Ontogeny of cells containing insulin, glucagon, pancreatic polypeptide hormone and somatostatin in the bovine pancreas

Antibodies to insulin, glucagon, pancreatic polypeptide hormone (PP) and somatostatin were used in the immunofluorescence histochemical procedure to study the ontogeny of pancreatic endocrine cells containing the four hormones in the bovine fetus of approximately 100 days gestation to term. Pancreatic sections from the bovine neonate and adult were also examined for the cellular distribution of the four hormones. Immunoreactive cells staining for insulin, glucagon, PP and somatostatin were present in the pancreas of all fetuses studied. Each endocrine cell type displayed a characteristic distribution within the developing pancreas and in the neonate and adult. The presence of the four islet hormones relatively early in bovine fetal life suggests that they may be important in intra- and extra-islet metabolism in the fetus.     

Mapping polypeptide hormone genes in the mouse: somatostatin, glucagon, calcitonin, and parathyroid hormone

Mouse-Chinese hamster hybrids segregating mouse chromosomes were analyzed by Southern hybridization techniques to map the genes for somatostatin (Smst), glucagon (Gcg), calcitonin (Calc), and parathyroid hormone (Pth). The mouse gene for somatostatin, detected on a 20-kb EcoRI fragment, is located on mouse chromosome 16. Glucagon cDNA hybridized to a 14-kb EcoRI fragment residing on chromosome 2. Calcitonin and parathyroid hormone genes, detected on 7.8-kb HindIII and 6.0-kb BamHI fragments, respectively, were on mouse chromosome 7. The calcitonin and parathyroid hormone genes appear to be part of a larger linkage group which has been conserved in mouse and man.     

Cellular distribution of insulin, glucagon, pancreatic polypeptide hormone and somatostatin in the fetal and adult pancreas of the guinea pig: a comparative immunohistochemical study

Antibodies to insulin, glucagon, pancreatic polypeptide hormone and somatostatin were utilized to demonstrate the cellular localization of the hormones in pancreatic tissue of fetal guinea pig of advanced gestation by immunofluorescence histochemistry. The topographical distribution of the 4 endocrine cell types was compared with those of the adult pancreas and was found to be significantly different particularly for cells immunostaining for insulin, glucagon and somatostatin. These observations suggest changes in histogenesis of pancreatic endocrine cells during transition from fetal to postnatal and adult life. The presence of the 4 islet hormones in the fetal pancreas of this species implies that they may be important in fetal metabolism and growth.     

Insulin, glucagon and somatostatin in fetal rat islets maintained free-floating in culture: immunocytochemistry and radioimmunoassay.

Fetal rat islets maintained free-floating in tissue culture represent a source of B-cells. Because we recently noted the occurrence of other cell types during long-term tissue culture, this in vitro model was used to examine the possible development of non B-cells. The changes in the numbers and percentages of B, A and D-cells in vitro were estimated by counting the hormone-positive cells after immunocytochemical staining. Insulin, glucagon, and somatostatin contents were determined in extracts of the cultured tissue. The experiments described here showed that the cultured islets maintained their viability over a two-week culture period, as evidenced by the increase of both the number of B-cells per islet and the DNA content per islet. During the first few days of culture, immunocytochemically stained free-floating islets indicated the presence of rare A- and D-cells at the periphery of B-cells; thereafter, numerous A- and D-cells were seen interdigitating with B-cells. Expressed per islet, the number of A- and D-cells increased during the culture; within the endocrine cell population, the percentage of these cells increased with time, at the expense of the percentage of B-cells. The glucagon and somatostatin contents of the free-floating islets were also increased. These converging observations suggest that additional non B-cells may have been produced by free-floating islets during long-term tissue culture.     

Insulin, glucagon and somatostatin localization in the pancreas of metamorphosed Xenopus laevis

