Immunocytochemical studies of the evolution of islet hormones.

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

Insulin in Invertebrates and Cyclostomes

It seems increasingly clear that insulin is a hormone that doesnot occur exclusively in vertebrates. Several independent reportsnow exist giving evidence of insulin production in the digestivetissues of both deuterostomian and protostomian invertebrates.Cells with some light-microscopical and ultrastructural characteristicsof vertebrate B-cells have also been observed. Recently, evidencehas been obtained that insulin can act as a hypoglycemic hormone,promoting glycogen synthesis, also in a protostomian invertebrate,the gastropod mollusc, Strophocheilus oblongus. The endocrine pancreas of the cyclostomes occupies a key positionin the comparative endocrinology of the islet parenchyma andin the evolution of insulin production. It may represent anevolutionary link between the presumably gut-connected dispersedinsulin-producing cells of deuterostomian invertebrates andthe pancreatic islets of gnathostomian vertebrates. This hypothesiswas supported by the fact that cells with light-microscopicaland ultrastructural similarities to the islet B-cells were observedin the bile duct mucosa of the hagfish, Myxine glutinosa. However,immunofluorescent studies with antisera against human insulinand C-peptide did not show any immunoreactive material outsidethe B-cells of the endocrine pancreas. Particular attentionwas paid to elucidate the biological significance of the largecystic cavities that are so typical for the cyclostomian isletparenchyma. The working hypothesis that they may contain storedinsulin, proinsulin (or even "proto-proinsulin") was not supportedby immunofluorescence, autoradiographic, or ultrastructuralinvestigations, nor by proinsulin assays. It is possible thathagfish islet B-cells contain zinc, despite the fact that theamino acid residue in B₁₀-position is aspartic acid insteadof histidine. The biosynthesis of hagfish insulin shows a patternsimilar to that in gnathostomes. Its rate is related to theambient temperature and at 11 C the conversion of proinsulinto insulin lasts several days.     

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.     

Gut-islet endocrinology-some evolutionary aspects

Immunological and biological studies have shown that many of the mammalian gastroenteropancreatic (GEP) hormones have counterparts in lower vertebrates. Hormonal localization in cyclostomes and fishes suggests that insulin was phylogenetically the first islet hormone, followed by somatostatin, glucagon and, last, pancreatic polypeptide (PP). Some of the GEP peptides are present in the central and peripheral nervous system of lower vertebrates as well as mammals. GEP hormone-like substances resembling insulin, somatostatin, glucagon, PP, gastrin, secretin, VIP, substance P and enkephalin also occur in protostomian invertebrates (Annelida, Arthropoda, Mollusca), particularly in their nervous system. These findings indicate that the vertebrate hormones may have originated in neural tissue before the development of the vertebrate line of evolution.     

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.     

Differential expression of neural cell adhesion molecule and cadherins in pancreatic islets, glucagonomas, and insulinomas.

The endocrine cells of the pancreas develop from the endoderm and yet display several characteristics of a neuronal phenotype. During embryonic life, ductal epithelial cells give rise to first the glugagon-producing cells (alpha-cells) and then cells that express insulin (beta-cells), somatostatin (delta-cells), and pancreatic polypeptide (PP-cells) in a sequential order. The endocrine cells are believed to arise from a stem cell with neuronal traits. The developmental lineage from a common neuron-like progenitor is evidenced by: transient coexpression of more than one cell type-specific hormone in immature cells, expression of neuronal markers during islet cell development, and the pluripotentiality of clones of insulinoma cells to develop into cells expressing other islet cell hormones. The four mature endocrine cell types assume a particular organization within the islets of Langerhans in a process where cell adhesion molecules are involved. In this study we have analyzed the expression of neural cell adhesion molecule (NCAM) and cadherin molecules in neonatal, young, and adult rat islet cells as well as in glucagonomas and insulinomas derived from a pluripotent rat islet cell tumor. Whereas primary islet cells at all ages express unsialylated NCAM and E-cadherin, as do insulinomas, the glucagonomas express the polysialylated NCAM, which is characteristic for developing neurons. The glucagonomas also lose E-cadherin expression and instead express a cadherin which is similar to N-cadherin in brain. Insulinoma cells express E-cadherin but differ from primary islet cells by expressing a second cadherin molecule, which is similar to N-cadherin. The expression of NCAM and cadherin isoforms in the glucagonoma suggest that this transformed alpha-cell type has converted to an immature phenotype with strong neuronal traits, reflecting the early palce of glucagon-producing cells in the islet cell lineage. In contrast, insulinoma cells are more islet-like in their phenotype and show less neuronal traits.     

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.     

