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.     

FMRF-amide immunoreactivity in the mammalian gastroenteropancreatic neuroendocrine system

The presence of FMRF-amide, a cardioactive tetrapeptide, was studied by immunocytochemistry in human and rat gastric antrum and pancreas, and in the ovine, bovine, canine and rabbit pancreas. In human and rat gastric antrum, numerous cells contained FMRF-amide immunoreactive material. By staining of serial sections and by double staining, colocalization of immunoreactivity for gastrin and FMRF-amide was observed in part of the gastrin cells. In the pancreas of these and the other species, immunoreactivity for FMRF-amide was located both in acinar and islet endocrine cells. Colocalization of FMRF-amide and pancreatic polypeptide was found in a proportion of pancreatic polypeptide cells in the pancreas. FMRF-amide immunoreactivity never colocalized with the other neurohormonal peptides which occur in the gastric antrum and the pancreas. Our observations show that neuroendocrine cells occur in the gastric antrum and pancreas which are exclusively immunoreactive or gastrin and for pancreatic polypeptide respectively. In addition cells occur which show immunoreactivity for FMRF-amide as well as for gastrin in the gastric antrum and with antiserum to FMRF-amide as well as for pancreatic polypeptide in the pancreas. It is concluded that FMRF-amide antibodies probably recognize a substance in G and PP cells which is not identical but may be structurally related to gastrin and pancreatic polypeptide.     

FMRF-amide immunoreactivity in the mammalian gastroenteropancreatic neuroendocrine system

Summary The presence of FMRF-amide, a cardioactiv tetrapeptide, was studied by immunocytochemistry in human and rat gastric antrum and pancreas, and in the ovine, bovine, canine and rabbit pancreas. In human and rat gastric antrum, numerous cells contained FMRF-amide immunoreactive material. By staining of serial sections and by double staining, colocalization of immunoreactivity for gastrin and FMRF-amide was observed in part of the gastrin cells. In the pancreas of these and the other species, immunoreactivity for FMRF-amide was located both in acinar and islet endocrine cells. Colocalization of FMRF-amide and pancreatic polypeptide was found in a proportion of pancreatic polypeptide cells in the pancreas. FMRF-amide immunoreactivity never colocalized with the other neurohormonal peptides which occur in the gastric antrum and the pancreas.Our observations show that neuroendocrine cells occur in the gastric antrum and pancreas which are exclusively immunoreactive or gastrin and for pancreatic polypeptide respectively. In addition cells occur which show immunoreactivity for FMRF-amide as well as for gastrin in the gastric antrum and with antiserum to FMRF-amide as well as for pancreatic polypeptide in the pancreas. It is concluded that FMRF-amide antibodies probably recognize a substance in G and PP cells which is not identical but may be structurally related to gastrin and pancreatic polypeptide.In honour of Prof. P. van Duijn     

Islet amyloid polypeptide (IAPP) in the gastrointestinal tract and pancreas of man and rat

Summary An immunohistochemical study for islet amyloid polypeptide (IAPP) was made on the gastrointestinal (GI) tract and pancreas of man and rat, using antisera raised against a synthetic peptide of C-terminal human IAPP (24–37) and a synthetic peptide of rat IAPP (18–37). A large number of IAPP-immunoreactive cells were found in the pyloric antrum, and a small number in the body of the stomach in both man and rat. Cytoplasmic processes extended out from the bipolar peripheral region of the immunoreactive cells, rather like neuronal processes, and some appeared to make contact with other immunoreactive cells. In addition, small numbers of immunoreactive cells were also seen in the duodenum and rectum, whereas they were absent from the jejunum, ileum and large intestine. An examination was made for evidence of colocalization of IAPP-immunoreactive material with material immunoreactive for gastrin, somatostatin, vasoactive intestinal polypeptide, pancreatic polypeptide, insulin, and glucagon, but none was found. IAPP-immunoreactive cells were also found in the pancreas of non-diabetic and non-insulin-dependent diabetic patients, but they were completely absent from a patient with insulin-dependent diabetes mellitus despite the presence of IAPP in the plasma. The results of these studies suggest that the peptide may have a biological role in situ in the GI tract and, in addition to the pancreas, may be a possible source of plasma IAPP.     

