Primary structure of frog PYY: implications for the molecular evolution of the pancreatic polypeptide family.

A peptide belonging to the pancreatic polypeptide (PP) family was isolated in pure form from the intestine of the European green frog (Rana ridibunda). The primary structure of the peptide was established as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu10-Asp-Ala- Ser-Pro-Glu-Glu-Met-Thr-Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile- Asn-Leu30-Val - Thr-Arg-Gln-Arg-Tyr-NH2. This amino acid sequence shows moderate structural similarity to human PYY (75% identity) but stronger similarity to the PP family peptides isolated from the pancreas of the salmon (86%) and dogfish (83%). The data suggest that the two putative duplications of an ancestral PP family gene that have given rise to PP, PYY and NPY in mammals had already taken place by the time of the appearance of the amphibia. In fish, however, only a single duplication has occurred, giving rise to NPY in nervous tissue and a PYY-related peptide in both pancreas and gut.     

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.     

The effect of diet on the Vervet monkey endocrine pancreas

Vervet monkeys (Cercopithecus aethiops) used for pancreatic endocrine cell distribution studies were found to have been maintained on different diets. Although the effect of dietary changes on the exocrine pancreas has been described in several animals, little, apart from the effect of malnutrition, has been reported for the endocrine pancreas. Reported here are pancreatic endocrine cell distributions in monkeys on a standard diet (n ? 3) compared with monkeys on an atherogenic diet (n = 3). Quantitation of immunolabelled pancreatic endocrine cell types revealed a significant 80% increase in A (glucagon) cell volume in monkeys on an atherogenic diet concomitant with a significant reduction in B (insulin) cell volume to approximately 60% of normal. This reflects a pattern of events that occurs in non-insulin dependent diabetes. An accompanying reduction in PP (pancreatic polypeptide) cell volumes supports our hypothesis that altering A and PP cell volumes could reflect differential gene expression in those cells in the adult in which glucagon and PP are co-localized.     

Energy homeostasis and gastrointestinal endocrine differentiation do not require the anorectic hormone peptide YY

The gastrointestinal hormone peptide YY is a potent inhibitor of food intake and is expressed early during differentiation of intestinal and pancreatic endocrine cells. In order to better understand the role of peptide YY in energy homeostasis and development, we created mice with a targeted deletion of the peptide YY gene. All intestinal and pancreatic endocrine cells developed normally in the absence of peptide YY with the exception of pancreatic polypeptide (PP) cells, indicating that peptide YY expression was not required for terminal differentiation. We used recombination-based cell lineage trace to determine if peptide YY cells were progenitors for gastrointestinal endocrine cells. Peptide YY(+) cells gave rise to all L-type enteroendocrine cells and to islet partial differential and PP cells. In the pancreas, approximately 40% of pancreatic alpha and rare beta cells arose from peptide YY(+) cells, suggesting that most beta cells and surprisingly the majority of alpha cells are not descendants of peptide YY(+)/glucagon-positive/insulin-positive cells that appear during early pancreagenesis. Despite the anorectic effects of exogenous peptide YY(3-36) following intraperitoneal administration, mice lacking peptide YY showed normal growth, food intake, energy expenditure, and responsiveness to peptide YY(3-36). These observations suggest that targeted disruption of the peptide YY gene does not perturb terminal endocrine cell differentiation or the control of food intake and energy homeostasis.     

Neuropeptide Y-related peptides from the pancreas of a teleostean (eel), holostean (bowfin) and elasmobranch (skate) fish

Homologous peptides belonging to the pancreatic polypeptide (PP) family were isolated from the pancreas of a teleostean fish, the American eel (Anguilla rostrata), an holostean fish, the bowfin (Amia calva) and an elasmobranch fish, the skate (Raja rhina), and their primary structures were determined. The peptides show stronger homology to neuropeptide Y, particularly in their COOH-terminal regions, than to peptide YY or pancreatic polypeptide and contain an alpha-amidated COOH-terminal tyrosine residue. The skate peptide Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Asp10-Asp-Ala-Ala-Pro-Glu-Glu- Leu-Ala-Lys- Tyr20-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Ile-Thr-Arg- Gln-Arg-Tyr-NH2 represents the first member of the PP family to be isolated from a cartilaginous fish. The primary structure of the pancreatic PP family peptide has been more strongly conserved among the phylogenetically more ancient holostean and elasmobranch fishes than among the teleosts. A comparison of the primary structures of all PP family peptides supports the hypothesis and evolution has acted to conserve features of tertiiary structure in the molecules (e.g., the polyproline- and alpha-helices) rather than individual amino acid residues.     

