Evidence of NPY Y5 receptor involvement in food intake elicited by orexin A in sated rats

Intracerebroventricular (icv) injections of orexin A stimulate feeding in sated rats. Since neuropeptide Y is a potent orexigenic peptide and orexin-containing neurons are morphologically linked with NPY-producing neurons in the hypothalamus, we evaluated the functional relationship between the two orexigenic peptides. The results show that whereas it was ineffective on its own, a selective NPY Y5 receptor antagonist, injected icv 15 min. before orexin A significantly suppressed orexin A-induced feeding. Since previous investigations demonstrated that an NPY Y1 receptor antagonist also inhibits feeding induced by orexin A, the current results further underscore the existence of a functional link between orexin and NPY producing neurons as the orexin network appears to be capable of influencing NPYergic signaling through Y1 and Y5 receptors to stimulate feeding.     

Transfected human neuropeptide Y cDNA expression in mouse pituitary cells. Inducible high expression, peptide characterization, and secretion

An expression vector was constructed that placed the cDNA for human neuropeptide Y (NPY) under the control of the mouse metallothionein promoter and was used to transfect the AtT-20 mouse anterior pituitary corticotrope cell line. AtT-20 cells normally process the pro-ACTH/endorphin precursor but do not produce detectable levels of NPY. The resulting AtT-20/NPY cell line (Mt.NPY1a) was used to study the ability of the corticotrope cells to synthesize, process, and secrete the foreign proNPY-related peptide products. The stable cell line created contains approximately 40 copies of proNPY cDNA per cell. NPY mRNA levels and proNPY synthesis were increased at least 35-fold when maximally induced with cadmium; proNPY synthesis was also induced by glucocorticoids. Upon induction the NPY secretion rate was equimolar to that of the endogenous peptides. ProNPY, NPY, and the COOH-terminal peptide produced by this cell line had molecular weight and amino acid-labeling pattern predicted from cDNA sequence data and from previous isolation of NPY-related molecules from NPY-producing cells. The structures of secreted proNPY, NPY, and COOH-terminal peptide, as well as determination of the site of proteolytic cleavage between NPY and the COOH-terminal peptide, were determined by tryptic mapping and Edman degradation of secreted biosynthetically labeled peptide products. The proNPY molecule appears to be processed in the same pathway responsible for cleavage of the endogenous pro-ACTH/endorphin precursor. Secretion of proNPY-derived peptides paralleled secretion of endogenous pro-ACTH/endorphin-derived products, under both basal and stimulated conditions. With induction proNPY expression there is a dose-dependent inhibition of both proNPY and pro-ACTH/endorphin proteolytic processing.     

Pertussis toxin inhibits neuropeptide Y-induced feeding in rats

Neuropeptide Y (NPY) is the most powerful peptide drug stimulating feeding in rats. Rats with paraventricular hypothalamic (PVH) cannulae were used to investigate the mechanisms involved in NPY-induced feeding. Consistent with previous reports, injection of 2 μg of NPY into the PVH significantly increased the cumulative food intake over 1-, 2- and 4-hr periods. Ad lib feeding decreased significantly two days after pertussis toxin (PT) administration, but recovered to nearly normal levels on the fourth day. PT had no immediate effect on NPY-induced feeding; however, four days after PT was injected NPY (2 μg) did not increase the food intake compared to control. In vitro investigations showed that isoproterenol-stimulated adenylate cyclase activity in the hypothalamus of control rats was inhibited by NPY. In PT-treated rats, however, no inhibition of cAMP production was observed. These results suggest that cAMP may mediate NPY-induced feeding and that a PT-sensitive G protein may be involved in this signal transduction.     

Re-evaluation of the effects of neuropeptide Y on aggregation of human platelets.

