An investigation of aldehyde fuchsin staining of unoxidized insulin

Aldehyde fuchsin is a standard stain for the secretion granules of pancreatic B cells. The participation of either insulin or proinsulin in aldehyde fuchsin staining is in dispute. There is some evidence that permanganate oxidized insulin is stained by aldehyde fuchsin. Aldehyde fuchsin staining of unoxidized insulin has not been investigated adequately despite excellent staining results with tissue sections. Unoxidized insulin and proinsulin suspended by electrophoresis in polyacrylamide gels were fixed with Bouin's fluid and placed in aldehyde fuchsin for one hour. Because the unoxidized proteins were not stained by aldehyde fuchsin, it was concluded that neither insulin or proinsulin are responsible for the intense aldehyde fuchsin staining of unoxidized pancreatic B cell granules in tissue sections. A series of controlled experiments was undertaken to test the effects of fixatives, oxidation and destaining procedures on aldehyde fuchsin staining of insulin, proinsulin and other proteins immobilized in polyacrylamide gels. It was demonstrated that only oxidized proteins were stained by aldehyde fuchsin and that cystine content of the proteins had no apparent relation to aldehyde fuchsin staining. It was concluded that neither insulin nor proinsulin is likely to be responsible for the intense aldehyde fuchsin staining of unoxidized pancreatic B cell granules in tissue sections.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cysteine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Summary Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cystine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

The ultrastructure of the islets of Langerhans in normal and obese-hyperglycemic mice

Summary The islets of Langerhans in normal and obese-hyperglycemic mice were studied by electron microscopy. Two different types of islet cells with ultrastructural features well distinguished from the majority of B cells were observed. While one of them was provided with complete cell membranes, the other appeared to be arranged in a syncytium. It was postulated that these islet cells may be identified with the argyrophil A₁ cells and the non argyrophil A₂ cells demonstrated in different species with light microscopy. In the normal mouse the fine structure of the islet B cells corresponds in the main to what has been observed previously in the rat. However, in addition to the great majority of spherical B cell granules some appear rectangular, i.e. show similar morphological characteristics as described for the granules in the dog. The long termada ptation of the B cells to increased functional demands in the obese-hyperglycemic syndrome was associated with a pronounced degranulation with margination of the granules and with obvious changes of some organelles. The mechanisms for formation and liberation of insulin from the B cells are discussed in the light of the ultrastructural appearance of these cells in the obese-hyperglycemic mice.Supported by the Swedish Medical Research Council and the research grant A-5759 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.     

The demonstration of B cells in pancreatic islets with Orcein

Summary Orcein stains granules in pancreatic islet cells selectively. The localization of Orcein-positive cells within islets differs from that of Grimelius-stained cells, but corresponds to the B cell type differentiated by Aldehyde Fuchsin.Usually there appear to be fewer Orcein-positive cells than Aldehyde Fuchsin-positive ones. This indicates either that Aldehyde Fuchsin is a more sensitive stain for B cells or that Orcein is a more selective stain for a B cell type subpopulation. The rationale of the Orcein reaction in B cells seems to depend on the oxidation of disulphide bonds present in insulin and its precursors rich in cystine.     

Exocyst Sec5 Regulates Exocytosis of Newcomer Insulin Granules Underlying Biphasic Insulin Secretion

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca²⁺ channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.     

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.     

An immunocytochemical study of the endocrine pancreas in the Australian fat-tailed dunnart (Sminthopsis crassicaudata)

Summary The endocrine pancreas of the Australian fattailed dunnart, Sminthopsis crassicaudata, was investigated by means of electron-microscopic immunocytochemistry using the protein A-gold technique on London resin (LR) white-embedded tissue. The primary antibodies used were raised against insulin, glucagon, somatostatin and pancreatic polypeptide. The morphology of the secretory granules differed in the four cell types. The insulin cells are pleomorphic, and the secretory granules composed of an electron-dense core surrounded by an electron-lucen halo. The glucago cells possess granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells have large, less dense secretory granules. The pancreatic polypeptide cells show small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborated by immunocytochemical data at the light-or preferably electron-microscopic level. Recent developments in immuno-electron-microscopic techniques have contributed to a better knowledge of cells responsible for the secretion of a wide variety of hormones, as in this study.     