The endocrine pancreas of Alligator mississippiensis

Summary Immunocytochemical methods for light and electron microscopy were used to demonstrate the regulatory peptides present in the endocrine pancreas of thealligator, Alligator mississippiensis.The peptides studied included insulin, glucagon (pancreatic and enteric), somatostatin, pancreatic polypeptide (avian, bovine and human), vasoactive intestinal polypeptide, substance P, metenkephalin, -endorphin, C-terminal gastrin/CCK and gastric inhibitory polypeptide. Endocrine cells were detected using antisera to insulin, pancreatic glucagon, somatostatin and avian pancreatic polypeptide, whereas peptidergic nerves were stained with antisera to vasoactive intestinal polypeptide. All other antisera were unreactive in the alligator pancreas. The peptide-containing structures were identified ultrastructurally by both the semithin/thin and immuno-gold methods. The results showed that five of the regulatory peptides commonly detected in the mammalian pancreas were immunologically recognisable in the alligator. In addition, the ultrastructural appearance of the peptide-containing cells was clearly distinct from that reported in mammals.     

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 secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control.

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical study of the distribuition of endocrine cells in the pancreas of the Brazilian sparrow species Zonotrichia Capensis Subtorquata (Swaison, 1837).

In the present study, we investigated types of pancreatic endocrine cells and its respective peptides in the Brazilian sparrow species using immunocytochemistry. The use of polyclonal specific antisera for somatostatin, glucagon, avian pancreatic polypeptide (APP), YY polypeptide (PYY) and insulin, revealed a diversified distribution in the pancreas. All these types of immunoreactive cells were observed in the pancreas with different amounts. Insulin-Immunoreactive cells to (B cells) were most numerous, preferably occupying the central place in the pancreatic islets. Somatostatin, PPA, PYY and glucagon immunoreactive cells occurred in a lower frequency in the periphery of pancreatic islets.     

A light-microscopic immunocytochemical study of the endocrine pancreas in the Australian brush-tailed possum (Trichosurus vulpecula).

The endocrine pancreas of the Australian brush-tailed possum (Trichosurus vulpecula) was investigated by means of immunocytochemistry using the avidin-biotin-peroxidase technique. This was a light microscopic study using this established technique. Serial paraffin sections were stained individually with primary antibodies for glucagon, insulin, somatostatin, and pancreatic polypeptide (PP), showing the same islet. Cells immunoreactive to glucagon, insulin, somatostatin and PP were found in endocrine islets. PP cells appear to be scattered amidst the exocrine portion also. Insulin immunoreactive cells were located in the central region of islet, glucagon in the periphery, somatostatin in periphery and had elongated processes. PP cells were more sparse and located both in the periphery of islet and amidst the exocrine tissue. These results can then be related to a similar study in the same marsupial, but using the immunofluorescence technique and to studies in other marsupials such as grey kangaroo (Macropus fuliginosus) fat-tailed dunnart (Sminthopsis crasicaudata) and the American opossum (Didelphis virginiana). These investigations are part of a study in Australian mammals.     

Immunohistochemical identification and localization of pancreatic polypeptide cells in the pancreas and gastrointestinal tract of the human fetus and adult man

Summary Pancreatic polypeptide (PP)-containing cells were detected by using anti-bovine PP (BPP) serum in the pancreas and gastrointestinal tract of human fetuses, premature infants and in the pancreas, antrum and jejunum of adult man obtained by biopsy from patients with normal gastroduodenal endoscopy. The localization was established by studying the distribution of PP cells in comparison to the distribution of glucagon-, somatostatin- and insulin cells. The first PP cells are seen in the pancreas at 10 weeks of gestation. They are located preferentially in the lower part of the head of the pancreas. The specificity of immunocytological reaction was ascertained by the inhibition of the reaction by bovine pancreatic polypeptide, glucagon and insulin did not modify the immunocytological reaction.     

