Insulin and other islet hormones (somatostatin, glucagon and PP) in the neuroendocrine system of some lower vertebrates and that of invertebrates--a minireview

Onto- and phylogenetical studies of the evolution of cells, producing regulatory peptides, belonging to the "hormone families" of insulin, somatostatin, glucagon, and PP (the pancreatic polypeptide), have shown that the islets of Langerhans in vertebrates form a substantial part of the large neuroendocrine system (NES). The NES consists of three major parts, viz. (i) neuronal cells of the central and peripheral nervous systems, (ii) disseminated cells in the mucosa of the alimentary tract (and that of other hollow organs), (iii) the parenchymal cells of the classical endocrine glands. In the NES of coelenterates no evidence of islet hormone production has been obtained, so far. In invertebrates, belonging to the protostomian evolution line, the neuronal parts of the NES predominate markedly, and in the most highly developed phyla, such as artropods and molluscs, clear-cut evidence has been obtained for the presence of cells producing members of the islet hormone families. A "brain-gut axis" for all the four islet hormones is well established in the NES of the pro-craniates, i.e. in the invertebrates of the deuterostomian evolution line. Here, the gut endocrine cells are cells of the disseminated type in the epithelium of the mucosa. A separate islet organ does not occur in the NES until the appearance of the first vertebrates, viz. the Agnatha, some 500 million years ago. Here, a grossly visible islet organ exists, free from exocrine, acinar, pancreatic parenchyma. It is a two-hormone organ with insulin and somatostatin cells only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative Aspects of Proinsulin and Insulin Structure and Biosynthesis

This review summarizes currently available information on thecomposition and structure of vertebrate insulins and proinsulins.Consideration is given to the important structural featuresof insulin and its precursor that are involved in the functionand formation of the active hormone. Studies on the biosynthesisof insulin in teleost fishes indicate the existence of largersingle chain precursor forms similar to the mammalian proinsulins.Preliminary results of experiments on insulin biosynthesis inthe hagfish (Myxine glutinosa), which has the most primitiveislet parenchyma of all vertebrates, indicate the existenceof a similar biosynthetic mechanism. The major storage productin the B-cells in all the vertebrate species studies thus faris insulin rather than proinsulin. In fishes an intracellulartryspin-like enzyme may suffice to convert proinsulin to insulin,while in mammals a more complex mechanism involving both anendopeptidase and an exopeptidase is probably required. Conversionoccurs within the Golgi apparatus and newly formed secretorygranules in the B-cells. The similarity to the higher vertebrates in the biosynthesisand molecular structure of insulin in the primitive hagfishindicates that the properties and biological role of this hormonehave remained fairly constant throughout several hundred millionyears, or that its evolution has followed the same pattern inmost extant organisms despite considerable differences in theirorigin and living conditions. A hypothesis for the evolutionof insulin and of the B-cells based on the biosynthetic mechanisminvolving proinsulin and its conversion to insulin is brieflyconsidered.     

The Avian Endocrine Pancreas

The avian endocrine pancreas is comprised of A-islets containingA₁- and A₂- cell types, and B-islets containing A₁- and B-celltypes. The function of the A₂- and B-cells is the secretionof glucagon and insulin, respectively, while that of the A₂-cellsis uncertain. The avian pancreas contains small amounts of insulin, has poorinsulinogenic potential, and releases the hormone "sluggishly"in response to high glucose load. Fasting, hormones, and/orvagal stimulation do not alter insulin release. Avian insulinis not anti-lipolytic and is poorly lipogenic in in vitro aviansystems. Both avian pancreas and plasma contain 5-10 times more glucagonthan observed in mammals; however, no studies have been reportedemploying the avian hormone. Birds are extremely sensitive tomammalian glucagon, exhibiting a rapid and marked hyperglycemia,hepatic glycogenolysis, hyperglycerolemia, and hypertriglyceridemia.The lipolytic effects of glucagon are intensified in ‘vitro’by insulin. A pancreatic polypeptide (APP) containing 36 amino acid residueshas been isolated from the avian pancreas, but not from gut,liver, proventriculus, or gizzard. APP circulates normally,fluctuates with nutritional manipulation, and is found in allavian species investigated. At high levels APP induces hepaticglycogenolysis and hypoglycerolemia. At low levels APP is apowerful "gastric" secretogogue, encouraging rapid proventricularvolume, acid, pepsin, and protein release.     

