Organ Culture of Foetal Rat Pancreas

The differentiation and growth of the foetal rat pancreas (20 days postcoitum) was studied in parabiotic organ culture with foetal adrenal tissue. In such co-cultures, characteristic pancreatic morphology was preserved and further acinar cell differentiation was fostered. Acinar cells continued to represent about 65% of the total explant volume following short-term incubation. The selective islet cell proliferation, previously observed in control pancreatic explants cultured alone, did not occur when adrenals were co-cultured. In addition, the amylase content of the incubation media and of the explanted pancreatic tissue remained high with adrenal co-culture, while the insulin content of the media and of the explanted tissue was markedly suppressed when compared to control pancreatic explants cultured alone. The effects of the adrenal in maintaining the differentiated acinar component of the pancreas and suppressing media insulin concentration diminished over extended incubation. The addition of adrenals to culture of foetal pancreatic explants after 6 days of control culture (at a time when differentiated acinar cells were not identifiable in the explant) did not result in redifferentiation of the acinar component, but did markedly depress media insulin content. Removal of adrenals after 4 days of co-culture resulted in an immediate rise in media insulin concentration and a rapid decline in pancreatic acinar mass. An adrenal-exocrine pancreatic axis is proposed and it is suggested that foetal adrenal secretions may play an important role in the development of the exocrine pancreas in vivo as well as in vitro.     

Fetal and neonatal rat pancreas in organ culture: age-related effects of corticosterone on the development of the islet cells

Fetal (18 days postcoitum) and neonatal (3-day) pancreatic explants were grown in organ culture with or without supplementation with corticosterone (0.1 micrograms/ml). After 0, 4, and 8 days of culture, the specific hormone-positive, islet cell volumes were determined by the use of immunocytochemical and morphometric methods. The insulin, glucagon, and somatostatin contents of the explants were estimated by radioimmunoassays. In the fetal explants, all of the islet cell populations increased in volume and the content of each of the hormones increased over an 8-day period of culture. Supplementation with corticosterone resulted in a restriction of the increases of the alpha and delta cell volumes and in the somatostatin content of the explants. In the neonatal explants, the volumes of the alpha and delta cells and the glucagon and somatostatin contents decreased over a 4-day culture period. The presence of corticosterone in the culture medium preserved these cells and their hormone content. Co-culture of 18-day fetal and 3-day neonatal pancreata in control medium for 8 days resulted in a significant decrease in the content of all three of the islet hormones in the fetal explants. These results suggest that a substance harmful to the islet cells is released from the degenerating acinar cells. Thus, the effects of the steroid on the islets may be mediated through its effects on the acinar tissue.     

Fetal rat pancreas in organ culture: effects of serum on the development of the endocrine cells

Eighteen-day-old rat fetal pancreas was grown in organ culture for four days on medium consisting of tissue culture Medium 199 and varying concentrations of chicken serum. The glucagon and somatostatin concentration of the explants was decreased when the serum concentration of the medium was reduced from 50 to 10%. There was a further reduction in these hormones when the explants were cultured on Medium 199 alone. Explant insulin content was reduced only when serum was omitted from the medium. A "serum factor" tripeptide was not able to substitute for this serum requirement. Heat-inactivation of the serum resulted in a significant increase in medium insulin content and an increase in both the insulin and glucagon contents of the explants. This increase in hormone content was directly correlated with increases in the beta and alpha cell volumes of the explants. There was no change in the somatostatin content or delta cell volume of the explants grown on heat-inactivated medium. It is suggested that the serum is an important component of the culture media and is apparently required in high concentration for the development of the islet cells in vitro. The islet cell types differ in their requirement for serum. The alpha and delta a higher concentration than do the beta cells.     

Fetal Rat Pancreas in Organ Culture: Effects of Serum on the Development of the Endocrine Cells

Eighteen-day-old rat fetal pancreas was grown in organ culture for four days on medium consisting of tissue culture Medium 199 and varying concentrations of chicken serum. The glucagon and somatostatin concentration of the explants was decreased when the serum concentration of the medium was reduced from 50 to 10%. There was a further reduction in these hormones when the explants were cultured on Medium 199 alone. Explant insulin content was reduced only when serum was omitted from the medium. A "serum factor" tripeptide was not able to substitute for this serum requirement. Heat-inactivation of the serum resulted in a significant increase in medium insulin content and an increase in both the insulin and glucagon contents of the explants. This increase in hormone content was directly correlated with increases in the beta and alpha cell volumes of the explants. There was no change in the somatostatin content or delta cell volume of the explants grown on heat-inactivated medium. It is suggested that the serum is an important component of the culture media and is apparently required in high concentration for the development of the islet cells in vitro. The islet cell types differ in their requirement for serum. The alpha and delta require a higher concentration than do the beta cells.     