Ontogeny and the effect of aging on pancreatic polypeptide and peptide YY

Pancreatic polypeptide (PP) and peptide YY (PYY) are related neuroendocrine peptides that are expressed in specialized cells. PP is found around the time of birth in different species. PYY in mice and rats has been extensively studied. PYY is the first peptide hormone to appear in both the pancreas and the colon and is initially expressed together with all other pancreatic islet and gut hormones. This suggests that there is a PYY-producing endocrine progenitor cell, at least in rodents. Whether the same is true for other species is unknown. In chickens, however, pancreatic insulin and glucagon cells appear before PYY. After birth, PYY levels in rats and humans reflect adaptation to enteral feeding. Whereas PYY cells increase with age in rodents, no such changes have been found in humans.     

Pancreatic polypeptide-like material in nerves and endocrine cells of the rat

A region-specific antiserum (AbS11) raised against the carboxyl-terminal hexapeptide of pancreatic polypeptide has been employed to measure rat pancreatic polypeptide specifically and to demonstrate apparent immunoreactivity in nerves and in endocrine cells outside the pancreas. The concentration of pancreatic polypeptide in the head of the rat pancreas measured with AbS11 (176 +/- 47 pmol/g) was 750 fold higher than that measured with a conventional anti-bPP antiserum (0.23 +/- 0.08 pmol/g). Column chromatographs of rat pancreatic extracts demonstrated two peaks of immunoreactivity both eluting after the porcine pancreatic polypeptide standard. AbS11 also detected specific immunoreactivity in rat brain (470 fmol/g) which went undetected in convention assays. Although immunohistochemical studies with AbS11 and human pancreatic polypeptide antiserum demonstrated immunoreactivity in the same population of pancreatic endocrine cells, immunoreactive nerve fibres and enteroglucagon cells were only demonstrable with AbS11. These studies demonstrate that the carboxyl terminus of rat pancreatic polypeptide is immunochemically similar to that of higher mammals. Furthermore, neural and extrapancreatic endocrine variants of this peptide share an immunochemical determinant contained within the carboxyl-terminal hexapeptide.     

Glycine-extended anglerfish peptide YG (aPY) a neuropeptide Y (NPY) homologue may be a precursor of a biologically active peptide

The 37 residue peptide YG (aPY), isolated from anglerfish endocrine pancreas, bears distinct sequence homology to the pancreatic polypeptide family of hormones. However, instead of a carboxyl-terminal tyrosine-amide, aPY has a free carboxyl-terminus ending with glycine. Towards studying the structure-activity relationship of this hormone, we have synthesized aPY by solid phase methodology using Boc-amino acid derivatives and phenylacetamidomethyl resin. The crude peptide was purified to homogeneity in 20% yield by reversed phase chromatography. The purified peptide had the expected amino acid composition and sequence, and was found to be identical with the natural aPY by analytical HPLC and peptide mapping of proteolytic digests. Neither the snythetic nor the natural aPY exhibited the characteristic vasoconstrictor activity of the related pancreatic polypeptide family of hormones. However, [Des37-Gly]-aPY, isolated from the anglerfish pancreas, caused vasoconstriction in rats. Based on these results and by analogy to the glycine-extended gastrin peptides, it may be suggested that aPY is a precursor of a biologically active peptide, namely [Des37-Gly]-aPY-amide.     

Canine pancreatic polypeptide complementary deoxyribonucleic acid sequence: pancreatic polypeptide and insulin messenger ribonucleic acid distribution in the lobes of the pancreas

The structure of the canine prepropancreatic polypeptide (preproPP) cDNA was determined. The nucleotide sequence conservation between human and canine preproPP is very high for the signal peptide (82%) and the region coding for the 36 amino acid pancreatic polypeptide (PP) (92%). The overall sequence homology for the C-terminal portion of proPP containing the icosapeptide and a C-terminal extension peptide is only 63% whereas the 3'-untranslated regions of human and canine PP mRNA share 73% homology after alignment for maximal homology. The only sequence conservation in icosapeptide is the region coding for the last 10 amino acids of the icosapeptide. Comparison of PP immunoactivity and PP mRNA concentrations in extracts of the developmentally distinct uncinate process and splenic lobes of the canine pancreas revealed the same ratio of mRNA concentrations (16 +/- 6.5) and PP peptide concentrations (18 +/- 7.0) in the uncinate process compared to the splenic lobe (n = 6). However, a similar comparison of insulin C-peptide (CP) immunoactivity and insulin mRNA concentration revealed a smaller ratio of CP immunoactivity (0.37 +/- 0.05) than insulin mRNA (0.58 +/- 0.10) between the same lobes (P less than 0.0074, n = 6). This increased steady state CP concentration relative to insulin mRNA in splenic lobe compared to the uncinate process was not observed for PP peptide and mRNA.     