The current study was designed to determine if insulin, glucagon and somatostatin-containing cells are present in the pancreas of adult Xenopus laevis. Localization methods utilized included cytochemical aldehyde fuchsin (AF) staining as well as the immunochemical peroxidase antiperoxidase (PAP) procedure for light microscopy. The results show numerous large clusters of AF-positive cells within a network of highly vascularized acinar tissue. PAP immunochemical localization with insulin antibody on adjacent sections demonstrates positive immunoreactivity to AF-positive cell groups and also the presence of immunoreactive insulin (IRI). Cells exhibiting this immunoreactivity are located in the central region of the islet-like structures. Serial sections not only show PAP immunoreactivity for IRI, but also for immunoreactive glucagon (IRG) and immunoreactive somatostatin (IRS) in the same islet-like structure. IRG and IRS-containing cells are situated around the periphery of the islet-like structures, surrounding the central core of IRI-containing cells. Antibody specificity was confirmed by homologous and heterologous antigen immuno-absorbance assays, as well as incubation of adjacent sections in preimmune sera. Based on this data we conclude that: the distribution of cells of the endocrine pancreas of metamorphosed Xenopus laevis is similar to that of many mammals and certain urodeles. Given the apparent specificity of the antigen-antibody reactions, it appears that Xenopus insulin, glucagon and somatostatin are structurally conserved.     

Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas

Proghrelin, the precursor of the orexigenic and adipogenic peptide hormone ghrelin, is synthetized in endocrine (A-like) cells in the gastric mucosa. During its cellular processing, proghrelin gives rise to the 28-amino acid peptide desacyl ghrelin, which after octanoylation becomes active acyl ghrelin, and to the 23-amino acid peptide obestatin, claimed to be a physiological opponent of acyl ghrelin. This study examines the effects of the proghrelin products, alone and in combinations, on the secretion of insulin, glucagon, pancreatic polypeptide (PP) and somatostatin from isolated islets of mice and rats. Surprisingly, acyl ghrelin and obestatin had almost identical effects in that they stimulated the secretion of glucagon and inhibited that of PP and somatostatin from both mouse and rat islets. Obestatin inhibited insulin secretion more effectively than acyl ghrelin. In mouse islets, acyl ghrelin inhibited insulin secretion at low doses and stimulated at high. In rat islets, acyl ghrelin inhibited insulin secretion in a dose-dependent manner but the IC(50) for the acyl ghrelin-induced inhibition of insulin release was 7.5 x 10(-8) M, while the EC(50) and IC(50) values, with respect to stimulation of glucagon release and to inhibition of PP and somatostatin release, were in the 3 x 10(-12)-15 x 10(-12) M range. The corresponding EC(50) and IC(50) values for obestatin ranged from 5 x 10(-12) to 20 x 10(-12) M. Desacyl ghrelin per se did not affect islet hormone secretion. However, at a ten times higher concentration than acyl ghrelin (corresponding to the ratio of the two peptides in circulation), desacyl ghrelin abolished the effects of acyl ghrelin but not those of obestatin. Acyl ghrelin and obestatin affected the secretion of glucagon, PP and somatostatin at physiologically relevant concentrations; with obestatin this was the case also for insulin secretion. The combination of obestatin, acyl ghrelin and desacyl ghrelin in concentrations and proportions similar to those found in plasma resulted in effects that were indistinguishable from those induced by obestatin alone. From the data it seems that the effects of endogenous, circulating acyl ghrelin may be overshadowed by obestatin or blunted by desacyl ghrelin.     

Beta-endorphin and somatostatin in the pancreatic D-cell colocalization by immunocytochemistry

Utilizing highly specific antisera, beta-endorphin and somatostatin immunoreactivity were identified simultaneously in the D-cell of the pancreas of the rat, guinea pig, and man by the fluorescence-immunocytochemical technique. Our observations are consistent with a modulating role of beta-endorphin either within the D-cell or upon A and B cells, thereby regulating the secretion of insulin and glucagon.     

Opossum insulin, glucagon and pancreatic polypeptide: Amino acid sequences

Pancreatic hormones have been purified from the opossum, a New World marsupial. Opossum insulin contains a Leu substitution at the N-terminus of the B-chain in place of the Phe that is generally present in mammalian insulins. In addition, there are two other amino acid substitutions in the opossum insulin A-chain (positions 8 and 18) compared to pig insulin. Opossum glucagon is identical to chicken glucagon with both differing from the usual mammalian glucagon by Ser in place of Asn at its penultimate C-terminal position. Opossum PP differs from the porcine peptide in only 3 sites (position 3, 19 and 30).     

