Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP- or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas. Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide- and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide- or glucagon-immunoreactivity were also found. Glucagon- and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.     

Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Summary Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP-or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas.Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide-and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide-or glucagon-immunoreactivity were also found. Glucagon-and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.     

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.     

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.     

Nerve Cells Associated with the Endocrine Pancreas in Young Mice: An Ultrastructural Analysis of the Neuroinsular Complex Type I

The neuroinsular complex type 1 is composed of pancreatic endocrine islet cells and nerve cell bodies intrinsic to the islet. The details of the relation between nerve cells and between endocrine cells and nerve cells in the complex are unknown. Pancreata from newborn and 18-day-old mice were analysed by electron microscopy to establish the ultrastructural morphology of the neuroinsular complex. Immunohistochemical staining for protein gene-product 9.5 was also performed. The study showed that nerve cell bodies were closely associated to each other in the periphery of the islets with no connective tissue separating the cells. The nerve cells were closely associated to both -cells and -cells. Direct intercellular contacts were observed between nerve cells and endocrine cells and between Schwann cells and endocrine cells. Varicose nerve endings were frequently observed in the neuroinsular complex. In the peripheral parts the varicosities were mostly being associated to the nerve cell bodies. The varicosities contained small clear or small clear and larger dense cored vesicles, suggesting cholinergic and peptidergic contents. The varicosities made specialized synaptic connections with adjacently located nerve cells. The study shows that the neuronal part of the neuroinsular complex is closely associated to the endocrine islet cells and that it is richly innervated, indicating an important regulatory function of the nerve cell component in the neuroinsular complex.     

Isolation and morphologic characterization of human ovarian carcinoma cell clusters present in effusions

Serous, mucinous, endometrioid and clear cell human ovarian carcinoma cells were isolated as multicellular aggregates from patient effusions by filtration on nylon mesh of defined porosity and examined by light microscopy. The cell clusters ranged from compact to loosely adherent groups of cells to spheroids with a central lumen surrounded by a cell monolayer. There was considerable variation in cluster morphology between effusions from different patients as well as within effusion from the same patient. Apparent budding of clusters was observed as well as different stages of cluster growth and development. This was observed for all histologic types studied. Electron microscopy of serous, mucinous and clear cell types showed that cells forming clusters were attached to each other by desmosomes, demonstrating that cluster formation did not result from a nonspecific stickiness of cells. Irregular microvilli were present on the external periphery of the various carcinoma cells and a prominent glycocalyx was present on the surface of mucinous carcinoma cells. Extensive interdigitation of cytoplasmic extensions and extended villi was present in mucinous and serous clusters which appeared to strengthen cluster cohesiveness. Nuclei were irregular with prominent nucleoli frequently present. The cell clusters usually remained intact and viable in culture but generally did not attach to glass or plastic substrata, whereas mesothelial cells and nonactivated histiocytes rapidly attached. When carcinoma cell clusters did attach, they were resistant to detachment by trypsin-EDTA treatment, in contrast to the nonmalignant cells.     

Innervation patterns of autonomic axons in the human endocrine pancreas

The autonomic nervous system regulates hormone secretion from the endocrine pancreas, the islets of Langerhans, thus impacting glucose metabolism. The parasympathetic and sympathetic nerves innervate the pancreatic islet, but the precise innervation patterns are unknown, particularly in human. Here we demonstrate that the innervation of human islets is different from that of mouse islets and does not conform to existing models of autonomic control of islet function. By visualizing axons in three dimensions and quantifying axonal densities and contacts within pancreatic islets, we found that, unlike mouse endocrine cells, human endocrine cells are sparsely contacted by autonomic axons. Few parasympathetic cholinergic axons penetrate the human islet, and the invading sympathetic fibers preferentially innervate smooth muscle cells of blood vessels located within the islet. Thus, rather than modulating endocrine cell function directly, sympathetic nerves may regulate hormone secretion in human islets by controlling local blood flow or by acting on islet regions located downstream.     

Pancreatic islet cytology of ictaluridae (Teleostei)

Summary The endocrine pancreas of the bullhead catfish, Ictalurus nebulosus, and the channel catfish, I. punctatas was studied by light and electron microscopy. In addition to the usual A, B and D cells, a fourth endocrine cell type was consistently observed in the electron microscope. All endocrine cell types were innervated. The vesicles of most of the nerve endings were ultrastructurally different from typical adrenergic and cholinergic vesicles, strongly suggesting the possibility of a third autonomic neurotransmitter serving as a regulator of catfish islet secretion.Supported in part by PHS grant AM 11407 awarded to Dr. Bryce Munger.     

