Ultrastructural studies of the ontogeny of fetal human and porcine endocrine pancreas, with special reference to colocalization of the four major islet hormones.

Most, if not all, endocrine cells seem capable of synthesizing and storing more than one hormone. Such cellular colocalization of hormones can be due either to the presence of two or more specific granules within the cells or to colocalization of the hormones within a single granule. The present study was performed to clarify the subcellular localization of insulin, glucagon, somatostatin, and pancreatic polypeptide within the endocrine cells of the human and porcine pancreas during fetal development, with special reference to possible colocalization of the hormones. The tissue specimens were processed for ultrastructural cytochemistry using Lowicryl as embedding medium. An immunogold labeling technique was used with two parallel, but not interacting, antibody chains. Sections from each specimen were double labeled in different combinations giving a complete covering of the four major islet hormones. During fetal life (50-90 days prenatally in porcine pancreas, 14 weeks gestation in the human pancreas) several hormones were demonstrated, not only in the same endocrine cells, but also in the same secretory granules (polyhormonal granules). Costorage of insulin, glucagon, somatostatin, and pancreatic polypeptide was demonstrated in granules in pancreatic endocrine fetal cells. At an early fetal stage, the endocrine cells contained either dense, round granules or pale, heteromorphous granules. With increasing age and maturation of the endocrine cells, structural differentiation of the secretory granules was found to be associated with a gradual disappearance of the polyhormonal granules. The first genuine monohormonal cell to appear in the porcine fetus was the pancreatic polypeptide cell (at 70 days gestation); it was followed by the somatostatin-producing endocrine cell. Mature insulin- and glucagon-producing cells were only demonstrated after birth. Thus, in the adult pancreatic endocrine cells, each specific endocrine cell type produced only one of the four classical hormones. The present investigation demonstrated that the endocrine cells of the fetal, but not the adult, pancreas are able to synthesize all the major islet hormones, and that these peptides are costored in the same granule. The data obtained support the concept of a common precursor stem cell for pancreatic hormone-producing cells.     

Cortisone-induced islet cell hyperplasia in hamsters

Pancreatic islet cell hyperplasia was studied in hamsters during one to eight weeks of cortisone treatment. Measurement of serum glucose and insulin; pancreatic insulin, glucagon, somatostatin, pancreatic polypeptide as well as islet tissue morphometry were performed. Serum glucose was highest at week 2, followed by mild to moderate hyperglycemia. Serum insulin was increasingly higher from week 1 to week 8. Pancreatic insulin was maximal at week 5 then declined through week 8 in the presence of beta cell neurosis in markedly hyperplastic islets. Pancreatic concentration of somatostatin and pancreatic polypeptide moderately increased more than the control levels; however, compared with the controls, glucagon was reduced by cortisone treatment. Effect of cortisone in the four types of islet cells is discussed, particularly on beta cell hyperplasia, which appears to be a response to decreased insulin binding to the target organs with no changes in receptor concentration.     

Ontogeny of Human Langerhans Islets. A Review of Some Light- and Electron-Microscopical, Immunohistochemical, and Functional Data on Fetal Development of the Endocrine Pancreas (Review)

The parenchymal cells of the islets of Langerhans belong to the extensive human neuroendocrine system. Its messenger substances are biogenic amines and neurohormonal peptides. Like other neuroendocrine cells, the islet cells might have originated from the neural crest. However, in the fetal life, their stem cells are located in the epithelium of the pancreatic ductuli. As early as at the 8th gestational week, these stem cells have been found to contain secretory granules of the neuroendocrine type. Evidences for production of insulin, somatostatin, glucagon, and PP (the pancreatic polypeptide) have been obtained immunohistochemically in the samples from the 10–12th gestational weeks. In the samples from the 14th week, cell clusters have been observed, which are outgrowing from the ductular epithelium and forming primitive Langerhans islets. The insulin cells predominate markedly and are shown to respond functionally to glucose stimulation. By the 16th week, the islets become vascularized, with the primary innervation. The completely formed endocrine pancreas, as it is observed at birth, is revealed at the 26th gestational week. Based on some light-microscopical, ultrastructural, and immunohistochemical characteristics of the islet parenchymal cells and their supply with blood vessels and nerves, three phases of the gland embryonal/fetal development are identified.     

Immunohistochemical study of the developing endocrine pancreas of the opossum (Didelphis virginiana)

Cells immunoreactive for insulin, glucagon, somatostatin, bovine pancreatic polypeptide and 5-hydroxytryptamine are found in the pancreas of the newborn opossum and of all later stages examined. All immunoreactive cell types are present in primary and secondary islets and within elements of the exocrine pancreas. Cells immunoreactive for glucagon, bovine pancreatic polypeptide, somatostatin and 5-hydroxytryptamine generally are confined to the periphery of secondary (intralobular) islets, whereas insulin-immunoreactive cells occupy the central region. Endocrine cells within primary (interlobular) islets are randomly scattered. A small number of pancreatic-polypeptide-immunoreactive cells are reactive for the amine 5-hydroxytryptamine also, but the reverse is not observed. The endocrine pancreas continues to differentiate and develop throughout postnatal life and into adulthood. Little difference was observed between the head and tail regions of the opossum pancreas for the measurements made.     