Several studies have shown that platelets are a major source of circulating neuropeptide Y (NPY) immunoreactivity in rats, but the effects of this vasoconstrictor peptide on platelets are not well-defined. Recently, it was reported that porcine NPY was an inhibitor of in vitro human platelet aggregation induced by epinephrine, an observation which would have important implications regarding platelet-vascular interactions during states involving platelet activation and thrombosis. Thus, we undertook the present studies, in an attempt to confirm the earlier report, and to extend those observations to human NPY. In contrast to the recent report, we found no inhibitory effect of either human or porcine NPY on epinephrine- or collagen-induced aggregation of human platelets from normal subjects. Likewise, specific NPY Y-1 and Y-2 agonists had no direct or indirect action on platelet aggregation. Finally, the effect of human NPY on intraplatelet cAMP was measured. The peptide had no effect on either basal or iloprost-stimulated cAMP levels. We hypothesize that the role of NPY in the platelet-vascular interaction is in promoting vasoconstriction associated with platelet aggregation, and does not include inhibition of further thrombosis.     

Synthesis of neuropeptide Y

Neuropeptide Y (NPY), a hexatriacontapeptide amide, was synthesized on benzhydrylamine resin. The peptide product obtained by HF treatment contained 63% of the target peptide, NPY. A comparison of the chemical, immunochemical and biological properties of the synthetic peptide with natural NPY indicated that they were identical.     

Rainbow trout (Oncorhynchus mykiss) neuropeptide Y.

Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Pro-Glu-Glu-Leu-Ala-Lys-Tyr-Tyr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchus kisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells.     

Selective activation of central NPY Y1 vs. Y5 receptor elicits hyperinsulinemia via distinct mechanisms

Central administration of neuropeptide Y (NPY) stimulates hyperphagia and hyperinsulinemia. Recent evidence has suggested that the Y1 and Y5 receptor subtypes may both mediate NPY-stimulated feeding. The present study attempts to further characterize the role of central NPY receptor subtypes involved in hyperinsulinemia. NPY and peptide analogs of NPY that selectively activated the NPY Y1 or Y5 receptor subtype induced feeding and hyperinsulinemia in satiated Long Evans rats, whereas NPY analogs that selectively activated the NPY Y2 or Y4 receptor subtype did not. To determine whether NPY-induced hyperinsulinemia is secondary to its hyperphagic effect, we compared the plasma insulin levels in the presence and absence of food after a 1-min central infusion of NPY and its analogs at 15, 60, and 120 min postinfusion. Our data suggest that selective activation of central NPY Y1 receptor subtype induced hyperinsulinemia independent of food ingestion, whereas the NPY Y5 receptor-induced hyperinsulinemia was dependent on food ingestion. Central administration of the selective Y1 receptor agonist D-Arg25 NPY eventually decreased plasma glucose levels 2 h postinfusion in Long Evans rats.     

Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundant in the brain and peripheral nervous system. NPY has a variety of effects when administered into the brain including a pronounced feeding effect, anxiolysis, regulation of neuroendocrine axes and inhibition of neurotransmitter release. These effects are mediated by up to 6 G protein coupled receptors designated Y1, Y2, Y3, Y4, Y5 and y6. To better understand the phylogeny and pharmacology of NPY in non-human primates, we have cloned and expressed the NPY Y1, Y2 and Y5 receptor subtypes from the Rhesus monkey. No cDNA sequence encoding a Y4 receptor was found suggesting substantial sequence differences when compared to the human sequence. Comparison of these sequences with those from human indicated strong sequence conservation of Y1, Y2 and Y5 between the two species. The displacement of (125)I-PYY binding to the Rhesus monkey and human receptors by various peptides was compared to evaluate the pharmacology of the two species. Similar pharmacologies were noted across the species at the various receptor subtypes. These results indicate the Rhesus monkey and human NPY receptor subtypes have a close amino acid sequence conservation and that the peptide recognition domains are conserved as well.     

The primary structure and tissue distribution of an amphibian neuropeptide Y.

