Journal articles on the topic 'Insulin secretory granule'
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1
Hutton, J. C. "The insulin secretory granule." Diabetologia 32, no. 5 (May 1989): 271–81. http://dx.doi.org/10.1007/bf00265542.
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Tompkins, Linda S., Kevin D. Nullmeyer, Sean M. Murphy, Craig S. Weber, and Ronald M. Lynch. "Regulation of secretory granule pH in insulin-secreting cells." American Journal of Physiology-Cell Physiology 283, no. 2 (August 1, 2002): C429—C437. http://dx.doi.org/10.1152/ajpcell.01066.2000.
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Luminal acidification is important for the maturation of secretory granules, yet little is known regarding the regulation of pH within them. A pH-sensitive green fluorescent protein (EGFP) was targeted to secretory granules in RIN1046-38 insulinoma cells by using a construct in which the EGFP gene was preceded by the nucleotide sequence for human growth hormone. Stimulatory levels of glucose doubled EGFP secretion from cell cultures, and potentiators of glucose-induced insulin secretion enhanced EGFP release. Thus this targeted EGFP is useful for population measurements of secretion. However, less than ∼4% of total cell EGFP was released after 1.5 h of stimulation. Consequently, when analyzed in single cells, fluorescence of the targeted EGFP acts as an indicator of pH within secretory granules. Glucose elicited a decrease in granule pH, whereas inhibitors of the V-type H+-ATPase increased pH and blocked the glucose effect. Granule pH also was modified by effectors of the protein kinase A pathway, with activation eliciting granule alkalinization, suggesting that potentiation of peptide release by cAMP may involve regulated changes in secretory granule pH.
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Grimaldi, K. A., J. C. Hutton, and K. Siddle. "Production and characterization of monoclonal antibodies to insulin secretory granule membranes." Biochemical Journal 245, no. 2 (July 15, 1987): 557–66. http://dx.doi.org/10.1042/bj2450557.
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Monoclonal antibodies to insulin secretory granule membranes were obtained following immunization of mice with granule membranes purified from a rat transplantable insulinoma. The specificities of the antibodies were investigated by using binding assays with different insulinoma subcellular fractions, by indirect immunofluorescence studies with intact and permeabilized cells, and by immunoblotting of granule membrane proteins fractionated by SDS/polyacrylamide-gel electrophoresis. Fifty-six antibodies were characterized initially, and 21 representative cell lines were cloned. The antibodies fell into four categories: (1) binding preferentially to secretory granules, and reacting with a component of approx. 80,000 Da on immunoblots (antigen designated SGM 80); (2) binding preferentially to secretory granules, and reacting with components of approx. 110,000 and 50,000 Da on immunoblots (antigen designated SGM 110); (3) binding preferentially to secretory granules but unreactive on immunoblots; (4) binding to membrane antigen(s) with a widespread intracellular distribution which included granules and plasma membranes. The antigens SGM 80 and SGM 110 were studied in more detail and both were shown to be integral membrane glycoproteins with antigenic determinants located on the internal face of the secretory granule membrane. These antigens were also present in normal rat islets of Langerhans and similar components were detected by immunoblotting in secretory granules from anterior pituitary and adrenal medulla. Proteins which were immunologically related to SGM 80 and SGM 110, but distinct in molecular size, were also identified in liver. It is concluded that secretory granules contain specific components which are restricted in subcellular location but widespread in tissue distribution. The antibodies obtained will be valuable reagents in the further investigation of the biogenesis and turnover of insulin secretory granules.
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Germanos, Mark, Andy Gao, Matthew Taper, Belinda Yau, and Melkam A. Kebede. "Inside the Insulin Secretory Granule." Metabolites 11, no. 8 (August 5, 2021): 515. http://dx.doi.org/10.3390/metabo11080515.
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The pancreatic β-cell is purpose-built for the production and secretion of insulin, the only hormone that can remove glucose from the bloodstream. Insulin is kept inside miniature membrane-bound storage compartments known as secretory granules (SGs), and these specialized organelles can readily fuse with the plasma membrane upon cellular stimulation to release insulin. Insulin is synthesized in the endoplasmic reticulum (ER) as a biologically inactive precursor, proinsulin, along with several other proteins that will also become members of the insulin SG. Their coordinated synthesis enables synchronized transit through the ER and Golgi apparatus for congregation at the trans-Golgi network, the initiating site of SG biogenesis. Here, proinsulin and its constituents enter the SG where conditions are optimized for proinsulin processing into insulin and subsequent insulin storage. A healthy β-cell is continually generating SGs to supply insulin in vast excess to what is secreted. Conversely, in type 2 diabetes (T2D), the inability of failing β-cells to secrete may be due to the limited biosynthesis of new insulin. Factors that drive the formation and maturation of SGs and thus the production of insulin are therefore critical for systemic glucose control. Here, we detail the formative hours of the insulin SG from the luminal perspective. We do this by mapping the journey of individual members of the SG as they contribute to its genesis.