Method for isolation of pancreatic blood vessels,their culture and coculture with islets of langerhans

The only cure available for Type 1 diabetes involves the transplantation of islets of Langerhans isolated from donor organs. However, success rates are relatively low. Disconnection from vasculature upon isolation and insufficient rate of revascularization upon transplantation are thought to be a major cause, as islet survival and function depend on extensive vascularization. Research has thus turned toward the development of pretransplantation culture techniques to enhance revascularization of islets, so far with limited success. With the aim to develop a technique to enhance islet revascularization, this work proposes a method to isolate and culture pancreas-derived blood vessels. Using a mild multistep digestion method, pancreatic blood vessels were retrieved from whole murine pancreata and cultured in collagen Type 1. After 8 days, 50% of tissue explants had formed anastomosed microvessels which extended up to 300 μm from the explant tissue and expressed endothelial cell marker CD31 but not ductal marker CK19. Cocultures with islets of Langerhans revealed survival of both tissues and insulin expression by islets up to 8 days post-embedding. Microvessels were frequently found to encapsulate islets, however no islet penetration could be detected. This study reports for the first time the isolation and culture of pancreatic blood vessels. The methods and results presented in this work provide a novel explant culture model for angiogenesis and tissue engineering research with relevance to islet biology. It opens the door for in vivo validation of the potential of these pancreatic blood vessel explants to improve islet transplantation therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2745, 2019.     

Characterization of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in pancreatic islets

Succinate dehydrogenase activities in homogenates of rat and ob/ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of alpha-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob/ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected alpha-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that alpha-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.     

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate enhances the long-term maintenance of pancreatic islet B cell differentiated function in tissue culture

A study was undertaken to examine the effects of the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, on pancreatic islet B cell differentiated function and number. Islets from 3-day-old rats were maintained in tissue culture for up to 30 days with or without the addition of the phorbol ester. Changes in the insulin content of the media and tissue were determined at 48 hr and weekly intervals, respectively, using radioimmunoassay. The 30-day cumulative output of insulin into the medium was increased almost 2-fold and the tissue insulin content was increased 4-fold by the phorbol ester. Counts of cell smears processed for the immunocytochemical localization of insulin indicated no effect on the total numbers of B cells or non-B cells in either control or treated cultures. This enhanced maintenance of islet B cell differentiated function by 12-O-tetradecanoylphorbol-13-acetate suggests that pancreatic islets may be a useful model with which to study the mechanisms of action of this and other tumor-promoters.     

Nonenzymatic isolation and culture of adult islets from atrophic pancreata of copper-deficient rats: A morphologic analysis

Summary The purpose of this study was to develop a nonenzymatic method of isolating adult islets using atrophied pancreata from copper-deficient rats and to analyze their morphologic characteristics and behavior in culture. This unusual model of isolation was studied because islets remain intact in the course of dietary copper deficiency while the acinar glandular component of the pancreas undergoes selective atrophy and lipomatosis. Small fragments containing islets were readily microdissected from atrophied glands and placed in culture. Within 24 h the fragments congealed into small irregular- to spherical-shaped masses within which the darker profile of islets could be distinguished. Within a period of 3 to 5 d, islet tissue began to bud from the lipocytic mass until by Day 7 spherical aggregates of intact islet tissue separated from the residual fragments. Subsequent to further in vitro treatment, these islets could be maintained as free viable spherical masses if periodically agitated, as attached stationary islets which developed monolayer growth if left undisturbed and as aggregated masses of islet tissue forming megaislets if combined in small groups. Grouped islets treated with actinomycin D and cycloheximide did not exhibit aggregation when incubated with these inhibitors. This suggests that megaislet formation was an active process requiring protein-RNA synthesis rather than passive clumping or aggregation that can accompany metabolically altered or dying islets undergoing cellular shedding and adhesion. Immunohistochemical localization demonstrated that insulin, glucagon, somatostatin, and pancreatic polypeptide-immunoreactive cell types were present within the islets derived from this technique. The cellular topography of these islets was not unlike that described by others for islets cultured from enzymatic isolation. This culture model may serve as a resource for mature, viable islets isolated without mechanical or enzymatic disaggregation which can have attenuating effects on islet function. This work was supported by a research grant from the Diabetes Research and Education Foundation.     

