A chromogranin peptide is co-stored with insulin in the human pancreatic islet B-cell granules

Summary The immunoreaction of a rabbit chromogranin A and B antiserum was studied in normal human pancreatic islets. By examination of consecutive light microscopical sections, it was revealed that, at high antiserum concentrations (1:2000 or less), the whole islet area was heavily labelled, although the peripheral glucagon (A)-cells were the most intense in their immunoreaction. At low antiserum concentrations (1:4000 or more) the A-cells still showed the same intense labelling reaction, but the central B-cells were weakly labelled. Electron microscopically, reactivity towards the chromogranin A and B antiserum and the monoclonal insulin antibodies was present in the same central electron-dense core of the B-cell secretory granules, as demonstrated after application of the immunogold technique at different antibody dilutions. In the A-cells, the chromogranin immunoreactivity was concentrated at the peripheral mantle of the secretory granules. The D-cell granules showed a weak immunolabelling. Examination of human islets with the monoclonal chromogranin A antibody LK2H10 revealed immunogold labelling only in the peripheal mantle of the A-cell granules, while the B-cell granules were unlabelled.The present results show that a chromogranin peptide is co-stored with insulin the in normal human B-cell secretory granules. Although the exact composition of this B-cell chromogranin is unknown, it is not identical to that of the chromogranin A present in the A-cell granules.     

Effects of pancreastatin on insulin, glucagon and somatostatin secretion by the perfused rat pancreas

Pancreastatin is a novel peptide, isolated from porcine pancreatic extracts, which has been shown to inhibit glucose-induced insulin release "in vitro". To achieve further insight into the influence of pancreastatin on pancreatic hormone secretion, we have studied the effects of this peptide on unstimulated insulin, glucagon and somatostatin output, as well as on the responses of these hormones to glucose and to tolbutamide in the perfused rat pancreas. Pancreastatin strongly inhibited unstimulated insulin release as well as the insulin responses to glucose and to tolbutamide. It did not significantly affect glucagon or somatostatin output under any of the above-mentioned conditions. These findings suggest that pancreastatin inhibits B-cell secretory activity directly, and not through an A-cell or D-cell paracrine effect.     

Correlation of 24-hour fluctuations in renin granules of juxtaglomerular cells and in renin and angiotensinogen in blood plasma of the rat

Summary Subcellular structures of juxtaglomerular (JG) cells in the rat kidney were morphometrically examined at six evenly spaced times over 24 h. Plasma renin activities and angiotensinogen concentrations were also measured at these times. The cell volumes were larger at 20.00 h and 04.00 h than at 00.00 h, whereas the nuclear volumes peaked at 20.00 h and 08.00 h, decreasing at 00.00 h and 16.00 h. The volume and surface densities of renin granules and their individual volumes and surface areas peaked at 16.00 h and 00.00 h, decreasing at 20.00 h and 08.00 h, whereas their numerical densities peaked at 20.00 h, decreasing at 12.00 h. The surface densities of the rough endoplasmic reticulum (rER) peaked at 20.00 h, decreasing at other times, except at 08.00 h, when rER volume and surface density were relatively high. The plasma renin activity was maximal at 20.00 h, whereas it was minimal at 08.00 h. The variation in plasma angiotensinogen concentrations was inversely correlated with that in plasma renin activities. These results suggest that JG cells actively synthesize and release renin during the dark period, especially at 20.00 h, whereas during the light period they gradually synthesize renin and produce the granules, most of which may be stored in the cells during this period.     

Homologous pancreastatin inhibits insulin secretion without affecting glucagon and somatostatin release in the perfused rat pancreas.

The identification of pancreastatin in pancreatic extracts prompted the investigation of its effects on islet cell function. However, in most of the investigations to date, pig pancreastatin was tested in heterologous species. Since there is great interspecies variability in the amino acid sequence of pancreastatin, we have investigated the influence of rat pancreastatin on insulin, glucagon and somatostatin secretion in a homologous animal model, namely the perfused rat pancreas. During 5.5 mM glucose infusion, pancreastatin (40 nM) inhibited insulin secretion (ca. 40%, P less than 0.025) as well as the insulin responses to 10 mM arginine (ca. 50%, P less than 0.025) and to 1 nM vasoactive intestinal polypeptide (ca. 50%; P less than 0.05). Pancreastatin failed to significantly modify glucagon or somatostatin release under any of the above experimental conditions. In addition, a lower pancreastatin concentration (15.7 nM) markedly suppressed the insulin release evoked by 11 mM glucose (ca. 85%, P less than 0.05). Our present observations reinforce the concept that pancreastatin is an effective inhibitor of insulin secretion, influencing the B-cell function directly and not through an A-cell or D-cell paracrine effect.     