Neuropeptide Y (NPY) has been isolated and sequenced from brain extracts of the European common frog, Rana temporaria. Plasma desorption mass spectroscopy of the purified peptide indicated a molecular mass of 4243.3 Da which was in agreement with that deduced from the sequence (4243.7 Da), incorporating a C-terminal amide. The primary structure of frog NPY was established as: YPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRY-NH2. Frog NPY contains a single, highly-conservative amino acid substitution (Lys for Arg at residue 19) with respect to human NPY. NPY immunoreactivity was localised exclusively in nerves within the brain, pancreas and gastrointestinal tract and reverse-phase HPLC of extracts of these tissues resolved a single immunoreactive peptide of identical retention time in each case. The primary structure of NPY has therefore been highly-conserved over a considerable evolutionary time-span.     

Neuropeptide Y mRNA and peptide in the night-migratory redheaded bunting brain

This study investigated the distribution of neuropeptide Y (NPY) in the brain of the night-migratory redheaded bunting (Emberiza bruniceps). We first cloned the 275-bp NPY gene in buntings, with ≥95 % homology with known sequences from other birds. The deduced peptide sequence contained all conserved 36 amino acids chain of the mature NPY peptide, but lacked 6 amino acids that form the NPY signal peptide. Using digosigenin-labeled riboprobe prepared from the cloned sequence, the brain cells that synthesize NPY were identified by in-situ hybridization. The NPY peptide containing cell bodies and terminals (fibers) were localized by immunocytochemistry. NPY mRNA and peptide were widespread throughout the bunting brain. This included predominant pallial and sub-pallial areas (cortex piriformis, cortex prepiriformis, hyperpallium apicale, hippocampus, globus pallidus) and thalamic and hypothalamic nuclei (organum vasculosum laminae terminalis, nucleus (n.) dorsolateralis anterior thalami, n. rotundus, n. infundibularis) including the median eminence and hind brain (n. pretectalis, n. opticus basalis, n. reticularis pontis caudalis pars gigantocellularis). The important structures with only NPY-immunoreactive fibers included the olfactory bulb, medial and lateral septal areas, medial preoptic nucleus, medial suprachiasmatic nucleus, paraventricular nucleus, ventromedial hypothalamic nucleus, optic tectum, and ventro-lateral geniculate nucleus. These results demonstrate that NPY is possibly involved in the regulation of several physiological functions (e.g. daily timing feeding, and reproduction) in the migratory bunting.     

Neuropeptide Y Stimulates Feeding but Inhibits Sexual Behavior in Rats*

The effects of neuropeptide Y (NPY), a tyrosine-rich peptide found in the rat brain, on feeding and sexual behavior were studied in male and female rats. Intraventricular (ivt) injections of NPY during the final hours of the light period induced feeding in a dose-related manner. While the lowest dose tested (0.02 nM) was without effect, higher doses (0.12, 0.47, 2.3 nM) uniformly elicited feeding with a latency of about 15 min in male rats. With the most effective dose, 0.47 nM, the increased food intake was due to an increased local eating rate. In contrast, the pattern of feeding behavior after a related peptide, rat pancreatic polypeptide (rPP), was quite different and less impressive. During the first hour, only one ivt dose of rPP (0.45 nM) evoked an increase in food intake, due to an increased time spent eating. Further, the effects of NPY on food intake were greater during the nocturnal period. Interestingly, increased food intake in nocturnal tests (4 h) was due solely to augmented intake during the first 60 min after ivt administration. In mating tests, initiated 2 h after the onset of darkness and 10 min after ivt administration of peptide, all but the lowest dose of NPY (0.01 nM) drastically suppressed ejaculatory behavior. Most rats treated with higher doses of NPY (0.02, 0.12, or 0.47 nM) mounted and intromitted only a few times before the cessation of sexual activity, and elongated latencies to the initial mount and intromission were observed. In contrast to the dramatic NPY-induced suppression of ejaculatory behavior, rPP (0.11 and 0.45 nM) was without effect on copulatory behavior. To substantiate further that the impairment of sexual behavior seen in NPY-treated rats was not due to an attenuated sexual ability, an additional experiment was performed. Penile reflexes, including erection, were monitored 10 min after ivt injection of NPY (0.12 nM), rPP (0.11 nM), or saline. No effect of NPY or rPP was observed on the proportion of rats showing erection or latency to initial erection, or in the number of erections per test. In fact, a slight facilitation of penile dorsiflexion responses was seen after NPY. These findings suggest that NPY selectively depresses sexual motivation in the male rat. In ovariectomized female rats responding to estrogen plus progesterone with a good level of sexual receptivity (lordosis quotient > 70), ivt saline and 0.01 nM NPY were without effect on sexual behavior. However, higher doses of NPY (0.12 and 0.47 nM) promptly suppressed sexual behavior in tests initiated 10 min after treatment. A significant 50% decrement in receptivity and a virtual elimination of proceptive behavior were observed. Further, although a low level of mounting (one to five mounts in 15 min) was seen in both the saline (33% mounting) and the 0.01 nM NPY (38% mounting) treated groups, none was observed in animals treated with the higher NPY doses. These observations indicate that NPY may also suppress female sexual behavior.     

