Amylin in pancreatic islets and pancreatic endocrine neoplasms

Amylin is a chief constituent of the amyloid present in insulinomas, and is colocalized in beta islet cells. By immunocytochemical staining, all four islet cells including insulin, glucagon, somatostatin (SRIF) and pancreatic polypeptide (PP) cells were positively stained for amylin. The strongly insulin-positive cells corresponded with the strongly amylin-positive cells, and glucagon cells appeared to be strongly positive for amylin, whereas SRIF and PP cells were weakly positive for amylin. Among 37 cases of pancreatic endocrine neoplasms, insulinomas were more stronger stained for amylin than other islet cell tumors; however, amylin staining was the same or weaker than insulin staining. Glucagonomas and PP-omas were weakly positive for amylin, whereas six of 11 gastrinomas were weakly positive for amylin. It is concluded that three orthoendocrine tumors including insulinomas, glucagonomas and PP-omas were all positive for amylin, whereas ectopic hormone secreting gastrinomas were positive for amylin in six of 11 cases (55%). This colocalization of amylin with insulin, glucagon and PP may support a structure-function relationship of amylin and pancreatic hormones. The lesser immunoreactive amylin in pancreatic endocrine neoplasms than in normal islet cells may contribute to autonomous hypersecretion of hormones by pancreatic endocrine neoplasms.     

Pancreatic endocrine carcinoma with multiple hormone production in a raccoon (Procyon lotor)

A pancreatic endocrine carcinoma with lung metastases was found in a 15-year-old male raccoon. This tumour was characterized by great variation in cell size and production of multiple polypeptide hormones. Endocrine markers expressed by the neoplastic cells were, in descending order of frequency, chromogranin A, pancreatic polypeptide (PP), glucagon, somatostatin and insulin. Co-expression of PP and glucagon, somatostatin or insulin was demonstrated immunohistochemically with consecutive sections, and cells with both glucagon and insulin were also observed. The pattern of co-expression was similar to that in the earliest stages of murine pancreatic development. Amyloid deposits in the islets of Langerhans, which reacted with antibody to islet amyloid polypeptide, were thought to be associated with age.     

The structure and hormone content of the endocrine pancreas of the one-humped camel (Camelus dromedarius)

Little is known of the blood sugar regulation in the camel and the morphology and function of its endocrine pancreas. The present paper describes the light microscopic structure and hormone content of the endocrine pancreas of the one-humped camel. Staining of pancreatic sections with haematoxylin-eosin or aldehyde-fuchsin showed numerous islets evenly distributed in all parts of the pancreas. Immunocytochemical staining for insulin or glucagon indicated that islets were predominantly composed of centrally located B-cells, surrounded by a peripheral rim of A-cells. Corresponding stainings for somatostatin or pancreatic polypeptide (PP) demonstrated that D-cells comprised only a small part of the islet volume while PP-cells were common both within and outside the islets. There were no obvious differences between the frequency of the various islet cells in different pancreatic regions. The pancreatic hormone concentrations roughly corresponded to the frequency of the different islet cell types. Insulin appeared most abundant followed by glucagon, PP and somatostatin in decreasing order. The concentrations of each of the hormones were similar in different regions of the gland. It is concluded that the endocrine pancreas of the one-humped camel is dispersed into islets of the same size and cellular composition as has been described in many other mammalian species.     

New markers for pancreatic islets and islet cell tumors

Islets of Langerhans account for 2 g of endocrine tissue in the pancreas, comprising approximately one million islets, with each containing 1000 endocrine cells. The major hormone secreted from the islets is insulin, which regulates blood glucose, the main fuel of the body. Islets also secrete glucagon, somatostatin and pancreatic polypeptide and all are involved in the paracrine mechanism. Islet cells can be stained immunohistochemically for the general endocrine markers, chromogranin A, synaptophysin, neuron-specific enolase and Leu7. Beta islet cells are well equipped with glucose transporter 2, which binds to glucose and regulates diffusion of glucose through the beta cell membrane. As all four islet hormones are initially synthesized as prohormones, all islet cells are equipped with prohormone convertase 1/3 and 2. In addition, islet cells also contain zinc-containing matrix metalloproteinases and their inhibitors, metallothionein, cyclin-dependent kinases and insulin-like growth factors, and many more hormones, peptides and enzymes. Thus, islets not only secrete insulin and other pancreatic hormones but are a complex organ whose major function is glucose homeostasis.     