Diurnal variations of food consumption, plasma glucose and plasma insulin concentrations in lean and obese hyperglycaemic mice.

Diurnal variations in food consumption and plasma concentrations of glucose and insulin were determined at 3-hourly intervals in obese hyperglycaemic mice (C57BL/6J ob/ob) and lean mice (C57BL/6J+/+). In lean mice, food consumption and plasma insulin concentrations increased during the light period and were reduced during the dark period, whereas plasma glucose concentrations were maximal at the beginning of the light period and declined to a minimum during the early dark period. In ob/ob mice, the plasma glucose concentration declined temporarily at the beginning of both the light and the dark period and became elevated towards the ends of these periods, but there were no significant diurnal variations of food consumption or plasma insulin concentrations. These observations indicate differences in the diurnal regulation of glucose homeostasis in lean and ob/ob mice.     

Morphology of the endocrine part of the pancreas of birds]

Methods of light and electron microscopy were used to study the structural organization of the endocrinous part of the pancreas in birds (domestic ducks and chickens). It has been established that the endocrinous part is formed by three types of pancreatic islands: dark, light and mixed islands. "Dark" islands consist of A- and D-cells, "light" ones--of B and D-cells and "mixed" islands--of A-, B- and D-cells. The index of B/A-cell ratio is equal to 0,39--0,4. This shows that each B-cell corresponds to 4 A-cells. On this basis one can say that the counterinsular apparatus of birds became stronger in the process of evolution and due to this a comparatively high level of glycemia is sustained in them. Availability of "dark" and "light" pancreatic islands also points out that in birds there occurred a partial disconnection of insular and counterinsular components of the endocrinous part of the pancreas, necessary for adaptation to principally new conditions of the environment.     

The Ultrastructure of the Endocrine Pancreas of the Grass Lizard,Mabuya quinquetaeniata*

Four endocrine cell types were identified ultrastructurally in the pancreas of the grass lizard, Mabuya quinquetaeniata. These cells were similar in shape, location and frequency to the previously described B-, A-, D- and PP-cells. The secretory granules of the B-cells were round or oval in profile, with an internal core of variable shape. The mean diameter of the B-cell granules was 780 nm (range 350–1000 nm). The A-cell granules were round, oval or irregular in shape and highly electron dense, with a narrow electron lucent space between the core and the limiting membrane. The mean diameter of these granules was 450 nm (range 200–750 nm). The D-cell granules were round, oval or irregular and of moderate electron density, with an average diameter of 340 nm (range 200–500 nm). The limiting membrane was closely apposed to the core or separated from it by a narrow lucent space. PP-cell granules were round with high electron density and with a narrow space between the core and the limiting membrane, and their average diameter was 150 nm (range 50–350 nm); these secretory granules accumulated at the cytoplasmic process. Tracing of the cytoplasmic processes of PP-cells in serially cut ultrathin sections revealed that most of these processes ended in the vicinity of blood capillaries, indicating that the PP-cells were endocrine rather than paracrine.     

Islet cell analysis and purification by light scatter and autofluorescence

Rat pancreatic A- and B-cells differ in light scatter and flavin-adenine-dinucleotide (FAD)-related fluorescence and are thus represented by two easily distinguishable populations in a fluorescence-activated cell sorter (FACS). Sorting of dissociated islet cells yields highly purified single A- and B-cell preparations. FACS-analysis of islet cells also indicated that FAD-fluorescence in 3-cells is reduced within a 5 minute exposure to 20 mM glucose, whereas no variations were observed in A-cell fluorescence nor with 3-0-methylglucose or fructose. FACS-analysis of blood cells and of dissociated liver, parotid, pituitary and pancreatic exocrine cells demonstrated a wide variation in the respective FAD-fluorescence intensities, which could be used for their purification as viable single cells as well as in studying their metabolic redox state.     

