Β-cell Granules Research Articles

Lack of cholesterol mobilization in islets of hormone-sensitive lipase deficient mice impairs insulin secretion

The observations that hormone-sensitive lipase (HSL) is located in close association to insulin granules in β-cells and that cholesterol ester hydrolase activity is completely blunted in islets of HSL null mice made us hypothesize that the role of HSL in β-cells is to provide cholesterol for the exocytosis of insulin. To test this hypothesis, wild type (wt) and HSL null islets were depleted of plasma membrane cholesterol using methyl-β-cyclodextrin (mβcd). A significant reduction in insulin secretion from HSL null islets was observed whereas wt islets were unaffected. Using synaptosomal protein of 25kDa (SNAP-25) as indicator of cholesterol-rich microdomains, confocal microscopy was used to show that HSL null β-cells treated with mβcd contained fewer clusters than wt β-cells. These results indicate that HSL plays an important role in insulin secretion by providing free cholesterol for the formation and maintenance of cholesterol-rich patches for docking of SNARE-proteins to the plasma membrane.

Insulin granule trafficking in β-cells: mathematical model of glucose-induced insulin secretion

A mathematical model that represents the dynamics of intracellular insulin granules in beta-cells is proposed. Granule translocation and exocytosis are controlled by signals assumed to be essentially related to ATP-to-ADP ratio and cytosolic Ca(2+) concentration. The model provides an interpretation of the roles of the triggering and amplifying pathways of glucose-stimulated insulin secretion. Values of most of the model parameters were inferred from available experimental data. The numerical simulations represent a variety of experimental conditions, such as the stimulation by high K(+) and by different time courses of extracellular glucose, and the predicted responses agree with published experimental data. Model capacity to represent data measured in a hyperglycemic clamp was also tested. Model parameter changes that may reflect alterations of beta-cell function present in type 2 diabetes are investigated, and the action of pharmacological agents that bind to sulfonylurea receptors is simulated.

A Subset of p23 Localized on Secretory Granules in Pancreatic β-cells

Proteins on the membrane of secretory granules (SGs) involved in their biogenesis and exocytosis are poorly characterized compared with those of synaptic vesicle in neurons. Thus the secretory granule membrane was prepared from a mouse pancreatic beta-cell line MIN6 by subcellular fractionation, and protein constituents were analyzed by microscale two-dimensional liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Using this proteomics approach, one of the p24 family proteins, p23, was unexpectedly found in the granule fraction, although p24 proteins are generally regarded as functioning in the early secretory pathways between the endoplasmic reticulum and the Golgi apparatus. We further showed that p23 is expressed at high levels in endocrine cells. Furthermore, immunocytochemical analyses of pancreatic beta-cells at the light and electron microscopic levels demonstrated that a significant amount of p23 is localized on the insulin granule membrane, although it is most intensely concentrated at the cis-Golgi compartment as previously shown in non-endocrine cells. These findings suggest that a fraction of p23 enters post-Golgi compartments and may function in the biogenesis and/or quality control of SGs.

Thea3 isoform of V-ATPase regulates insulin secretion from pancreatic β-cells

Vacuolar-type H(+)-ATPase (V-ATPase) is a multi-subunit enzyme that has important roles in the acidification of a variety of intracellular compartments and some extracellular milieus. Four isoforms for the membrane-intrinsic subunit (subunit a) of the V-ATPase have been identified in mammals, and they confer distinct cellular localizations and activities on the proton pump. We found that V-ATPase with the a3 isoform is highly expressed in pancreatic islets, and is localized to membranes of insulin-containing secretory granules in beta-cells. oc/oc mice, which have a null mutation at the a3 locus, exhibited a reduced level of insulin in the blood, even with high glucose administration. However, islet lysates contained mature insulin, and the ratio of the amount of insulin to proinsulin in oc/oc islets was similar to that of wild-type islets, indicating that processing of insulin was normal even in the absence of the a3 function. The insulin contents of oc/oc islets were reduced slightly, but this was not significant enough to explain the reduced levels of the blood insulin. The secretion of insulin from isolated islets in response to glucose or depolarizing stimulation was impaired. These results suggest that the a3 isoform of V-ATPase has a regulatory function in the exocytosis of insulin secretion.