Ghrelin Expression in the Mouse Pancreas Defines a Unique Multipotent Progenitor Population

Pancreatic islet cells provide the major source of counteractive endocrine hormones required for maintaining glucose homeostasis; severe health problems result when these cell types are insufficiently active or reduced in number. Therefore, the process of islet endocrine cell lineage allocation is critical to ensure there is a correct balance of islet cell types. There are four endocrine cell types within the adult islet, including the glucagon-producing alpha cells, insulin-producing beta cells, somatostatin-producing delta cells and pancreatic polypeptide-producing PP cells. A fifth islet cell type, the ghrelin-producing epsilon cells, is primarily found during gestational development. Although hormone expression is generally assumed to mark the final entry to a determined cell state, we demonstrate in this study that ghrelin-expressing epsilon cells within the mouse pancreas do not represent a terminally differentiated endocrine population. Ghrelin cells give rise to significant numbers of alpha and PP cells and rare beta cells in the adult islet. Furthermore, pancreatic ghrelin-producing cells are maintained in pancreata lacking the essential endocrine lineage regulator Neurogenin3, and retain the ability to contribute to cells within the pancreatic ductal and exocrine lineages. These results demonstrate that the islet ghrelin-expressing epsilon cells represent a multi-potent progenitor cell population that delineates a major subgrouping of the islet endocrine cell populations. These studies also provide evidence that many of hormone-producing cells within the adult islet represent heterogeneous populations based on their ontogeny, which could have broader implications on the regulation of islet cell ratios and their ability to effectively respond to fluctuations in the metabolic environment during development.     

Structure of neuro-endocrine and neuro-epithelial interactions in human foetal pancreas

In the pancreas of many mammals including humans, endocrine islet cells can be integrated with the nervous system components into neuro-insular complexes. The mechanism of the formation of such complexes is not clearly understood. The present study evaluated the interactions between the nervous system components, epithelial cells and endocrine cells in the human pancreas. Foetal pancreas, gestational age 19–23 weeks (13 cases) and 30–34 weeks (7 cases), were studied using double immunohistochemical labeling with neural markers (S100 protein and beta III tubulin), epithelial marker (cytokeratin 19 (CK19)) and antibodies to insulin and glucagon. We first analyse the structure of neuro-insular complexes using confocal microscopy and provide immunohistochemical evidences of the presence of endocrine cells within the ganglia or inside the nerve bundles. We showed that the nervous system components contact with the epithelial cells located in ducts or in clusters outside the ductal epithelium and form complexes with separate epithelial cells. We observed CK19-positive cells inside the ganglia and nerve bundles which were located separately or were integrated with the islets. Therefore, we conclude that neuro-insular complexes may forms as a result of integration between epithelial cells and nervous system components at the initial stages of islets formation.     

Phylogenetic study of serotonin-immunoreactive structures in the pancreas of various vertebrates

Summary The distribution pattern of serotonin (5HT) in the pancreas was studied immunohistochemically by using a 5HT monoclonal antibody in various vertebrates including the eel, bullfrog, South African clawed toad, turtle, chicken, mouse, rat, guinea-pig, cat, dog and human. In all species examined, except the bullfrog, 5HT-like immunoreactivity was observed in nerve fibers, in endocrine cells, or in both. Positive nerve fibers were found in the eel, turtle, mouse, rat and guinea-pig. These fibers ran mainly along the blood vessels and partly through the gap between the exocrine glands. In the eel and guinea-pig, positive fibers invaded the pancreatic islet. Occasionally, these positive fibers were found adjacent to the surface of both exocrine and endocrine cells, suggesting a regulatory role of 5HT in pancreatic function. 5HT-positive endocrine cells were observed in the pancreas of all species except for the bullfrog and rat. In the eel and in mammals such as the mouse, guinea-pig, cat, dog and human, 5HT-positive cells were mainly observed within the pancreatic islet. In the South African clawed toad, turtle and chicken, the positive cells were mainly in the exocrine region. The present study indicates that the distribution patterns of 5HT in the pancreas varies considerably among different species.     

Pancreatic islet immunoreactivity to the Reg protein INGAP.

The Reg-related protein family member INGAP (islet neogenesis-associated protein) is a pleiotropic factor enhancing islet neogenesis, neurite growth, beta-cell protection, and beta-cell function. Using an antibody to the N-termini of INGAP, we have identified that immunoreactivity to INGAP localized to the pancreatic endocrine cells in mouse. INGAP- and insulin-immunoreactive cells are mutually exclusive, with INGAP-immunoreactive cells being preserved after streptozotocin-mediated destruction of beta-cells. Glucagon- and INGAP-immunoreactive cells colocalize, although respective antigen expression occurs in different intracellular locations. These data suggest that INGAP-immunoreactive cells include alpha-cells; however, detection of single INGAP-immunoreactive/glucagon-negative cells indicates that this may not be exclusive. In addition to mouse, detection of islet endocrine cells that were INGAP immunoreactive/glucagon immunoreactive/insulin negative was also observed in islets from human, monkey, and rat. These findings reveal that INGAP and/or related group 3 Reg proteins have a conserved expression in the pancreatic islet.     