Isolation and characterization of neuropeptide Y from porcine intestine

The isolation and primary structure of intestinal neuropeptide Y (NPY) is described. The peptide was purified from porcine intestinal extracts using a chemical assay and radioimmunoassay for NPY. The amino acid sequence of this peptide is: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala- Arg-Tyr-Tyr- Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. This the structure of intestinal NPY is identical to the NPY of brain origin.     

Neuropeptide Y induces fasted pattern of duodenal motility via Y(2) receptors in conscious fed rats

Neuropeptide Y (NPY), a 36-amino acid peptide abundantly expressed in the brain, has been implicated in the regulation of feeding and visceral functions. The present study was designed to investigate whether or not NPY specifically regulates duodenal motility. The manometric method was used to measure duodenal motility in conscious, freely moving rats. The rat duodenum showed phasic contractions mimicking the migrating motor complex in the fasted state that were replaced by irregular contractions after the ingestion of food. NPY powerfully affected the contractile activity after intracerebroventricular (i.c.v.) administration, changing fed (postprandial) patterns into phasic contractions characterized as fasted (interdigestive) patterns. This effect was mediated via receptors with pharmacological profiles similar to rat Y(2) and Y(4) receptors, although neither Y(1) nor Y(5) agonists had any effects on motility despite potent feeding-stimulatory effects. Immunoneutralization with anti-NPY antiserum administered i.c.v. abolished fasted patterns and induced fed-like motor activities. An i.c.v. dose of peptide YY produced a different effect from NPY, with increase in the motor activities of both fed and fasted patterns. These results indicate that fasted and fed motor activities are regulated processes and that NPY induces fasted activity through Y(2), and possibly Y(4), receptors, which may represent an integrated mechanism linked to the onset of feeding behavior.     

Interaction between norepinephrine, NPY and VIP in the ovarian artery.

The in vitro effect and the interaction between norepinephrine (NE), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were studied in dissected segments of the rabbit ovarian artery. In addition, the structural requirement of the NPY receptor was investigated using NPY peptide analogs. NE induced a dose-dependent vasoconstriction with an Emax of 131.4 +/- 2.9% of K(+)-induced constriction. The vasoconstrictor effect of NPY was less than 5% of K(+)-induced vasoconstriction. Incubation of the artery with 10(-7) M NPY for 4 min induced a significant potentiation of NE-induced contractions. The selective NPY Y1 receptor agonist [Leu31, Pro34]NPY was also able to potentiate the NE response at the half-maximum contraction level, but not NPY(11-36), an NPY peptide fragment predominantly stimulating the NPY Y2 receptor. NPY exerted a dose-dependent vasoconstrictor effect on vessels contracted for 20 min with 10(-6) M NE. VIP induced a dose-dependent relaxation of vessels contracted with 10(-6) M NE. The VIP-induced relaxation could be reversed by NPY. In conclusion, receptors capable of interacting with NPY, presumably of the Y1 type, and VIP are present in the rabbit ovarian artery, and activation of these receptors may profoundly influence the response of the artery to norepinephrine.     

