Colocalization of glucagon-like peptide and glucagon immunoreactivities in pancreatic islets and intestine of salmonids

Summary Pancreatic islets of salmon contain at least two peptides of the glucagon family: 29-amino acid glucagon and 31-amino acid glucagon-like peptide (GLP). Both peptides were recently isolated from the pancreatic islets of coho salmon and sequenced (Plisetskaya et al. 1986). Antibodies generated against these two peptides and against human glucagon were used as immunocytochemical probes to investigate whether glucagon and GLP are processed in the same, or in different cell types in the pancreatic islets and the gut of salmon. Two salmonid species, rainbow trout and coho salmon, were studied. All islet A-cells in the two species were immunoreactive toward both anti-salmon (s)-glucagon and anti-s-GLP. Similar colocalization of glucagon and GLP immunoreactivities was found in open-type endocrine cells in mucosae of the small intestine (including the pyloric coecae) and the large intestine close to the vent of rainbow trout. None of the antibodies stained mucosal cells of the body of the stomach. These results suggest that in the pancreas and the gut of salmonid fish the same cells produce both glucagon and GLP. These peptides are most likely the products of a single gene coding for the preproglucagon sequence.     

Immunocytochemical studies of the evolution of islet hormones.

By using both immunofluorescence and peroxidase-anti-peroxidase procedures to detect cells producing the four islet hormones, supplemented by biochemical, biological, and radioimmunological assays of tissue extracts, it has been shown that insulin seems to be the most original hormone, apparently occurring already in invertebrates in cells of open type in the alimentary tract mucosa. Insulin cells also predominate in the first islet organ, namely that of the cyclostomes. The order of appearance in the endocrine pancreas during the subsequent evolution is: somatostatin; glucagon; and the pancreatic polypeptide. Even in lower vertebrates pancreatic polypeptide cells occur in those parts of the pancreas situated in close proximity to the gut.     

Specific detection of human and rabbit glucagon mRNA using a synthetic oligodeoxynucleotide

A unique 14 base oligodeoxynucleotide dTTCATCAGCCACTG complementary to glucagon mRNA was deduced from the amino acid sequence of the hormone (residues 24–28; GLN-TRP-LEU-MET-ASN). The oligonucleotide specifically hybridized to RNA from rabbit pancreas and human pancreatic islet cells. No detectable hybridization was observed with either yeast or rat liver RNA. The melting temperature of the hybrids was 50 ± 5°C indicating no significant mismatch for human or rabbit glucagon mRNA. Hybridization kinetics followed a single pseudofirst-order reaction (Cot_{0 · 5} = 2.5 × 10^?4 M sec). From the extent of reaction at completion there are a minimum of 43 fmol glucagon mRNA/mg RNA (total pancreas).     

Comparison of insulin and glucagon pulsatile secretion between the rat and dog pancreas in-vitro

Sustained pulses of insulin and glucagon were obtained from the isolated perfused in vitro rat pancreas. The respective periodicity of hormone release (peak to peak interval) was calculated by the Pulsar computer algorithm as insulin 5.8 +/- 0.3 min and glucagon 6.5 +/- 0.25 min. Because pulsatile insulin secretion is absent in type II diabetics, pulsatile islet hormone secretion could theoretically be regulated directly by intra-islet hormone interactions or indirectly by hormone sensitive nerve feedback, possibly from a venous hormone sensitive receptor system within the pancreas. To test the possible contributions of these systems in pulse regulation, the direction of perfusion was reversed in both rat and dog pancreata to prevent hormone contact with putative venous hormone receptors. The periodicity of hormone secretion was unchanged by reversed perfusion in both species. As vascular perfusion of islet cells is normally B to A to D, these results suggest that neither intra-islet hormone interactions nor intra-pancreatic insulin or glucagon sensitive nerve feedback systems are responsible, on an acute basis, for the regulation of pulsatile insular secretion from the normal pancreas. Insulin regulates net glucagon secretion but does not acutely influence glucagon pulses. The presence of pulses during retrograde perfusion may be the result of the entrainment of the pacemaker-islet system. These observations are consistent with the presence of an independent pacemaker and neural coordinating system within the dog and rat pancreas which may influence both the A- and B-cell.     

