Isolation and properties of secretory granules from rat islets of Langerhans. II. Ultrastructure of the beta granule.

Beta granules isolated from rat islets of Langerhans and subjected only to phosphotungstic acid had, in negatively stained images, a 50-A periodicity. This periodicity was also observed in thin-section profiles of beta granules in intact cells. In shadowed preparations, the granules were spherical in shape and had irregular edges and surface structure. The presence of such a periodicity in the beta granule indicates that its matrix may be composed of a crystalline material.

Isolating the elusive islet.

The lack of a technique that allows mass isolation of intact, viable human islets is part of the reason that islet transplantation has not become available to the human diabetic. This report outlines the history of islet isolation and presents two new technical modifications that have been developed in the dog. Many of the current problems in islet isolation are presented, including the difficulty in obtaining enough human pancreatic tissue with minimal warm-ischemia time; inadequate distention of the pancreas to provide sufficient disruption for maximal enzymatic reaction to release intact islets; inefficient chopping methods; the use of collagenase of variable composition; different digestion methods for obtaining isolated islets; and inefficient methods for separating and purifying the islets from the ductal, acinar, and fibrous components. The first new modification involves distention of the dog pancreas through the venous system of the gland rather than the ductal system. This results in improved intralobular disruption, which improved the yield of isolated dog islets by permitting more efficient collagenase digestion. The second new modification eliminates the concept of isolating intact islets: the dog pancreas is digested by trypsin to a single-cell preparation that is partially purified by Ficoll gradients; further purification of the endocrine cells results from selective aggregation using rotational culture. This process produces pseudoislets that contain all the islet cell types and can be kept in culture for up to 4 wk, releasing their hormones in response to appropriate stimuli. These modifications may assist in the struggle to isolate the elusive human islet for safe and effective islet transplantation in the diabetic patient.

Regional distribution and concentration of pancreatic polypeptide in the human and canine pancreas.

The regional concentrations of pancreatic polypeptide (PP), insulin, and glucagon and the cellular distribution of PP were studied in 13 human and nine canine pancreases by radioimmunoassay, immunoperoxidase localization, and cell quantitation. PP concentration was highest in both the uncinate process and the head of the human pancreas and in the right lobe of the canine pancreas. In contrast, glucagon and insulin levels were higher in the body and tail of both the human and canine pancreases. Human F-cells, which contain PP, were located primarily at the periphery of the islets, although a few F-cells were scattered throughout the ducts and acini. Canine F-cells were located in ducts, acini, and islets; the relative proportion of canine F-cells in the endocrine and exocrine tissues differed according to location. Cellular quantitation of F-cells in both species correlated significantly with the tissue concentration of PP in all regions studied, validating the use of morphometric techniques to quantitate the regional distribution of PP.

Pancreatic perinuclear inclusions in diabetes mellitus and other diseases.

Unusual perinuclear inclusions in pancreatic duct cells were observed by electron microscopy in 23 diabetic pancreases, in nine pancreases with islet hyperplasia, and in 10 of 27 pancreases without islet pathology. The origin and significance of the perinuclear inclusions could not be determined, but it is suggested that they may be of viral origin or may represent a cellular response to an unknown stimulus.

Neonatal pig pseudo-islets. A product of selective aggregation.

Pseudo-islets hve been formed form single cell preparations of neonatal porcine pancreas by selective aggregation using the gyrotational tissue culture method for 7 days. Small, distinct aggregates present on the second day of culture gradually enlarged by the seventh day and at that time were taken off for assessment of secreting capabilities and cell type identification. Provocative stimulation and immunocytochemical examination confirmed viable, well-preserved pseudo-islets which contain all four islet cell types. Neonatal pig pseudo-islet formation will allow up to pursue the suitability and growth potential of this source of islet tissue under appropriate immunoaltered conditions as xenograft source of future transplantation studies.

Gastrin in the perinatal rat pancreas and gastric antrum: immunofluorescence localization of pancreatic gastrin cells and gastrin secretion in monolayer cell cultures.