Immunocytochemical study of tyrosine hydroxylase and dopamine β-hydroxylase immunoreactivities in the rat pancreas

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory acitivity of the acinar cells, duct cells and endocrine cells.     

Immunohistochemical localization of human kallikreins 6 and 10 in pancreatic islets

Tissue kallikreins are thought to be present in the pancreatic islets of Langerhans and to aid in the conversion of proinsulin to insulin. In recent immunohistochemical studies, we observed strong staining of the newly identified human kallikreins 6 and 10 (hK6 and hK10) in the islets of Langerhans. Here, we examine hK6 and hK10 immunoexpression in different types of islet cells of the endocrine pancreas, in order to obtain clues for hK6 and hK10 function in these cells. Ten cases of normal pancreatic tissue, two cases of nesidioblastosis, five insulin-producing tumours and one case of multiple endocrine neoplasia 1 syndrome, containing an insulin-, a somatostatin- and several glucagon-producing tumours, as well as tiny foci of endocrine dysplasia with different predominance of the secreted hormones (mainly glucagon and pancreatic polypeptide) were included in the study. A streptavidin–biotin–peroxidase and an alkaline phosphatase protocol, as well as a sequential immunoenzymatic double staining method were performed, using specific antibodies against hK6, hK10, insulin, glucagon, somatostatin, pancreatic polypeptide, and serotonin. hK6 and hK10 immunoexpression was observed in the islets of Langerhans, including the pancreatic polypeptide-rich islets, in the normal pancreas. Scattered hK6 and hK10 positive cells were localized in relationship with pancreatic acinar cells. In the exocrine pancreas, a cytoplasmic and/or brush border hK6 and hK10 immunoexpression was observed in the median and small sized pancreatic ducts, while the acinar cells were negative. Foci of nesidioblastosis and endocrine dysplasia expressed both kallikreins. hK6 and hK10 were also strongly and diffusely expressed throughout all insulin-, glucagon- and somatostatin-producing tumours. The double staining method revealed co-localization of each hormone and hK6/hK10 respectively, in the same cellular population, in the normal as well as in the diseased pancreas. Our results support the view that hK6 and hK10 may be involved in insulin and other pancreatic hormone processing and/or secretion, as well as in physiological functions related to the endocrine pancreas.     

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.     

An immunocytochemical study of endocrine pancreas of snakes

Summary The pancreas from eleven species of snakes representing both advanced and primitive families has been investigated for the presence of eleven regulatory peptides reported to occur in the mammalian endocrine pancreas. Of the eleven peptides studied, insulin, pancreatic glucagon and somatostatin were present in endocrine cells within the islets of all the species investigated. The neuropeptide, vasoactive intestinal polypeptide, was located within nerve terminals innervating the islets in the Boidinae, Colubrinae, Elaphidae and Crotalidae but absent from the Natricinae investigated.No immunoreactivity was demonstrable with the antisera to substance P, met-enkephalin, C-terminal gastrin, bombesin, glicentin and gastric inhibitory polypeptide. Pancreatic polypeptide-like immunoreactivity was demonstrable only in the boid snakes and exclusively stained by a C-terminal specific antiserum.     

Somatostatin inhibits insulin and glucagon release by monolayer cell cultures of rat endocrine pancreas

Dihydrosomatostatin (0.001–1.0 ug/ml) inhibited both insulin and glucagon secretion by monolayer cell cultures of newborn rat pancreas. When cultures were incubated with somatostatin and then rinsed, the effect of somatostatin appeared to last longer on the pancreatic alpha cell than on the beta cell as indicated by a more prolonged inhibition of glucagon secretion than of insulin release. Submaximal inhibition of glucose-stimulated insulin release by somatostatin was partially reversed by increasing the concentration of glucose. We conclude that the effect of somatostatin appears to be mediated directly on the pancreatic endocrine cells.     

