Glucokinase is concentrated in insulin-secretory granules of pancreatic B-cells

We immunohistochemically examined the distribution of glucokinase (GK) in the B-cells of pancreatic islets of normal rats. GK was stained punctately in the cytoplasm of B-cells when examined under the light microscope. By use of a double-immunostaining technique, most of the GK immunoreactivity was observed to be colocalized with insulin immunoreactivity. Electron microscopic examination by the immunogold method revealed that GK immunoreactivity was predominantly located within insulin-secretory granules of pancreatic B-cells. Accepted: 20 April 1999     

Insulin, glucagon and somatostatin in fetal rat islets maintained free-floating in culture: immunocytochemistry and radioimmunoassay.

Fetal rat islets maintained free-floating in tissue culture represent a source of B-cells. Because we recently noted the occurrence of other cell types during long-term tissue culture, this in vitro model was used to examine the possible development of non B-cells. The changes in the numbers and percentages of B, A and D-cells in vitro were estimated by counting the hormone-positive cells after immunocytochemical staining. Insulin, glucagon, and somatostatin contents were determined in extracts of the cultured tissue. The experiments described here showed that the cultured islets maintained their viability over a two-week culture period, as evidenced by the increase of both the number of B-cells per islet and the DNA content per islet. During the first few days of culture, immunocytochemically stained free-floating islets indicated the presence of rare A- and D-cells at the periphery of B-cells; thereafter, numerous A- and D-cells were seen interdigitating with B-cells. Expressed per islet, the number of A- and D-cells increased during the culture; within the endocrine cell population, the percentage of these cells increased with time, at the expense of the percentage of B-cells. The glucagon and somatostatin contents of the free-floating islets were also increased. These converging observations suggest that additional non B-cells may have been produced by free-floating islets during long-term tissue culture.     

Decrease in insulin-containing secretory granules and mitochondrial gene expression in mouse pancreatic islets maintained in culture following streptozotocin exposure.

We have previously described a preferential reduction in the secretory response to nutrient secretagogues in pancreatic mouse islets maintained in culture after in vitro exposure to streptozotocin (SZ). This reduction was associated with an impaired substrate metabolism at the mitochondrial level. To further clarify this issue, mouse pancreatic islets were exposed in vitro to 2.2 mM SZ for 30 min. At 4 h after SZ treatment ultrastructural changes were apparent in the endoplasmic reticulum and Golgi areas of the B-cells. However, 2 and 6 days following SZ exposure the B-cells appeared well preserved, except for a marked decrease in the number of insulin-containing secretory granules. A morphometric analysis of the B-cells 6 days after SZ exposure showed a normal B-cell size and a normal volume fraction of B-cell mitochondria. However, there was a decrease in total islet size and a 13% decrease in the volume fraction of B-cells in the islets. These mouse islets exhibited a decreased content of the mitochondrial DNA-encoded cytochrome b mRNA, as evaluated by dot-blot analysis. As a whole, the data obtained indicate that SZ treatment does not induce a decrease in the number of mitochondria or long-lasting ultrastructural damage to this organelle. However, there is a clear decrease in the cytochrome b mRNA, suggesting that SZ can induce damage to the mitochondrial DNA.     

Decrease in insulin-containing secretory granules and mitochondrial gene expression in mouse pancreatic islets maintained in culture following streptozotocin exposure

We have previously described a preferential reduction in the secretory response to nutrient secretagogues in pancreatic mouse islets maintained in culture after in vitro exposure to streptozotocin (SZ). This reduction was associated with an impaired substrate metabolism at the mitochondrial level. To further clarify this issue, mouse pancreatic islets were exposed in vitro to 2.2 mM SZ for 30 min. At 4 h after SZ treatment ultrastructural changes were apparent in the endoplasmic reticulum and Golgi areas of the B-cells. However, 2 and 6 days following SZ exposure the B-cells appeared well preserved, except for a marked decrease in the number of insulin-containing secretory granules. A morphometric analysis of the B-cells 6 days after SZ exposure showed a normal B-cell size and a normal volume fraction of B-cell mitochondria. However, there was a decrease in total islet size and a 13% decrease in the volume fraction of B-cells in the islets. These mouse islets exhibited a decreased content of the mitochondrial DNA-encoded cytochrome b mRNA, as evaluated by dot-blot analysis. As a whole, the data obtained indicate that SZ treatment does not induce a decrease in the number of mitochondria or long-lasting ultrastructural damage to this organelle. However, there is a clear decrease in the cytochrome b mRNA, suggesting that SZ can induce damage to the mitochondrial DNA.     