Engineering pancreatic islets

Pancreatic islets are neuroendocrine organs that control blood glucose homeostasis. The precise interplay of a heterogeneous group of cell populations (beta, alpha, delta and PP cells) results in the fine-tuned release of counterbalanced hormones (insulin, glucagon, somatostatin and pancreatic polypeptide respectively). Under the premises of detailed knowledge of the physiological basis underlying this behaviour, two lines of investigation might be inferred: generating computational and operational models to explain and predict this behaviour and engineering islet cells to reconstruct pancreatic endocrine function. Whilst the former is being fuelled by new computational strategies, giving biophysicists the possibility of modelling a system in which new "emergent" properties appear, the latter is benefiting from the useful tools and strategic knowledge achieved by molecular, cell and developmental biologists. This includes using tumour cell lines, engineering islet cell precursors, knowledge of the mechanisms of differentiation, regeneration and growth and, finally, therapeutic cloning of human tissues. Gaining deep physiological understanding of the basis governing these processes is instrumental for engineering new pancreatic islets.     

Ontogeny, postnatal development and ageing of endocrine pancreas in Bubalus bubalis

The ontogenesis, postnatal development and ageing of the endocrine pancreas in mammals have not been extensively studied. In order to improve understanding of this organ, we studied the buffalo pancreas during fetal and postnatal development. Glucagon, insulin and somatostatin immunoreactive cells (i.c.) were first seen in 2-mo-old embryos. Pancreatic polypeptide (PP) i.c. were observed during the 3rd month of gestation. The early embryo pancreas was almost totally composed of endocrine tissue. The endocrine portion only slightly increased in mass with animal growth, whereas the exocrine portion noticeably increased in mass during the late fetal and postnatal periods. In adults, therefore, the exocrine portion was more evident than the endocrine portion. Three types of islet were observed in fetal and young buffalos: small, large and PP-islets. The small islets were composed of insulin, glucagon, somatostatin and PP i.c. The large islets were primarily composed of insulin i.c. and a few glucagon, somatostatin and PP i.c. The PP islets were mostly composed of PP i.c. with a few somatostatin, insulin and glucagon i.c. The number of large islets greatly diminished by adulthood. Glucagon, insulin, somatostatin and PP i.c. were also seen scattered in the exocrine parenchyma and along the duct epithelium. In the duct epithelium, these cells were either single or grouped, and they sometimes formed a protrusion projecting towards the connective tissue. These morphological features were primarily observed in fetuses and young buffalos.     

Peptidergic hormones and neuropeptides, and aminergic neurotransmitters of the pancreatic islets of the Houbara bustard (Chlamydotis undulata)

Immunoreactivity to insulin (Ins), somatostatin (Som), glucagon (Glu) and pancreatic polypeptide (PP) was found in 70%, 22%, 15% and 11% respectively of Houbara pancreatic endocrine islet cells. Whilst Ins occurred centrally and SOM was observed both in peripherally and centrally located islets, the other hormones were localised in peripheral islet cells; Som was also observed in neuronal cell bodies and nerve fibres. In addition, the islet cells contained substance P (SP) (65%) in the centre and vasoactive intestinal polypeptide (VIP) (2%) at the periphery. Immunoreactivity to choline acetyltransferase (ChAT), VIP and galanin (Gal) occurred in the walls of blood vessels located mainly at the periphery of islets. Occasionally, VIP and Gal immunoreactive varicose nerve terminals and ChAT immunoreactive cell bodies were also observed in the centre of islets. SP neuronal cell bodies were not observed but prominent SP immunoreactive varicose terminals were discernible in capillary walls within the islets. Neuropeptide Y (NPY) immunoreactive neurons were detected in neuronal cell bodies located mainly peripherally. Neuronal nitric oxide synthase (nNOS) immunoreactivity occurred in neuronal cell bodies and nerve fibres mainly at the periphery and also in centrally located islet endocrine cells. Immunoreactivity to tyrosine hydroxylase (TH) was similar in distribution to that of ChAT. In comparison with other avian species, the islets of the dorsal pancreatic lobe of the bustard contain all the peptidergic hormones normally present in the islets of other avian species, but are not segregated into dark A and light B cells. Many of the insulin containing cells also contained SP. The islets also contained several neuropeptides which are probably involved in their regulation.     