Neuropeptide Y and Peptide YY Immunoreactivities in the Pancreas of Various Vertebrates

Ding, W.-G., H. Kimura, M. Fujimura and M. Fujimiya. Neuropeptide Y and peptide YY immunoreactivities in the pancreas of various vertebrates. Peptides 18(10) 1523–1529, 1997.—NPY-like immunoreactivity was observed in nerve fibers and endocrine cells in pancreas of all species examined except the eel, which showed no NPY innervation. The density of NPY-positive nerve fibers was higher in mammals than in the lower vertebrates. These nerve fibers were distributed throughout the parenchyma, and were particularly associated with the pancreatic duct and vascular walls. In addition, the density of NPY-positive endocrine cells was found to be higher in lower vertebrates than mammals; in descending order; eel = turtle = chicken > bullfrog > mouse = rat = human > guinea pig = dog. These NPY-positive cells in the eel and certain mammals tended to be localized throughout the islet region, whereas in the turtle and chicken they were mainly scattered in the exocrine region. PYY-immunoreactivity was only present in the pancreatic endocrine cells of all species studied, and localized similarly to NPY. Thus these two peptides may play endocrine or paracrine roles in the regulation of islet hormone secretion in various vertebrate species.     

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)     

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.     

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

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

Ontogeny of 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.     

The ghrelin cell: a novel developmentally regulated islet cell in the human pancreas

OBJECTIVES: Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), was recently identified in the stomach. Ghrelin is produced in a population of endocrine cells in the gastric mucosa, but expression in intestine, hypothalamus and testis has also been reported. Recent data indicate that ghrelin affects insulin secretion and plays a direct role in metabolic regulation and energy balance. On the basis of these findings, we decided to examine whether ghrelin is expressed in human pancreas. Specimens from fetal to adult human pancreas and stomach were studied by immunocytochemistry, for ghrelin and islet hormones, and in situ hybridisation, for ghrelin mRNA. RESULTS: We identified ghrelin expression in a separate population of islet cells in human fetal, neonatal, and adult pancreas. Pancreatic ghrelin cells were numerous from midgestation to early postnatally (10% of all endocrine cells). The cells were few, but regularly seen in adults as single cells at the islet periphery, in exocrine tissue, in ducts, and in pancreatic ganglia. Ghrelin cells did not express any of the known islet hormones. In fetuses, at midgestation, ghrelin cells in the pancreas clearly outnumbered those in the stomach. CONCLUSIONS: Ghrelin is expressed in a quite prominent endocrine cell population in human fetal pancreas, and ghrelin expression in the pancreas precedes by far that in the stomach. Pancreatic ghrelin cells remain in adult islets at lower numbers. Ghrelin is not co-expressed with any known islet hormone, and the ghrelin cells may therefore constitute a new islet cell type.     

Endocrine Aspects of Homeostasis

SYNOPSIS. Recent findings in endocrine research have greatlyincreased our understanding of the relationship between hormonesand homeostasis. The present paper reviews selected major advancesin such areas as neuropeptides, peptide biosynthesis in endocrineand neuronal cells, peptide receptors and intracellular pathwaysin target cells, "new" peptide hormones, and evolutionary considerationsof peptide hormones. Further understanding of hormone interactionsand of relationships between nervous, endocrine, and immunesystems has added to the growing complexity of the mechanismsof fine tuning and regulating our internal environment. Moreover,discovery of the same or similar peptides throughout the courseof evolution, i.e., from unicellular organisms through vertebrates,has led to a new unifying theory of intercellular communication.Endocrine aspects of homeostasis is an expanding and excitingfield of biology.     

Development of hormonal peptides and processing enzymes in the embryonic avian pancreas with special reference to co-localisation

Studies on the developing mammalian pancreas have suggested that insulin and glucagon co-exist in a transient cell population and that peptide YY (PYY) marks the earliest developing endocrine cells. We have investigated this in the embryonic avian pancreas, which is characterised by anatomical separation of insulin and glucagon islets. Moreover, we have compared the development of the endocrine cells to that of processing enzymes involved in pancreatic hormone biosynthesis. PYY-like immunoreactivity occurred in islet cells from the youngest stages examined: it increased in amount from approximately 5 days of incubation and was co-localised with glucagon and to a lesser extent with insulin. Insulin and glucagon cells were numerous: co-existence of the two peptides in the same cells was but rarely observed. From the youngest stages examined, prohormone convertase (PC) 1/3-like immunoreactivity was detected in insulin cells and PC2-, 7B2- and carboxypeptidase E-like immunoreactivity in both glucagon and insulin cells. We conclude that: (1) PYY-like immunoreactivity occurs in avian islet cells but generally in lesser amounts than in mammals at the earlier stages, (2) the paucity of cells co-expressing insulin and glucagon indicate that all avian insulin cells do not pass through a stage where they co-express glucagon and (3) the early expression of the enzymes responsible for the processing of prohormones suggests that this process is initiated soon after islet cells first differentiate.     

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.     

Microvascular development: learning from pancreatic islets

Microvascular development is determined by the interplay between tissue cells and microvascular endothelial cells. Because the pancreatic islet is an organ composed mainly of endothelial and endocrine cells, it represents a good model tissue for studying microvascular development in the context of a tissue. In this review, we will describe the special morphology of islet capillaries and its role in the physiologic function of islets: secretion of insulin in response to blood glucose levels. We will speculate on how islet-secreted VEGF-A generates a permeable endothelium that allows insulin to pass quickly into the blood stream. In addition, we speculate on how endothelial cells might form a capillary lumen within the islets. At the end, we look at the islet microvasculature from a medical point of view, thus describing its critical role during type I diabetes and islet transplantation.