Involvement of cellular calcium in exocrine pancreatic insufficiency during streptozotocin-induced diabetes mellitus

This study investigates the effects of the islet hormones insulin (Ins), glucagon (Glu), and somatostatin (Som) with nerve stimulation (EFS) acetylcholine (ACh) and cholecytokinin-octapeptide (CCK-8) on amylase secretion and intracellular free calcium concentration [Ca(2+)](i) in the pancreas of age-matched control and diabetic rats. Either Ins, Glu or Som elicited small increases in amylase secretion from the pancreas of age-matched control animals compared to a much larger increase in amylase secretion with either EFS, ACh or CCK-8. Combining the islet hormones with either EFS, ACh or CCK-8 resulted in marked potentiation of amylase output. In the diabetic pancreas, the islet hormones had no effect on amylase secretion compared to diabetic control. Moreover, either EFS, ACh or CCK-8 evoked a much smaller increase in amylase output compared to age-matched control. In addition, the islet hormones failed to potentiate the secretory effects of either EFS, ACh or CCK-8. In fura-2 loaded acinar cells from age-matched control pancreas either Ins or Glu elicited a small increase in [Ca(2+)](i) whereas Som had no effect. Both ACh and CCK-8 evoked large increases in [Ca(2+)](i) compared to control. Combining either Ins, Glu or Som with either ACh or CCK-8 resulted in a marked elevation in [Ca(2+)](i) compared to the responses obtained with either the islet hormones, ACh or CCK-8 alone. In diabetic fura-2 loaded pancreatic acinar cells, the islet hormones had no effect on [Ca(2+)](i) compared to control and moreover, the responses were much smaller than those obtained in acinar cells from age-matched control. Both ACh and CCK-8 induced large increases in [Ca(2+)]( i) in diabetic acinar cells. However, combining the islet hormones with either ACh or CCK-8 failed to enhance [Ca(2+)](i) compared to the reponses obtained in acinar cells from age-matched control. The results suggests that [Ca(2+)](i) homeostasis is deranged during diabetes mellitus and this in turn is probably associated with reduced pancreatic amylase secretion.     

Novel expression of liver FBPase in Langerhans islets of human and rat pancreas

Several reports have indicated the absence of gluconeogenic enzymes in pancreatic islet cells. In contrast, here we demonstrate that liver fructose-1,6-bisphosphatase (FBPase) is highly expressed both in human and rat pancreas. Interestingly, pancreatic FBPase is active and functional, and is inhibited by AMP and fructose-2,6-bisphosphate (Fru-2,6-P2). These results suggest that FBPase may participate as a component of a metabolic sensing mechanism present in the pancreas. Immunolocalization analysis showed that FBPase is expressed both in human and rat Langerhans islets, specifically in beta cells. In humans, FBPase was also located in the canaliculus and acinar cells. These results indicate that FBPase coupled with phosphofructokinase (PFK) plays a crucial role in the metabolism of pancreatic islet cells. The demonstration of gluconeogenic recycling of trioses as a new metabolic signaling pathway may contribute to our understanding of the differences between the insulin secretagogues trioses, fructose, and glucose in pancreas.     

Effect of media conditioned by concanavalin A activated spleen cells on pancreatic islet cells

The effect of media conditioned by concanavalin A-activated spleen cells (C-sup) on insulin release and its islet cell cytotoxicity were studied. In a functional study, C-sup significantly inhibited both basal insulin release and glucose-stimulated insulin release. Morphologically, C-sup had a destructive effect on isolated islets after 72 h incubation. Islet cell cytotoxicity was shown by lactate dehydrogenase (LDH) release assay after 5 days incubation with C-sup in a dose-dependent manner. These results suggest that acceleration of the onset of diabetes in young diabetes prone (DP) Bio-Breeding/Worcester (BB/W) rats following the injection of C-sup may depend on the suppressive and cytotoxic effects of C-sup on pancreatic islet cells.     

