Insulin, glucagon, pancreatic polypeptide hormone and somatostatin in the goat pancreas: demonstration by immunocytochemistry

Insulin, glucagon, pancreatic polypeptide hormone (PP) and somatostatin immunoreactive cells were demonstrated in the islet of the goat pancreas by the immunofluorescence procedure. Islet cells showing immunostaining for the hormones appeared to have a characteristic distribution. The demonstration of PP and somatostatin within the pancreas of the goat suggests they may be significant in modulating intra- and extra-islet function in this ruminant species.     

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.     

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.     

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.     

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.     

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).     

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.     

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.     

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.     

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.     

Effects of cholecystokinin, secretin, and pancreatic polypeptide on secretion of gastric inhibitory polypeptide, insulin, and glucagon

Although the capacity of food components to cause more insulin secretion when given orally than when given intravenously is related significantly to increased plasma concentration of gastric inhibitory polypeptide (GIP), stimulated only by the oral route, questions arise as to what extent other gastrointestinal hormones modify insulin secretion either directly or by influencing the secretion of GIP. The triacontatriapeptide form of cholecystokinin (CCK₃₃), infused in dose gradients intravenously in dogs increases insulin secretion, and comparably to equimolar doses of the carboxy-terminal octapeptide of cholecystokin (CCK₈); neither compound changes fasting plasma levels of GIP or glucose. Glucagon was increased only by the largest dose of CCK₈ (0.27 ug/kg). Unlike the situation with GIP, it is not necessary to increase the plasma glucose above fasting level to obtain the insulin-releasing action of CCK. When glucose is infused intravenously (2 g in 0.5 min) at the beginning of a 15-minute infusion of CCK₈ (10 ng/kg/min), the amount of insulin release is greater than is produced by CCK₈ or glucose alone. In the same type of experiment, the infusion of GIP, in equimolar amounts as CCK₈, plus glucose causes no more insulin secretion than is stimulated by glucose alone. Secretin has only a small stimulating action on insulin release, and pancreatic polypeptide (PP) has no effect. Neither secretin nor PP affects GIP secretion, whether either is given alone, or together, or with CCK₈. Either secretin or CCK₈ inhibits oral glucose-stimulated increase in plasma GIP. These inhibitory effects are probably very much related to the hormone-induced decrease in gastric emptying, but changes in somatostatin secretion and other hormones possibly exert contributory actions. In conclusion, GIP in certain dose ranges has been reported to cause major increase in insulin secretion, but we showed that the insulin-releasing action of a small dose of glucose (2 g) infused intravenously was not augmented by GIP (44.5 ng/kg/min), although it was significantly increased by an equimolar dose of CCK₈. When plasma glucose was maintained at a fasting level, gradient equimolar dosages of CCK₈ and CCK₃₃ had comparable insulin-releasing action; GIP had no effect.     

Ontogeny of rat pancreatic polypeptide (PP) cells

Summary Cells storing pancreatic polypeptide (PP) appear in rat pancreas at the time of parturition, much later than insulin and glucagon cells. At this stage, the pancreatic polypeptide (PP) cells occur scattered in the exocrine parenchyma and in the islets. Subsequently, 5–7 days postnatally, an abrupt increase in the number of PP cells occurs. At this stage, they are fairly numerous in the islets and comparatively rare in the exocrine parenchyma. Not until 8–10 days after birth is the number of PP cells similar to that in the adult pancreas. A few PP cells were seen in the antral mucosa during the first 10 days after birth. They were not seen elsewhere in the gut.     

Immunohistochemical study on the endocrine pancreas of cattle with special reference to coexistence of serotonin and glucagon or bovine pancreatic polypeptide

Bovine pancreatic endocrine cells were investigated by light microscopic immunohistochemistry. Serotonin-immunoreactive cells as well as insulin-, glucagon-, somatostatin-, bovine pancreatic polypeptide (BPP)-immunoreactive cells were detected in the pancreatic islets. Generally, insulin-immunoreactive cells were distributed throughout the islet and the others took peripheral location. Since the distribution and shape of serotonin-immunoreactive cells were very similar to glucagon- and BPP-immunoreactive cells, serial sections were restained by using the elution method. All glucagon- and BPP-immunoreactive cells also showed serotonin immunoreactivity but glucagon and BPP immunoreactivities were never observed to be colocalized in the same cell. A small number of serotonin-immunoreactive cells were observed that showed serotonin immunoreactivity only.     

Inhibitory effect of somatostatin-28 on pancreatic polypeptide, glucagon and insulin secretion in normal man

We have compared the effects of equimolar doses of intravenous somatostatin-28 (SS-28) and somatostatin-14 (SS-14) (250 micrograms and 125 micrograms, respectively) on the secretion of pancreatic polypeptide (PP), glucagon and insulin evoked by a protein-rich meal in normal subjects. Both peptides reduced the fasting plasma levels of these hormones and completely abolished their responses to the alimentary stimulus; in addition, they caused an early decrease of plasma glucose followed by a hyperglycemic phase. As compared to SS-14, SS-28 elicited a longer-lasting inhibition of PP and insulin secretion and displayed greater hypo- and hyperglycemic effects. A somatostatin-like component, similar to SS-28, has been identified in pancreatic extracts as well as in peripheral plasma. Thus, it might be hypothesized that this peptide plays a role in the control of pancreatic hormone release.