Porcine NPY Research Articles

In vitro effect of leptin on somatolactin release in the European sea bass ( Dicentrarchus labrax): dependence on the reproductive status and interaction with NPY and GnRH

The aim of the present work was to investigate the neuroendocrine control of pituitary somatolactin (SL) release using dispersed pituitary cell culture obtained from male European sea bass ( Dicentrarchus labrax) at different stages of sexual development. The effect of mouse recombinant leptin, sea bream gonadotropin releasing-hormone (sbGnRH) and porcine neuropeptide Y (pNPY) and their potential interaction on the SL release were investigated. High doses of leptin (10 −8–10 −6 M) were differentially effective in inducing SL release depending on the sexual developmental stage. Porcine NPY alone was not effective on basal SL release, but it dose-dependently (0.1 and 1 nM) enhanced SL release induced by leptin (10 −6 and 10 −8 M) in late pre-pubertal but not in post-pubertal stages. No effect of sbGnRH in association or not with leptin was observed on SL release. These findings are the first evidences that leptin and pNPY can play an important role in the neuroendocrine control of pars intermedia function and SL release in fish. In addition, the sensitivity of SL producing cells to leptin and NPY only in prepubertal and pubertal stages, provides the potential role of SL in the nutritional control of the onset of puberty.

Neuropeptide Y inhibits spontaneous alpha-melanocyte-stimulating hormone (alpha-MSH) release via a Y(5) receptor and suppresses thyrotropin-releasing hormone-induced alpha-MSH secretion via a Y(1) receptor in frog melanotrope cells.

In amphibians, the secretion of alpha-MSH by melanotrope cells is stimulated by TRH and inhibited by NPY. We have previously shown that NPY abrogates the stimulatory effect of TRH on alpha-MSH secretion. The aim of the present study was to characterize the receptor subtypes mediating the action of NPY and to investigate the intracellular mechanisms involved in the inhibitory effect of NPY on basal and TRH-induced alpha-MSH secretion. Y(1) and Y(5) receptor mRNAs were detected by RT-PCR and visualized by in situ hybridization histochemistry in the intermediate lobe of the pituitary. Various NPY analogs inhibited in a dose-dependent manner the spontaneous secretion of alpha-MSH from perifused frog neurointermediate lobes with the following order of potency porcine peptide YY (pPYY) > frog NPY (fNPY) > porcine NPY (pNPY)-2-36) > pNPY-(13-36) > [D-Trp(32)]pNPY > [Leu(31),Pro(34)]pNPY. The stimulatory effect of TRH (10(-8)6 M) on alpha-MSH release was inhibited by fNPY, pPYY, and [Leu(31),Pro(34)]pNPY, but not by pNPY-(13-36) and [D-Trp(32)]pNPY. These data indicate that the inhibitory effect of fNPY on spontaneous alpha-MSH release is preferentially mediated through Y(5) receptors, whereas the suppression of TRH-induced alpha-MSH secretion by fNPY probably involves Y(1) receptors. Pretreatment of neurointermediate lobes with pertussis toxin (PTX; 1 microg/ml; 12 h) did not abolish the inhibitory effect of fNPY on cAMP formation and spontaneous alpha-MSH release, but restored the stimulatory effect of TRH on alpha-MSH secretion, indicating that the adenylyl cyclase pathway is not involved in the action of fNPY on TRH-evoked alpha-MSH secretion. In the majority of melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca(2+) concentration. Preincubation of cultured cells with fNPY (10(-7) M) or omega-conotoxin GVIA (10(-7) M) suppressed the plateau phase of the Ca(2+) response induced by TRH. However, although fNPY abrogated TRH-evoked alpha-MSH secretion, omega-conotoxin did not, showing dissociation between the cytosolic Ca(2+) concentration increase and the secretory response. Collectively, these data indicate that in frog melanotrope cells NPY inhibits spontaneous alpha-MSH release and cAMP formation through activation of a Y(5) receptor coupled to PTX- insensitive G protein, whereas NPY suppresses the stimulatory effect of TRH on alpha-MSH secretion through a Y(1) receptor coupled to a PTX-sensitive G protein-coupled receptor.

Action of leptin on in vitro luteinizing hormone release in the European sea bass (Dicentrarchus labrax).