Imaging Docking and Fusion of Insulin Granules Induced by Antidiabetes Agents

Sulfonylurea and glinide drugs, commonly used for antidiabetes therapies, are known to stimulate insulin release from pancreatic beta-cells by closing ATP-sensitive K+ channels. However, the specific actions of these drugs on insulin granule motion are largely unknown. Here, we used total internal reflection fluorescence (TIRF) microscopy to analyze the docking and fusion of single insulin granules in live beta-cells exposed to either the sulfonylurea drug glibenclamide or the glinide drug mitiglinide. TIRF images showed that both agents caused rapid fusion of newcomer insulin granules with the cell membrane in both control and diabetic Goto-Kakizaki (GK) rat pancreatic beta-cells. However, in the context of beta-cells from sulfonylurea receptor 1 (SUR1) knockout mice, TIRF images showed that only mitiglinide, but not glibenclamide, caused fusion of newcomer insulin granules. Compositely, our data indicate that 1) the mechanism by which both sulfonylurea and glinide drugs promote insulin release entails the preferential fusion of newcomer, rather than previously docked, insulin granules, and that 2) mitiglinide can induce insulin release by a mechanism independent of mitiglinide binding to SUR1.

Anti-Diabetic Activity of Fruits of Terminalia chebula on Streptozotocin Induced Diabetic Rats

The present study was aimed to evaluate the anti-diabetic potential of Terminalia chebula (T. chebula) fruits on streptozotocin (STZ)-induced experimental diabetes in rats. Oral administration of ethanolic extract of the fruits (200 mg/kg body weight/rat/day) for 30 days significantly reduced the levels of blood glucose and glycosylated hemoglobin in diabetic rats. Determination of plasma insulin levels revealed the insulin stimulating action of the fruit extract. Also, the alterations observed in the activities of carbohydrate and glycogen metabolising enzymes were reverted back to near normal after 30 days of treatment with the extract. Electron microscopic studies showed significant morphological changes in the mitochondria and endoplasmic reticulum of pancreatic β cells of STZinduced diabetic rats. Also, a decrease in the number of secretory granules of β-cells was observed in the STZinduced diabetic rats and a these pathological abnormalities were normalized after treatment with T. chebula extract. The efficacy of the fruit extract was comparable with glibenclamide, a well known hypoglycemic drug.

Expression and physicochemical characterization of an extracellular segment of the receptor protein tyrosine phosphatase IA-2

The receptor protein tyrosine phosphatase superfamily (RPTP) includes proteins with a single transmembrane, one or more intracellular phosphatase, and a variety of extracellular domains. The 106-kDa insulinoma-associated protein (IA-2, ICA512) receptor is unique among RPTP members because: (a) it has a single, phosphatase-like intracellular domain identified as one of the most prominent self antigens in autoimmune diabetes; (b) its extracellular region bears no sequence similarity to known domains; (c) it is present in the membrane of secretory granules in neurons and pancreatic β-cells where it suffers a complex processing; and (d) it has very poorly understood biological properties. In this work, we describe the expression, purification, and physicochemical characterization of residues 449–576 of IA-2 (IA-2ec 449–576). Judging from CD, fluorescence, hydrodynamic, and thermal unfolding analyses, this fragment forms an autonomously folding unit with tight packing and well-defined secondary and tertiary structure. CD analysis suggests that about 25% of IA-2ec 449–576 residues are α-helical, whereas about the same amount are in β-sheet structure. The availability of soluble and folded IA-2ec 449–576 is a step forward toward the characterization of a part of IA-2 at atomic detail, which may provide new insight in the biology of diabetes, the neurotransmission process, and the dynamic of secretory granules.