Electron-microscopic immunocytochemical study of the endocrine pancreas of sea bass (Dicentrarchus labrax)

Insulin (B)-, somatostatin 25 (SST-25) (D1)-, somatostatin 14 (SST-14) (D2)-, glucagon (A)-, and glucagon PP/PYY/NPY (PP-like)-immunoreactive cells in islets of sea bass (Dicentrarchus labrax) were characterized according to their ultrastructure and immunogold labeling. Cells labeled with antisera to bonito and salmon insulin had numerous secretory granules with a small halo and round core, and a few with wide halo and round or crystalloid core. Gold particles were found throughout the granule in tissue labeled with the former but only in the core in tissue labeled with the latter. D1 cells had large granules with a medium electron-dense content and some with a darker core. D2 had smaller medium or high electron-dense secretory granules than D1 cells, located mainly in cell periphery. Glucagon-immunoreactive cells contained some granules with a polygonal core that was heavily labeled and other granules with a round core with no or hardly any labeling. Glucagon and PP-like immunoreactivity were co-localized in secretory granules, in which the gold particles showed no different distribution with the various antisera used. PYY-immunoreactive granules were also found in nerve endings. All the pancreatic endocrine cell types showing involutive characteristics are found.     

Glucocorticoid-induced changes in insulin secretion related to the metabolism and ultrastructure of pancreatic islets

The effect of adrenalectomy and dexamethasone-treatment on insulin secretion was studied and related to the changes observed in the glucose oxidation, calcium uptake, cAMP and insulin content, as well as the ultrastructure of pancreatic rat islets. It was found that adrenalectomy was followed by a decreased glucose-induced insulin secretion, glucose oxidation, calcium uptake, cAMP and insulin content without any remarkable change observed at the ultrastructural level. Conversely, adrenalectomized-rats supplemented with dexamethasone showed an increased glucose-induced insulin secretion, glucose oxidation, calcium uptake and cAMP content but a diminished islet insulin content. At the ultrastructural level, a clear picture of increased secretory activity was found, with diminished number of mature B granules and greater number of pale granules, while rough endoplasmic reticulum and Golgi complex frequently appeared hypertrophic. These changes were only observed in the B cells. On account of our results, we might suggest that insulin secretion is partially controlled by glucocorticoid circulating levels throughout their effect on pancreatic islet metabolism.     

A morphometric study of the secretory process in the endocrine pancreas of the foetal rat

The present study concerns pancreatic beta cells from rat foetus at 18, 19 and 21 days of gestation. On micrographs, the cytoplasm of beta cells was subdivided into 3 zones: one zone corresponding roughly to the cell web, a second zone just underlying the cell web, and a third zone comprising the remaining cytoplasm. The secretory granules present in each zone were counted; in the cell web, granules fused with plasma membrane were counted separately. During later foetal stages the increase in the frequency of granule to plasma membrane fusions parallels the increase in blood insulin levels, and the total number of granules in beta cells increases in parallel with the pancreatic insulin content. Therefore, as the beta cell matures, both secretion and biosynthesis of insulin increase sharply. The observed changes in the distribution of the granules in the different zones of the cytoplasm with the foetal age suggests that the cell web controls the access of the granules to the plasma membrane. The morphometric technique used allows a direct determination, at the cellular level, of even small variations in exocytosis-mediated secretory discharge and suggests a regulatory role of the cell web.     

Immunocytochemical localisation of GABA in endocrine cells of the rat entero-pancreatic system

The occurence of GABA-containing cells in the rat entero-pancreatic system was investigated by using anti-GABA-glutaraldehyde antibodies at the light and electron microscope level. In the pancreas, the B cells showed intense immunoreactivity, contrary to non-B and exocrine cells. Moreover, post-embedding immunogold staining was localised mostly in mitochondria, close to rough endoplasmic reticulum and in the nucleus. The insulin granules appeared non-significantly stained, which suggests the lack of cosecretion of GABA together with insulin. In the duodenum, GABA immunoreactivity was detected in certain endocrine cell types, suggesting a possible interaction with this amino acid. The well established GABAergic innervation in the enteric system was also confirmed by immunolabelling.     