Some peptide-like colocalizations in endocrine cells of the pyloric caeca and the intestine of Oncorhynchus mykiss (Teleostei)

Summary The coexistence of immunoreactivities to cholecystokinin, glucagon, glucagon-like peptide 1, salmon pancreatic polypeptide, neuropeptide tyrosine, and peptide tyrosine tyrosine was studied immunocytochemicaly, revealing for the first time in fish intestine the existence in the same cell of immunoreactivities to cholecystokinin-glucagon/glucagon-like peptide 1, cholecystokinin-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-neuropeptide tyrosine, salmon pancreatic polypeptide tyrosine tyrosine, and glucagon/glucagon-like peptide 1-peptide tyrosine tyrosine. Colocalization of cholecystokinin-salmon pancreatic polypeptide was observed only in the pyloric caeca of the rainbow trout Oncorhynchus mykiss, while the other colocalizations also occurred in proximal and middle intestinal segments. In all cases, endocrine cells immunoreactive to only one of the paired antisera were detected except for anti-glucagon and anti-glucagon-like peptide 1, which always immunostained the same cells.     

Brain-gut axis in pancreatic secretion and appetite control.

The stimulation of exocrine pancreatic secretion that has been attributed by Pavlov exclusively to various reflexes (nervism), was then found that it depend also on numerous enterohormones, especially cholecystokinin (CCK) and secretin, released by duodeno-jejunal mucosa and originally believed to act via an endocrine pathway. Recently, CCK and other enterohormones were found to stimulate the pancreas by excitation of sensory nerves and triggering vago-vagal and entero-pancreatic reflexes. Numerous neurotransmitters and neuropeptides released by enteric nervous system (ENS) of gut and pancreas have been also implicated in the regulation of exocrine pancreas. This article was designed to review the contribution of vagal nerves and entero-hormones, especially CCK and other enterohormones, involved in the control of appetitive behavior such as leptin and ghrelin and pancreatic polypeptide family (peptide YY and neuropeptide Y). Basal secretion shows periodic fluctuations with peals controlled by ENS and by motilin and Ach. Plasma ghrelin, that is considered as hunger hormone, increases under basal conditions, while plasma leptin falls to the lowest level. Postprandial pancreatic secretion, classically divided into cephalic, gastric and intestinal phases, involves predominantly CCK, which under physiological conditions acts almost entirely by activation of vago-vagal reflexes to stimulate the exocrine pancreas, being accompanied by the fall in plasma ghrelin and increase of plasma leptin, reflecting feeding behavior. We conclude that the major role in postprandial pancreatic secretion is played by vagus and gastrin in cephalic and gastric phases and by vagus in conjunction with CCK and secretin in intestinal phase. PP, PYY somatostatin, leptin and ghrelin that affect food intake appear to participate in the feedback control of postprandial pancreatic secretion via hypothalamic centers.     

The complete primary structure of pancreatic polypeptide from the European common frog, Rana temporaria

Using an antiserum directed against the highly-conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP), numerous immunoreactive endocrine cells were identified within the pancreas of the European common frog, R. temporaria. An acidified ethanolic extract of pancreatic tissue (0.859 g, n = 35) contained 26.2 nmol equivalents/g of tissue. Gel permeation chromatography of the extract resolved a single peak of immunoreactivity co-eluting with synthetic bovine PP standard. Reverse phase HPLC of this material resolved a single peak of immunoreactivity which was purified to homogeneity following chromatography on a semipreparative C-18 column and an analytical C-8 column. Plasma desorption mass spectrometry (PDMS) of the purified peptide resolved a single component with a molecular mass of 4240.9 Da. Direct gas phase sequencing established the sequence of the first 26 residues. Following incubation of the peptide with endopeptidase Asp-N and direct application of the digest to the sequencer, the entire primary structure of the peptide was established as: APSEPHHPGDQATQDQLAQYYSDLYQYITFVTRPRF. The derived molecular mass of this peptide, incorporating a C-terminal amide, was 4240.6 Da which is entirely consistent with that obtained by PDMS.     

Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius)

The primary structure of pancreatic polypeptide from the teleostean fish, Cottus scorpius (daddy sculpin) was established as: YPPQPESPGGNASPEDWAKYHAAVRHYVNLITRQRYNH2 The presence of a COOH-terminally alpha-amidated amino acid was established using an HPLC method of general applicability. Although the peptide shows strong homology towards anglerfish pancreatic polypeptide (86%), homology towards porcine peptide YY (PYY) (61%) and porcine neuropeptide Y (NPY) (61%) was greater than towards porcine pancreatic polypeptide (PP) (47%). This result supports suggestions that the gene duplication events which led to PP, NPY and PYY formation took place after the time of divergence of fish and mammals.     

Molecular evolution of the neuropeptide Y (NPY) family of peptides: cloning of three NPY-related peptides from the sea bass (Dicentrarchus labrax)

Neuropeptide Y (NPY) is a 36-amino-acid peptide that is widely and abundantly expressed in the central nervous system of all vertebrates investigated. Related peptides have been found in various vertebrate groups: peptide YY (PYY) is present in gut endocrine cells of many species and pancreatic polypeptide (PP) is made in the pancreas of all tetrapods. In addition, a fish pancreatic peptide called PY has been reported in three species of fishes. The evolutionary relationships of fish PY have been unclear and it has been proposed to be the orthologue (species homologue) of each of the three tetrapod peptides. We demonstrate here with molecular cloning techniques that the sea bass (Dicentrarchus labrax), an acanthomorph fish, has orthologues of both NPY and PYY as well as a separate PY peptide. Sequence comparisons suggest that PY arose as a copy of the PYY gene, presumably in a duplication event separate from the one that generated PP from PYY in tetrapods. PY sequences from four species of fish indicate that, similar to PP, PY evolves much more rapidly than NPY and PYY. The physiological role of PY is unknown, but we demonstrate here that sea bass PY, like NPY and PYY but in contrast to the tetrapod PP, is expressed in brain.     

Gastrointestinal satiety signals III. Glucagon-like peptide 1, oxyntomodulin, peptide YY, and pancreatic polypeptide

Many peptides are synthesized and released from the gastrointestinal tract and pancreas, including pancreatic polypeptide (PP) and the products of the gastrointestinal L cells, glucagon-like peptide 1 (GLP-1), oxyntomodulin, and peptide YY (PYY). Whereas their roles in regulation of gastrointestinal function have been known for some time, it is now evident that they also influence eating behavior. This review considers the anorectic peptides PYY, PP, GLP-1, and oxyntomodulin, which decrease appetite and promote satiety in both animal models and humans.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax)

Summary The development of the endocrine pancreas of the teleost sea bass (Dicentrarchus labrax, L.) was examined from hatching to 61 days, using the peroxidase-antiperoxidase technique for light microscopy. Mammalian and bonito insulin (mI and bI)-, salmo somatostatin-25 (SST-25)-, somatostatin-14 (SST-14a and b)-, glucagon-, bovine pancreatic polypeptide (PP)-, peptide tyrosine-tyrosine (PYY)- and salmo neuropeptide Y (NPY)-like immunoreactivity was demonstrated. Four ontogenetic stages were established according to the organization and immunostaining of the endocrine cells. One cell strand or primordial cord showing mI/bI- and SST-25/SST-14a-like immunoreactivity was first found at hatching in the dorsal epithelium of the anterior zone of the midgut (stage 1). One primitive islet, comprising outer SST-25/SST-14a- and inner mI/bI- and SST-14a/ SST-14b-immunoreactive cells, was found in 2- to 5-day-old larvae (stage 2). One single islet, in which glucagon-immunoreactive cells appear in the periphery, was found in larvae from 9 to 20 days after hatching (stage 3). One big islet containing, in addition, PP-immunoreactive cells in the outer region and slender cell processes which showed PYY-like immunoreactivity, was found from 25 to 61 days after hatching. During this period, primordial islets, composed of SST-25- and bI-immunoreactive cells, and clustered or isolated pancreatic endocrine cells, close to the pancreatic duct, as well as small and intermediate islets (secondary islets), in which glucagon, PP, PYY and NPY seem to be co-localized, were progressively found (stage 4). The origin of the endocrine pancreas of sea bass, and the ontogenetic and phylogenetic significance, are discussed.     