The presence and development of immunoreactive gastrin (IRGa) in the fetal and neonatal pancreas and pyloric antrum of the rat were studied. IRGa appeared in both organs at least as early as the 16th day of fetal life. Antral IRGa increased rapidly and continuously in the neonatal period, while pancreatic IRGa concentration increased and was maintained at a relatively constant level from days 5 to 35. Monolayer cell cultures of the neonatal rat pancreas were used to evaluate the role of cyclic AMP mediated release of gastrin. The addition of N6,O2'-dibutyryl cyclic AMP (4 mM) or theophylline (4 mM) to the culture medium induced significant release of gastrin. The stimulation of adenylate cyclase with cholera toxin (10 ng/ml) also resulted in significant gastrin release. Long-term cultures (18-24 days) were shown to release gastrin continuously at a relatively constant rate. The cellular localization of pancreatic gastrin in 7-day-old cultures was performed by immunological techniques, using fluorescein-labeled antibodies to gastrin. The gastrin-containing cells were located at the periphery of most of the endocrine cell clusters. Immunofluorescence techniques for insulin and glucagon also showed that the alpha cells had a similar peripheral distribution, although they were more frequent in number. In contrast, insulin-containing cells were numerous and were present in all areas of the endocrine cell clusters. The studies support the following conclusions: a) Gastrin is present in the rat pancreas, even as early as late fetal life; b) Gastrin-producing cells are present and functionally competent in monolayer cell cultures of the neonatal rat pancreas for prolonged periods of time (24 days); c) Gastrin is released from these cells when intracellular levels of cyclic AMP are increased; d) By immunofluorescence methods, the gastrin-producing cells in pancreatic cell cultures are found to be located at the periphery of the endocrine cell clusters.

Acromegaly and Cushing's syndrome associated with a foregut carcinoid tumor.

We report an 18-yr-old youth with a metastatic foregut carcinoid tumor, Cushing's syndrome, and hypersomatotropic gigantism. Administration of cyproheptadine caused a dramatic fall in urinary cortisol excretion and plasma ACTH levels associated with clinical remission of the Cushing's syndrome. GH secretion was not affected by cyproheptadine administration. Ectopic ACTH secretion was confirmed by RIA of tumor extracts and immunohistochemical demonstration of ACTH-containing cells in hepatic metastases. There were two sources of GH production demonstrated in this patient. Ectopic secretion of GH by the carcinoid hepatic metastases was documented by both RIA and immunohistochemical techniques. A somatotrophic pituitary tumor was also present. The histological characteristics of this tumor suggest adenomatous hyperplasia rather than de novo neoplastic change as the likely mechanism of its pathogenesis. GH releasing factor-like activity was demonstrated in extracts of plasma and in extracts of the carcinoid tumor. We conclude that cyproheptadine exerted an effect on the ectopic ACTH-producing cells but not on the ectopic GH-producing cells or on adenohypophyseal GH secretion. Production of a GH releasing factor-like activity by the carcinoid tumor may have caused the pituitary somatotrophic tumor.

Retrospective study of 77 pancreatic endocrine tumors using the immunoperoxidase method.

Immunocytochemical stains for various pancreatic hormones were performed on 77 pancreatic endocrine tumors from 59 patients [17 with hypoglycemia, three with glucagonoma syndrome, 18 were Zollinger-Ellison syndrome, six with WDHA (watery, diarrhea, hypokalemia, and achlorhydria) syndrome and 15 without endocrine symptoms]. In all tumors that caused either hypoglycemia or glucagonoma syndrome, insulin and glucagon were respectively identified. On the other hand, only 10 tumors from 18 patients with Zollinger-Ellison syndrome were positive for gastrin, and only four of six patients with WDHA syndrome had a vasoactive intestinal peptides-positive tumor. Ten of 15 clinically silent tumors contained hormone-producing cells but without a consistent pattern. Ten neoplasms were negative for all hormones tested. Twenty-six tumors showed positively for more than one hormone and usually one cell type predominated. Four patients had multiple tumors which showed variation in the architecture and cellular composition. The tumors were classified into three major histopathologic groups: solid, gyriform, and glandular. The correlation between the pattern of growth and the hormonal production was generally poor. However, a pure gyriform pattern was often associated with insulin production, and glandular differentiation was commonly seen in tumors associated with Zollinger-Ellison syndrome. This study demonstrates the reliability of the immunocytochemical method for the specific identification of cell types in pancreatic endocrine tumors.

Ultrastructural localization of pancreatic polypeptide in the F cell of the dog pancreas.