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Haddad, A., B. Kopriwa, and G. Pelletier. "Localization of glycoproteins in insulin secretory granules by ultrastructural autoradiography." Journal of Histochemistry & Cytochemistry 35, no. 10 (October 1987): 1059–62. http://dx.doi.org/10.1177/35.10.3305700.
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To determine whether or not the secretory granules of insulin-secreting cells contained glycoproteins, isolated rat pancreatic islets were incubated for 2 and 4 hr in a medium containing L-[3H]-fucose. Quantitative analysis of high-resolution electron microscopic autoradiographs of the insulin-secreting beta cells demonstrated that glycoproteins with fucose residues are contained within the insulin secretory granule.
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Waselle, Laurent, Thierry Coppola, Mitsunori Fukuda, Mariella Iezzi, Aziz El-Amraoui, Christine Petit, and Romano Regazzi. "Involvement of the Rab27 Binding Protein Slac2c/MyRIP in Insulin Exocytosis." Molecular Biology of the Cell 14, no. 10 (October 2003): 4103–13. http://dx.doi.org/10.1091/mbc.e03-01-0022.
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Rab27a is a GTPase associated with insulin-containing secretory granules of pancreatic β-cells. Selective reduction of Rab27a expression by RNA interference did not alter granule distribution and basal secretion but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors of the GTPase revealed that the Rab27a-binding protein Slac2c/MyRIP is associated with secretory granules of β-cells. Attenuation of Slac2c/MyRIP expression by RNA interference did not modify basal secretion but severely impaired hormone release in response to secretagogues. Although β-cells express Myosin-Va, a potential partner of Slac2c/MyRIP, no functional link between the two proteins could be demonstrated. In fact, overexpression of the Myosin-Va binding domain of Slac2c/MyRIP did not affect granule localization and hormone exocytosis. In contrast, overexpression of the actin-binding domain of Slac2c/MyRIP led to a potent inhibition of exocytosis without detectable alteration in granule distribution. This effect was prevented by point mutations that abolish actin binding. Taken together our data suggest that Rab27a and Slac2c/MyRIP are part of a complex mediating the interaction of secretory granules with cortical actin cytoskeleton and participate to the regulation of the final steps of insulin exocytosis.
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Brüning, Dennis, Kirstin Reckers, Peter Drain, and Ingo Rustenbeck. "Glucose but not KCl diminishes submembrane granule turnover in mouse beta-cells." Journal of Molecular Endocrinology 59, no. 3 (October 2017): 311–24. http://dx.doi.org/10.1530/jme-17-0063.
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KCl depolarization is widely used to mimic the depolarization during glucose-stimulated insulin secretion. Consequently, the insulin secretion elicited by KCl is often regarded as the equivalent of the first phase of glucose-induced insulin secretion. Here, the effects of both stimuli were compared by measuring the secretion of perifused mouse islets, the cytosolic Ca2+ concentration of single beta-cells and the mobility of submembrane insulin granules by TIRF microscopy of primary mouse beta-cells. Two cargo-directed granule labels were used namely insulin-EGFP and C-peptide-emGFP. The granule behaviour common to both was used to compare the effect of sequential stimulation with 40 mM KCl and 30 mM glucose and sequential stimulation with the same stimuli in reversed order. At the level of the cell secretory response, the sequential pulse protocol showed marked differences depending on the order of the two stimuli. KCl produced higher maximal secretion rates and diminished the response to the subsequent glucose stimulus, whereas glucose enhanced the response to the subsequent KCl stimulus. At the level of granule behaviour, a difference developed during the first stimulation phase in that the total number of granules, the short-term resident granules and the arriving granules, which are all parameters of granule turnover, were significantly smaller for glucose than for KCl. These differences at both the level of the cell secretory response and granule behaviour in the submembrane space are incompatible with identical initial response mechanisms to KCl and glucose stimulation.
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Kemter, Elisabeth, Andreas Müller, Martin Neukam, Anna Ivanova, Nikolai Klymiuk, Simone Renner, Kaiyuan Yang, et al. "Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs." Proceedings of the National Academy of Sciences 118, no. 37 (September 10, 2021): e2107665118. http://dx.doi.org/10.1073/pnas.2107665118.
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β cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of β cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo–labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.