Microtubules Regulate Localization and Availability of Insulin Granules in Pancreatic Beta Cells

Two key prerequisites for glucose-stimulated insulin secretion (GSIS) in β cells are the proximity of insulin granules to the plasma membrane and their anchoring or docking to the plasma membrane (PM). Although recent evidence has indicated that both of these factors are altered in the context of diabetes, it is unclear what regulates localization of insulin granules and their interactions with the PM within single cells. Here, we demonstrate that microtubule (MT)-motor-mediated transport dynamics have a critical role in regulating both factors. Super-resolution imaging shows that whereas the MT cytoskeleton resembles a random meshwork in the cells’ interior, MTs near the cell surface are preferentially aligned with the PM. Computational modeling suggests two consequences of this alignment. First, this structured MT network preferentially withdraws granules from the PM. Second, the binding and transport of insulin granules by MT motors prevents their stable anchoring to the PM. These findings suggest the MT cytoskeleton may negatively regulate GSIS by both limiting the amount of insulin proximal to the PM and preventing or breaking interactions between the PM and the remaining nearby insulin granules. These results predict that altering MT network structure in β cells can be used to tune GSIS. Thus, our study points to the potential of an alternative therapeutic strategy for diabetes by targeting specific MT regulators.     

Ultrastructural studies of the ontogeny of fetal human and porcine endocrine pancreas, with special reference to colocalization of the four major islet hormones.

Most, if not all, endocrine cells seem capable of synthesizing and storing more than one hormone. Such cellular colocalization of hormones can be due either to the presence of two or more specific granules within the cells or to colocalization of the hormones within a single granule. The present study was performed to clarify the subcellular localization of insulin, glucagon, somatostatin, and pancreatic polypeptide within the endocrine cells of the human and porcine pancreas during fetal development, with special reference to possible colocalization of the hormones. The tissue specimens were processed for ultrastructural cytochemistry using Lowicryl as embedding medium. An immunogold labeling technique was used with two parallel, but not interacting, antibody chains. Sections from each specimen were double labeled in different combinations giving a complete covering of the four major islet hormones. During fetal life (50-90 days prenatally in porcine pancreas, 14 weeks gestation in the human pancreas) several hormones were demonstrated, not only in the same endocrine cells, but also in the same secretory granules (polyhormonal granules). Costorage of insulin, glucagon, somatostatin, and pancreatic polypeptide was demonstrated in granules in pancreatic endocrine fetal cells. At an early fetal stage, the endocrine cells contained either dense, round granules or pale, heteromorphous granules. With increasing age and maturation of the endocrine cells, structural differentiation of the secretory granules was found to be associated with a gradual disappearance of the polyhormonal granules. The first genuine monohormonal cell to appear in the porcine fetus was the pancreatic polypeptide cell (at 70 days gestation); it was followed by the somatostatin-producing endocrine cell. Mature insulin- and glucagon-producing cells were only demonstrated after birth. Thus, in the adult pancreatic endocrine cells, each specific endocrine cell type produced only one of the four classical hormones. The present investigation demonstrated that the endocrine cells of the fetal, but not the adult, pancreas are able to synthesize all the major islet hormones, and that these peptides are costored in the same granule. The data obtained support the concept of a common precursor stem cell for pancreatic hormone-producing cells.     

