The identification of the F-cell in the dog pancreas as the pancreatic polypeptide producing cell.

The endocrine cells of the processus uncinatus in the dog pancreas were investigated with special reference to the formerly known F-cell. The F-cell was detected frequently in the periphery of pancreatic islets as well as among exocrine tissue. In both localizations the F-cell shows similar ultrastructural features. Membrane-bound irregularly shaped secretory granules of variable electron density were seen. The cell possesses all features of an endocrine polypeptide secreting cell. Using the immunofluorescence and immunoperoxidase technique in the uncinate processus of the dog, we could reveal that the anti-sera against bovine pancreatic polypeptide (BPP) reacts with the cell which is localized at the same sites as the F-cell. We therefore conclude that the pancreatic F-cell is identical to the pancreatic polypeptide-producing cell. The other endocrine cell types of the dog pancreas are glucagon-producing A-cells, insulin-producing B-cells, and somatostatin-producing D-cells, as well as serotonin-producing EC-cells which are regularly present in the dog pancreatic islets and also scattered among exocrine tissue and the duct epithelial cells.     

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.     

Innervation of the pancreas by vasoactive intestinal polypeptide (VIP) immunoreactive nerves

The vasoactive intestinal polypeptide (VIP) has been shown to exert effects on endocrine and exocrine pancreatic secretion. Immunocytochemistry reveals that VIP immunoreactive nerves occur in the porcine, canine, feline and avian pancreas. In the pancreas of pig and cat VIP nerves are abundant around non-immunoreactive nerve cell bodies of the intrapancreatic ganglia but scarce in the islets and in the exocrine parenchyma. In the dog pancreas, however, the intrapancreatic ganglia contain strongly immunoreactive VIP nerve cell bodies which give off axons that seem to heavily innervate vessels as well as endocrine and exocrine cells. We suggest that in the pig and cat the pancreatic VIP nerves mainly affect the activity of a second type of intrapancreatic neuron, whose transmitter is unknown, whereas in the dog pancreas VIP nerves directly contact their putative effector structures.     

Nestin-expressing cells in the pancreatic islets of Langerhans

The pancreatic islets of Langerhans produce several peptide hormones, predominantly the metabolically active hormones insulin and glucagon, which are critical for maintaining normal fuel homeostasis. Some evidence exists that pancreatic endocrine cells turn over at a slow rate and can regenerate in certain conditions. This could be due to the presence of pluripotent cells residing in the pancreas. Recently the intermediate filament protein nestin has been identified to be a marker for a multipotent stem cell in the central nervous system. Given the similarity between the pancreatic islets and neuronal cells, we hypothesized that stem cells expressing nestin might be present in the pancreas. Here we present evidence that a subset of cells in the pancreatic islets express the stem cell marker nestin. These cells might serve as precursors of differentiated pancreatic endocrine cells.     

Glutaminase activity is confined to the mantle of the islets of Langerhans

Glutamatergic signalling plays an important role in the coordination of hormone secretion from the endocrine pancreas. Thus, glutamate production must be a process exquisitely regulated to ensure a proper transmitter function. Recently we have reported that the endocrine pancreas co-expresses two isoforms of the protein glutaminase (GA), denoted as kidney-type (KGA) and liver-type (LGA). However, how GA activity, and therefore glutamate production, is regulated in the islets represents a critical issue that remains unresolved. Since the purification of these enzymes from rat islets is a daunting task, in order to characterize each isoform we have taken advantage of the spatial segregation of these isoenzymes in pancreas. To assist us with this goal, we have developed a new procedure that enables us to assay GA activity in situ. The assay is highly specific for GA as indicated by its dependence on glutamine and orthophosphate. Surprisingly, LGA, which is abundantly expressed by beta-cells, did not show detectable activity under the assay conditions. All the GA activity detected in pancreatic islets was attributed to KGA and was confined to the mantle of the islets. Double labelling analyses strongly suggested that alpha-cells should be regarded as the site of glutamate production in the endocrine pancreas.     

