptors and presynaptic Y2 receptors based on the preference of Pro34-substituted analogues for the Y1 receptors and carboxyl-terminal fragments for the Y2. A Y1 receptor has been cloned, and this receptor appears to mediate several effects of NPY, including vasoconstriction and anxiolysis in animal models. We report the cloning of a human brain Y2 receptor from a human brain library. Pools of clones were transiently expressed in COS-1 cells, and 125I-PYY binding pools were identified by autoradiography. After a single positive pool was detected in the original screening, a single clone was isolated by four rounds of sequential enrichment. The clone encoded a 381-amino acid protein of the heptahelix (seven TM) type. Amino acid identity of this receptor with the Y1 receptor was 31% overall with 40% identity in the TM regions. Comparison with the human PP1 receptor indicated 33% overall amino acid identity with 42% identity in the TM regions. Pharmacologically, the receptor exhibited high affinity for NPY, PYY, and carboxyl-terminal fragments of NPY and PYY. In addition, Pro34-substituted analogues had very low affinity. With the use of Northern blot analysis, high levels of Y2 mRNA were detected in a variety of brain regions with little expression in peripheral tissues. Thus, the receptor protein has the pharmacological properties and distribution of the human Y2 receptor.     

Interactions between neuropeptide Y and gamma-aminobutyric acid in stimulation of feeding: a morphological and pharmacological analysis

Neuropeptide Y (NPY) produced in neurons in the arcuate nucleus and brain stem and released in the paraventricular nucleus (PVN) and surrounding areas is involved in stimulation of feeding in rats. We recently reported that gamma-aminobutyric acid (GABA) is coexpressed in a subpopulation of NPY neurons in the arcuate nucleus. To determine whether GABA is colocalized in NPY terminals in the PVN, the site of NPY action, light and electron microscopic double staining for NPY and GABA using pre- and postembedding immunolabeling was performed on rat brain sections. GABA was detected in NPY-immunopositive axons and axon terminals within both the parvocellular and magnocellular divisions of the PVN. These morphological findings suggested a NPY-GABA interaction in the hypothalamic control of feeding. Therefore, the effects of muscimol (MUS), a GABA(A) receptor agonist, on NPY-induced food intake were examined in sated rats. When injected intracerebroventricularly, both NPY and MUS elicited dose-dependent feeding responses that were blocked by the administration of 1229U91 (a putative Y1 receptor antagonist) or bicuculline (a GABA(A) receptor antagonist), respectively. Coadministration of NPY and MUS intracerebroventricularly amplified the feeding response over that evoked by NPY or MUS alone. Similarly, microinjection of either NPY or MUS into the PVN stimulated food intake in a dose-related fashion, and coinjection elicited a significantly higher response than that evoked by either individual treatment. These results suggest that GABA and NPY may coact through distinct receptors and second messenger systems in the PVN to augment food intake.     

Characterization of the neuropeptide Y5 receptor in the human hypothalamus: a lack of correlation between Y5 mRNA levels and binding sites

Neuropeptide Y (NPY) is a 36-amino-acid peptide that appears to play a central role in the control of feeding behavior. Recently, a cDNA encoding a novel NPY receptor subtype (Y5) was cloned from the rat and human hypothalamus, and shown to have a pharmacology consistent with NPY-induced feeding. We have subsequently cloned this cDNA from human hypothalamus and stably expressed it in CHO cells. Consistent with earlier reports, hY5 has a high affinity for NPY, [Leu31, Pro34]NPY, and NPY(3-36), but low affinity for larger C-terminal deletions of NPY and BIBP3226. High levels of hY5 mRNA were found in the human testis, brain, spleen and pancreas, with lower levels in several other tissues. In the human brain, hY5 mRNA levels were typically higher than hY2, but lower in comparison to hY1 receptor mRNA. To quantify the relative amounts of hY1, hY2 and hY5 mRNA in the human hypothalamus, we employed competitive RT-PCR. Interestingly, the relative amount of hY5 mRNA was substantially higher than either hY1 or hY2. However, pharmacological characterization of NPY binding sites in human hypothalamus membranes revealed predominantly the hY2 subtype. These data establish that while hY5 mRNA levels are very high in the human hypothalamus, conventional radioligand binding techniques do not detect hY5-like binding site. Whether hY5-like binding sites exist in the other human tissues that express hY5 mRNA (and what function hY5 has in those tissues) awaits future investigation.     

Central injection in rats of alpha-melanocyte-stimulating hormone analog: effects on food intake and brain Fos

Lateral cerebroventricular (LCVT) administration of the alpha-MSH agonist analog Nle4DPhe7alpha-MSH (NDP-MSH) inhibited food intake in food-deprived rats, but did not inhibit water intake in water-deprived rats. When NDP-MSH was administered into the fourth ventricle (4CVT), comparable suppressions of food intake were observed. LCVT and 4CVT administration of NDP-MSH also reduced spontaneous 24 h food intake. LCVT injection of NDP-MSH greatly attenuated food intake stimulated in sated rats by acute CVT administration of neuropeptide Y (NPY). These and other data suggest that alpha-MSH is an important endogenous regulator of food intake, possibly acting downstream of NPY. In an attempt to assess further the sites of action of NDP-MSH, a qualitative mapping study of Fos-like immunoreactive (Fos-ir) neurons was performed following LCVT administration of NDP-MSH. LCVT injection of NDP-MSH induced Fos-ir in several forebrain regions including cortex, striatum, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus and arcuate nucleus. The combination of NPY and NDP-MSH did not produce obvious antagonism or cancellation of effects in any region examined. Thus, the site(s) of action of NDP-MSH on food intake remain to be clarified.     