The presence and actions of NPY in the canine endocrine pancreas

Immunofluorescent staining for neuropeptide Y (NPY) in canine pancreatic tissue was performed together with an evaluation of the effects of synthetic NPY on the release of insulin (IRI), glucagon (IRG) and somatostatin (SLI) from the duodenal lobe of the canine pancreas in situ. NPY-like immunoreactivity was localized in perivascular nerve fibers throughout the acinar tissue. NPY-immunoreactive fibers were also demonstrated in the islets, usually surrounding blood vessels but also occasionally in fibers associated with endocrine cells, primarily at the periphery of islets. In addition, the ganglia dispersed in the pancreatic parenchyma were densely innervated by NPY-immunoreactive fibers, and these ganglia regularly contained cell bodies staining for NPY. Direct infusion of NPY into the pancreatic artery (p.a.) produced a dose-dependent decrease of pancreatic SLI output and of pancreatic venous blood flow. Low-dose p.a. infusion of NPY (50 pmol/min) had no effect on basal IRI or IRG output or on the islet response to glucose (5-g bolus, i.v.). High-dose p.a. infusion of NPY (500 pmol/min) transiently stimulated IRI output and modestly increased IRG output. However, the comparatively sparse innervation of canine islets with NPY-like immunoreactive fibers and the relatively minor effects of large doses of synthetic NPY on pancreatic hormone release lead us to conclude that this peptide is not an important neuromodulator of islet function in the dog.     

Establishment and immunocharacterization of an immortalized pancreatic cell line derived from the H-2Kb-tsA58 transgenic mouse

Summary This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2K^b-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products α-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.     

Copper deprivation in rats induces islet hyperplasia and hepatic metaplasia in the pancreas

BACKGROUND INFORMATION: Prolonged copper deprivation in rats followed by refeeding with a normal diet has previously been used to induce the appearance of hepatocyte-like cells in the pancreas, but the effects on islet size and morphology have not been determined. RESULTS: In the present study we investigated the distribution of pancreatic alpha- and beta-cells and of hepatocytes in adult rats fed a copper-deficient diet followed by refeeding with a normal diet. Immunohistochemical staining for insulin and glucagon showed that the islets of the copper-deficient group were up to 2.4 times larger in mass compared with controls. The islets were disorganized, with alpha-cells found in multiple layers at the periphery of the islet and sometimes deep in the core. Isolated alpha- and beta-cells were also found in increased numbers in the ductular system. Copper deprivation caused almost complete ablation of the acinar cells, and refeeding induced adipogenesis, acinar regeneration and hepatocyte-like cells. Ductular proliferation and nerve hyperplasia were also present. The hepatocytes tended to be associated with islets or with ducts, rather than with residual pancreatic exocrine tissue. CONCLUSIONS: These data show that copper deficiency in rats, as well as inducing the appearance of hepatocytes, is capable of causing islet hyperplasia.     

Islet-neogenesis-associated protein enhances neurite outgrowth from DRG neurons

Islet-neogenesis-associated protein, INGAP, is a 175-amino-acid pancreatic acinar protein that stimulates pancreatic duct cell proliferation in vitro and islet neogenesis in vivo. To date, the mitogenic activity of INGAP has been identified only in nonneural tissues. The aim of this study was to examine the effects of a pentadecapeptide of INGAP (INGAP peptide), the biologically active portion of the native protein, in cultured dorsal root ganglia (DRG) explants from C57BL/6 mice. The present study provides evidence that INGAP peptide acts as a mitogen in the peripheral nervous system (PNS), and that it enhances neurite outgrowth from DRGs in vitro in a time- and dose-dependent manner. The neuritogenic action of INGAP peptide correlates with an increase in [(3)H]thymidine incorporation (P < 0.0001) and mitochondrial activity (P < 0.001). Results from these studies suggest that INGAP peptide promotes Schwann cell proliferation in the DRG which releases trophic factors that promote neurite outgrowth.     