The stimulus-secretion coupling of glucose-induced insulin release. Cationic and secretory effects of menadione in the endocrine pancreas.

1. Menadione (2-methyl-1,4-naphthoquinone) inhibits insulin release evoked in the rat endocrine pancreas by glucose or glyceraldehyde, but fails to affect the secretory response to Ca2+, Ba2+, theophylline or gliclazide. The inhibitory effect of menadione upon glucose-induced insulin release is a dose-related, rapid and reversible phenomenon, menadione and glucose acting apparently as competitive antagonists. Menadione affects both the early and late phase of the secretory response to glucose. Menadione also antagonizes in a dose-related fashion the ability of glucose to reduce 86Rb efflux, to provoke 86Rb accumulation, to cause biphasic changes in 45 Ca efflux and to stimulate 45 Ca net uptake in pancreatic islets. 2. It is concluded that menadione impairs the insulinotropic action of glucose and other nutrients by impeding the remodelling of cationic fluxes normally provoked by these secretagogues in islet cells. Menadione, however, does not affect the capacity of divalent cations to activate the effector system which controls the release of secretory granules. Menadione may therefore represent a valuable tool to elucidate the mechanism by which glucose normally modifies the movement of cations in the pancreatic B-cell.     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

Altered dipeptidyl peptidase-4 activity during the progression of hyperinsulinemic obesity and islet atrophy in spontaneously late-stage type 2 diabetic rats

Altered dipeptidyl peptidase-4 (DPP4) activity during the progression of late-stage type 2 diabetes was measured in Otsuka Long-Evans Tokushima fatty (OLETF) rats. Compared with OLETF rats subjected to 30% food restriction, food-satiated OLETF rats exhibited spontaneous hyperphagic obesity, insulin resistance, hyperglycemia, hyperinsulinemia, and increased plasma DPP4 activity during the early phase of the experiment (up to ～30 wk). Subsequently, their plasma DPP4 activity as well as their body weight, body fat, and plasma insulin concentration declined to control levels during the late phase, resulting in excessive polyuria, proteinuria, dyslipidemia, pancreatic islet atrophy, hypoinsulinemia, and diabetes, which changed from insulin-resistant diabetes to hypoinsulinemic diabetes secondary to progressive islet insufficiency, and their fasting blood glucose level remained high. Since plasma DPP4 activity demonstrated significant positive correlations with body weight and the fasting plasma insulin level but not with the fasting blood glucose level during the late stage of diabetes, body fat and fasting plasma insulin levels may be useful factors for predicting the control of plasma DPP4 activity. In contrast, pancreatic DPP4 activity was significantly increased, and the expression of pancreatic insulin was significantly reduced in late-stage diabetic OLETF rats, suggesting that a relationship exists between the activation of pancreatic DPP4 and insulin depletion in pancreatic islet atrophy. In conclusion, it is suggested that plasma DPP4 activity changes in accordance with the progression of hyperinsulinemic obesity and pancreatic islet atrophy. DPP4 activity may play an important role in insulin homeostasis.     

Biological actions of peptide YY: effects on endocrine pancreas, pituitary-adrenal axis, and plasma catecholamine concentrations in the dog

The present study was designed to gather information on the biological activity of peptide YY (PYY) in conscious dogs. PYY was infused intravenously at a dose of 238 pmol/kg X h, and plasma concentrations of glucose, insulin, pancreatic polypeptide (PP), ACTH, cortisol and catecholamines (norepinephrine-NE; epinephrine-E; dopamine-DA) were subsequently measured. PYY significantly increased plasma insulin levels transiently without effect on plasma glucose, but decreased plasma PP levels during all infusion periods. PYY stimulated both plasma ACTH and cortisol secretion, and this action of PYY was also shared by PP, with PP being less potent in ACTH-cortisol release. PYY further elicited specific changes in plasma catecholamine concentrations, i.e. an increase of NE but not of E, which were in contrast to the effects of insulin-induced hypoglycemia. PP failed to alter plasma insulin and catecholamine concentrations. These results suggest that PYY can affect anterior pituitary hormone secretion, sympathetic nervous outflow and pancreatic endocrine activity in addition to its known actions on gastric and pancreatic secretion in the dog.     