Amylin in human pancreatic islets

AIMS: Double and triple immunocytochemical staining was used to co-localise amylin with four pancreatic hormones, including insulin, glucagon, somatostatin and pancreatic polypeptide. METHODS: Normal human adult islets were initially stained for amylin using the avidin-biotin alkaline phosphatase method. The stained sections were subsequently stained for four pancreatic hormones, respectively, with the immunoperoxidase method. RESULTS: The same islet cells that were positively stained for amylin, were also positively stained for insulin, and were positively stained for glucagon, somatostatin and pancreatic polypeptide in lesser cell numbers. CONCLUSIONS: Amylin is known to co-secrete and is co-localised with insulin and three other islet hormones are co-localised with amylin. This co-localisation of amylin with all four pancreatic hormones may support a role for amylin in secretory processes of these hormones.     

Analysis of pancreatic islet cells and hormone content in the spontaneously diabetic KKAy mouse by morphometry,immunocytochemistry and radioimmunoassay

Summary The splenic pancreas of 165 day old diabetic KKAy and age-matched nondiabetic C57BL/ 6 mice was examined by morphometry and immunocytochemistry at the light microscopic level and by radioimmunoassay to evaluate the morphology, surface area, endocrine cell composition and hormone content of the pancreatic islets. The insulin cells of the diabetic mice were severely degranulated and many of the glucagon, somatostatin and pancreatic polypeptide cells were displaced from the mantle to the core of the islet tissue where the non-insulin cells appeared to lose their continuity. The topography of some of the islets of KKAy mice was further deranged by acinar cells among the endocrine tissue. Morphometric analysis revealed that the surface area of the islets of KKAy mice was significantly expanded in comparison with that of C57BL/6 mice. The volume and numerical percents of the insulin cells were significantly increased whereas those of the glucagon and somatostatin cells were decreased in the KKAy mice. Since only the mean absolute number of insulin cells was elevated in the diabetic mice, the alteration in the relative proportions of the noninsulin cells and hypertrophy of the islets seemed to be a manifestation of insulin cell hyperplasia. Pancreatic insulin and somatostatin contents were markedly diminished in the islets of KKAy compared with those of C57BL/6 mice. These results demonstrate that the microscopic anatomy, endocrine cell populations and hormone content of the pancreatic islets are deranged in the KKAy mouse with severe hyperinsulinemia and hyperglycemia.     

A molecular inventory of human pancreatic islets: sequence analysis of 1000 cDNA clones

The islets of Langerhans play a central role in glucose homeostasisby secreting the polypeptide hormones insulin and glucagon.They are comprised primarily of four endocrine cell types: insulin-secretingß-cells which represent about 70% of the cells inthe islet along with smaller number of cells secreting glucagon,somatostatin and pancreatic polypeptide. Diabetes mellitus resultsfrom the specific loss or dysfunction of the ß-cells.Because of the central role of the islets of Langerhans in theregulation of glucose homeostasis, we are preparing a databaseof genes expressed in this tissue. One thousand cDNA clonesrandomly isolated from a human pancreatic islet library werepartially sequenced yielding 280 kilobases of sequence. Databasesearches indicated that 397 of the cDNAs represented known humangenes or human homologs of genes identified in other speciesand a further 58 sequences corresponded to expressed sequencetags identified in other tissues or cells (contamination byexocrine pancreatic tissue was estimated to be less than 10%).545 of the cDNAs were not related to any other sequences inthe databases. The islet cDNA collection provides a unique sourceof genes for genetic studies of diabetes as well as for molecularstudies of islet function in normal and diabetic states.     

Morphological changes in pancreatic islets of KATP channel-deficient mice: the involvement of KATP channels in the survival of insulin cells and the maintenance of islet architecture

The ATP-sensitive potassium channel (KATP channel) is an essential ion channel involved in glucose-induced insulin secretion. The KATP channel is composed of an inwardly rectifying potassium channel, Kir6.2, and the sulfonylurea receptor (SUR 1); in the pancreas it is reported to be shared by all endocrine cell types. A previous study by our research group showed that Kir 6.2-knockout mice lacked KATP channel activities and failed to secrete insulin in response to glucose, but displayed normal blood glucose levels and only mild impairment in glucose tolerance at younger ages. In some aged knockout mice, however, obesity and hyperglycemia were recognizable. The present study aimed to reveal morphological changes in pancreatic islets of Kir 6.2-knockout mice throughout life. At birth, there were no significant differences in the islet cell arrangement between the knockout mice and controls. At 14 postnatal weeks glucagon cells appeared in the central parts of islets, and this image became more pronounced with aging. In animals older than 50 weeks insulin cells decreased in numbers and intensity of insulin immunoreactivity; most islets in 70- and 80-week-old mice were predominantly composed of glucagon cells and peptide YY (PYY)-containing cells. Staining of serial sections and double staining of single sections from these old mice demonstrated the frequent coexpression of glucagon and PYY, which is a phenotype for the earliest progenitor cells of pancreatic endocrine cells. These findings suggest that the KATP channel is important for insulin cell survival and also regulates the differentiation of islet cells.     