Metabolic action of neuropeptide Y in relation to its effect on feeding

Because energy homeostasis depends on a continuous balance between food intake, energy expenditure, and energy storage, it was expected that neuropeptide Y (NPY) could act not only on food intake but also on metabolic parameters. Using an original calorimetric device that allows the computation of the background metabolism (energy expenditure free from the cost of locomotor activity), we assessed the effect of a microinjection of NPY upon the quantitative (background metabolism, thermic effect of food) and qualitative (respiratory quotient) components of energy metabolism. NPY was injected into the juxtafornical hypothalamus at a dose that promotes feeding behavior (1 microg/0.5 microL) and enhances locomotor activity. Although total metabolism was increased proportionally to locomotion, no effect of NPY on background metabolism was observed when no food was available. Only following a calibrated meal given 30 min after the microinjection did NPY induce a delayed decrease in respiratory quotient whereas the postprandial background metabolism remained unaffected. In conclusion, only the new-generation calorimeters can show that the NPY-induced rise in overall metabolic rate is entirely accounted for by the unavoidable enhancement in locomotor activity and that the only metabolic effect of NPY is the delayed postprandial respiratory quotient decrease, suggesting a postabsorptive orientation toward more lipid utilization.     

Fluorescent labelled analogues of neuropeptide Y for the characterization of cells expressing NPY receptor subtypes

Porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, and subtype selective analogues have been synthesized by solid phase peptide synthesis. The peptides were labelled with Cy3, a commercially available fluorescent marker based on a cyanine dye, by solid phase strategy. During the cleavage a partial fragmentation of the fluorescent marker occurred. This has been investigated by means of HPLC and electrospray mass spectrometry. The labelled analogues of NPY showed high affinity to the NPY receptor subtypes Y1 and Y2. Thus, Cy3-NPY, Y1-selective Cy3-[Pro34] NPY and Y2 selective Cy3-[Ahx5-24] NPY were used to label SK-N-MC- and SMS-KAN-cells, which are stably expressing the Y1-(SK-N-MC) and the Y2-receptor subtype (SMS-KAN). The binding of the labelled analogues to the receptors was reversible and specific. The photoactivatable analogue, [(Tmd)Phe27] NPY, which showed high affinity to both receptor subtypes was labelled with Cy3 in solution. Whereas the fluorescent labelling of the cells with analogues without photoactivatable amino acid was reversible, successful photocrosslinking could be investigated by the irreversible staining of the cells using Cy3-[(Tmd)Phe27] NPY. These subtype selective analogues are exciting tools to trace receptors in tissues and to identify the pharmacologically characterized subtypes without radioactivity.     

Inhibition of stimulated cyclic AMP production by multiple neuropeptide Y receptors in the rat brainstem

Neuropeptide Y (NPY) has been shown to modulate blood pressure, heart rate and to inhibit the baroreceptor reflex at the level of nucleus tractus solitarius (NTS). The aim of this study was to examine effects of NPY and its related peptides on forskolin (1 microM)-stimulated cyclic AMP production in slices of the rat NTS. Each peptide was present at 0.3 microM. Pretreatment with NPY inhibited the stimulated increase in cyclic AMP levels in slices of rat NTS. Also [Pro34]NPY, an analog, which activates Y1, Y3 (and Y5) receptors inhibited the stimulated increase in cyclic AMP levels. However, pretreatment with the Y1 receptor-selective antagonist BIBP3226 (3 microM) did not affect the [Pro34]NPY-evoked inhibition of cyclic AMP levels. In addition, [Leu31,Pro34]NPY, an Y1 (and PP1/Y4 and Y5) receptor agonist did not inhibit the stimulated increase in cyclic AMP production. Also the Y2 receptor-selective agonist C2-NPY inhibited the stimulated elevation of cyclic AMP levels, while peptide YY, which does not recognize Y3 receptors did not significantly affect the stimulated cyclic AMP production. In conclusion, it seems that Y2 and Y3 receptors are coupled to inhibition of adenylate cyclase activity in the rat NTS.     

Evidence suggesting that galanin (GAL), melanin-concentrating hormone (MCH), neurotensin (NT), proopiomelanocortin (POMC) and neuropeptide Y (NPY) are targets of leptin signaling in the hypothalamus

Leptin (OB protein) reduces food intake by acting at the hypothalamic level. The purpose of the present study was to identify potential targets of leptin signaling in the hypothalamus in ad-lib fed rats. Central administration of leptin (5 microg) for 3 days decreased food intake and body weight gain in association with a decrease in hypothalamic galanin (GAL), melanin-concentrating hormone (MCH), proopiomelanocortin (POMC) and neuropeptide Y (NPY) gene expression and with an increase in neurotensin (NT) gene expression. In pair-fed rats, NPY gene expression was increased and there was no change in either MCH, GAL, POMC or NT gene expression. This study identifies GAL, MCH, POMC and NT as non-NPY targets of leptin signaling and suggests that leptin's action on food intake and body weight is most likely mediated by inhibiting excitatory (e.g. NPY, MCH, GAL, POMC) and stimulating inhibitory (e.g., NT) signals in the feeding circuitry.