Fine structure and immunocytochemistry of cells within the endocrine pancreas of larval and adult sea lampreys, Petromyzon marinus L

Immunocytochemistry with protein A-gold and routine electron microscopy were used to identify cell types within the endocrine pancreas of larvae, juvenile adults, and upstream-migrant adults of the sea lamprey, Petromyzon marinus. The larval pancreatic islets are composed only of insulin-immunoreactive B-cells, which are uniform in their fine structure. The cranial and caudal pancreatic tissue in both adult periods contains three cell types: B-cells, somatostatin-immunoreactive D-cells, and a third cell type of unknown content. No glucagon-immunoreactive cells are present in lampreys, but B- and D-cells exist in equal numbers in the pancreatic tissue of adults. The B-cells of adults have a fine structure similar to those in larvae. D-cells have secretory granules that are distinctly different from those both in B-cells and in the third cell type. Although B- and D-cells in lamprey pancreatic tissues have a basic morphological similarity to these cells in other vertebrates, their granules are generally of smaller dimensions. The inclusion of granules within large pleomorphic bodies in many D-cells indicates that granule turnover is common. Immunocytochemistry will be a useful tool for showing the relationship between the cells in the degenerating bile ducts and those of the developing adult pancreas.     

Role of cholecystokinin in the control of gastric somatostatin in the rat: in vivo and in vitro studies.

Cholecystokinin (CCK) has been shown to be a powerful stimulus for somatostatin release from isolated canine fundic D-cells in short-term culture. The influence of the CCK analogue caerulein on the secretory activity of the D-cell in the intact stomach in vitro and the effect of elevated plasma levels of endogenous CCK on gastric somatostatin stores in vivo were investigated in the rat. Basal somatostatin secretion from the isolated, vascularly perfused rat stomach preparation was not affected by various doses of caerulein. Slight stimulation of somatostatin-like immunoreactivity (SLI) release by epinephrine was significantly inhibited by caerulein, whereas caerulein did not alter half-maximal stimulation of SLI secretion by isoproterenol. Rats with chronically elevated plasma CCK levels induced by experimental exocrine pancreatic insufficiency did not show any change in tissue concentrations of SLI or in D-cell number, both in the antrum and corpus. These data suggest that CCK--in contrast to dogs--is not an important modulator of gastric somatostatin in the rat.     

Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography

Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.     

Basal, oxytocin-, and insulin-stimulated glucose oxidation in human endometrium

Samples of endometrium from regularly cycling women (28 +/- 2 days cycle) were assayed for [U-14C]glucose oxidation activity in the presence or absence of 100 nM oxytocin or 1.7 nM insulin. The basal rate of glucose oxidation in the tissues obtained from women in early and midfollicular phase and late luteal phase was approximately 125 pmol/(h X mg tissue). Late follicular and midluteal phases had higher basal rates, up to 400 pmol/(h X mg tissue). Oxytocin increased glucose oxidation by 50-100 pmol X h-1 X mg-1 in early and midfollicular phase and in early luteal phase endometrial fragments. Insulin did not stimulate glucose oxidation in these tissues. In samples of late luteal phase, glucose oxidation was stimulated by both oxytocin and insulin. High and low basal glucose oxidation activity in the endometrium corresponded, respectively, to reported periods of high and low plasma estradiol in normal menstruating women. In contrast, oxytocin stimulated glucose oxidation in endometria from women with anticipated low plasma estradiol.     

Different responses of circulating ghrelin, obestatin levels to fasting, re-feeding and different food compositions, and their local expressions in rats

Obestatin, a sibling of ghrelin derived from preproghrelin, opposes several physiological actions of ghrelin. Our previous study has demonstrated that both plasma ghrelin and obestatin levels were decreased significantly 2h after food intake in human. To further expand current knowledge, we investigated the temporal profiles of their levels in ad libitum fed rats, 48h fasted rats and 48h fasted rats refed 2h with a standard chow, crude fiber, 50% glucose or water, and their expressions in stomach, liver and pancreatic islets immunohistochemically. Plasma ghrelin and obestatin levels were measured by EIA. Plasma leptin, insulin and glucose levels were also evaluated. Both plasma ghrelin and obestatin levels increased significantly in fasted rats compared with ad libitum fed rats. The ingestion of standard chow produced a profound and sustained suppression of ghrelin levels, whereas plasma obestatin levels decreased significantly but recovered quickly. Intake of crude fiber or 50% glucose, however, produced a more profound and sustained suppression of obestatin levels, though they had relatively less impact on ghrelin levels. Plasma glucose was the only independent predictor of ghrelin levels, obestatin levels, and ghrelin to obestatin ratios. Obestatin immunoreactivity was detected in the fundus of stomach, liver and pancreatic islets, with roughly similar patterns of distribution to ghrelin. These data show quantitative and qualitative differences in circulating ghrelin and obestatin responses to the short-term feeding status and nutrient composition, and may support a role for obestatin in regulating metabolism and energy homeostasis.     