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)     

The duct-ligated pancreas transplant and its effect on the islet cellular composition of the host pancreas

Summary Rats rendered diabetic by streptozotocin were subjected to pancreas transplantation. After twenty weeks, the duct-ligated pancreas transplant was studied morphometrically to determine the effect of duct occlusion on the various cell populations of the islets. Concomitantly, the streptozotocin-treated host pancreas was examined for a possible influence of the graft on the diabetic pattern of islet cell population. Twenty weeks after pancreas transplantation, the volume fractions of insulin, glucagon, somatostatin and pancreatic polypeptide cells in the graft islets did not differ from those of the normal control pancreas. In the pancreas of nontransplanted diabetic rats, insulin-positive B cells were reduced from 60–65% to less than 10% of the islet volume, whereas non-B cells were significantly increased in volume density. The changes in fractional volume of the various islet cells correlated fairly well with changes in plasma concentration of the corresponding pancreas hormones. In the recipient's own pancreas, the relative volumes of glucagon and somatostatin cells were unaffected by the pancreas transplant. However, the insulin cell mass was significantly increased, and comprised about 20% of the islet volume, while cells containing pancreatic polypeptide were found only sporadically.Supported by Nordic Insulin Fund, The Swedish Diabetes Association, and MFR, proj. no. 4499. The technical assistance by M. Maxe and M. Carlesson is gratefully acknowledged     

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.     

Development of the endocrine cells in the rat pancreatic and bile duct system

Summary Morphological features of the endocrine cells in the duct system of the pancreas and the biliary tract have been recently characterized in the adult animal with respect to their physiological roles. In the present study, we have investigated their chronological appearance as well as their developmental progress at various stages of the rat fetal and postnatal life. On day 12 of gestation, glucagon and insulin, as well as CCK cells, were identified in the pancreatic primordium. On day 14, glucagon and CCK cells were first detected in the epithelial lining of the common hepatic and the hepatic ducts. These cells remained the dominant endocrine type in the duct system during the fetal period. Insulin and pancreatic polypeptide cells were first observed in the common hepatic duct only on days 16 and 18 of gestation respectively. In spite of their presence in the islets, somatostatin cells were not detected in the duct system during fetal life. They started to appear in the accessory pancreatic duct of the neonate, and subsequently in the common hepatic duct as well as in the small pancreatic ones on day 7 after birth. During postnatal development, the endocrine cells showed progressive or retrogressive changes in different portions of the duct system according to the cell type. In general, somatostatin, CCK and pancreatic polypeptide cells showed an increase, while glucagon and insulin cells gradually dwindled in number up to the adult stage. Somatostatin cells exhibited a significant increase in number, becoming the highest population among the duct endocrine cells in the adult. Throughout the developmental progress, the endocrine cells appear to be allocated in regions relevant to their possible influence modulating the exocrine secretion as well as the drainage of the pancreatic and bile fluid. To whom correspondence should be address.     

Immunocytochemical localization of hormone-producing cells within the pancreatic islets of the rainbow trout (Salmo gairdneri)

Summary A histological study of the pancreatic islets in rainbow trout, Salmo gairdneri, was undertaken in which polypeptide hormone-producing cells were localized, using immunocytochemical staining techniques. Four different celltypes were identified in this manner. These were the insulin, somatostatin, pancreatic polypeptide and glucagon/gastric inhibitory polypeptide (GIP) cells. The glucagon/GIP cell was designated thus as antisera to both hormones crossreacted with a common population of cells. A fifth cell-type, commonly referred to as a clear cell, was also identified although its secretory product is as yet undetermined. These functional cell types were compared to the standard tinctorial properties of pancreatic endocrine cells. The relationships of the various cell types with each other was also observed.     

Immunocytochemical study of argyrophil (Sevier Munger) endocrine cells of adult human pancreas

Bouin-fixed tissues from non-diabetic adult human pancreata display an argyrophil reaction mainly in the periphery of the islets with the silver technique of Sevier-Munger. The nature of these argyrophil cells was examined after restaining by an indirect immunocytochemical method using antibodies against insulin, glucagon, somatostatin and pancreatic polypeptide. After this procedure the argyrophil cells were identified as glucagon (A-) cells and pancreatic polypeptide (PP-) cells, although the latter exhibited a weaker reaction. The insulin (B-) cells and somatostatin (D-) cells were unreactive. The results show that the Seiver-Munger stain is of equal value to the Grimelius silver nitrate stain in adult human pancreatic islets after fixation in Bouin's fluid.     

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.     