Slp4-a/Granuphilin-a Interacts with Syntaxin-2/3 in a Munc18-2-dependent Manner

Slp4-a/granuphilin-a was originally described as a protein specifically associated with insulin-containing granules in pancreatic beta-cells, but it was subsequently found to be present on amylase-containing granules in parotid acinar cells. Although Slp4-a has been suggested to control insulin secretion through interaction with syntaxin-1a and/or Munc18-1, nothing is known about the binding partner(s) of Slp4-a during amylase release from parotid acinar cells, which do not endogenously express either syntaxin-1a or Munc18-1. In this study we systematically investigated the interaction between syntaxin-1-5 and Munc18-1-3 by co-immunoprecipitation assay using COS-7 cells and discovered that Slp4-a interacts with a closed conformation of syntaxin-2/3 in a Munc18-2-dependent manner, whereas Munc18-2 itself hardly interacts with Slp4-a at all. By contrast, Slp4-a was found to strongly interact with Munc18-1 regardless of the presence of syntaxin-2/3, and syntaxin-2/3 co-immunoprecipitated with Slp4-a only in the presence of Munc18-1/2. Deletion analysis showed that the syntaxin-2/3 (or Munc18-1/2)-binding site is a linker domain of Slp4-a (amino acid residues 144-354), a previously uncharacterized region located between the N-terminal Rab27A binding domain and the C2A domain. We also found that the Slp4-a.syntaxin-2 complex is actually present in rat parotid glands and that introduction of the antibody against Slp4-a linker domain into streptolysin O-permeabilized parotid acinar cells severely attenuates isoproterenol-stimulated amylase release, possibly by disrupting the interaction between Slp4-a and syntaxin-2/3 (or Munc18-2). These results suggest that Slp4-a modulates amylase release from parotid acinar cells through interaction with syntaxin-2/3 on the apical plasma membrane.

Fatty Acid Translocase (FAT/CD36) Is Localized on Insulin-Containing Granules in Human Pancreatic β-Cells and Mediates Fatty Acid Effects on Insulin Secretion

The membrane receptor FAT/CD36 facilitates the major fraction of long-chain fatty acid (FA) uptake by muscle and adipose tissues. In line with the well-known effects of FA metabolism on carbohydrate utilization and insulin responsiveness, altered expression of CD36 has been linked to phenotypic features of the metabolic syndrome including insulin resistance and dyslipidemia. FA metabolism is also known to significantly affect insulin secretion. However, the role of CD36 in this process remains unknown, since its expression levels and function in the pancreas have not been explored. In the present study, freshly isolated human islets and a mouse-derived beta-cell line (MIN6) were shown positive for CD36 expression by RT-PCR, Western blot, and immunofluorescence. The identity of the PCR product was confirmed by microsequencing. The identified transcript was translated and the protein was expressed and subjected to the known posttranslational glycosylation. Fluorescence resonance energy transfer analysis and subcellular protein fractionation indicated that insulin and CD36 are colocalized in the secretory granules of beta-cells. Islet CD36 functioned in FA uptake because this process was blocked by the irreversible CD36 inhibitor sulfosuccinimidyl-oleate. More importantly, sulfosuccinimidyl-oleate reversed enhancing and inhibiting effects, respectively, of acute and long-term palmitate incubations on glucose-dependent insulin secretion. In conclusion, our study demonstrates that human islets express CD36 in the plasma membrane as well as in the insulin secretory granules. CD36 activity appears important for uptake of FA into beta-cells as well as for mediating their modulatory effects on insulin secretion.

Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in β-cells

Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic β-cells. Here, we show that exocytosis of SGs and insertion of ICA512 in the plasma membrane promotes the Ca2+-dependent cleavage of ICA512 cytoplasmic domain by μ-calpain. This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression. Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in β-cells, whose impaired insulin production and secretion causes diabetes.