The presence and actions of NPY in the canine endocrine pancreas

Immunofluorescent staining for neuropeptide Y (NPY) in canine pancreatic tissue was performed together with an evaluation of the effects of synthetic NPY on the release of insulin (IRI), glucagon (IRG) and somatostatin (SLI) from the duodenal lobe of the canine pancreas in situ. NPY-like immunoreactivity was localized in perivascular nerve fibers throughout the acinar tissue. NPY-immunoreactive fibers were also demonstrated in the islets, usually surrounding blood vessels but also occasionally in fibers associated with endocrine cells, primarily at the periphery of islets. In addition, the ganglia dispersed in the pancreatic parenchyma were densely innervated by NPY-immunoreactive fibers, and these ganglia regularly contained cell bodies staining for NPY. Direct infusion of NPY into the pancreatic artery (p.a.) produced a dose-dependent decrease of pancreatic SLI output and of pancreatic venous blood flow. Low-dose p.a. infusion of NPY (50 pmol/min) had no effect on basal IRI or IRG output or on the islet response to glucose (5-g bolus, i.v.). High-dose p.a. infusion of NPY (500 pmol/min) transiently stimulated IRI output and modestly increased IRG output. However, the comparatively sparse innervation of canine islets with NPY-like immunoreactive fibers and the relatively minor effects of large doses of synthetic NPY on pancreatic hormone release lead us to conclude that this peptide is not an important neuromodulator of islet function in the dog.     

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.     

New views on the identification of the various cell types in the pancreatic islets of the rat. An ultrastructural and morphometrical study

Islets of Langerhans taken from different parts of the pancreas have been studied ultrastructurally in adult rats. Five different islet cell types were identified in each islet with the aid of morphometrical analysis of their specific secretory granules. Previous immunohistochemical findings concerning the amount and location of insulin-, glucagon-, somatostatin- and pancreatic-polypeptide-containing cells and their ultrastructurally recognizable counterparts were compared, and it was possible to identify four main islet cell types with the electron microscope. Moreover, cells quite similar to the enterochromaffine cells described elsewhere in the exocrine pancreas and in the gastrointestinal tract were found to normally occur in the pancreatic islets of the rat.     

Glucose dependent stimulation by prostaglandin D2 of glucagon and insulin in perfused rat pancreas

Effects of prostaglandin D2 on pancreatic islet function in perfused rat pancreas were examined in comparison with those of prostaglandin E2, which has hitherto been suggested to be a modifier of pancreatic hormone release. In the presence of 2.8 mM glucose, only glucagon release was strongly stimulated by 14 microM of prostaglandin D2, while release of both glucagon and insulin was augmented by 14 microM of prostaglandin E2. When the glucose concentration was elevated to 11.2 mM, insulin release was accelerated by 14 microM of prostaglandin D2 but there was no effect upon glucagon release. Again, release of both glucagon and insulin was augmented by 14 microM of prostaglandin E2 in the presence of 11.2 mM of glucose. The regulation of glucagon and insulin release through prostaglandin D2 is apparently adapted to glycemic changes, and may be a physiological modulator of pancreatic islet function.     

Ghrelin is expressed in a novel endocrine cell type in developing rat islets and inhibits insulin secretion from INS-1 (832/13) cells.