The F cell of the dog pancreas has been identified as the specific cell type containing pancreatic polypeptide. This localization of pnacreatic polypeptide was accomplished by immunocytochemical staining of ultrathin sections and direct electron microscopic identification. Verification of the specificity of the reaction was obtained by blocking experiments on serial sections of the same cell. It is proposed that the name F cell be used for defining in all species the islet cell that contains pancreatic polypeptide.

Elevated levels of pancreatic polypeptide in obese-hyperglycemic mice.

The concentration and distribution of pancreatic polypeptide (PP) was studied in hyperglycemic obese (ob/ob) mice and homozygous (+/+) controls. By radioimmunoassay, the concentration of PP in the pancreas of ob/ob mice was greater both in the duodenal lobe (70.7%) and in the body and tail (66.3%) when compared to identical areas of control pancreases. Analysis of immunostained pancreatic tissue indicated that the number of PP cells per islet cross-section was significantly increased (58%) in the ob/ob tissue. The calculated total number of PP cells per average islet was elevated three times in the ob/ob islet. However, the proportion PP cell volume relative to the islet volume in ob/ob was one-half that of control islets. These results clearly demonstrate an elevation in both the number of PP cells per islet and the concentration of PP in the diabetic ob/ob murine pancreas.

Enhanced preservation of endosecretory granules in PP-cells of the canine pancreas.

Standard fixation techniques commonly used for light and electron microscopic studies have resulted in reported differences in the ultrastructural appearance of endosecretory granules of the pancreatic polypeptide (PP) cell. To clarify these differences, canine pancreatic tissues of intact and cultured pseudoislets were studied using a variety of ingredients, additives and fixatives in an effort to better preserve the endosecretory granules of PP cells. Results show that preservation of PP granules is enhanced by addition in zinc chloride (0.5%) to a glutaraldehyde-paraformaldehyde fixative in 0.1 M cacodylate buffer, followed by osmium tetroxide fixation. This fixative is recommended for all light and electron microscopic studies of the pancreatic polypeptide cell.

A new in vitro model for studies of pancreatic polypeptide secretion and biochemistry.

A new model tissue (pseudoislet) is described for studies of pancreatic polypeptide (PP) secretion and biochemistry. It consists of islet-like aggregates of canine pancreatic endocrine cells which are formed and maintained on tissue culture. Immunocytochemical staining revealed that pseudoislets prepared from the duodenal end of the pancreas contained a predominance (40-60%) of F cells (the PP secreting cell). Also present were 10-25% exocrine cells and an equal proportion of A, B and D cells. Several studies were conducted to characterize the pseudoislets' capacity to secrete PP. Basal rates of PP release and the concentration of PP per pseudoislet remained constant during four weeks of culture. Stimulation at weekly intervals by carbachol (0.1 mM) resulted in a stable secretory rate for 2 weeks, that declined progressively at weeks 3 and 4. When studied in a perfusion system, carbachol-stimulated PP release occurred in a biphasic pattern, similar to the well-recognized biphasic release of insulin from perifused rat islets. Dose-response curves of four cholinergic agonists revealed clear differences in secretagogue activity. Acetylcholine and methacholine were found to be equipotent, followed in order of potency by carbachol and bethanechol. These histologic and secretory data show that canine pseudoislets are healthy tissues composed of a high proportion of F cells which secrete PP in response to cholinergic stimulation. The data suggest that the cultured canine pseudoislet model provides an excellent system useful in studies of PP secretion and biosynthesis.

Psammoma bodies in endocrine tumors of the gastroenteropancreatic axis: a rather common occurrence.

Seventeen of 204 cases of gastroenteropancreatic tumors contained psammoma bodies (8%). Of these 17, ten were B cell tumors, representing 33% of the B cell tumors studied. Three tumors of the appendix and four tumors of the small bowel also had psammoma bodies. Electron microscopic studies revealed that the bodies appeared to have two possible sites of origin: intracellular (type I) and intraluminal from secretory products of the tumor cells (type II). Awareness by surgical pathologists of the fact that variable degrees of calcification in endocrine tumors of the gastroenteropancreatic system is a rather common occurrence may be important, especially in frozen section diagnosis.

The juvenile human endocrine pancreas: normal v idiopathic hyperinsulinemic hypoglycemia.