Insulin Secretion

Insulin Secretion

Desensitization of Insulin Secretion by Depolarizing Insulin Secretagogues

Prolonged stimulation of insulin secretion by depolarization and Ca2+ influx regularly leads to a reversible state of decreased secretory responsiveness to nutrient and nonnutrient stimuli. This state is termed "desensitization." The onset of desensitization may occur within 1 h of exposure to depolarizing stimuli. Desensitization by exposure to sulfonylureas, imidazolines, or quinine produces a marked cross-desensitization against other ATP-sensitive K+ channel (KATP channel)-blocking secretagogues. However, desensitized beta-cells do not necessarily show changes in KATP channel activity or Ca2+ handling. Care has to be taken to distinguish desensitization-induced changes in signaling from effects due to the persisting presence of secretagogues. The desensitization by depolarizing secretagogues is mostly accompanied by a reduced content of immunoreactive insulin and a marked reduction of secretory granules in the beta-cells. In vitro recovery from a desensitization by the imidazoline efaroxan was nearly complete after 4 h. At this time point the depletion of the granule content was partially reversed. Apparently, recovery from desensitization affects the whole lifespan of a granule from biogenesis to exocytosis. There is, however, no direct relation between the beta-cell granule content and the secretory responsiveness. Even though a prolonged exposure of isolated islets to depolarizing secretagogues is often associated with the occurrence of ultrastructural damage to beta-cells, we could not find a cogent link between depolarization and Ca2+ influx and apoptotic or necrotic beta-cell death.

The Cytokine Interleukin-1β Reduces the Docking and Fusion of Insulin Granules in Pancreatic β-Cells, Preferentially Decreasing the First Phase of Exocytosis

The prediabetic period in type I diabetes mellitus is characterized by the loss of first phase insulin release. This might be due to islet infiltration mediated by mononuclear cells and local release of cytokines, but the mechanisms involved are unknown. To determine the role of cytokines in insulin exocytosis, we have presently utilized total internal reflection fluorescence microscopy (TIRFM) to image and analyze the dynamic motion of single insulin secretory granules near the plasma membrane in live beta-cells exposed for 24 h to interleukin (IL)-1beta or interferon (IFN)-gamma. Immunohistochemistry observed via TIRFM showed that the number of docked insulin granules was decreased by 60% in beta-cells treated with IL-1beta, while it was not affected by exposure to IFN-gamma. This effect of IL-1beta was paralleled by a 50% reduction in the mRNA and the number of clusters of SNAP-25 in the plasma membrane. TIRF images of single insulin granule motion during a 15-min stimulation by 22 mm glucose in IL-1beta-treated beta-cells showed a marked reduction in the fusion events from previously docked granules during the first phase insulin release. Fusion from newcomers, however, was well preserved during the second phase of insulin release of IL-1beta-treated beta-cells. The present observations indicate that IL-1beta, but not IFN-gamma, has a preferential inhibitory effect on the first phase of glucose-induced insulin release, mostly via an action on previously docked granules. This suggests that beta-cell exposure to immune mediators during the course of insulitis might be responsible for the loss of first phase insulin release.

Hypothesis: one rate-limiting step controls the magnitude of both phases of glucose-stimulated insulin secretion.

The biphasic secretory response of pancreatic beta-cells to abrupt and sustained exposure to glucose is well documented. Some of the ATP-sensitive K(+) (K(ATP)) channel-dependent mechanisms underlying the first phase of insulin release are known; the mechanisms underlying the second phase are less well known. The hypothesis we propose is that one rate-limiting step, controlling the conversion of granules in a readily releasable (RR) docked granule pool to an immediately releasable (IR) pool, is responsible for the magnitude of both phases of release. Furthermore, we propose that the K(ATP) channel-independent signaling pathway regulates this rate-limiting step. The size of the IR pool of granules that constitutes the first phase is determined under resting conditions by the forward and reverse rates of conversion of granules in the RR and IR pools. The resulting equilibrium position determines the maximum number of beta-cell granules available for release during the first phase upon exposure to glucose. At the nadir between the two phases, the IR pool has been depleted so that the rate of granule release is equal to the low forward rate for the conversion of RR to IR granules. After the nadir, the forward rate is accelerated during the rising portion of the second phase until it reaches a maximum rate at the plateau.

Synaptotagmin V and IX isoforms control Ca2+ -dependent insulin exocytosis.

