Organogenesis and differentiation of the pancreas in the toad Bufo bufo L.

Summary In Bufo bufo at stage III₆ the first endocrine islets appear in the part of the pancreas corresponding to the dorsal anlage. At stage IV₂, 5 days later, the pancreatic duct develops and new islets arise by budding off from the ductal epithelium. The ultrastructural study of the secretory granules morphology of endocrine cells has distinguished four different cell types: B-cells (stage III₉), A-cells (stage IV₃), D-cells (stage IV₃) and a fourth type not yet identified (stage IV₃). By immunocytology insulin and corticotropin-releasing factor (CRF) cells have been demonstrated at stage III₉, and glucagon and somatostatin cells at stage IV₁. Lastly, endocrine islets can be homogeneous (predominantly containing insulin cells, rarely glucagon cells) or heterogeneous (insulin cells at the centre and glucagon or somatostatin cells at the periphery). Hypotheses are put forward for the origin and the constitution of the different generations of endocrine islets and isolated cells.     

Polyhormonal aspect of the endocrine cells of the human fetal pancreas

Histological studies were performed on 30 pancreases obtained from normal human fetuses aged between the 9th and 38th week. For immunocytochemistry, the avidin-biotin-peroxidase method was used to identify and colocalise insulin, glucagon, somatostatin, pancreatic polypeptide and proliferating cell nuclear antigen. In the 9th week, cells containing all investigated peptides were present. During the fetal period, two populations of endocrine cells have been distinguished, Langerhans islets and freely dispersed cells. The free cells were polyhormonal, containing insulin, glucagon, somatostatin and pancreatic polypeptide, and were localised in the walls of pancreatic ducts throughout the whole gland. During the development of the islets we have observed four stages: (1) the scattered polyhormonal cell stage (9th–10th week), (2) the immature polyhormonal islet stage (11th–15th week), (3) the insulin monohormonal core islet stage (16th–29th week), in which zonular and mantle islets are observed, and (4) the polymorphic islet stage (from the 30th week onwards), which is characterised by the presence of monohormonal cells expressing glucagon or somatostatin. Bigeminal and polar islets also appeared during this last stage. The islets consisted of an insulin core surrounded by a thick (in the part developing from the dorsal primordium) or thin rim (part of the pancreas concerned with the ventral primordium) of intermingled mono- or dihormonal glucagon-positive or somatostatin-positive cells. The most externally located polyhormonal cells exhibited a reaction for glucagon, somatostatin and pancreatic polypeptide. Apart from the above-mentioned types of islets, all arrangements observed in earlier stages were present. Proliferating cell nuclear antigen-positive cells (single in the large islets and more numerous in the smaller ones) were predominantly observed in the outermost layer. Taken together our data indicate that, during the human prenatal development of the islet, endocrine cells are able to synthesise several different hormones. Maturation of these cells involved or depended on a change from a polyhormonal to a monohormonal state and is concerned with decreasing proliferative capacity. This supports the concept of a common precursor stem cell for the hormone-producing cells of the fetal human pancreas. Accepted: 1 June 1999     

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.     

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.     

Embryogenesis of the murine endocrine pancreas; early expression of pancreatic polypeptide gene.

By immunofluorescence on cytospin preparations and on semithin sections of mouse pancreatic buds, we have found glucagon and pancreatic polypeptide (PP)-containing cells at embryonal day 10.5 (E 10.5) in dorsal buds and at E 11.5 in ventral buds. Insulin-containing cells appear in dorsal buds at E 11.5, and one to two days later in ventral buds. Somatostatin-containing cells are detectable from E 13.5 in both dorsal and ventral buds. A quantitative analysis shows that up to E 15.5, PP-containing cells are relatively abundant in both buds. By PCR amplification of oligo(dT)-primed cDNAs prepared from total pancreatic RNA, we also detect PP mRNA from E 10.5 onwards, thus confirming the early expression of the PP gene in the developing mouse pancreas. Analysis of endocrine cells in situ suggests three major patterns of cell distribution in embryonic pancreas. First, individual hormone-containing cells are located within the epithelium of pancreatic ducts. In both dorsal and ventral buds, the majority of these endocrine cells contain PP, but many also contain glucagon, insulin or somatostatin. Secondly, clusters of endocrine cells are found in the pancreatic interstitium. Many of these cells contain both glucagon and PP which, by immunogold labelling of consecutive thin sections, can be shown to co-exist within individual secretory granules. Finally, starting on E 18.5, typical islets are formed with centrally located B cells and with the adult 'one cell-one hormone' phenotype. These results suggest an intriguing ontogenic relationship between A- and PP-cells, and also indicate that PP-containing cells may occupy a hitherto unexpected place in the lineage of endocrine islet cells.     