Subtotal pancreatectomy specimens from 11 pediatric patients with idiopathic hyperinsulinemic hypoglycemia (IHH) were studied by conventional light and electron microscopic methods and by morphometric methods applied to sections immunostained specifically for A, B, D, and PP cells. The results were compared with corresponding studies of pancreata obtained at autopsy of 31 infants and children without abnormalities in carbohydrate homeostasis. In the control tissue, the total volume density of islet cells in live born premature infants (n = 12) was about 20%, in live born term newborn infants (0 to 1 months, n = 9) between 17.5% and 20%, in infants (1 to 7 months, n = 5) about 10%, and in children (1.5 to 11 years, n = 5) about 7.5%. Endocrine tissue was as abundant in the body and head as in the tail of the pancreata. The contribution of PP cells to total islet cell mass increased with age, and for the relative contribution of PP cell compared with total pancreatic parenchyma it remained relatively constant, while that of A, B, and D cells decreased with age. A wide spectrum of islet cell aggregates was a normal feature of development in the control tissue, an observation accentuated by specific immunocytochemical staining. Islet cells of all types were present singly and in small clusters in pancreatic ductal structures and intimately related to acini; nesidioblastosis, therefore, is a feature of normal maturation of the pancreas. Seven of the 11 cases of IHH had pancreata that were morphologically and morphometrically normal for age. No anatomic basis for hyperinsulinism in these cases was apparent. Four pancreata from patients with IHH contained discrete foci of proliferation of islet cells of all types but in which B cells greatly predominated. We conclude that nesidioblastosis as a morphologic diagnosis cannot be viewed as the structural basis of endocrine dysfunction since it can be absent in IHH, or many of its features present in control pancreata.

Studies on gastrin secretion in vitro from cultures of rat pyloric antrum: effects of agents modifying the microtubular-microfilament system.

The purpose of the present study was to examine the effects of microtubular-microfilament (MT-MF) modifying agents on gastrin release from antral mucosal cells maintained for 3 days under tissue culture conditions. Gastrin levels in the media, determined by radioimmunoassay, were monitored at hourly intervals for 6-8-hr periods. Colchicine, 1 mM, produced mild depression in media gastrin levels and completely inhibited dcAMP-theophylline-induced gastrin release. Ethyl alcohol depressed secretion of gastrin. Conversely, colchicine at 0.01 mM, significantly increased levels of gastrin. Gastrin levels were also significantly increased by tetracaine, 1 mM, and by deuterium oxide, 75% and 37.5%. Cytochalasin D had little effect on resting gastrin levels. Results of these experiments indicate response of gastrin-producing cells maintained under these conditions to a number of test agents; that the secretory responses of gastrin cells to the MT-MF modifying agents are similar to those of other endocrine cells in which concommitant changes in the MT-MF system have been studied, providing support for the proposal that this system is involved in the sequence of intracellular events which leads to gastrin secretion.

Studies of the functional and morphologic status of islets maintained at 24 C for four weeks in vitro.

Isolated rat islets were maintained in vitro at 24 C for 1-4 weeks in tissue culture medium containing D-glucose (1.5 mg/ml). The rate of insulin release at 24 C remained stable for three weeks (2.2 muU/islet/hr) and decreased to 1.2 muU/islet/hr during the fourth week. Increasing the temperature from 24 C to 37 C at the end of 1, 2, 3, or 4 weeks produced a 5--7-fold increase in the rate of insulin release in the presence of glucose (1.5 mg/ml). This rate of secretion was comparable to control islets maintained at 37 C for 1--4 weeks. Light- and electron-microscopic studies revealed minimal central necrosis of large islets maintained at 24 C for 3 weeks. In contrast, extensive central necrosis was present in large islets maintained at 37 C for only 1 week. Degranulation of B cells occurred at 24 C with almost complete degranulation at 28 days. Regranulation occurred when the temperature was increased to 37 C. These findings indicate that isolated islets maintained at 24 C remain functionally and morphologically intact for 4 weeks. Initial studies have shown that maintenance of islets at 24 C for 1 week in conjunction with a single injection of antilymphocyte serum will produce marked prolongation of survival of islet allografts. The finding that isolated islets will survive for prolonged periods of time at 24 C should be of importance to future studies on islet transplatation, immune rejection, and investigations on hormonal release from islets maintained under these conditions.