Leucyl-β-naphthylamide-splitting enzymes in the mammalian endocrine pancreas

The proteolytic capacity of the endocrine pancreas in obese-hyperglycaemic mice was evaluated by using the chromogenic substrate l-leucyl-beta-naphthylamide (LNA). The analytical sensitivity obtained with this substrate in photometric and fluorimetric assays permitted quantitative determinations of C-N-bond-splitting activity in both crude islet homogenates and electrophoretic fractions thereof. The following observations were made: (1) The rate of LNA cleavage was maximal at about pH7 in islets as well as in acinar tissue. An apparent K(m) of 4x10(-5)-6x10(-5)m was calculated for both the endocrine and exocrine pancreas. (2) The level of LNA-splitting enzyme activity was of the same magnitude in the islets as in the liver, and significantly higher in the islets than in the exocrine pancreas. Starving the animals for 7 days did not affect the enzyme activity levels. (3) Two distinct LNA-splitting enzymes could be separated from the pancreatic islets by means of disc electrophoresis, the most rapidly migrating band representing the highest activity. Though similar electrophoresis patterns were obtained with acinar tissue and liver, only one enzyme could be demonstrated in serum. The data suggest that the beta-cells, in addition to being highly specialized for the production of a specific protein, contain a comparatively high capacity for protein catabolism.     

Immunohistochemical expression of rabphilin-3A-like (Noc2) in normal and tumor tissues of human endocrine pancreas

Involvement of rabphilin-3A-like (RPH3AL), or Noc2, the potential effector of Ras-associated binding proteins Rab3A and Rab27A in the regulation of exocytotic processes in the endocrine pancreas has been demonstrated in experimental models. Noc2 expression together with other regulatory molecules of the exocytotic machinery in human tissues, however, has not been studied. We evaluated immunohistochemical expression of the key molecules of the exocytotic machinery, Noc2, Rab3A, Rab27A, and RIM2, together with the characteristic islet cell hormones, insulin and glucagon in normal and endocrine tumor tissues of human pancreas. Normal pancreatic islets were stained for all of these proteins and showed strong cytoplasmic localization. A similar pattern of strong cytoplasmic expression of these proteins was observed in the majority of endocrine tumors. By contrast, the exocrine portions of normal appearing pancreas completely lacked Rab27A staining and showed decreased expression of the proteins, Noc2, Rab3A, and RIM2. The staining pattern of Noc2 and Rab27A was similar to the staining pattern of glucagon-producing cells within the islets. The concomitant expression of Noc2 with these molecules suggests that Noc2 may serve as an effector for Rab3A and Rab27A and that it is involved in the regulation of exocytosis of the endocrine pancreas in humans.     

An immunocytochemical study of fetal cells at the maternal-placental interface using monoclonal antibodies to keratins,vimentin and desmin

Summary Thioredoxin and thioredoxin reductase (NADPH-oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) have been proposed to be involved in several thioldependent reduction-oxidation reactions in cells. Both proteins have been immunohistochemically demonstrated in the periphery of the cytoplasm and in cytoplasmic granules of acinar and islet cells in mouse pancreas. In animals fed ad libitum, the staining for thioredoxin was more intense in the exocrine acinar cells than in the islet cells, whereas that for thioredoxin reductase was more intense in the endocrine than in the exocrine pancreas. In the islets of fed mice all endocrine cell types showed about the same staining intensity for thioredoxin, while thioredoxin reductase was greatly enriched in the somatostatin-containing D cells. Starvation overnight caused an increased staining for both proteins in the acinar cells as well as in the islets. Under conditions of starvation, thioredoxin reductase, in contrast to thioredoxin, appeared to increase preferentially in the islet B cells, as compared with the D cells. Cysteamine treatment reduced the staining for somatostatin and for thioredoxin reductase in the D cells without any obvious effect on the other pancreatic cells. The results are compatible with a role for thioredoxin and thioredoxin reductase in secretion.     

Impaired differentiation of endocrine and exocrine cells of the pancreas in transgenic mouse expressing the truncated type II activin receptor

Activin A is expressed in endocrine precursor cells of the fetal pancreatic anlage. To determine the physiological significance of activins in the pancreas, a transgenic mouse line expressing the truncated type II activin receptor under the control of beta-actin promoter was developed. Histological analyses of the pancreas revealed that the pancreatic islets of the transgenic mouse were small in size and were located mainly along the pancreatic ducts. Immunoreactive insulin was detected in islets, some acinar cells, and in some epithelial cells in the duct. In addition, there were abnormal endocrine cells outside the islets. The shape and the size of the endocrine cells varied and some of them were larger than islets. These cells expressed immunoreactive insulin and glucagon. In the exocrine portion, there were morphologically abnormal exocrine cells, which did not form a typical acinar structure. The cells lacked spatial polarity characteristics of acinar cells but expressed immunoreactive amylase, which was distributed diffusely in the cytoplasm. Plasma glucose concentration was normal in the transgenic mouse before and after the administration of glucose. The insulin content of the pancreas in transgenic and normal mice was nearly identical. These results suggest that activins or related ligands regulate the differentiation of the pancreatic endocrine and exocrine cells.     