The discovery of leptin has sparked a rapidly growing number of publications concerning the role of leptin in the regulation of body adiposity, feeding, and reproductive system in mammals. To date, there have been no reports on the presence of leptin-related peptide, and functional studies on the role of leptin remain limited in fishes. We investigated the effect of mouse recombinant leptin on basal and sea bream (sb) GnRH-induced LH release from dispersed pituitary cells obtained from male European sea bass (Dicentrarchus labrax) at different stages of sexual development. The potential interaction of leptin with the porcine neuropeptide Y (pNPY), known to play a dual role in feeding and reproduction in vertebrates, was also investigated. High doses of leptin (10(-8)-10(-6) M) and/or pNPY (0.1 and 1 nM) had different effects on LH release at various stages of sexual development. Porcine NPY alone was weakly effective on basal LH release, but it enhanced LH release induced by leptin (10(-6) M) in late prepuberty but not in early postpuberty. Additive or inhibitory effects of leptin were observed on sbGnRH-induced LH release depending on sbGnRH dose and stage of sexual development. The direct action of leptin on LH release at the pituitary level in sea bass suggests that leptin is a regulator of the reproductive system in fishes.

Chronic blockade of the melanocortin 4 receptor subtype leads to obesity independently of neuropeptide Y action, with no adverse effects on the gonadotropic and somatotropic axes.

Neuropeptide Y (NPY) is a powerful orexigenic factor, and alphaMSH is a melanocortin (MC) peptide that induces satiety by activating the MC4 receptor subtype. Genetic models with disruption of MC4 receptor signaling are associated with obesity. In the present study, a 7-day intracerebroventricular infusion to male rats of either the MC receptor antagonist SHU9119 or porcine NPY (10 nmol/day) was shown to strongly stimulate food and water intake and to markedly increase fat pad mass. Very high plasma leptin levels were found in NPY-treated rats (27.1 +/- 1.8 ng/ml compared with 9.9 +/- 0.9 ng/ml in SHU9119-treated animals and 2.1 +/- 0.2 ng/ml in controls). As expected, NPY infusion induced hypogonadism, characterized by an impressive decrease in seminal vesicle and prostate weights. No such effects were seen with the SHU9119 infusion. Similarly, whereas the somatotropic axis of NPY-treated rats was fully inhibited, this axis was normally activated in the obese SHU9119-treated rats. Chronic infusion of SHU9119 strikingly reduced hypothalamic gene expression for NPY (65.2 +/- 3.6% of controls), whereas gene expression for POMC was increased (170 +/- 19%). NPY infusion decreased hypothalamic gene expression for both POMC and NPY (70 +/- 9% and 75.4 +/- 9.5%, respectively). In summary, blockade of the MC4 receptor subtype by SHU9119 was able to generate an obesity syndrome with no apparent side-effects on the reproductive and somatotropic axes. In this situation, it is unlikely that hyperphagia was driven by increased NPY release, because hypothalamic NPY gene expression was markedly reduced, suggesting that hyperphagia mainly resulted from loss of the satiety signal driven by MC peptides. NPY infusion produced hypogonadism and hyposomatotropism in the face of markedly elevated plasma leptin levels and an important reduction in hypothalamic POMC synthesis. In this situation NPY probably acted both by exacerbating food intake through Y receptors and by reducing the satiety signal driven by MC peptides.

Molecular biology and pharmacology of multiple NPY Y 5 receptor species homologs

NPY is a 36-amino acid peptide which exerts its physiological effects through the activation of a family of G-protein coupled receptors. In vivo and in vitro characterization of the recently cloned rat Y 5 receptor suggests that it is a primary mediator of NPY-induced feeding (Gerald et al., Nature 1996;382:168–171). We now report the molecular cloning and pharmacological characterization of the human, dog and mouse homologs of the Y 5 receptor. With the exception of a 21 amino acid repeat in the amino terminus of the mouse Y 5 receptor, the sequence of the four species homologs appear to be highly conserved, with 88% to 97% amino acid identities between any two species. Similarly, the pharmacological profiles of the four species homologs as determined in porcine 125I-PYY binding assays show a great deal of conservation, with the following rank order of affinity: human or porcine NPY, PYY, [Leu 31,Pro 34]NPY, NPY 2–36, human PP>human [ d-Trp 32]NPY>rat PP, C2-NPY. Northern blot analysis reveals that the Y 5 receptor is widely distributed in the human brain, with the strongest signals detected in the cortex, putamen and caudate nucleus. The chromosomal localization of the human Y 5 receptor, previously shown to be overlapping and in the opposite orientation to the Y 1 receptor, is determined to be 4q31, the same locus as previously demonstrated for the human Y 1 receptor (Herzog et al., J Biol Chem 1993;268:6703–6707), suggesting that these receptors may be coregulated. These Y 5 species homologs along with corresponding animal models may be useful in the search for novel therapeutics in the treatment of obesity and related feeding disorders.