Synaptotagmin (Syt) is involved in Ca2+ -regulated secretion and has been suggested to serve as a general Ca2+ sensor on the membrane of secretory vesicles in neuronal cells. Insulin exocytosis from the pancreatic beta-cell is an example of a Ca2+ -dependent secretory process. Previous studies have yielded conflicting results as to which Syt isoform is present on the secretory granules in the native beta-cell. Here we show by western blotting and RT-PCR analysis, the presence of both Syt V and Syt IX in rat pancreatic islets and in the clonal beta-cell line INS-1E. The subcellular distribution of the two Syt isoforms was assessed by confocal microscopy and by sedimentation in a continuous sucrose density gradient in INS-1E cells. These experiments show that both proteins colocalize with insulin-containing secretory granules but are absent from synaptic-like microvesicles. Further immunofluorescence studies performed in primary pancreatic endocrine cells revealed that Syt V is present in glucagon-secreting alpha-cells, whereas Syt IX is associated with insulin granules in beta-cells. Transient overexpression of Syt V and Syt IX did not alter exocytosis in INS-1E cells. Finally, reduction of the expression of both Syt isoforms by RNA interference did not change basal secretion. Remarkably, hormone release in response to glucose was selectively and strongly reduced, indicating that Syt V and Syt IX are directly involved in the Ca2+ -dependent stimulation of exocytosis.

Effects of thioacetamide on pancreatic islet B-cell function.

Thioacetamide (0.01-1.3 mM) fails to exert any significant immediate effect upon insulin release from rat isolated islets. However, when administered (4 micromol/g body wt) intraperitoneally 24 h before sacrifice, it reduced food intake and body weight and affected the secretory response of isolated islets to several secretagogues, despite unaltered insulin content of such islets. This coincided with a decrease in D-[U-14C]glucose oxidation, total islet calcium content and the ionized calcium content of secretory granules in islet B-cells, and changes in both 133Ba and 45Ca net uptake. Likewise, in islets prepared from thioacetamide-injected rats and prelabeled with 45Ca before perifusion, the cationic and insulin secretory responses to D-glucose or gliclazide, but not to the association of Ba2+ and theophylline in the absence of extracellular Ca2+, often differed from that otherwise found in islets prepared from control rats. These findings are interpreted as indicative of an impaired capacity of Ca2+ sequestration by intracellular organelles in the islet B-cells of thioacetamide-treated rats.

AAA award winners.

AAA award winners.

The mode of action of acute and chronic concentrations of waterborne Cd in the digestive gland of the acclimated infested freshwater crab ( Potamonautes warreni).

Cadmium (Cd) uptake, transport and accumulation were investigated in the digestive gland of the freshwater crab, Potamonautes warreni, acclimated in its natural habitat to stresses, such as microbial gill infestations, Cd(2+) and NH(4)(+), and subsequently exposed to increasing concentrations of Cd in the laboratory for up to 21 days. Cd exposure (0.2 mg l(-1)) for 7-14 days led to Cd permeating cell membranes in a particulate form; it was adsorbed intracellularly to endocytotic circulating amoebocytes, lipid droplets and Golgi vesicles in R-cells. Cd also caused dissociation of the fibrillar rough endoplasmic reticulum (RER) and an increase in phagocytotic activity in F- and B-cells. After 21 days, Cd accumulated as crystal deposits on the basal membranes of cells in the haemolymph space and along the microvilli of cells lining the tubular lumen. Elevated Cd concentrations were found in the cytosol, amoebocytes, Golgi vesicles and P/Ca granules in R-cells. Chronic exposure to higher concentrations of Cd (0.5 and 1.0 mg l(-1)) increased crystal deposition, whereas concentrations of Cd, copper and iron decreased in the cell membranes and in amoebocytes and increased in Golgi vesicles. Reduced lipid content, swollen nuclei with vesiculated nucleoli and enhanced activity of RER in R-cells were also noted. Cd was stored in the P/Ca and Ca granules of B-cells. Acute exposure to Cd (2.0 mg l(-1) for 48 h) caused metal granule accumulation along cells lining the tubular lumen and cellular dissociation, with acidosis and necrosis in the cytoplasm and Cd deposits in mitochondria. Cd accumulated in the cells of the digestive gland in a time-, concentration- and cell-type-specific fashion.

Identification of a chromogranin A domain that mediates binding to secretogranin III and targeting to secretory granules in pituitary cells and pancreatic beta-cells.

Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca(2+)-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48-111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214-373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells.

Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic β-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy

The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic β-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH7.4, but not at approximately pH5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin—pHluorin, time-lapse confocal laser scanning microscopy (5 or 10s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50mM KCl and 22mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin—pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.