An immunocytochemical study of endocrine pancreas of snakes

Summary The pancreas from eleven species of snakes representing both advanced and primitive families has been investigated for the presence of eleven regulatory peptides reported to occur in the mammalian endocrine pancreas. Of the eleven peptides studied, insulin, pancreatic glucagon and somatostatin were present in endocrine cells within the islets of all the species investigated. The neuropeptide, vasoactive intestinal polypeptide, was located within nerve terminals innervating the islets in the Boidinae, Colubrinae, Elaphidae and Crotalidae but absent from the Natricinae investigated.No immunoreactivity was demonstrable with the antisera to substance P, met-enkephalin, C-terminal gastrin, bombesin, glicentin and gastric inhibitory polypeptide. Pancreatic polypeptide-like immunoreactivity was demonstrable only in the boid snakes and exclusively stained by a C-terminal specific antiserum.     

Nestin is expressed in vascular endothelial cells in the adult human pancreas.

In this study we examined the expression of nestin in islets, the exocrine part, and the big ducts of the adult human pancreas by immunofluorescent double staining. Two different anti-nestin antisera in combination with various pancreatic and endothelial markers were employed. Nestin-immunoreactive cells were found in islets and in the exocrine portion. All nestin-positive cells co-expressed the vascular endothelial markers PECAM-1 (CD31), endoglin (CD105), and CD34 as well as vimentin. Endocrine, acinar, and duct cells did not stain for nestin. We also demonstrated that in the area of big pancreatic ducts, nestin-positive cells represent small capillaries scattered in the connective tissue surrounding the duct epithelium and do not reside between the duct cells. We detected nestin-expressing endothelial cells located adjacent to the duct epithelium where endocrine differentiation occurs. We have shown that nestin is expressed by vascular endothelial cells in human pancreas, and therefore it is unlikely that nestin specifically marks a subpopulation of cells representing endocrine progenitors in the adult pancreas.     

Demonstration by immunofluorescence of somatostatin cells in the islets of Langerhans in 3 species of monkeys]

Indirect immunofluorescence technique with anti-somatostatin serum has allowed us to detect somatostatin containing cells in the Langerhans' islets of three species of monkeys (Macacus irus, Papio hamadryas, Cercopithecus aethiops). These cells are in close relationship with another cells, but occupy a more central position in the islets. Thus, the distribution of somatostatin-containing cells in the monkey's endocrine pancreas has not a similar pattern as in rodent's pancreas; they have a comparable topography as in the human islets but these cells are always more elongated than in the human islets. Only slight morphological differences are observed in the three species of monkeys.     

Immunocytochemical localization of muscarinic acetylcholine receptors in the rat endocrine pancreas

Summary Immunocytochemical application of the antimuscarinic acetylcholine receptor antibody M35 to pancreas tissue revealed the target areas for the parasympathetic nervous system. Immunoreactivity in the endocrine pancreas was much higher than that in the exocrine part. Moreover, the endocrine cells at the periphery of the islets of Langerhans displayed the highest level of immunoreactivity. Based on these findings in the mantle of the islets, two types of islets have been distinguished: type-I islets with intensely stained mantle cells, and type-II islets with a much lower concentration of these cells. On average, type-I islets were larger (244.8 m±6.1 SEM) than type-II islets (121.5 m±3.8 SEM). M35-immunoreactivity was present on the majority of D cells, which were characterized by their immunoreactivity to somatostatin [of 446 D cells 356 (79.8%) were M35-immunopositive]. However, only a small proportion of the intensely stained mantle cells belonged to the D cell population. Therefore, it is concluded that the majority of the intensely stained mantle cells represent glucagon-secreting A and/or pancreatic polypeptide-secreting F cells. The intensity of M35-immunoreactivity at the periphery and central core of the islets paralleled the density of cholinergic innervation, suggesting a positive correlation between the intensity of cholinergic transmission and the number of muscarinic acetylcholine receptors at the target structures. The present study further revealed some striking parallels for the muscarinic acetylcholine receptor characteristics between the (endocrine) pancreas and the central nervous system.     