Influence of membrane potential changes on cytoplasmic Ca2+ concentration in an electrically excitable cell, the insulin-secreting pancreatic B-cell.

Glucose stimulation of insulin release involves metabolism of the sugar and elevation of cytoplasmic calcium (Ca2+i) in pancreatic B-cells. We compared the dynamic changes of metabolism (fluorescence of endogenous reduced pyridine nucleotides, NAD(P)H), membrane potential (intracellular microelectrodes), and Ca2+i (fura-2 technique), in intact mouse islets. Glucose (15 mM) sequentially triggered an increase in NAD(P)H fluorescence, a depolarization with electrical activity, and a rise in Ca2+i. The change in NAD(P)H was monophasic and regular, whereas the changes in membrane potential and Ca2+i were multiphasic, with steady-state regular oscillations of similar average frequencies (about 2.2/min). Digital image analysis revealed that Ca2+i oscillations were synchronous in all regions of the islets. Omission of extracellular Ca2+ abolished the rise in Ca2+i but not the increase in NAD(P)H. Both electrical and Ca2+i oscillations disappeared in low external Ca2+ (1 mM), and became larger but slower in high Ca2+ (10 mM). Sustained depolarization (by tolbutamide, arginine, or high K+) and hyperpolarization (by diazoxide) of B-cells caused sustained increases and decreases of Ca2+i, respectively. In conclusion, the changes in membrane potential induced by various secretagogues trigger synchronous changes in Ca2+i in all B-cells of the islets. The oscillatory pattern of the electrical and Ca2+i responses induced by glucose is not accompanied by and thus probably not due to similar oscillations of metabolism.     

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.     

Ultrastructural characteristics of the "light" and "dark" B-cells in the pancreatic islets of rats]

The ultrastructure of B-cells in the rat pancreatic islets has been studied under various experimental conditions (thyroidectomy, continuous thyroxine treatment, regeneration after partial pancreatectomy, thyroidectomy with partial pancreatectomy, partial pancreatectomy, partial pancreatectomy with continuous thyroxine treatment). Five types of B-cells have been distinguished. It has been supposed that "light" B-cell 1 is related to the stage of secretory granule extrusion, "light" B-cell 2 reflects the extrusion of secretory material and the early stages of secretory granule synthesis; "dark" B-cell 1 is involved in the intensive synthesis, formation and extrusion of secretory material, and "dark" B-cell 2 in the intensive secretory granule synthesis, formation and storage.     

Fine structural studies of the islets of Langerhans in the Djungarian hamster (Phodopus sungorus)

Summary A study of the pancreatic islets of Langerhans in the Djungarian hamster (Phodopus sungorus) was initiated by the observation that 98 percent of the animals of a recently established colony showed ketoacidosis soon after birth, about ten percent of which later developed persistent hyperglycemia. The islets are made up of a centrally located mass of insulin-producing B-cells surrounded by a peripheral rim of A-and D-cells. Most islets are richly supplied by unmyelinated nerve fibers which terminate on all three cell types with cholinergic synaptic endings. Early changes in islet fine structure due to ketosis comprise degranulation of A-cells combined with signs of crinophagia of -granules. After the manifestation of hyperglycemia, degranulation of B-cells is followed by deposition of glycogen which in the late phase of the diabetic syndrome forms large masses obscuring the regular cellular organelles. In six-to nine-month hyperglycemic animals degenerative changes are also observed in D-cells in the form of autophagic vacuoles.Dedicated to Professor Dr. Helmut Ferner, Vienna on the occasion of his 65th birthday     

Ultrastructural analysis of rat pancreatic islet B-cells following thyroidectomy]

Electron microscopic investigations have shown that after 5 days the pancreatic B-cells of thyroidectomized rats are characterized by marked endoplasmic reticulum dilatations, the reduction of Golgi complex, and the secretory granule extrusion into pericapillary spaces. Later (after 30 days), the insuline granule extrusion from B-cells is retarded, and an increased number of mielin figures in the cytoplasm is observed.     