Ghrelin is produced mainly by endocrine cells in the stomach and is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). It also influences feeding behavior, metabolic regulation, and energy balance. It affects islet hormone secretion, and expression of ghrelin and GHS-R in the pancreas has been reported. In human islets, ghrelin expression is highest pre- and neonatally. We examined ghrelin and GHS-R in rat islets during development with immunocytochemistry and in situ hybridization. We also studied the effect of ghrelin on insulin secretion from INS-1 (832/13) cells and the expression of GHS-R in these cells. We found ghrelin expression in rat islet endocrine cells from mid-gestation to 1 month postnatally. Islet expression of GHS-R mRNA was detected from late fetal stages to adult. The onset of islet ghrelin expression preceded that of gastric ghrelin. Islet ghrelin cells constitute a separate and novel islet cell population throughout development. However, during a short perinatal period a minor subpopulation of the ghrelin cells co-expressed glucagon or pancreatic polypeptide. Markers for cell lineage, proliferation, and duct cells revealed that the ghrelin cells proliferate, originate from duct cells, and share lineage with glucagon cells. Ghrelin dose-dependently inhibited glucose-stimulated insulin secretion from INS-1 (832/13) cells, and GHS-R was detected in the cells. We conclude that ghrelin is expressed in a novel developmentally regulated endocrine islet cell type in the rat pancreas and that ghrelin inhibits glucose-stimulated insulin secretion via a direct effect on the beta-cell.     

Ontogeny of immunoreactive glicentin in the human gastrointestinal tract and endocrine pancreas

The gestational time of appearance and distribution of immunoreactive glicentin was compared to that of immunoreactive glucagon in the gastrointestinal tract and endocrine pancreas of human fetuses, aged between 5 and 24 weeks, by an indirect immunoperoxidase method. With the glicentin antiserum No. R 64, the first immunoreactive cells were detected at the 10th week of gestation in the oxyntic mucosa and proximal small intestine, at the 8th week in the ileum and at the 12th week in the colon. In the endocrine pancreas, the first immunoreactive cells were observed as early as 8 weeks within the walls of the primitive pancreatic ductules. At a more advanced stage of development (12 weeks), they were found interspersed among the islet cell clusters and still later (16 weeks) inside the recognizable islets of Langerhans. With the glucagon antiserum No. GB 5667, no immunoreactive cells were demonstrated in the gastrointestinal tract whatever the age of the fetuses. In the endocrine pancreas, the first immunoreactive cells were observed at the 8th week of gestation in the pancreatic parenchyma. The distribution of glucagon-containing cells in the pancreas was similar to that of glicentin immunoreactivity throughout ontogenesis. In the pancreatic islets of one 18-week-old human fetus, the study of consecutive semithin sections treated by both antisera showed that the same cells were labelled. The significance of these findings concerning the role of glicentin as a glucagon precursor is discussed.     

An immunocytochemical investigation with monoclonal antibodies to somatostatin

Summary Four monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin.The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.     

An immunocytochemical investigation with monoclonal antibodies to somatostatin

Four monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin. The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.     

Phe-met-arg-phe-amide (FMRF-NH2) inhibits insulin and somatostatin secretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells in the rat islet

FMRF-NH2-like immunoreactivity was localized in the pancreatic polypeptide containing cells of the rat islet. FMRF-NH2 was investigated with regard to its effect on insulin, somatostatin and glucagon secretion from the isolated perfused rat pancreas. FMRF-NH2 (1 microM) significantly inhibited glucose stimulated (300 mg/dl) insulin release (p less than 0.005) and somatostatin release (p less than 0.01) from the isolated perfused pancreas. FMRF-NH2 (1 and 10 microM) was without effect on glucagon secretion, either in low glucose (50 mg/dl), high glucose (300 mg/dl), or during arginine stimulation (5 mM). These findings indicate that these FMRF-NH2 antisera recognize a substance in the pancreatic polypeptide cells of the islet which may be capable of modulating islet beta and D cell activity.     