Coexistence of glucagon and pancreatic polypeptide in human and rat pancreatic endocrine cells

Pancreatic islet cells containing both glucagon and pancreatic polypeptide simultaneously (glucagon/PP cells) were identified in the rat and human normal pancreas using immunocy-tochemical staining on consecutive serial sections and double-immunolabeling techniques on the same sections. Numerous glucagon/PP cells were found at the periphery of the islets in all regions of the pancreas, particularly in the rat. As a whole, these bipeptide-containing cells appeared in higher proportions than the cells secreting glucagon or PP separately. Double immunogold labeling performed on both surfaces of the thin tissue sections allowed differentiation between the glucagon/PP cells and the single-labeled glucagon or PP cells at the ultra-structural level. The pancreatic glucagon/PP cells displayed the morphological characteristics of either A cells or PP cells. Both peptides were found in the same secretory granules of the glucagon/PP cells and, in the human pancreas, the glucagon/PP cells displayed secretory granules with a dense core in which both peptides were rather concentrated. The coexistence of glucagon and PP is assumed to originate from the simultaneous expression of the two different genes in the same cell and suggests that the cellular processing through the rough en-doplasmic reticulum-Golgi apparatusgranule secretory pathway for both peptides takes place in parallel.     

Immunohistochemical localization of polypeptide hormones in pancreatic endocrine cells of a dipnoan fish, Protopterus aethiopicus.

Light microscopical immunohistochemistry was used to demonstrate the regulatory peptides present in the endocrine pancreas of Protopterus aethiopicus. The peptides studied included insulin, glucagon, pancreatic polypeptide and somatostatin. The results showed that the 4 regulatory peptides commonly detected in the mammalian endocrine pancreas were immunologically discernible in this dipnoan fish. Large amounts of insulin-immunoreactive cells, in the centre of the pancreatic islets, were surrounded by a small rim of glucagon-or pancreatic polypeptide-immunoreactive cells. In addition, adjacent sections stained with anti-glucagon and anti-pancreatic polypeptide revealed that these hormones could be found in the same cells. Somatostatin-positive cells were scattered throughout the islets. Their processes were seen to contact many different endocrine pancreatic cells, suggesting that the somatostatin-immunoreactive cells control the functions of other endocrine pancreatic cells.     

Nestin expression in pancreatic endocrine and exocrine cells of mice lacking glucagon signaling.

Nestin, a marker of neural stem cells, is also expressed by cells located in the epithelium of the pancreatic primordium and by a subpopulation of exocrine cells but not by endocrine cells. These findings raised the possibility that the pancreatic epithelium is heterogeneous and comprised of subpopulations of exocrine/nestin-positive and endocrine/nestin-negative precursor cells. We examined this issue in two mutant mouse models characterized by protracted expression of several embryonal properties in islet cells. One mutant line comprises mice lacking mature glucagon due to abrogation of proprotein convertase-2 (PC2(-/-)), responsible for the conversion of proglucagon into glucagon, while the second line consists of mice with a global deletion of the glucagon receptor (Gcgr(-/-)). We demonstrate that nestin is transiently expressed by acinar cells and by insulin and glucagon cells of islets of both lines of mice. In addition, the lack of glucagon signaling increased nestin mRNA levels in pancreas of mutant embryos and adult mice. We conclude that nestin+ cells located in the pancreatic primordium generate the cells of the endocrine and exocrine lineages. Furthermore, our results suggest that nestin expression is regulated by glucagon signaling.     