Streptozotocin-induced suppression of FAD-linked glycerophosphate dehydrogenase in pancreatic islets of adult rats.

Injection of streptozotocin (30-40 mg/kg body weight) to adult rats caused within 4-6 days a sizeable decrease in the activity of FAD-linked glycerophosphate dehydrogenase in pancreatic islets, with little change in either glutamate dehydrogenase or 2-oxoglutarate dehydrogenase activity. The severity of the enzymatic defect was related to that of the diabetic state, although a decreased enzymic activity was also observed in islets from virtually normoglycemic animals examined 2-3 weeks after streptozotocin injection. The administration of nicotinamide prior to that of streptozotocin prevented the change in enzymic activity. It is proposed that the enzymatic defect, rather than being attributable to a genomic effect of streptozotocin, may reflect the preferential impairment of a subpopulation of pancreatic B-cells.     

Functional and morphological study of cultured pancreatic islets treated with cyclosporine

Cyclosporine A (CsA), a potent immunosuppressive drug, has been found to induce glucose intolerance through its toxic effect on the endocrine pancreas. It is not exactly known whether CsA has a direct effect on the endocrine pancreas or induces its effect indirectly. The present study was therefore undertaken to examine the function and morphology of isolated pancreatic islets when they are directly exposed in vitro to CsA. Pancreatic islets were isolated from adult male Lewis rats using collagenase ductal perfusion technique. The islets were separated with the discontinuous Ficoll gradient technique and further purified by hand picking of the non-islet tissue. The islets were cultured in RPMI-1640, pH 7.4 and maintained at 37 degrees C in a humid atmosphere of 5% (v/v) carbon dioxide in air. Cyclosporine was added to the culture medium to give a final concentration of 1 microg/ml (therapeutic dose), 5 microg/ml (toxic dose), or vehicle (control). Islets were harvested at 1, 4 and 10 days of culture and processed for functional or histological study. The functional study of the islets cultured with 1 microg/ml CsA showed insulin and C-peptide contents similar to those of the control islets. The islets cultured with 5 microg/ml CsA showed a marked decrease in insulin and C-peptide contents. Glucose-dependent insulin release was variable. C-peptide release was lower than that of the control following both the therapeutic and toxic doses of CsA. Phase contrast microscopy showed that the islets cultured with 1 microg/ml CsA were mostly normal looking with a well-defined regular periphery; a few islets had ill-defined or irregular peripheries. The islets cultured with 5 microg/ml CsA had ill-defined irregular peripheries at 1 day, and were dense and forming clumps at 4 and 10 days following culture. There was a decrease in the islet number following the therapeutic dose; the decrease was more following the toxic dose of CsA. The islet diameters increased after the therapeutic dose, but slightly decreased following the toxic dose of CsA. Islets showed a weakly positive immunoperoxidase reaction for insulin that was weaker following the toxic dose of CsA. It is concluded that CsA has a direct effect on B-cells that was proved by the functional and morphological changes seen in the pancreatic islets cultured in vitro.     

Effects of glucose and glucagon on the fructose 2,6-bisphosphate content of pancreatic islets and purified pancreatic B-cells. A comparison with isolated hepatocytes.

Glucose caused a sustained and dose-related increase in the fructose 2,6-bisphosphate content of isolated pancreatic islets, as well as of purified pancreatic B-cells. With isolated B-cells, the glucose saturation curve was sigmoidal and superimposable on that obtained with hepatocytes isolated from unfed rats. However, the response to glucose was notably faster in purified B-cells than in isolated hepatocytes. In contrast again with the situation prevailing in the liver, glucagon failed to decrease significantly the concentration of fructose 2,6-bisphosphate in either islets or purified B-cells. It is proposed that, in the process of glucose-stimulated insulin secretion, an early increase in fructose 2,6-bisphosphate formation may, by causing activation of 6-phosphofructo-1-kinase, allow glycolysis to keep pace with the rate of glucose phosphorylation.     