Isolation and structure of the second of two major peptide products from the precursor to an anglerfish peptide homologous to neuropeptide Y

The peptide hormone recently isolated from anglerfish endocrine pancreas (aPY) (Andrews, P. C., Hawke, D., Shively, J.E., and Dixon, J.E. (1985) Endocrinology 116, 2677-2681), is a member of a family of peptide hormones which includes pancreatic polypeptide, neuropeptide Y, and the gut peptide YY. A 30-residue carboxyl-terminal fragment of the precursor to aPY has been purified from anglerfish endocrine pancreas in two steps using both classical chromatographic methods and reversed-phase high pressure liquid chromatography. It was identified by sequence homology with the analogous peptide from human preproneuropeptide Y. The sequence was found by Edman degradation and fast atom bombardment mass spectrometry to be SSPEEAVAWLLFKADPSQDIEPRLDDDNAW. The high yield of this fragment (6.5 nmol . g-1) is similar to that previously reported for aPY (7.9 nmol . g-1) and suggests that it is a major product of pro-aPY processing. The data indicate that pro-aPY is proteolytically processed into two major products: the 37-residue aPY and the 30-residue carboxyl-terminal fragment.     

Immunoreactive pancreatic polypeptide (PP) occurs in the central and peripheral nervous system: Preliminary immunocytochemical observations

Summary Pancreatic polypeptide (PP) is a candidate hormone of unknown physiological significance. It is produced by a population of endocrine cells in the pancreas. In the present study a PP-like peptide was found to occur in the mammalian and avian central and peripheral nervous systems. Immunoreactive nerve fibres and nerve cell bodies were widely distributed in the brain. Dense accumulations of nerve fibres occurred in the following areas: nucleus accumbens, interstitial nucleus of the stria terminalis, para- and periventricular hypothalamic nuclei, and medial preoptic area. In addition, nerve fibres were regularly seen in cortical areas. Immunoreactive perikarya were observed in the following regions: cortex, nucleus accumbens, neostriatum and septum. In the gut, immunoreactive nerve fibers were distributed in the myenteric plexus, in smooth muscle, around blood vessels, and in the core of the villi. Immunoreactive perikarya occurred in the submucosal and myenteric plexus, suggesting that PP immunoreactive nerves are intrinsic to the gut.In the species examined, the neuronal PP-like peptide could be demonstrated with an antiserum raised against avian PP, but not with those raised against bovine or human PP. Thus, neuronal PP is distinct from the PP that occurs in pancreatic endocrine cells.     

Morphological changes in the human endocrine pancreas induced by chronic excess of endogenous glucagon.

The non-tumoral endocrine pancreas from a patient with elevated plasma levels of glucagon due to a malignant glucagonoma was studied immunocytochemically, ultrastructurally and morphometrically. Compared with normal pancreatic islets from control subjects, those of the pancreas from the patient with a glucagonoma showed an almost complete disappearance of A cells, a decrease in immunoreactive insulin in B cells associated with cytological features indicating enhanced synthesis and secretion of this hormone, and an increase in immunoreactive somatostatin and pancreatic polypeptide (PP) accompanied by unusually high numbers of D and PP cells. In addition, numerous B cells were found outside the islets, either forming micro-islets or scattered in the exocrine tissue (nesidioblastosis). The possible mechanisms involved in determining the changes in the secretory activity of B cells and the alterations in the cell composition of the islets are discussed.     

Receptors for NPY in peripheral tissues bioassays

Neuropeptide Y (NPY) and its congeners, peptide YY (PYY) and the pancreatic polypeptide (PP), have a large spectrum of peripheral actions. NPY is found in peripheral neurons, co-localized or not with noradrenaline; PYY and PP are expressed in endocrine cells of the pancreas and in the intestine of vertebrates. NPY is the most abundant peptide in the brain and is involved in the regulation of food intake and of circadian rhythm. It intervenes also in the process of anxiety and memory. NPY is a potent vasoconstrictor, a cardiac stimulant, and may affect the gut through enteric neurons. PYY and PP act mainly on the gastrointestinal system; however, when in blood, they can cross-react with functional sites elsewhere and replace NPY in some parts of the brain (e.g. regions involved in feeding behavior). These peptides act through G protein coupled receptors (GPCR) of which five different types are known and have been cloned (1,2); functional sites (receptors) for NPY have been found in vessels, the gut, and in vasa deferentia (3-6).