Cholesterol Accumulation Increases Insulin Granule Size and Impairs Membrane Trafficking

The formation of mature secretory granules is essential for proper storage and regulated release of hormones and neuropeptides. In pancreatic β cells, cholesterol accumulation causes defects in insulin secretion and may participate in the pathogenesis of type 2 diabetes. Using a novel cholesterol analog, we show for the first time that insulin granules are the major sites of intracellular cholesterol accumulation in live β cells. This is distinct from other, non‐secretory cell types, in which cholesterol is concentrated in the recycling endosomes and the trans‐Golgi network. Excess cholesterol was delivered specifically to insulin granules, which caused granule enlargement and retention of syntaxin 6 and VAMP4 in granule membranes, with concurrent depletion of these proteins from the trans‐Golgi network. Clathrin also accumulated in the granules of cholesterol‐overloaded cells, consistent with a possible defect in the last stage of granule maturation, during which clathrin‐coated vesicles bud from the immature granules. Excess cholesterol also reduced the docking and fusion of insulin granules at the plasma membrane. Together, the data support a model in which cholesterol accumulation in insulin secretory granules impairs the ability of these vesicles to respond to stimuli, and thus reduces insulin secretion.     

SNAP-25 is expressed in islets of Langerhans and is involved in insulin release

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP- 25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.     

Low-molecular-weight constituents of isolated insulin-secretory granules. Bivalent cations, adenine nucleotides and inorganic phosphate.

The concentrations of Zn2+, Ca2+, Mg2+, Pi and adenine nucleotides were determined in insulin-secretory granules prepared from a transplantable rat insulinoma. Differential and density-gradient centrifugation analyses revealed that Zn2+ in this tissue was principally localized in the secretory granule, a second major fraction being found in association with cytosolic proteins. Pi was principally recovered in the latter fraction, whereas Ca2+ and Mg2+ were more widely distributed. Intragranular ion-distribution experiments suggested that Zn2+ was complexed mainly to insulin and its precursor forms and remained in the granule in an insoluble state. The Zn2+/insulin ratio (0.54) was greater than that expected for insulin molecules having two centrally co-ordinated Zn2+ atoms/hexamer, but less than the maximal Zn2+-binding capacity of the molecule. Most of the granular Ca2+, Mg2+ and Pi was released in a soluble form when granules were disrupted by sonication. Simulation in vitro of the ionic composition of the granule suggested that up to 90% of its Ca2+ was complexed to Pi and adenine nucleotides. Granular macromolecules also bound Ca2+, as shown by equilibrium-dialysis studies of granule lysates. However, such binding was displaced by Mg2+. Examination of the efflux of Ca2+ from granules incubated in iso-osmotic suspensions at 37 degrees C suggested that the passive permeability of the granule membrane to Ca2+ was very low. Nevertheless, more than 50% of the granular Ca2+ was rapidly released in an ionized form on hypo-osmotic or detergent-induced disruption of the granule membrane. This may represent a potentially mobilizable pool of Ca2+ in vivo.     

Low density lipoprotein degradation by rat mast cells. Demonstration of extracellular proteolysis caused by mast cell granules

The interaction between rat serosal mast cells and low density lipoproteins (LDL) was studied in vitro. When rat 125I-LDL was incubated with mast cells, it was bound to a binding site on the mast cell surface but was not internalized by the cells. Even though 125I-LDL was not internalized, its protein component, apolipoprotein B, was rapidly degraded. The proteolytic activity responsible for the degradation of apolipoprotein B was present in the extracellular fluid of mast cells. It could be shown that the degradation was caused entirely by specific cell organelles of mast cells, the granules, which were spontaneously released into the extracellular fluid during preparation and incubation of the cells. In contrast to uncontrolled spontaneous degranulation, a controlled specific degranulation of mast cells can be induced by treating the cells with the compound 48/80. When increasing amounts of 48/80 were added to mast cell suspensions, a dose-dependent release of granules was observed and an increase in the rate of 125I-LDL degradation resulted. The increase in 125I-LDL degradation closely followed the increase in granule release. Thus, a quantitative relationship between the amount of granules present in the extracellular fluid and the amount of degradation of 125I-LDL could be established. The apolipoprotein part of LDL was extensively degraded by isolated mast cell granules. Analysis by polyacrylamide gel electrophoresis showed that upon incubation of LDL with isolated granules, the apolipoprotein B band rapidly disappeared with simultaneous appearance of several low molecular weight bands. The degradation of 125I-LDL by mast cell granules proceeded optimally at neutral pH and at physiological ionic strength. The results show that mast cell granules are able to efficiently degrade LDL in vitro, once released from mast cells into the extracellular fluid.