Histochemical demonstration of pancreatic B-cells by means of the colloidal iron (Hale) reaction (author's transl)]

The stainability of B-cells in islets of Langerhans by means of colloidal iron reaction has been examined using two standard modifications (Graumann resp. Mowry) of the original Hale-reaction. After previous oxidation of the sections with performic acid a strong and selective staining of B-cells was obtained by the use of a colloidal iron reaction based upon Graumann's method. With Mowry's technique B-cells remained unstained. The demonstration of B-cells using a performic acid-colloidal iron reaction has been compared with methods of known selectivity (Aldehyde Fuchsin, Dichlorpseudoisocyanine). By restaining procedures it could be shown that with the three methods the same type of cell i.e. B-cell is stained.     

Age and changes in the proportions of cells in the pancreatic islets of the human pancreas

Morphometric investigations of the pancreas have been performed in 74 persons died from different disease at the age from birth up to 85 years. In sections impregnated after Grimelius the amount of argyrophil glucagon-producing A-cells has been counted, as well as in non-impregnated sections--insulin-producing B-cells. Total mass of the islets in the pancreas have been calculated, with a special reference to A- and B-cells and their quantitative relationships. There is not any significant differences of these parameters in men and women. At the same time, a regular increase in A-cells with age is noted, which together with a slight decrease in B-cells results in certain changes in ratio between A- and B-cells. With age, these changes produce a decreased tolerance to glucose, and predominance of A-cells over B-cells results in diabetes mellitus. This is proved when the results of the morphometric investigations are compared with those of intravital studies on glucose contents in blood.     

Localisation of islet amyloid peptide in lipofuscin bodies and secretory granules of human B-cells and in islets of type-2 diabetic subjects

Summary Islet amyloid peptide (or diabetes-associated peptide), the major component of pancreatic islet amyloid found in type-2 diabetes, has been identified by electronmicroscopic immunocytochemistry in pancreatic B-cells from five non-diabetic human subjects, and in islets from five type-2 diabetic patients. The greatest density of immunoreactivity for islet amyloid peptide was found in electrondense regions of some lysosomal or lipofuscin bodies. The peptide was also localised by quantification of immunogold in the secretory granules of B-cells, and was present in cytoplasmic lamellar bodies. Acid phosphatase activity was also demonstrated in these organelles. Immunoreactivity for insulin was found in some lysosomes. These results suggest that islet amyloid peptide is a constituent of normal pancreatic B-cells, and accumulates in lipofuscin bodies where it is presumably partially degraded. In islets from type-2 diabetic subjects, amyloid fibrils and lipofuscin bodies in B-cells showed immunoreactivity for the amyloid peptide. Abnormal processing of the peptide within B-cells could lead to the formation of islet amyloid in type-2 diabetes.     

Effect of glucocorticoids on the endocrine system of the pancreas of the pigeon and rat after a food load

The level of 35S-methionine incorporation (in 15, 30 min, 1, 2, 3, 6, 12, 24 h) has been investigated in A- and B-cells of the pigeon and rat pancreatic islets against the background of excessive injection of hydrocortisone. The pigeon and rat A- and B-endocrinocytes respond in a similar was to the excess of hydrocortisone. An accelerated elimination of the isotope from the pigeon A- and B-endocrinocytes is noted, while in the rat the effect of the excessive hydrocortisone is opposite.     