Light and Electron Microscopical Characterization of Heterophilic Granulocytes in the Intestinal Wall and Islet Parenchyma of the Hagfish,Myxine glutinosa (Cyclostomata)

Heterophilic granulocytes were studied in the blood, intestinal wall, and islet parenchyma of the Atlantic hagfish (Myxine glutinosa) by light and electron microscopical methods. The granulocytes are pseudoeosinophils and show a PAS-positive cytoplasmic reaction. Ultrastructurally, the cells contain evenly distributed pleomorphic cytoplasmic granules with the granule membrane close to the osmiophilic core. Emigrated blood granulocytes are found extra-vascularly in the submucous connective tissue, and obviously they can pass the basal lamina and migrate into the epithelium of the intestine, bile duct, and islet parenchyma. Though the staining characteristics of hagfish granulocytes are different from those of endocrine cells in the intestinal mucosa and islet parenchyma, intraepithelial granulocytes in some locations may sometimes be difficult to distinguish ultrastructurally from insulin-containing B-cells, since heterophil granules have both a size and a shape close to those of secretion granules in B-cells. However, in contrast to B-cells the granulocytes show the following ultrastructural features: a lobated nucleus with peripherally arranged electron-dense chromatin; cytoplasmic processes and often rod-like granules with no clear space between the granule membrane and core; prominent cytoplasmic vacuoles and microtubules; and sparse mitochondria and endoplasmic reticulum. Furthermore, immigrated granulocytes lack desmosomes and annulate lamellae. Some of the intraepithelial granulocytes in the mucosa show signs of disintegration and cell death. Degenerative cell processes are also described in the islet parenchyma.     

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.     

New types of islet cells in a cyclostome,Petromyzon marinus L.

Four types of acidophilic granular cells, in addition to B-cells, are identified in the islet organ of anadromous specimens of two subspecies of Petromyzon marinus by light and electron microscopy. Three of these acidophils (PI, PII and PIV-cells) occur in both the cranial and hepatic islets while a fourth type (PIII-cell) has only been found in the hepatic islet of some animals. The granules of the PI-cells stain with ponceau de xylidine, give a distinct tryptophan reaction and in ultrastructural examination show large, dense granules. The PII-cells contain unusual crystals and appear to be a non-secretory stage of the PI. The PIII-cells stain deep-red and acid fuchsin. They contain very large, dense granules and some lysosomes. PIV-cells stain selectively with phosphotungstic acid-hematoxylin and ultrastructurally, contain small, more or less dense granules. It appears that PI- and PIV-cells develop directly from B-cells, while the PIII-cells derive from PI-cells. despite their direct or indirect origin from B-cells, the PI-, PIII- and PIV-cells show characteristic features of functionally independent endocrine cells. Petromyzon marinus may be an ideal model for the understanding of phylogenetic and pathological interrelationships between islet and gastrointestinal hormones. It is clear that the interpretation of the islet organ of the cyclostomes, which has been generally considered a source of insulin only, requires a revaluation.     

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.     

Localization of calcitonin gene-related peptide and islet amyloid polypeptide in the rat and mouse pancreas

Summary It was previously demonstrated that the two chemically related peptides calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) both occur in the pancreas. We have now examined the cellular localization of CGRP and IAPP in the rat and the mouse pancreas. We found, in both the rat and the mouse pancreas, CGRP-immunoreactive nerve fibers throughout the parenchyma, including the islets, with particular association with blood vessels. CGRP-immunoreactive nerve fibers were regularly seen within the islets. In contrast, no IAPP-immunoreactive nerve fibers were demonstrated in this location. Furthermore, in rat islets, CGRP immunoreactivity was demonstrated in peripherally located cells, constituting a major subpopulation of the somatostatin cells. Such cells were lacking in the mouse islets. IAPP-like immunoreactivity was demonstrated in rat and mouse islet insulin cells, and, in the rat, also in a few non-insulin cells in the islet periphery. These cells seemed to be identical with somatostatin/CGRP-immunoreactive elements. In summary, the study shows (1) that CGRP, but not IAPP, is a pancreati neuropeptide both in the mouse and the rat; (2) that a subpopulation of rat somatostatin cells contain CGRP; (3) that mouse islet endocrine cells do not contain CGRP; (4) that insulin cells in both the rat and the mouse contain IAPP; and (5) that in the rat, a non-insulin cell population apparently composed of somatostatin cells stores immunoreactive IAPP. We conclude that CGRP is a pancreatic neuropeptide and IAPP is an islet endocrine peptide in both the rat and the mouse, whereas CGRP is an islet endocrine peptide in the rat.     

Biogenic amines in the guinea pig endocrine pancreas.

Pancreata of guinea-pigs were investigated for the presence and cellular distribution of biogenic amines. Out of the established endocrine cell types only insulin (B-) cells contained immunoreactivity for serotonin and noradrenaline. However, the B-cells' content of both amines was quite variable. Serotonin was also confined to enterochromaffin (EC-) cells. No immunoreactivity for dopamine or histamine was present in any islet cell. Treatment of guinea-pigs with Ro-4-4602 led to a marked decrease of serotonin and noradrenaline in pancreatic endocrine cells. The present findings suggest that serotonin and noradrenaline are involved in the function of the endocrine pancreas, particularly of islet B-cells.     

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.     

Cellular and subcellular expression of Golf/Gs and Gq/G11 alpha-subunits in rat pancreatic endocrine cells.