Analysis of rat insulin II promoter-ghrelin transgenic mice and rat glucagon promoter-ghrelin transgenic mice

We developed and analyzed two types of transgenic mice: rat insulin II promoter-ghrelin transgenic (RIP-G Tg) and rat glucagon promoter-ghrelin transgenic mice (RGP-G Tg). The pancreatic tissue ghrelin concentration measured by C-terminal radioimmunoassay (RIA) and plasma desacyl ghrelin concentration of RIP-G Tg were about 1000 and 3.4 times higher than those of nontransgenic littermates, respectively. The pancreatic tissue n-octanoylated ghrelin concentration measured by N-terminal RIA and plasma n-octanoylated ghrelin concentration of RIP-G Tg were not distinguishable from those of nontransgenic littermates. RIP-G Tg showed suppression of glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion, pancreatic insulin mRNA and peptide levels, beta cell mass, islet architecture, and GLUT2 and PDX-1 immunoreactivity in RIP-G Tg pancreas were not significantly different from those of nontransgenic littermates. Islet batch incubation study did not show suppression of insulin secretion of RIP-G Tg in vitro. The insulin tolerance test showed lower tendency of blood glucose levels in RIP-G Tg. Taking lower tendency of triglyceride level of RIP-G Tg into consideration, these results may indicate that the suppression of insulin secretion is likely due to the effect of desacyl ghrelin on insulin sensitivity. RGP-G Tg, in which the pancreatic tissue ghrelin concentration measured by C-RIA was about 50 times higher than that of nontransgenic littermates, showed no significant changes in insulin secretion, glucose metabolism, islet mass, and islet architecture. The present study raises the possibility that desacyl ghrelin may have influence on glucose metabolism.     

The effect of hypophysectomy on pancreatic islet hormone and insulin-like growth factor I content and mRNA expression in rat

The growth arrest after hypophysectomy in rats is mainly due to growth hormone (GH) deficiency because replacement of GH or insulin-like growth factor (IGF) I, the mediator of GH action, leads to resumption of growth despite the lack of other pituitary hormones. Hypophysectomized (hypox) rats have, therefore, often been used to study metabolic consequences of GH deficiency and its effects on tissues concerned with growth. The present study was undertaken to assess the effects of hypophysectomy on the serum and pancreatic levels of the three major islet hormones insulin, glucagon, and somatostatin, as well as on IGF-I. Immunohistochemistry (IHC), in situ hybridization (ISH), radioimmunoassays (RIA), and Northern blot analysis were used to localize and quantify the hormones in the pancreas at the peptide and mRNA levels. IHC showed slightly decreased insulin levels in the cells of hypox compared with normal, age-matched rats whereas glucagon in cells and somatostatin in cells showed increase. IGF-I, which localized to cells, showed decrease. ISH detected a slightly higher expression of insulin mRNA and markedly stronger signals for glucagon and somatostatin mRNA in the islets of hypox rats. Serum glucose concentrations did not differ between the two groups although serum insulin and C-peptide were lower and serum glucagon was higher in the hypox animals. These changes were accompanied by a more than tenfold drop in serum IGF-I. The pancreatic insulin content per gram of tissue was not significantly different in hypox and normal rats. Pancreatic glucagon and somatostatin per gram of tissue were higher in the hypox animals. The pancreatic IGF-I content of hypox rats was significantly reduced. Northern blot analysis gave a 2.6-, 4.5-, and 2.2-fold increase in pancreatic insulin, glucagon, and somatostatin mRNA levels, respectively, in hypox rats, and a 2.3-fold decrease in IGF-I mRNA levels. Our results show that the fall of serum IGF-I after hypophysectomy is accompanied by a decrease in pancreatic IGF-I peptide and mRNA but by partly discordant changes in the serum concentrations of insulin and glucagon and the islet peptide and/or mRNA content of the three major islet hormones. It appears that GH deficiency resulting in a low IGF-I state affects translational efficiency of these hormones as well as their secretory responses. The maintenance of normoglycemia in the presence of reduced insulin and elevated glucagon serum levels, both of which would be expected to raise blood glucose, may result mainly from the enhanced insulin sensitivity, possibly due to GH deficiency and the subsequent decrease in IGF-I production.