G protein-coupled receptor signaling and sphingosine-1-phosphate play a phylogenetically conserved role in endocrine pancreas morphogenesis

During development pancreatic endocrine cells migrate in a coordinated fashion. This migration is necessary to form fully functional islets, but the mechanisms involved remain unknown. Therapeutic strategies to restore β-cell mass and islet functionality by reprogramming endogenous exocrine cells would be strengthened from simultaneous treatments that enhance endocrine cell clustering. We found that endocrine progenitors respond to and regulate G protein-coupled receptor (GPCR) signaling in order to cluster in islets. Rgs4, a dedicated regulator of GPCR signaling, was specifically expressed in early epithelial endocrine progenitors of both zebrafish and mouse, and its expression in the mouse endocrine progenitors was strictly dependent upon Ngn3, the key specification gene of the endocrine lineage. Rgs4 loss of function resulted in defects in islet cell aggregation. By genetically inactivating Gα(i)-mediated GPCR signaling in endocrine progenitors, we established its role in islet cell aggregation in both mouse and zebrafish. Finally, we identified sphingosine-1-phosphate (S1P) as a ligand mediating islet cell aggregation in both species acting through distinct but closely related receptors.     

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.     

Ultrastructural and autoradiographic analysis of the cellular reactivity of the exo- and endocrine epithelium of the frog pancreas under the action of cobalt chloride]

The frog pancreatic epithelium was studied using morphometry, autoradiography and electron microscopy during the treatment with cobalt chloride (10 mg/100 g body wieght). In response to this treatment, generative changes in some exocrine cells were revealed. At the same time, those of cells, whose nuclei were in the state of DNA synthesis, increased in number. Cobalt chloride resulted in endocrine A-cells damage. A compensation of the broken endocrine A-cell function was maintained in a transformation of the exocrine epithelium into endocrine A-cells and in the appearance of intermediate acinar-islet cells, as well as in islet A-cell hyperplasia.     

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.     

Nesidioblastosis and islet cell changes related to endogenous hypergastrinemia

The endocrine pancreas from four hypergastrinemic patients with recurrent peptic ulceration has been studied by light and electron microscopy. Greatly increased numbers of ducts and centroacinar cells have been observed associated with a striking increase in the number of islets and endocrine cells scattered in the acinar tissue (nesidioblastosis). The islet cells scattered throughout the exocrine parenchyma are of all the known islet cell types, with a prevalence of B and especially A cells. Many islets, probably formed de novo, are of a considerable size, have irregular contours and are in close apposition to centroacinar cells and ducts. The degree of nesidioblastosis and islet hyperplasia does not seem to be related to the plasma gastrin levels. Cytological changes have also been found in the islet cells of the hypergastrinemic patients compared with controls. These changes mainly affect the B cells and consist of a striking decrease in the number of mature secretory granules associated with a fairly extended ergastoplasm and Golgi apparatus and with a relevant increase in the number of immature granules. In two of the four patients examined, who had more severe hypergastrinemia, cytological signs of enhanced secretion are also recognized in A cells. The features indicating hypersecretion of B and A cells seem to be related to the plasma gastrin levels. The above findings indicate that chronic endogenous hypergastrinemia promotes proliferation and differentiation of islet cells and stimulates the secretory function of B cells and, to a lesser extent, of A cells, thus providing evidence for a trophic and secretagogue action of gastrin on the endocrine pancreas.     

Neonatal pancreatic cells redifferentiate into both neural and pancreatic lineages

Studies of islet neogenesis have suggested that the regeneration of islet cells can be achieved through redifferentiation of pre-existing islet cells. However, this hypothesis is largely unproven and fails to account for the diversity of observed islet neogenesis. Here we show that cultured neonatal pancreatic cells dedifferentiate into betaIII tubulin-expressing precursors, which then expand and redifferentiate into both neural and pancreatic lineage progenies. Redifferentiation happens not only in the islet cells, but also in the ductal cells that may represent what are called ductal origin "pancreatic stem cells". The in vitro redifferentiation of neonatal pancreatic cells recapitulates the embryonic development by sequential endocrine differentiation accompanied by the coexpression of neuronal marker betaIII tubulin with endocrine hormones until terminal differentiation. The neuronal differentiation of pancreatic cells, however, occurs prior to endocrine differentiation and gradually becomes dominant, thus implying a default neuronal lineage specification for cultured pancreatic cells.     

Beta cells within single human islets originate from multiple progenitors

Background

In both humans and rodents, glucose homeostasis is controlled by micro-organs called islets of Langerhans composed of beta cells, associated with other endocrine cell types. Most of our understanding of islet cell differentiation and morphogenesis is derived from rodent developmental studies. However, little is known about human islet formation. The lack of adequate experimental models has restricted the study of human pancreatic development to the histological analysis of different stages of pancreatic development. Our objective was to develop a new experimental model to (i) transfer genes into developing human pancreatic cells and (ii) validate gene transfer by defining the clonality of developing human islets.

Methods and Findings

In this study, a unique model was developed combining ex vivo organogenesis from human fetal pancreatic tissue and cell type-specific lentivirus-mediated gene transfer. Human pancreatic progenitors were transduced with lentiviruses expressing GFP under the control of an insulin promoter and grafted to severe combined immunodeficient mice, allowing human beta cell differentiation and islet morphogenesis. By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets.

Conclusion

The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development. This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.     

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.