Ontogenetic appearance of immunoreactive endocrine cells in rat pancreatic islets

Summary Chronological development of immunoreactive, pancreatic endocrine cells was immunohistochemically studied in rats. The first immunoreaction occurs for glucagon on day 11.5 and for insulin on day 12.5 of gestation, respectively, in the cells located within the cap-like or tubular pancreatic primordium derived from the gut wall. Immunoreactive somatostatin cells appear first at the periphery of primitive islets on day 15.5. On day 18.5, the cells of the primitive islets obtain their definitive arrangement and the islets are now separated from the tissue of the exocrine pancreas. Decapitation or encephalectomy performed on day 16.5 embryos fails to influence the ensuing further development of endocrine pancreas. This suggests that the hypothalamus or pituitary does not play an essential role in the histogenesis of the pancreatic islets.     

Immunohistochemical localization of somatostatin and pancreatic polypeptide in smokers with chronic pancreatitis

Recent studies have demonstrated a significant role of tobacco smoking in the development of chronic pancreatitis. Although there are published papers on the effects of cigarette smoking on insulin secretion in patients, no data are available on the effects of smoking on pancreatic endocrine cells secreting somatostatin and pancreatic polypeptide. The aim of the study was to evaluate the effects of cigarette smoking on endocrine pancreatic function by immunolocalization of somatostatin and pancreatic polypeptide in the pancreas from smokers and non-smoking patients with chronic pancreatitis in comparison with healthy controls. The LSAB2-HRP technique with polyclonal antibodies was used for the immunolocalization of somatostatin and pancreatic polypeptide in histological preparations of the pancreas. The intensity of immunohistochemical reaction was calculated with digital image analysis. The study demonstrated increased numbers of somatostatin (D) secreting cells and pancreatic polypeptide (PP) cells and their altered location in pancreatic islets and parenchyma of smoking patients with chronic pancreatitis, as compared to non-smoking patients and healthy controls. Smoking patients showed significantly higher immunostaining of the hormones in the pancreas compared to non-smoking patients and healthy persons. This study indicates that smoking may play a significant role in the development of endocrine disturbances in the development of chronic pancreatitis.     

Keratinocyte growth factor induces pancreatic ductal epithelial proliferation.

Keratinocyte growth factor (KGF) causes a proliferation of pancreatic ductal epithelial cells in adult rats after daily systemic administration for 1 to 2 weeks. Even before the proliferation of intralobular ducts is histologically evident, KGF also induces proliferating cell nuclear antigen expression within the ductal epithelium of intercalated, intralobular, and interlobular ducts. KGF also causes incorporation of 5-bromodeoxyuridine in ductal epithelial cells. Epithelial cell proliferation is histologically most prominent at the level of the intralobular ducts adjacent to and within the islets of Langerhans. Pancreatic ductal proliferation is not histologically apparent in rats sacrificed 7 to 10 days after the cessation of KGF administration. The pancreatic hormones insulin, glucagon, somatostatin, and pancreatic polypeptide are normally distributed within islets that demonstrate intrainsular ductal proliferation. The proliferating ductal epithelium does not show endocrine differentiation as evidenced by the lack of immunoreactivity for pancreatic hormones. KGF is a potent in vivo mitogen for pancreatic ductal epithelial cells.     

Anatomopathologic pictures of chronic pancreatitis

Morphology of pancreas (either esocrine, either endocrine) was studied in 29 cases of surgically treated chronic pancreatitis (27 cases of chronic calcifying pancreatitis and 2 cases of chronic obstructive pancreatitis). Parenchymal sclerosis in chronic calcifying pancreatitis (CCP) which represents the goal of our study was graded as mild (10 cases), moderate (10) and severe (7). Immunoperoxidase staining (PAP method) for insulin, glucagon, somatostatin, pancreatic polipeptide (PP), vasoactive intestinal polipeptide (VIP) and gastrin, was used to investigate endocrine pancreas. Acinar sclerosis and endocrine damage were closely related. Progression of sclerosis into islet appears to follow vascular pedicles producing a fragmentation into small cell groups as final result. In all cases of moderate or severe sclerosis, A/B cell ratio was increased due to the reduction of insulin positive cells. "Adenoma-like complexes", i.e., apparent concentration of islets, resulting from the loss of the acinar component, were observed in 7 cases with moderate or severe sclerosis. Nesidioblastosis was a prominent feature in all cases but one, with a positivity for insulin in 11 cases, for glucagon in 13, for somatostatin in 6 and for PP in 17. No positivity for gastrin was observed, while VIP was detected in a few ganglia. An increased amount of PP cells in islet and budding from the ducts was noticed and their presence outside the pancreatic head was demonstrable in 4 out of the 7 distal pancreatectomy specimens. Our data confirm the secondary involvement of the endocrine pancreas in the sclerotic acinar process.