Development of endocrine pancreatic islets in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

Differentiation of the pancreatic islets in grass snake Natrix natrix embryos, was analyzed using light, transmission electron microscopy, and immuno-gold labeling. The study focuses on the origin of islets, mode of islet formation, and cell arrangement within islets. Two waves of pancreatic islet formation in grass snake embryos were described. The first wave begins just after egg laying when precursors of endocrine cells located within large cell agglomerates in the dorsal pancreatic bud differentiate. The large cell agglomerates were divided by mesenchymal cells thus forming the first islets. This mode of islet formation is described as fission. During the second wave of pancreatic islet formation which is related to the formation of the duct mantle, we observed four phases of islet formation: (a) differentiation of individual endocrine cells from the progenitor layer of duct walls (budding) and their incomplete delamination; (b) formation of two types of small groups of endocrine cells (A/D and B) in the wall of pancreatic ducts; (c) joining groups of cells emerging from neighboring ducts (fusion) and rearrangement of cells within islets; (d) differentiated pancreatic islets with characteristic arrangement of endocrine cells. Mature pancreatic islets of the grass snake contained mainly A endocrine cells. Single B and D or PP–cells were present at the periphery of the islets. This arrangement of endocrine cells within pancreatic islets of the grass snake differs from that reported from most others vertebrate species. Endocrine cells in the pancreas of grass snake embryos were also present in the walls of intralobular and intercalated ducts. At hatching, some endocrine cells were in contact with the lumen of the pancreatic ducts.     

Insulin-, glucagonand somatostatin-like immunoreactivity in the endocrine pancreas of the lungfish,Neoceratodus forsteri

Summary The endocrine pancreas of the Australian lungfish,Neoceratodus forsteri, was investigated immunocytochemically for the presence of polypeptide hormone-producing cells. Three cell types were identified, namely insulin-, glucagon-, and somatostatin-immunoreactive elements. The insulin cells are confined solely to the center of the islets. Glucagon and somatostatin cells are distributed peripherally around the central mass of the insulin cells. Isolated cells or clusters of glucagon and somatostatin cells are also dispersed within the exocrine parenchyma. The immunoreactive cell types are compared with those staining with standard histological procedures. The spatial relationships of the different cell populations are examined.     

Genetic analysis of early endocrine pancreas formation in zebrafish

Endocrine pancreas of zebrafish consist of at least four different cell types that function similarly to mammalian pancreatic islet. No mutants specifically affecting formation of the endocrine pancreas have been identified during the previous large-scale mutagenesis screens in zebrafish due to invisibility of a pancreatic islet. We combined in situ hybridization method to visualize pancreatic islet with an ethyl-nitroso-urea mutagenesis screen to identify novel genes involved in pancreatic islet formation in zebrafish. We screened 900 genomes and identified 11 mutations belonging to nine different complementation groups. These mutants fall into three major phenotypic classes displaying severely reduced insulin expression, reduced insulin expression with abnormal islet morphology, or abnormal islet morphology with relatively normal number of insulin expressing cells. Seven of these mutants do not have any other visible phenotypes associated. These mutations affect different processes in pancreatic islet development. Additional analysis on glucagon and somatostatin cell specification revealed that somatostatin cells are specified at a separate domain from insulin cells whereas glucagon cells are specified adjacent to insulin cells. Furthermore, glucagon cells and somatostatin cells are always associated with insulin cells in mutants that have scattered insulin expression. These data indicate that there are separate mechanisms regulating endocrine cell migration, proliferation, and differentiation. Further study on these mutants will reveal important information on novel genes involved in pancreatic islet cell specification and morphogenesis.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax)