Localization and characterization of neuropeptide Y binding sites in porcine and human colon

1. We have used quantitative receptor autoradiography to investigate the localization and characteristics of binding sites for 125Iodine-Bolton Hunter-labelled human neuropeptide Y ([125I]-BH-NPY) in porcine and human colon, and compared the binding characteristics with those found in porcine spleen. 2. Saturable, specific, high affinity [125I]-BH-NPY binding was localized to myenteric ganglia in porcine and human colons, and to submucosal ganglia in porcine colon. 3. Specific [125I]-BH-NPY binding to porcine myenteric ganglia was reversible in the presence of guanosine 5'-O-(3-thiotriphosphate) and was inhibited by related peptides with the rank order of potency; porcine NPY = human NPY = peptide tyrosine tyrosine (PYY) >> pancreatic polypeptide. 4. The Y2 selective analogue, NPY (13-36), competed for [125I]-BH-NPY binding to porcine myenteric ganglia with greater potency than the Y1 selective analogue, [Leu31, Pro34] NPY, the difference being small, but significant. 5. The characteristics of [125I]-BH-NPY binding to porcine myenteric ganglia were similar to those observed concurrently to porcine splenic red pulp. 6. The small difference in inhibitory potencies between NPY(13-36) and [Leu31, Pro34]NPY observed in this study in comparison with previous studies was not explained by differential ligand depletion during incubations, but may be due to differences in methodology between binding studies performed on tissue sections and on membranes. 7. We conclude that specific [25I]-BH-NPY binding sites are present in the myenteric and submucosal ganglia of the colon and that these sites may act as functional receptors by which NPY and PYY modulate colonic motility and electrolyte transport.

Characterization of neuropeptide Y-like immunoreactivity in vertebrate retina

We have determined the endogenous levels of neuropeptide Y-like immunoreactivity (NPY-LI) in retinal extracts from pigs, cats, rabbits, chickens, and frogs by radioimmunoassay (RIA). The NPY-LI levels varied among the species. The highest concentration was found in frog retina, where seasonal variations were seen, 861 ± 31 pmol g −1 wet weight in the autumn and 334 ± 26 pmol g −1 wet weight in the spring. Lower levels were demonstrated in chicken and pig retina, 4·1 ± 0·4 and 3·6 ± 0·3 pmol g −1 wet weight. respectively. The lowest concentration was demonstrated in rabbit retina, 2·0 ± 0·3 pmol g −1 wet weight. (All values are expressed as mean ± s.e.m.) The NPY-LI in pig, rabbit, chicken and frog retina was characterized by high-performance liquid chromatography (HPLC). The main part of the extracted NPY-LI had an elution volume close to that to the porcine NPY. We have also analysed the evoked release of endogenous NPY from frog retina, induced either with light flashes (3 Hz, 300 lx), or with potassium depolarization of the neurons (40 m m). Light flashes and potassium induced an increased release of NPY-LI of 61 and 77%, respectively. NPY-LI in the efflux had the same HPLC retention time as that extracted directly from the retina.

Solution synthesis of human neuropeptide Y (hNPY).