Immunoreactive GABA transaminase within the pancreatic islet is localized in mitochondria of the B-cell

Subcellular localization of gamma aminobutyrate-alpha-ketoglutarate transaminase (GABA-T) in the pancreatic islets of Langerhans was determined by use of an electron microscopic, immunogold post-embedding protocol. The objective of this study was to define the islet cell distribution and subcellular localization of GABA-T. Within the islet, GABA-T was found only in the B-cells and was localized in mitochondria; 78 mitochondria contained 336 gold particles, whereas 245 secretory granules contained only 18 gold particles. Although studies utilizing either the isolated perfused pancreas or cultured islets have shown that exogenous GABA modulates D-cell secretion, in this study immunoreactive GABA-T, the catabolic enzyme for GABA, was not detectable in A- and D-cells of the islet. Control studies substituting normal rabbit serum for the GABA-T antiserum resulted in absence of labeling. These results indicate that the high concentration of GABA present in islet B-cells is catabolized by GABA-T in the mitochondrial compartment, consistent with the possibility that GABA functions as a mediator of B-cell activity.     

Increase of gap junctions between pancreatic B-cells during stimulation of insulin secretion

The development of gap junctions between pancreatic B-cells was quantitatively assessed in freeze-fracture replicas of isolated rat islets under different conditions of insulin secretion. The results show that in resting B-cells, gap junctions are small and scarce but that these junctions increase when insulin secretion is stimulated. Both a short (90 min) stimulation by glucose in vitro and a prolonged (2.5 d) stimulation by glibenclamide in vivo raise the number of gap junctions; in addition, the glibenclamide stimulation causes an increase in the size of individual gap junctions. As a consequence, the total area occupied by gap junctions on the B-cell membrane and the ratio of this area to the cell volume were found significantly increased in the latter condition. The slight increase of these values observed after the glucose stimulation did not reach significance. These data indicate a change of gap junctions during the secretory activity of the pancreatic B-cells. The possibility that the coupling of the cells is affected by the treatment is discussed.     

Pancreatic lesions of small-obese mice (C57BL/6 J-ob/ob) with hyperglucagonemia

Male Small-obese mice (Small-ob) which derived from a C57BL/6 J-ob/ob mouse colony were examined histopathologically at 13-, 39-, and over 52-week-old. C57BL/6 J-ob mice (?/+: Non-ob, ob/ob: Ob) were studied as controls. In Small-ob mice, plasma glucagon concentration was higher than that of the Ob mice (this difference was highly significant), and serum levels for insulin was within normal limits. Microscopically, hypertrophy and hyperplasia of the islets of Langerhans were found only in the pancreas of Ob mice. The increase in the number of A-cells and the decrease in the number of B-cells were revealed immunohistochemically in the islets of Small-ob mice. These changes were more severe with advance of age. In the aged Small-ob mice, perivascular and periductular cell infiltration were found, but inflammatory change of islet tissue was not confirmed in any animals examined. Diabetic symptoms in Small-ob mice seems to stem from the disparity in insulin/glucagon (I/G) ratio associated with hyperglucagonemia which result from increased number of A-cells of pancreatic islets.     

Morphological aspects on pancreatic islets of non-obese diabetic (NOD) mice

The pancreatic islets of female non-obese diabetic (NOD) mice (a model of insulin-dependent diabetes mellitus), have been examined by both light and electron microscopy. At about the age of 2 weeks, mononuclear cells began to infiltrate in or near the islets and some of these cells were in contact with the islet cells. Following this degeneration of islet B-cells took place, the process occurring in two ways. In many cells numerous secretory granules with extremely dense cores occupied the cytoplasm. Other cells, however, were filled with low-density secretory granules and the nuclei of these cells became pycnotic. After degeneration of B-cells, the islets were effaced by numerous mononuclear cells. With the onset of the diabetic state these mononuclear cells gradually disappeared, and thereafter small islets remained. By electron microscopy, retrovirus-like particles were observed in cisternae of the rough endoplasmic reticulum in islet B-cells at all stages. With an anti-retrovirus serum (goat anti-KiMSV-NIHxeno serum), positive immunofluorescence was observed in some pancreatic islet cells of NOD mice aged 1 day and 4, 6, 8, 9, 10 and 14 weeks. It is suggested that these virus particles may be intimately related to the inflammatory reaction occurring in the islets and to the development of diabetes mellitus.