Summary The development of the endocrine pancreas of the teleost sea bass (Dicentrarchus labrax, L.) was examined from hatching to 61 days, using the peroxidase-antiperoxidase technique for light microscopy. Mammalian and bonito insulin (mI and bI)-, salmo somatostatin-25 (SST-25)-, somatostatin-14 (SST-14a and b)-, glucagon-, bovine pancreatic polypeptide (PP)-, peptide tyrosine-tyrosine (PYY)- and salmo neuropeptide Y (NPY)-like immunoreactivity was demonstrated. Four ontogenetic stages were established according to the organization and immunostaining of the endocrine cells. One cell strand or primordial cord showing mI/bI- and SST-25/SST-14a-like immunoreactivity was first found at hatching in the dorsal epithelium of the anterior zone of the midgut (stage 1). One primitive islet, comprising outer SST-25/SST-14a- and inner mI/bI- and SST-14a/ SST-14b-immunoreactive cells, was found in 2- to 5-day-old larvae (stage 2). One single islet, in which glucagon-immunoreactive cells appear in the periphery, was found in larvae from 9 to 20 days after hatching (stage 3). One big islet containing, in addition, PP-immunoreactive cells in the outer region and slender cell processes which showed PYY-like immunoreactivity, was found from 25 to 61 days after hatching. During this period, primordial islets, composed of SST-25- and bI-immunoreactive cells, and clustered or isolated pancreatic endocrine cells, close to the pancreatic duct, as well as small and intermediate islets (secondary islets), in which glucagon, PP, PYY and NPY seem to be co-localized, were progressively found (stage 4). The origin of the endocrine pancreas of sea bass, and the ontogenetic and phylogenetic significance, are discussed.     

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.     

Effect of galanin on arginine-stimulated pancreatic hormone release from isolated perifused rat islets.

The effect of galanin on pancreatic hormone release was studied using isolated perifused rat pancreatic islets. In the presence of 100 mg/dl glucose, 10(-8) mol/L galanin significantly inhibited the basal somatostatin release compared with the perifusion without galanin, whereas there was no significant change in the basal insulin and glucagon release. However, under stimulation of 20 mmol/L arginine, 10(-8) mol/L galanin significantly enhanced glucagon release and suppressed insulin and somatostatin release. These effects disappeared immediately after cessation of galanin infusion. Additionally, 10(-8) mol/L galanin significantly enhanced the first and second phase of glucagon release stimulated by arginine, whereas arginine-stimulated insulin and somatostatin releases were significantly inhibited in both phases. In the cysteamine-treated rat islets, neither enhancement of glucagon release nor suppression of insulin release by galanin was reproducible. These findings indicate two possible explanations. First, it is suggested that the effects of galanin on insulin and glucagon release may be direct and reversed by non-specific effect of cycteamine. Secondly, it seems likely that galanin-enhanced glucagon release may be indirect and in part due to the concomitant somatostatin suppression. Galanin may have an important regulatory function on endocrine pancreas.     

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.     

Morphological changes in the human endocrine pancreas induced by chronic excess of endogenous glucagon.

The non-tumoral endocrine pancreas from a patient with elevated plasma levels of glucagon due to a malignant glucagonoma was studied immunocytochemically, ultrastructurally and morphometrically. Compared with normal pancreatic islets from control subjects, those of the pancreas from the patient with a glucagonoma showed an almost complete disappearance of A cells, a decrease in immunoreactive insulin in B cells associated with cytological features indicating enhanced synthesis and secretion of this hormone, and an increase in immunoreactive somatostatin and pancreatic polypeptide (PP) accompanied by unusually high numbers of D and PP cells. In addition, numerous B cells were found outside the islets, either forming micro-islets or scattered in the exocrine tissue (nesidioblastosis). The possible mechanisms involved in determining the changes in the secretory activity of B cells and the alterations in the cell composition of the islets are discussed.     