Human neuropeptide Y (hNPY) was synthesized in a conventional manner by assembling seven peptide fragments followed by reduction of the Met(O) residue with phenylthiotrimethylsilane and subsequent deprotection with 1 M trimethylsilyl trifluoromethanesulfonate (TMSOTf)-thioanisole in trifluoroacetic acid (TFA). Alternatively, deprotection was performed in a two-step manner; first, treatment with 1 M trimethylsilyl bromide-thioanisole in TFA, and then with 1 M TMSOTf-thioanisole in TFA. After purification by gel-filtration on Sephadex G-25, followed by reversed-phase high-performance liquid chromatography, a highly purified sample of synthetic hNPY was obtained in both cases. When administered in dogs, synthetic hNPY was as active as porcine NPY in terms of the effects on systemic arterial blood pressure, pancreatic blood flow, and superior mesentric artery (SMA) blood flow. Met(O)17-hNPY was found to be as active as the parent sample in these bioassays.

Distribution and characterization of neuropeptide Y-like immunoreactivity in the brain of the crested newt.

The newt brain represents a simplified model for the increasingly complex vertebrate neuronal organization. The localization of neuropeptide Y-like (NPY-like) containing neurons in the brain of Triturus cristatus was studied by means of indirect immunofluorescence, peroxidase-antiperoxidase, and avidin-biotin techniques using a highly specific antiserum. NPY-like positive cell bodies were observed in several areas, most notably in the telencephalon (primordium hippocampi and amygdaloid complex), the preoptic and suprachiasmatic areas, the hypothalamus, the dorsal thalamus, the tegmentum, and the rhombencephalon (laterolateral grey column and raphe area). Nerve fibres were particularly abundant in the pallium, striatum, septum, amygdaloid, preoptic neuropils, and pars intercalaris diencephali. Bundles of NPY-immunoreactive fibres also were visualized in the dorsal thalamus and in the posterior hypothalamus. The pars intermedia lacked any NPY-like positive fibres. Neuronal processes also were found in the tectum mesencephali and in the body of the cerebellum. A prominent NPY-like fibre network was observed in the octavolateralis. Concentrations of NPY measured by means of a specific radioimmunoassay were threefold higher in the hypothalamus (15.2 +/- 1.3 ng/mg proteins) than in the rhombencephalon (4.9 +/- 0.3) and the mesencephalon (4.3 +/- 0.2). The concentration found in the telencephalon was 2.1 +/- 0.3 ng/mg proteins. Sephadex G-50 gel chromatography of whole brain extracts indicated the presence of high molecular weight forms of NPY-like material in addition to the authentic peptide. Both amphibian and mammalian NPY peptides had an apparent molecular weight of 4,000 daltons, as evidenced by immunoblotting analysis. High-performance liquid chromatography demonstrated, however, that the newt peptide was slightly less hydrophobic than porcine NPY. The present findings indicate that NPY-immunoreactive neurons are widely distributed in the brain of urodeles. Our data indicate that the NPY molecule has been relatively well preserved during evolution.

NPY-like peptides occur in the nervous system and midgut of the migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sarcophaga bullata

The distribution of the NPY-like substances in the nervous system and the midgut of the migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sarcophaga bullata was determined by immunocytochemistry using an antiserum directed against synthetic porcine NPY. The peroxidase-antiperoxidase procedure revealed that NPY immunoreactive cell bodies and nerve fibers were observed in the brain, optic lobes, corpora cardiaca, suboesophageal ganglion and ventral nerve cord of the locust and in the brain, optic lobes and suboesophageal ganglion of the fleshfly. In the locust midgut, numerous endocrine cells and nerve fibers penetrating the outer musculature contained NPY-like immunoreactivity. The concentrations of NPY immunoreactive material in acetic acid extracts of locust brain, optic lobes, thoracic ganglia, ovaries and midguts was measured using a specific radioimmunoassay technique. The dilution curves of the crude tissue extracts were parallel to the standard curve. The highest amount of NPY-like immunoreactivity was found in the locust ovary and midgut. Reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay were used to characterize the NPY-like substances in the locust brain and midgut. HPLC-analysis revealed that NPY-immunoreactivity in the locust brain eluted as three separate peaks. The major peak corresponded to a peptide less hydrophobic than synthetic porcine NPY. RP-HPLC analysis of midgut extracts revealed the presence of an additional NPY-immunoreactive peak which had a retention time similar to the porcine NPY standard. The present data show the existence of a widespread network of NPY immunoreactive neurons in the nervous system of the locust and the fleshfly. Characterization of the immunoreactive substances indicates that peptides similar but not identical to porcine NPY are present in the central nervous system and midgut of insects.