Immunocytochemical study of argyrophil (Sevier Munger) endocrine cells of adult human pancreas

Summary Bouin-fixed tissues from non-diabetic adult human pancreata display an argyrophil reaction mainly in the periphery of the islets with the silver technique of Sevier-Munger. The nature of these argyrophil cells was examined after restaining by an indirect immunocytochemical method using antibodies against insulin, glucagon, somatostatin and pancreatic polypeptide. After this procedure the argyrophil cells were identified as glucagon (A-) cells and pancreatic polypeptide (PP-) cells, although the latter exhibited a weaker reaction. The insulin (B-) cells and somatostatin (D-) cells were unreactive. The results show that the Sevier-Munger stain is of equal value to the Grimelius silver nitrate stain in adult human pancreatic islets after fixation in Bouin's fluid.Supported by grants from the Swedish Medical Research Council (Project No. 102)     

Localization of four polypeptide hormones in the saurian pancreas.

Glucagon, insulin, somatostatin, and pancreatic polypeptide have been localized in the anolian pancreas using peroxidase-antiperoxidase immunocytochemistry. The most abundant endocrine cell type contains glucagon. Insulin-containing cells are the next most numerous. Somatostatin-immunoreactive cells tend to be localized at the periphery of the islet cords. Pancreatic polypeptide-containing cells are a minor endocrine component scattered throughout the exocrine pancreas and occasionally within the islet areas. No staining was observed after application of antigastrin serum.     

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.     

Response of pancreatic immunoreactive somatostatin to arginine

Perfusion of isolated dog pancreases with arginine (20 mM) was associated with a prompt and sustained increase in immunoreactive somatostatin (IRS) in the venous effluent while insulin and glucagon rose promptly but soon receded from their peak levels. These results are compatible with a postulated feedback relationship between somatostatin-, glucagon-, and perhaps insulin-secreting cells of the islets in which somatostatin, stimulated by local glucagon, restrains glucagon secretion and perhaps glucagon-mediated insulin release as well.The demonstration that D-cells of the pancreatic islets contain immunoreactive somatostatin (1, 2, 3) which is probably biologically active (4), and are situated topographically between the A-cells and B-cells in the heterocellular region of the islet (5) has suggested a functional role for these components of the islet of Langerhans (6). In view of the inhibitory action of somatostatin upon both insulin and glucagon secretion (7, 8, 9), it was postulated that the D-cell might serve to restrain glucagon and/or insulin secretion (6). We have since reported that the release of IRS from the isolated dog pancreas increases promptly during the perfusion of high concentrations of glucagon whereas high concentrations of insulin do not appear to stimulate IRS release (10). In this study we examine the effect of perfusion with arginine, a potent stimulus of both glucagon and insulin secretion, upon pancreatic IRS release.     

Redifferentiation of insulin-secreting cells after in vitro expansion of adult human pancreatic islet tissue

Cellular replacement therapy holds promise for the treatment of diabetes mellitus but donor tissue is severely limited. Therefore, we investigated whether insulin-secreting cells could be differentiated in vitro from a monolayer of cells expanded from human donor pancreatic islets. We describe a three-step culture protocol that allows for the efficient generation of insulin-producing cell clusters from in vitro expanded, hormone-negative cells. These clusters express insulin at levels of up to 34% that of average freshly isolated human islets and secrete C-peptide upon membrane depolarization. They also contain cells expressing the other major islet hormones (glucagon, somatostatin, and pancreatic polypeptide). The source of the newly differentiated endocrine cells could either be indigenous stem/progenitor cells or the proliferation-associated dedifferentiation and subsequent redifferentiation of mature endocrine cells. The in vitro generated cell clusters may be efficacious in providing islet-like tissue for transplantation into diabetic recipients.     

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.     

The endocrine cells in the gastroenteropancreatic system of the bowfin, Amia calva L.: an immunohistochemical, ultrastructural, and immunocytochemical analysis.

The gastroenteropancreatic (GEP) endocrine system of bowfin (Amia calva) was described using light and electron microscopy and immunological methods. The islet organ (endocrine pancreas) consists of diffusely scattered, mostly small islets and isolated patches of cells among and within the exocrine acini. The islets are composed of abundant, centrally located B cells immunoreactive to bovine and lamprey insulin antisera and D cells showing a widespread distribution and specificity to somatostatin antibodies. A and F cells are present at the very periphery of the islets and are immunoreactive with antisera against glucagon (and glucagon-like peptide) and several peptides of the pancreatic polypeptide (PP)-family, respectively. The peptides of the two families usually collocates within the same peripheral islet cells and are the most common immunoreactive peptides present in the extra-islet tissue. Immunocytochemistry and fine structural observations characterised the granule morphology for B and D cells and identified two cell types with granules immunoreactive to glucagon antisera. These two putative A cells had similar granules, which were distinct from either B or D cells, but one of the cells had rod-shaped cytoplasmic inclusions within cisternae of what appeared to be rough endoplasmic reticulum. The inclusions were not immunoreactive to either insulin or glucagon antisera. Only small numbers of cells in the stomach and intestine immunoreacted to antisera against somatostatin, glucagon, and PP-family peptides. The paucity of these cells was reflected in the low concentrations of these peptides in intestinal extracts. The GEP system of bowfin is not unlike that of other actinopterygian fishes, but there are some marked differences that may reflect the antiquity of this system and/or may be a consequence of the ontogeny of this system in this species.