Immunohistochemical localization of glucagon and pancreatic polypeptide on rat endocrine pancreas: coexistence in rat islet cells

We used immunofluorescence double staining method to investigate the cellular localization of glucagon and pancreatic polypeptide (PP) in rat pancreatic islets. The results showed that both A-cells (glucagon-secreting cells) and PP-cells (PP-secreting cells) were located in the periphery of the islets. However, A-cells and PP-cells had a different regional distribution. Most of A-cells were located in the splenic lobe but a few of them were in the duodenal lobe of the pancreas. In contrast, the majority of PP-cells were found in the duodenal lobe and a few of them were in the splenic lobe of the pancreas. Furthermore, we found that 67.74% A-cells had PP immunoreactivity, 70.92% PP-cells contained glucagon immunoreactivity with immunofluorescence double staining. Our data support the concept of a common precursor stem cell for pancreatic hormone-producing cells.Key words: glucagon, pancreatic polypeptide, rat, pancreas, Immunofluorescence double staining histochemistry.The pancreatic islet is comprised of numerous cell types that synthesize and secrete distinct peptide hormones. Four major cell types are recognized in pancreatic islets of many mammalian species including rat, A-cells which contain glucagon, B-cells which contain insulin, D-cells which contain somatostatin, and PP-cells which contain the pancreatic polypeptide (PP) (Erlandsen, 1980; Reddy et al., 1988).Previous studies have revealed coexistence of glucagon- and PP-like immunoreactivity in endocrine pancreas cells of frog, rat, baboon, murine, monkey, and fish (Kaung and Elde, 1980; Kaung, 1985a, 1985b; Wolfe-Coote et al., 1988; Herrera et al., 1991; Lozano et al., 1991; Park and Bendayan, 1992; Louw et al., 1997). However, those experiments were performed by staining adjacent ultrathin sections with anti-glucagon serum and anti-PP serum respectively by peroxidase antiperoxidase (PAP) or immuno-gold labeling or avidin-biotin-peroxidase method, and the situation of two kinds of positive cells were compared.It is still not clear whether one cell type contains two or more peptides. Therefore, we used immunofluorescence double staining to identify the peptides secreted by single specific cells.This is the first time that coexistence of glucagon and PP in rat islet cells has been detected by an immunofluorescence double staining method.     

Bombesin stimulates the release of insulin and glucagon, but not pancreatic somatostatin, from the isolated perfused dog pancreas.

Synthetic bombesin, perfused in the isolated canine pancreas at a rate of 340-380 ng/min for 10 min, elicited a 4-fold rise in insulin to a peak at 2 min; a rapid decline followed discontinuation of bombesin. Glucagon rose by 50% to a peak at 6 min, but remained elevated after discontinuation of the bombesin. Somatostatin-like immunoreactivity was not significantly affected by perfusion with bombesin.     

Effects of pancreastatin on insulin, glucagon and somatostatin secretion by the perfused rat pancreas

Pancreastatin is a novel peptide, isolated from porcine pancreatic extracts, which has been shown to inhibit glucose-induced insulin release "in vitro". To achieve further insight into the influence of pancreastatin on pancreatic hormone secretion, we have studied the effects of this peptide on unstimulated insulin, glucagon and somatostatin output, as well as on the responses of these hormones to glucose and to tolbutamide in the perfused rat pancreas. Pancreastatin strongly inhibited unstimulated insulin release as well as the insulin responses to glucose and to tolbutamide. It did not significantly affect glucagon or somatostatin output under any of the above-mentioned conditions. These findings suggest that pancreastatin inhibits B-cell secretory activity directly, and not through an A-cell or D-cell paracrine effect.     

Pancreatic polypeptide and glucagon: Non-random distribution in pancreatic islets

When the technique of immunofluorescence is applied to rat pancreas to detect insulin, glucagon, somatostatin and pancreatic polypeptide (PP), two populations of islets having distinct cellular content and topographical distribution can be recognized. Islets from the lower part of the head show a well-defined rim of PP-containing cells, but very few or no glucagon-containing cells. Islets from the body and tail display the familiar rim of glucagon-containing cells and possess very few or no PP-containing cells. This inverse relationship between glucagon and PP-cells in different parts of the pancreas means that caution must be exercised when interpreting functional or morphological observations using different pancreatic fractions.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.     