NPY stimulates nett absorption across rat intestinal mucosa in vivo

NPY is a peptide recently isolated from porcine brain which has been seen to be widely distributed in the central and peripheral nervous system. The peptide is know to be present in substantial quantities in the extrinsic and intrinsic innervation of the gut but few studies have yet been done on the functional activity of the peptide in this site. In this study, intravenous infusion of porcine NPY was seen to cause nett absorption in the proximal duodenum. In addition, NPY was found to inhibit the nett secretory effect of stimultaneously infused VIP in the proximal duodenum and jejunum. This evidence of functional activity of NPY in the gut further supports a role for this peptide in controlling or modulating intestinal function.

Innervation of the large arteries and heart of the toad (Bufo marinus) by adrenergic and peptide-containing neurons

The innervation of the major arteries and heart of the toad (Bufo marinus) was examined by use of glyoxylic acid-induced catecholamine fluorescence and peptide immunohistochemistry. All arteries possessed a moderate to dense plexus of adrenergic axons, which also showed neuropeptide Y-like immunoreactivity (NPY-LI). Some adrenergic axons in the intracardiac vagal trunks showed NPY-LI, but the varicose adrenergic axons innervating the cardiac muscle of the atria and ventricle, and the coronary blood vessels did not display NPY-LI. About half of the nerve cell bodies in the anterior sympathetic chain ganglia with dopamine-beta-hydroxylase-LI (DBH-LI) also contained NPY-LI. The nerve cell bodies with DBH-LI alone were generally larger (median diameter 30 micron) than those with both DBH-LI and NPY-LI (median diameter 20 micron). Some cell bodies showing DBH-LI alone were surrounded by boutons with NPY-LI but not DBH-LI. Axons that displayed simultaneously both substance P-LI (SP-LI) and calcitonin gene-related peptide-LI (CGRP-LI) also formed a plexus around all arteries studied, being particularly dense around the mesenteric and pulmonary arteries. These axons are most likely sensory since SP-LI was reduced by capsaicin treatment, and nerve cell bodies with both SP-LI and CGRP-LI were found in dorsal root ganglia and the vagal ganglion. A dense plexus of axons showing somatostatin-LI was located around the pulmonary artery and its main intrapulmonary branches. A few nerves with vasoactive intestinal polypeptide-LI were found around the dorsal aorta and pulmonary artery. No perivascular nerves with enkephalin-LI were observed. Reversed-phase, high-pressure liquid chromatography of acid extracts of the large arteries showed that the major peaks of NPY-LI and SP-LI co-eluted with porcine NPY (1-36) and synthetic SP (1-11), respectively. Thus, the location and structure of these peptides in perivascular nerves has been highly conserved during vertebrate evolution.

Guanethidine-sensitive release of neuropeptide Y-like immunoreactivity in the cat spleen by sympathetic nerve stimulation

Splenic nerve stimulation (10 Hz for 2 min) caused a perfusion-pressure increase, a volume reduction and an increase in the output of neuropeptide Y-like immunoreactivity (NPY-LI) from the isolated blood-perfused cat spleen. Gel-filtration HPLC analysis revealed that plasma NPY-LI collected during nerve stimulation was similar to the NPY-LI in the spleen and synthetic porcine NPY. Combined propranolol and phenoxybenzamine pretreatment enhanced NPY output upon nerve stimulation by about 60%. Forty percent of the perfusion-pressure increase and 25% of the volume reduction seen during control stimulations remained after adrenoceptor blockade. Guanethidine abolished the release of NPY-LI, the perfusion-pressure increase and the volume reduction normally seen upon splenic nerve stimulation. Infusion of synthetic porcine NPY caused a long-lasting increase in perfusion pressure and a relatively moderate volume reduction. Noradrenaline (NA) both increased perfusion pressure and induced a marked volume reduction. The NPY effects were resistant to adrenoceptor antagonists in doses which abolished the NA response. In conclusion, the present data show that NPY-LI is released upon sympathetic nerve stimulation by a guanethidine-sensitive mechanism. Furthermore, the sympathetic response is partially resistant to adrenoceptor antagonists and NPY has powerful vasoconstrictor effects. This provides further evidence for a role of NPY in sympathetic vascular control.