Regulatory peptides in the pancreas of two species of elasmobranchs and in the Brockmann bodies of four teleost species

Summary Immunohistochemistry was used to localize regulatory peptides in endocrine cells and nerve fibres in the pancreas of two species of elasmobranchs (starry ray,Raja radiata and spiny dogfish,Squalus acanthias), and in the Brockmann bodies of four teleost species (goldfish,Carassius auratus, brown troutSalmo trutta, rainbow trout,Oncorhynchus mykiss and cod,Gadus morhua). In the elasmobranchs, the classical pancreatic hormones somatostatin, glucagon and insulin were present in endocrine cells of the islets. In addition, endocrine cells were labelled with antisera to enkephalins, FMRF-amide, gastrin/cholecystokinin-(CCK)/caerulein, neurotensin, neuropeptide Y (NPY), and peptide YY (PYY). Nerve fibres were demonstrated with antisera against bombesin, galanin and vasoactive intestinal polypeptide (VIP). These nerve fibres innervated the walls of blood vessels, in the exocrine as well as the endocrine tissue. In the four teleost species immunoreactivity to somatostatin, insulin and glucagon was intense in the Brockmann bodies. Cells were labelled with antisera to enkephalin, neurotensin, FMRFamide, gastrin/CCK/ caerulein, NPY, PYY and VIP. Only a few nerve fibres were found with antisera against dopamine--hydroxylase (DBH, cod), enkephalin (met-enkephalin-Arg-Phe, cod), bombesin (cod), gastrin/CCK/caerulein (cod) and VIP. Galanin-like-immunoreactive fibres were numerous in the Brockmann bodies of all teleosts examined. Immunoreactivity to calcitonin gene-related peptide (CGRP), substance P, tyrosine hydroxylase (TH), and phenyl-N-methyl transferase (PNMT) could not be found in any of the species studied.     

Somatostatin-induced inhibition of insulin secretion from isolated islets of rat pancreas in presence of glucagon.

In order to study the oeffect of somatostatin on the endocrine pancreas directly, islets isolated from rat pancreas by collagenase were incubated for 2 hrs 1) at 50 and 200 mg/100 ml glucose in the absence and presence of somatostatin (1, 10 and 100 mg/ml) and2) at 200 mg/100 ml glucose together with glucagon (5 mug/ml), with or without somatostatin (100 ng/ml). Immunologically measurable insulin was determined in the incubation media at 0, 1 and 2 hrs. Insulin release was not statistically affected by any concentration stomatostatin. On the other hand, somatostatin exerted a significant inhibitory action on glucagon-potentiated insulin secretion (mean +/- SEM, mu1/2 hrs/10 islets: glucose and glucagon: 1253 +/- 92; glucose, glucagon and somatostatin: 786 +/- 76). The insulin output in th epresence of glucose, glucagon and somatostatin was also significantly smaller than in thepresence of glucose alone (1104 +/- 126) or of glucose and somatostatin (1061 +/- 122). The failure of somatostatin to affect glucose-stimulated release of insulin from isolated islets contrasts its inhibitory action on insulin secretion as observed in the isolated perfused pancreas and in vivo. This discrepancy might be ascribed to the isolation procedure using collagenase. However, somatostatin inhibited glucagon-potentiated insulin secretion in isolated islets which resulted in even lower insulin levels than obtained in the parallel experiments without glucagon. It is concluded that the hormone of the alpha cells, or the cyclic AMP system, might play a part in the machanism of somatostatin-induced inhibition of insulin release from the beta-cell.     

Expression of amylase and other pancreatic genes in Xenopus

To better understand the relationship between the endocrine and exocrine cell types in the Xenopus pancreas, we have cloned the Xenopus amylase cDNA and compared its expression profile with that of four other pancreatic markers: insulin, glucagon, elastase and trypsinogen. Our results demonstrate that the first pancreatic marker to be expressed is insulin, exclusively in the dorsal pancreas. These insulin-expressing cells form small groups which resemble islets, but no insulin is detected in the ventral pancreas until stage 47. In contrast, the exocrine markers, amylase, elastase and trypsinogen are first expressed only in the ventral pancreas beginning at stage 41; by stage 45 their expression extends into the dorsal pancreas. Glucagon, on the other hand, is not expressed in the pancreas until stage 45. In the endocrine cell clusters we do not find glucagon-expressing cells surrounding insulin-expressing cells, either in the tadpole or in the mature frog pancreas.     

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.