Insulin, glucagon, and somatostatin secretion by cultured rat islet cell tumor and its clones

Cells derived from rat islet tumor and grown in culture (parent cells-RIN-m) and two clones obtained from them were used to study the effect of various secretagogues on insulin, glucagon, and somatostatin secretion. Parent cells secreted all three hormones in various quantities, while clone 5F secreted predominantly insulin and clone 14B secreted predominantly somatostatin. The secretory behavior of these cells were compared to each other and to that of normal islets. In general, as in the case of normal islets, insulin secretion was stimulated by calcium, potassium, tolbutamide, theophylline, and glucagon. It was inhibited by somatostatin. Glucagon secretion was stimulated by calcium, arginine, and theophylline. Somatostatin secretion was stimulated in clone 14B by arginine, tolbutamide, theophylline, and insulin. These cells differ from normal islets, in that they do not respond to glucose or arginine with increased insulin secretion. Also somatostatin failed to inhibit glucagon secretion. The similarity in insulin secretory responses of parent cells and clone 5F suggests that local or paracrine islet hormone secretion plays only a negligible role in the control of other hormone secretion in these cells.     

Identification of pancreatic glucagon cells in the cartilaginous fish Scyliorhinus canicula by ultrastructural immunocytochemistry

The ultrastructural localization of glucagon in the presence of Scyliorhinus canicula was investigated. We used a post-embedding immunoelectron microscopy method on pancreatic samples fixed in glutaraldehyde and osmicated before embedding. Contrasting with uranyl acetate and lead citrate was also performed after immunolabelling, but best results were obtained with uranyl acetate only. Glucagon-like immunoreactivity was located in round granules (300-600 nm) surrounded by a limiting membrane. The matrix varied in electron density and exhibited a dense core surrounded by a less dense mantle. The granules were seen in two different cell types, which differed in the electron density of their cytoplasm. Glucagon-immunoreactive cells were the largest pancreatic cells types and were often localized near somatostatin-containing cells.     

Effect of obestatin on insulin, glucagon and somatostatin secretion in the perfused rat pancreas

Obestatin is a 23-amino acid peptide derived from preproghrelin, purified from stomach extracts and detected in peripheral plasma. In contrast to ghrelin, obestatin has been reported to inhibit appetite and gastric motility. However, these effects have not been confirmed by some groups. Obestatin was originally proposed to be the ligand for GPR39, a receptor related to the ghrelin receptor subfamily, but this remains controversial. Obestatin and GPR39 are expressed in several tissues, including pancreas. We have investigated the effect of obestatin on islet cell secretion in the perfused rat pancreas. Obestatin, at 10 nM, inhibited glucose-induced insulin secretion, while at 1 nM, it potentiated the insulin response to glucose, arginine and tolbutamide. The potentiated effect of obestatin on glucose-induced insulin output was not observed in the presence of diazoxide, an agent that activates ATP-dependent K(+) channels, thus suggesting that these channels might be sensitive to this peptide. Obestatin failed to significantly modify the glucagon and somatostatin responses to arginine, indicating that its stimulation of insulin output is not mediated by an alpha- or delta-cell paracrine effect. Our results allow us to speculate about a role of obestatin in the control of beta-cell secretion. Furthermore, as an insulinotropic agent, its potential antidiabetic effect may be worthy of investigation.     

Localization of insulin, glucagon and somatostatin in the pancreas of the adult newt, Notophthalmus viridescens

The current study is designed to demonstrate the presence of immunoreactive insulin (IRI), glucagon and somatostatin in the adult pancreas. Methods include aldehyde fuchsin (AF) staining and peroxidase anti-peroxidase (PAP) immunochemical localization for light microscopy as well as protein A gold (PAG) staining for scanning electron microscopy (SEM) in conjunction with backscattered electron imaging (BEI). Our results show the presence of large clusters of AF-positive cells within networks of highly vascularized pancreatic acinar tissue. PAP immunochemistry of pancreas serial sections exhibit positive immunoreactivity to the same AF-positive structure, thus demonstrating the presence of IRI. This immunoreactivity is found in a high percentage of cells in the islet-like structures. These cells tend to be centrally located within the cluster. Antibody specificity controls, including homologous antigen immunoabsorbance, as well as incubation of sections in normal guinea pig serum give negative immunoreactivity. Immunoreactive glucagon-containing cells and somatostatin-containing cells are distributed around the periphery of the central core of IRI-containing cells. SEM in conjunction with BEI confirm the presence of PAG within these cell clusters. We conclude that: (a) newt pancreatic IRI reacts in a specific manner with bovine antibody, suggesting a partial structural similarity to mammalian antigen; (b) IRI is localized within within pancreatic islet-like cell clusters and these IRI-containing cells form a central mass which is surrounded by glucagon and somatostatin-containing cells; this cellular distribution is similar to that found in many mammals. PAG conjugated insulin antibody is detectable by SEM in conjunction with BEI in islet cells of the newt pancreas.