Anglerfish islets contain NPY immunoreactive nerves and produce the NPY analog aPY

It has recently been demonstrated that aPY, a peptide which has significant homology with neuropeptide Y (NPY) is present in extracts of anglerfish islets. The purpose of this study was to determine whether cells or nerves which contain NPY-like immunoreactivity could be identified in anglerfish islet tissue and whether aPY is synthesized by this tissue. Antisera against bovine pancreatic polypeptide (BPP), NPY and the 200 kd neurofilament polypeptide were used for immunohistochemical analysis of islets. Identical cells were stained by both the NPY and BPP antisera. The NPY and 200 kd neurofilament antisera also labeled nerve fibers in the tissue which were not stained with the BPP antiserum. The nature of the NPY-like peptide synthesized in islet cells was determined by subjecting differentially radioactively labeled Mr 2,500-8,000 peptides from islet extracts to reverse phase HPLC. Labeled aPY was unequivocally identified in the extracts and was labeled appropriately (as predicted from its sequence) with 13 different radioactive amino acids. These results demonstrate that one form of NPY-like peptide synthesized in anglerfish islets is aPY. The form of NPY-like peptide which was immunolocalized in nerves remains to be determined.     

Decrease of hypothalamic neuropeptide Y gene expression by vanadyl sulfate in streptozotocin-induced diabetic rats.

In an attempt to elucidate the effect of vanadium compounds on the gene expression of neuropeptide Y (NPY), vanadyl sulfate (VOSO4) was orally administrated at the dose of 1 mg/kg body weight into streptozotocin-induced diabetic rats (STZ-diabetic rats) three times daily for 1 week. We found a marked lowering of plasma glucose with a significant decrease of food and water intake in these STZ-diabetic rats treated with VOSO4, although the weight gain was unaffected. The increase of hypothalamic NPY, both the mRNA level and peptide concentration, in STZ-diabetic rats was also reduced by this oral treatment of VOSO4. However, similar treatment of VOSO4 in normal rats failed to modify the feeding behavior and hypothalamic NPY gene expression. These data suggest that decrease of hypothalamic NPY gene expression by VOSO4 is related to the recovery of hyperphagia in diabetic rats lacking insulin.     

NPY-leptin: opposing effects on appetitive and consummatory ingestive behavior and sexual behavior

Many studies have indicated that neuropeptide Y (NPY) stimulates and leptin inhibits food intake. In line with this, intracerebroventricular injection of NPY (10 microg) stimulated and leptin (10 microg) inhibited intake of a sucrose solution when female rats were required to obtain the solution from a bottle. However, NPY inhibited and leptin stimulated intake if the solution was infused intraorally. Thus NPY stimulates the responses used to obtain food but inhibits those used to consume food, and leptin has the opposite effects. To test the specificity of these responses the sexual behavior of male rats was examined. NPY-treated males showed minor deficits in sexual behavior but chose to ingest a sucrose solution rather than copulate with a female if offered the choice. By contrast, leptin-treated males ingested little sucrose and displayed an increase in ejaculatory frequency if given the same choice. It is suggested that NPY is not merely an orexigenic peptide, but one that directs attention toward food. Similarly, leptin may not be an anorexic peptide, but one that diverts attention away from food toward alternate stimuli.     