Cardiac and pancreatic lesions in guinea pigs infected with encephalomyocarditis (EMC) virus.

Cardiac and pancreatic lesions were observed in guinea pigs infected with 2 variants (B and D) of encephalomyocarditis (EMC) virus. Cardiac changes were characterized by focal myocardial necrosis and subsequent replacement by immature granulation tissue, and the pancreatic ones by vacuolar degeneration of acinar cells. In the electron microscopic examinations, the affected cardiomyocytes showed intracellular oedema, swelling and/or partial destruction of mitochondria, and distortion and disruption of myofibrils. Intracellular vacuolization and dilatation of rough-surfaced endoplasmic reticulum (rER) were conspicuous in the damaged pancreatic acinar cells. In addition, intracisternal granules were found in dilated rER with a high frequency. These changes were common to animals infected with the B and D variants. On the contrary, B cell alterations; i.e. degranulation and degeneration of insulin granules, were detected only in animals infected with the D variant.     

Cellular and subcellular immunolocalization of L-glutamate decarboxylase in rat pancreatic islets

The cellular and subcellular distribution of L-glutamate decarboxylase (GAD), the biosynthetic enzyme for gamma-aminobutyric acid (GABA), was determined immunohistochemically in rat pancreatic islet using light and electron microscopic techniques. The cellular distribution of GAD was determined at the light microscopic level using an elution/re-staining protocol and a computerized digital image processing technique. At this level of resolution, immunofluorescent GAD was observed to be co-localized with immunofluorescent insulin in the islet B-cells and absent in both the A-cells, which contained glucagon, and the D-cells, which contained somatostatin. Subcellular localization of GAD was determined using an electron microscopic, colloidal gold post-embedding protocol and was compared to insulin immunoreactivity in serial sections of the same B-cell. In the same islet B-cell, GAD immunoreactivity appeared predominantly in the extragranular cytoplasm, whereas insulin immunoreactivity was associated with the secretory granules. Quantitative analysis of GAD immunoreactivity in the B-cell revealed 15.3 +/- 1.8 gold particles/micron2 in the cytoplasm, 1.7 +/- 0.2 gold particles/micron2 in the secretory granules, and 0.4 +/- 0.4 gold particles/micron2 in the mitochondria. The results of this study, localization of the biosynthetic enzyme for GABA to the B-cell cytoplasmic compartment and its absence in the secretory granules which contain insulin, are compatible with the hypothesis that GABA functions as an intracellular mediator of B-cell activity.     

电损毁海马CA3区及连合前穹窿对大鼠血浆胰岛素水平...

Bilateral electrical lesioning of the hippocampal CA3 region (HCA3-EL) or anterior commissura hippocampi (ACHF-EL) caused marked elevations in plasma basal levels of insulin. 2 weeks later, fasting blood glucose levels were also augmented with decreased glucose tolerance. In contrast, the secretory response of pancreatic B cells to glucose stimulation was markedly enhanced. Following intravenous glucose tolerance test (IVGTT), the relative amounts of glucagon-like and insulin-like immunoreactants were reduced in the pancreatic islets of both HCA3-EL and ACHF-EL rats in comparison with the controls. In the HCA3-EL group, the relative amounts of somatostatin-like immunoreactants and gross numbers of such immunostained cells in islets were also decreased as compared with the control. No difference was seen in pancreatic-polypeptide-like immunoreactivities as assessed by immunohistochemistry plus microphotometry method. The above results suggest strongly that HCA3 and ACHF exert a tonic inhibitory action on the insulin secretion in the rat.