We studied the cellular and subcellular localization of Galpha-subunits in pancreas by immunocytochemistry. Golfalpha and G11alpha were specifically localized in islet insulin B-cells and glucagon A-cells, respectively. Gsalpha and Gqalpha labeling was more abundant in B-cells. The presence of Golfalpha in B-cells was confirmed by in situ hybridization. In B-cells, Golfalpha and Gsalpha were found in the Golgi apparatus, plasma membrane (PM) and, remarkably, in mature and immature insulin secretory granules, mainly at the periphery of the insulin grains. Gqalpha was detected on the rough endoplasmic reticulum (RER) near the Golgi apparatus. In A-cells, the Galpha-subunits were mostly within the glucagon granules: G11alpha gave the strongest signal, Gsalpha less strong, Gq was scarce, and Golf was practically absent. Gqalpha and Gsalpha immunoreactivity was detected in acinar cells, although it was much weaker than that in islet cells. The cell-dependent distribution of the Galpha-subunits indicates that the stimulatory pathways for pancreatic function differ in acinar and in islet B- and A-cells. Furthermore, the G-protein subunits in islet cell secretory granules might be functional and participate in granule trafficking and hormone secretion.     

Inhibition of proinsulin biosynthesis and conversion by a putative insulin mediator

The phospho-oligosaccharide (POS), presumed to act at the postreceptor level as the insulin second messenger, was recently reported to inhibit glucose-stimulated insulin release from rat pancreatic islets. In the present study, POS was also found to inhibit glucose-stimulated proinsulin biosynthesis and conversion in rat islets. By comparison with prior findings on the effects of both exogenous insulin and anti-insulin serum upon proinsulin synthesis, these results argue against the view that insulin would normally exert a negative feedback control upon the biosynthetic and secretory activities of islet B-cells.     

Histochemistry of the pancreatic islets in golden hamsterMesocricetus auratus waterhouse 1839

Summary The histological picuture of the Langerhans' islets in hamster pancreas is quite similar to that in white rat pancreas, i.e. the B-cells are located in the middle of the islet, while the A-cells in its periphery. Very often the argyrophil cells (D-cells) are located between the A- and B-cells forming a peculiar “barrier”. The histochemical studies reveal differences between the endocrine tissue and exocrine parenchyma. In general, the islet cells are richer in enzymes, as compared with the acini. The histochemical characteristic of hamster pancreas is closest to that of white rat pancreas. Like in rat, alkaline phosphomonoesterase reaction is very strong in the A-cells, while G-6-P reaction is negative. But, concerning zinc localization, there are differences between hamster and rat. Zinc reaction is very strong in the peripheral A-cells in white rat pancreas, while in hamster this reaction is much stronger in the B-cells (the reaction is negative in the A-cells). The D-cells can not be differentiated from the other endocrine pancreatic cells by means of hystochemical studies. But these studies permit certain conclusion on the possible role of the enzymes and substances investigated in cytophysiology of the islet cells.     

Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.     

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.     

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.     

Acid-Base Regulation and Temperature in Selected Invertebrates as a Function of Temperature

The pH of the hemolymph of selected invertebrates decreasesas their body temperature increases. The magnitude of this change(pH/°C) is very similar to the change of the pH of waterwith temperature (pN/°C) and suggests that these invertebrates,like poikilothermous vertebrates, regulate the pH of their extracellularfluid so that its degree of alkalinity relative to the pH ofwater remains constant. The degree of alkalinity (pH_blood-pN)varies between species, but seems to be fixed for any givenspecies. In Limulus pH-pN was essentially the same for in vivosamples, measured after the whole animal had been acclimatedto different temperatures, as it was for in vitro samples inwhich the hemolymph was cooled or warmed anaerobically, suggestingthat the CO₂ content of the extracellular fluid is constantas the temperature changes. The P_CO2 of the hemolymph is invariablylower in animals breathing water than in those breathing air.In the invertebrates, as in the vertebrates, manipulation ofP_CO2 and HCO₃- is probably the major mechanism in the regulationof the relative alkalinity of the extracellular fluid.     

Differences in glucose handling by pancreatic A- and B-cells

Glucose exerts opposite effects upon glucagon and insulin release from the endocrine pancreas. Glucose uptake and oxidation were therefore compared in purified A- and B-cells. In purified B-cells, the intracellular concentration of glucose or 3-O-methyl-D-glucose equilibrates within 2 min with the extracellular levels, and, like in intact islets, the rate of glucose oxidation displays a sigmoidal dose-response curve for glucose. In contrast, even after 5 min of incubation, the apparent distribution space of D-glucose or 3-O-methyl-D-glucose in A-cells remains much lower than the intracellular volume. In A-cells, both the rate of 3-O-methyl-D-glucose uptake and glucose oxidation proceed proportional to the hexose concentration up to 10 mM and reach saturation at higher concentrations. Addition of insulin failed to affect 3-O-methyl-D-glucose or D-glucose uptake and glucose oxidation by purified A-cells. Glucose releases 30-fold more insulin from islets than from single B-cells, but this marked difference is not associated with differences in glucose handling. The rate of glucose oxidation is virtually identical in single and reaggregated B-cells and is not altered after addition of glucagon or somatostatin. It is concluded that the dependency of glucose-induced insulin release upon the functional coordination between islet cells is not mediated through changes in glucose metabolism.