Dietary zinc restriction promotes degeneration of the endocrine pancreas in mice

Zinc is a key component of several proteins, interacting with the pancreatic hormones insulin and amylin. The role of zinc in insulin oligomerization and crystallinity is well established, although the effects of dietary zinc restriction on both energetic metabolism and β-pancreatic hormonemia and morphology remain unexplored. Here we report the effects of dietary zinc restriction on the endocrine pancreas and metabolic phenotype of mice. Nontransgenic male Swiss mice were fed a low-zinc or control diet for 4 weeks after weanling. Growth, glycemia, insulinemia and amylinemia were lower and pancreatic islets were smaller in the intervention group despite the preserved insulin crystallinity in secretory granules. We found strong immunostaining for insulin, amylin and oligomers in apoptotic pancreatic islet. High production of β-pancreatic hormones in zinc-restricted animals counteracted the reduced islet size caused by apoptosis. These data suggest that zinc deficiency is sufficient to promote islet β-cell hormonal disruption and degeneration.     

Turnover of Acinar and Islet Cells in the Pancreas of Monosodium Glutamate‐Treated Obese Mice

Objective: Subcutaneous administrations of monosodium glutamate (MSG) to neonatal animals result in obesity and induce the toxicity on the central nervous system, and furthermore, have an effect on entero‐pancreatic hormone. The effect of MSG on the cell turnover of organs, especially the pancreas, has received little attention until now. This study was designed to examine the effect of MSG on pancreatic cell turnover by immunohistochemistry and [³H]thymidine autoradiography. Research Methods and Procedures: Male JcI‐ICR strain mice were SC injected with MSG (2 mg/g body weight daily) for 5 days after birth, received 112 repeated injections of [³H]thymidine at 6‐hour intervals for 28 days after birth, and then were killed immediately thereafter, or 30, 60, or 120 days after the last injection. Autoradiography was performed on sections immunostained for glucagon, insulin, and somatostatin. Results: After continuous labeling, most pancreatic cells were labeled, and thereafter, labeling of cells decreased in control and MSG‐treated mice. The mean grain counts of acinar cells in MSG‐treated mice decreased more slowly than those in control mice. On the other hand, those of islet cells, including glucagon, insulin, and somatostatin cells, decreased more rapidly in MSG‐treated mice than those in control mice. Discussion: Cell turnover of acinar cells was decelerated and that of islet cells including glucagon, insulin, and somatostatin cells was accelerated in MSG‐treated mice pancreas. MSG‐induced hypothalamic lesions exert the contrary influences on the cell turnover of acinar and islet cells.     

Evidence for secretion of 7B2 by A- and B-cells of hamster pancreatic islets.

7B2 is a neuroendocrine protein, and in the pancreatic islets the presence of 7B2 in A- and B-cells was immunohistochemically demonstrated. In order to examine 7B2 secretion by A- and B-cells of pancreatic islets, we prepared isolated hamster pancreatic islet cells as well as an A-cell-rich culture, and studied 7B2 secretion under certain stimulations. 7B2 was secreted by isolated hamster pancreatic islet cells. This secretion was stimulated by theophylline and arginine, but glucose had a weak effect on the 7B2 secretion. Such a response of 7B2 to the stimulations was different from that of insulin or glucagon. 7B2 secretion was also noted in the A-cell-rich culture. These results suggest that 7B2 is secreted by both A- and B-cells of the hamster pancreatic islets and its secretion is regulated under certain conditions.     

Co-expression and regulation of connexins 36 and 43 in cultured neonatal rat pancreatic islets

Fetal and neonatal pancreatic islets present a lower insulin secretory response as compared with adult islets. Prolonged culturing leads to an improvement of the glucose-induced insulin secretion response in neonatal pancreatic islets that may involve regulation of gap junction mediated cell communication. In this study, we investigated the effect of culturing neonatal islet cells for varying periods of time and with different glucose medium concentrations on the cellular expression of the endocrine pancreatic gap junction associated connexin (Cx) 36 and Cx43. We report here that the 7-d culture induced upregulation of the expression of these junctional proteins in neonatal islets in a time-dependent manner. A correlation was observed between the increased mRNA and protein expression of Cx36 and Cx43 and the increased insulin secretion following islet culturing. In addition, increasing glucose concentration within the culture medium induced a concentration-dependent enhancement of Cx36 islet expression, but not of Cx43 expression in cultured neonatal islets. In conclusion, we suggest that the regulation of gap junctional proteins by culture medium containing factors and glucose may be an important event for the maturation process of beta cells observed at in vitro conditions.