Expression and Regulation of Nampt in Human Islets

Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the mammalian NAD+ biosynthesis of a salvage pathway and exists in 2 known forms, intracellular Nampt (iNampt) and a secreted form, extracellular Nampt (eNampt). eNampt can generate an intermediate product, nicotinamide mononucleotide (NMN), which has been reported to support insulin secretion in pancreatic islets. Nampt has been reported to be expressed in the pancreas but islet specific expression has not been adequately defined. The aim of this study was to characterize Nampt expression, secretion and regulation by glucose in human islets. Gene and protein expression of Nampt was assessed in human pancreatic tissue and isolated islets by qRT-PCR and immunofluorescence/confocal imaging respectively. Variable amounts of Nampt mRNA were detected in pancreatic tissue and isolated islets. Immunofluorescence staining for Nampt was found in the exocrine and endocrine tissue of fetal pancreas. However, in adulthood, Nampt expression was localized predominantly in beta cells. Isolated human islets secreted increasing amounts of eNampt in response to high glucose (20 mM) in a static glucose-stimulated insulin secretion assay (GSIS). In addition to an increase in eNampt secretion, exposure to 20 mM glucose also increased Nampt mRNA levels but not protein content. The secretion of eNampt was attenuated by the addition of membrane depolarization inhibitors, diazoxide and nifedipine. Islet-secreted eNampt showed enzymatic activity in a reaction with increasing production of NAD+/NADH over time. In summary, we show that Nampt is expressed in both exocrine and endocrine tissue early in life but in adulthood expression is localized to endocrine tissue. Enzymatically active eNampt is secreted by human islets, is regulated by glucose and requires membrane depolarization.     

Characterization of monoamine oxidase isoforms in human islets of Langerhans.

In this paper, we describe the characterization of the expression of monoamine oxidase (MAO) in whole pancreas and in isolated islets of Langerhans from human. Classical monamine oxidase activity assays reveal that both isoforms A & B are present in human pancreas. Two complementary approaches indicated that both MAO A and B are expressed in isolated islet: RT-PCR using specific primers revealed amplification products with the expected size for MAO-A and MAO-B: two peptides corresponding to MAO A (approximately 61 kDa) and B (approximately 55 kDa) were detected using a polyclonal anti MAO-A/MAO-B antiserum. Western blotting and subsequent densitometric analysis indicate that whole and endocrine pancreas express the two isoforms with different relative proportions. Islets appear to express almost twice as much MAO protein as whole pancreas, in near equal proportions of the two isoforms, whereas whole pancreas expresses more MAO-A than the B isoform. The expression of MAO A and B in islets could be the first step toward the characterization of the functional properties of these enzymes in the endocrine pancreas.     

Upregulation of the expression of tight and adherens junction-associated proteins during maturation of neonatal pancreatic islets in vitro

Cell–cell contacts mediated by intercellular junctions are crucial for proper insulin secretion in the endocrine pancreas. The biochemical composition of the intercellular junctions in this organ and the role of junctional proteins in endocrine pancreatic dysfunctions are still unclear. In this study, we investigated the expression and cellular location of junctional and cytoskeletal proteins in cultured neonatal rat pancreatic islets. Neonatal B-cells had an impaired insulin secretion compared to adult cells. Cultured neonatal islets showed a time-dependent increase in the glucose-induced secretory response. The maturation of B-cells in vitro was accompanied by upregulation of the expression of some junctional proteins in islet cells. Neonatal islets cultured for only 24 h showed a low expression and a diffuse cytoplasmic location of the tight junctional proteins occludin and ZO-1 and of the adherens junctional proteins - and -catenins, as demonstrated by immunoblotting and immunocytochemistry. Culturing islets for up to 8 days significantly increased the cell expression of these junctional proteins but not of the cytoskeletal proteins vinculin and -actinin. A translocation of ZO-1 and catenins to the cell–cell contact region, as well as a higher association of F-actin with the intercellular junction, were also observed in neonatal islets following prolonged culturing. ZO-1 and -catenin were immunolocated in the endocrine pancreas of adult rats indicating that these junctional proteins are also expressed in this organ in situ. In conclusion, endocrine pancreatic cells express several junctional proteins that are upregulated following differentiation of the endocrine pancreas in vitro.