Peptide S is a novel potent inhibitor of voluntary and fast-induced food intake in rats

Peptide S (NPS or PEPS) and its cognate receptor have been recently identified both in the central nervous system and in the periphery. NPS/PEPS promotes arousal and has potent anxiolytic-like effects when it is injected centrally in mice. In the present experiment, we tested by different approaches its central effects on feeding behaviour in Long-Evans rats. PEPS at doses of 1 and 10 microg injected in the lateral brain ventricle strongly inhibited by more than 50% chow intake in overnight fasted rats with effects of longer duration with the highest dose (P<0.0001). A similar decrease was observed for the spontaneous intake of a high-energy palatable diet (-48%; P<0.0001). This anorexigenic effect was comparable to that induced by corticotropin-releasing hormone in fasted rats at equimolar doses. However, peptide S did not modify food intake stimulated by neuropeptide Y (NPY) at equimolar doses. It also did not affect the fasting concentrations of important modulators of food intake like leptin, ghrelin, and insulin in circulation. This study therefore showed that peptide S is a new potent anorexigenic agent when centrally injected. Its inhibitory action appears to be independent of the NPY, ghrelin, and leptin pathways. Development of peptide S agonists could constitute a new approach for the treatment of obesity.     

The effect of dexamethasone on neuropeptide Y concentrations in specific hypothalamic regions.

Neuropeptide Y (NPY) is a major hypothalamic peptide which is implicated in the regulation of energy balance and in the activation of the hypothalamo-pituitary adrenal axis. This study aimed primarily to determine the effects on regional hypothalamic NPY levels, of catabolism and weight loss induced in rats by the synthetic glucocorticoid, dexamethasone, injected daily at a dose of 0.4 mg/kg for 7 days. NPY concentrations were significantly raised in the paraventricular nucleus (PVN) of male Wistar rats (45%, p = 0.009; n = 10) compared with saline-injected controls (n = 10). Body weight (p less than 0.001) and food intake (p less than 0.001) were significantly reduced, plasma insulin concentrations were increased (p less than 0.001), but there was no change in glucose concentrations. Chronic dexamethasone treatment did not cause the marked NPY increases in the arcuate nucleus (ARC) and other hypothalamic regions which have been observed in other catabolic states causing weight loss. One possible explanation is the high insulin levels induced by dexamethasone, which may have prevented compensatory hyperphagia by suppressing an increase in hypothalamic NPYergic activity. We also examined the acute effects of a single dexamethasone injection on regional hypothalamic levels, to determine whether the drug had a direct action separate from that due to sustained weight loss. In the acute study, groups of rats (n = 7) were examined at 4 h after a single injection of dexamethasone or saline. NPY concentrations were significantly increased in the lateral hypothalamic area (LHA), (60%, p = 0.008) when compared with saline-injected controls, but there was no change in body weight or glucose or insulin concentrations during the 4h interval. Altered transport or release of NPY in the lateral hypothalamic area may be a result of acute feedback regulation by glucocorticoids on the hypothalamus.     

The role of previous exposure in the appetitive and consummatory effects of orexigenic neuropeptides

The ingestion of foods is comprised of two distinct phases of behavior: appetitive and consummatory. While most food intake paradigms include both phases, the intraoral intake test emphasizes the stereotyped consummatory-phase by infusing a liquid food directly into the oral cavity. Several hypothalamic peptides have been shown to increase intake of chow in standard food intake paradigms and the current experiments sought to test whether these peptides would increase food intake in the intraoral intake paradigm. NPY, melanin-concentrating hormone (MCH) and orexin-A were infused into the third ventricle (i3vt) in a counterbalanced latin-square design just prior to rats getting 0.1M sucrose solution infused via indwelling intraoral catheters and compared it to intake on bottle tests with access to the same sucrose solution. On the first day, each peptide increased intraoral intake relative to saline in the between-subjects comparison. Moreover, intake of sucrose following i3vt saline increased as a function of training. By the final day of the experiment, rats receiving saline consumed as much sucrose as rats receiving NPY, MCH, or orexin-A. This finding was conceptually replicated in the second experiment in which rats drank sucrose freely from a bottle on the home cage. A third experiment directly assessed the role of previous exposure in the sucrose intake induced by NPY. Those results confirm that repeated exposure to sucrose increases baseline intake and attenuates the hyperphagic effect of NPY. These results are consistent with two conclusions: (1) NPY, MCH, and orexin-A increase both appetitive and consummatory-phase ingestive behaviors on initial exposures; (2) repeated training interacts with the effects of these orexigenic peptides.