Immunocytochemical characterization of glucagon-immunoreactive cells using monoclonal antibodies to pancreatic glucagon

Monoclonal antibodies raised to pancreatic glucagon were tested for their ability to detect glucagon-containing endocrine cells in material processed for light and electron microscopy. Samples from man, baboon and rat were used in this investigation. Two antibodies were specific for the pancreatic islet A cells, the remainder detected both pancreatic and enteric endocrine cells. In man and baboon the glucagon-containing cells were confined to the pancreas, lower small intestine and colon. In the rat the distribution was extended to include the corpus of the stomach and the jejunum. The cells identified in the ileum and colon were of three morphological types endocrine, paracrine (type 1) with a single basal process and paracrine (type 2) with multiple small cytoplasmic processes. These antibodies also detected cells in material fixed by conventional methods for electron microscopy. The ultrastructural appearance of the baboon pancreatic glucagon-containing ultracellular secretory granules were demonstrated to be clearly distinct from those described previously in man and rat. The secretory granules averaged 330 +/- 23 nm and lacked the characteristic clear outer halo seen in the other two species.     

In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract

Recent reports have suggested that mesenchymal cells derived from bone marrow may differentiate into not only mesenchymal lineage cells but also other lineage cells. There is possibility for insulin-producing cells (IPCs) to be differentiated from mesenchymal cells. We used self-functional repair stimuli of stem cells by partial injury. Rat pancreatic extract (RPE) from the regenerating pancreas (2 days after 60% pancreatectomy) was treated to rat mesenchymal cells. After the treatment of RPE, they made clusters like islet of Langerhans within a week and expressed four pancreatic endocrine hormones; insulin, glucagon, pancreatic polypeptide, and somatostatin. Moreover, IPCs released insulin in response to normal glucose challenge. Here we demonstrate that the treatment of RPE can differentiate rat mesenchymal cells into IPCs which can be a potential source for the therapy of diabetes.     

Hybrid insulin genes reveal a developmental lineage for pancreatic endocrine cells and imply a relationship with neurons

Insulin appears in the developing mouse pancreas at embryonic day 12 (e12). Transgenic mice harboring three distinct hybrid genes utilizing insulin gene regulatory information first express the transgene product two days earlier, at e10, in a few cells of the pancreatic bud. Throughout development and postnatal life, all of the insulin-producing (beta) cells coexpress the hybrid insulin gene. In addition, islet cells containing glucagon, somatostatin, pancreatic polypeptide, and the neuronal enzyme tyrosine hydroxylase coexpress the transgene when they first arise. Similarly, coexpression of these normally distinct islet cell markers occurs during differentiation of the four endocrine cell types. The transgene product also appears transiently during embryogenesis in cells of the neural tube and in neural crest. The results suggest a common precursor for the endocrine cells of the pancreas. Moreover, they imply a relationship between neural and pancreatic endocrine tissue.     

Somatostatin-containing and other endocrine cells in the pancreas of the spectacled caiman

Light-microscopic immunocytochemistry was used to localize 4 major pancreatic hormones in the pancreas of the spectacled caiman, Caiman fuscus. Somatostatin, insulin, glucagon and pancreatic polypeptide were localized by the peroxidase-antiperoxidase complex technique. A relatively large population of somatostatin-containing D cells was present. The D cells were nearly as numerous as the insulin-containing B cells and glucagon-containing A cells which were the most common cell types. All three cell types were commonly intermingled with one another in endocrine cell areas. Pancreatic polypeptide-reactive F cells were absent from some regions of the pancreas, but where present were related to other endocrine cell types. Functional properties of the pancreatic endocrine cells in this anatomical variant remain to be determined.