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Yau, Belinda, Lori Hays, Cassandra Liang, D. Ross Laybutt, Helen E. Thomas, Jenny E. Gunton, Lindy Williams, et al. "A fluorescent timer reporter enables sorting of insulin secretory granules by age." Journal of Biological Chemistry 295, no. 27 (April 27, 2020): 8901–11. http://dx.doi.org/10.1074/jbc.ra120.012432.
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Within the pancreatic β-cells, insulin secretory granules (SGs) exist in functionally distinct pools, displaying variations in motility as well as docking and fusion capability. Current therapies that increase insulin secretion do not consider the existence of these distinct SG pools. Accordingly, these approaches are effective only for a short period, with a worsening of glycemia associated with continued decline in β-cell function. Insulin granule age is underappreciated as a determinant for why an insulin granule is selected for secretion and may explain why newly synthesized insulin is preferentially secreted from β-cells. Here, using a novel fluorescent timer protein, we aimed to investigate the preferential secretion model of insulin secretion and identify how granule aging is affected by variation in the β-cell environment, such as hyperglycemia. We demonstrate the use of a fluorescent timer construct, syncollin-dsRedE5TIMER, which changes its fluorescence from green to red over 18 h, in both microscopy and fluorescence-assisted organelle-sorting techniques. We confirm that the SG-targeting construct localizes to insulin granules in β-cells and does not interfere with normal insulin SG behavior. We visualize insulin SG aging behavior in MIN6 and INS1 β-cell lines and in primary C57BL/6J mouse and nondiabetic human islet cells. Finally, we separated young and old insulin SGs, revealing that preferential secretion of younger granules occurs in glucose-stimulated insulin secretion. We also show that SG population age is modulated by the β-cell environment in vivo in the db/db mouse islets and ex vivo in C57BL/6J islets exposed to different glucose environments.
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Georgiadou, Eleni, and Guy A. Rutter. "Age matters: Grading granule secretion in beta cells." Journal of Biological Chemistry 295, no. 27 (July 3, 2020): 8912–13. http://dx.doi.org/10.1074/jbc.h120.014586.
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Insulin is stored in secretory granules to facilitate rapid release in response to rising glucose levels, but the mechanisms by which these granules are identified and prioritized for secretion remains unclear. Using a fluorescent timer and flow cytometry–assisted organelle sorting, Yau et al. develop an elegant approach to assess insulin secretion as a function of granule age in pancreatic islet beta cells. Their findings supply quantitative evidence supporting the age-dependent release of different granule pools and confirm earlier models of preferential release of younger granules.
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Rohli, Kristen E., Cierra K. Boyer, Sandra E. Blom, and Samuel B. Stephens. "Nutrient Regulation of Pancreatic Islet β-Cell Secretory Capacity and Insulin Production." Biomolecules 12, no. 2 (February 20, 2022): 335. http://dx.doi.org/10.3390/biom12020335.
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Pancreatic islet β-cells exhibit tremendous plasticity for secretory adaptations that coordinate insulin production and release with nutritional demands. This essential feature of the β-cell can allow for compensatory changes that increase secretory output to overcome insulin resistance early in Type 2 diabetes (T2D). Nutrient-stimulated increases in proinsulin biosynthesis may initiate this β-cell adaptive compensation; however, the molecular regulators of secretory expansion that accommodate the increased biosynthetic burden of packaging and producing additional insulin granules, such as enhanced ER and Golgi functions, remain poorly defined. As these adaptive mechanisms fail and T2D progresses, the β-cell succumbs to metabolic defects resulting in alterations to glucose metabolism and a decline in nutrient-regulated secretory functions, including impaired proinsulin processing and a deficit in mature insulin-containing secretory granules. In this review, we will discuss how the adaptative plasticity of the pancreatic islet β-cell’s secretory program allows insulin production to be carefully matched with nutrient availability and peripheral cues for insulin signaling. Furthermore, we will highlight potential defects in the secretory pathway that limit or delay insulin granule biosynthesis, which may contribute to the decline in β-cell function during the pathogenesis of T2D.
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POULI, Aristea E., Evaggelia EMMANOUILIDOU, Chao ZHAO, Christina WASMEIER, John C. HUTTON, and Guy A. RUTTER. "Secretory-granule dynamics visualized in vivo with a phogrin–green fluorescent protein chimaera." Biochemical Journal 333, no. 1 (July 1, 1998): 193–99. http://dx.doi.org/10.1042/bj3330193.
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To image the behaviour in real time of single secretory granules in neuroendocrine cells we have expressed cDNA encoding a fusion construct between the dense-core secretory-granule-membrane glycoprotein, phogrin (phosphatase on the granule of insulinoma cells), and enhanced green fluorescent protein (EGFP). Expressed in INS-1 β-cells and pheochromocytoma PC12 cells, the chimaera was localized efficiently (up to 95%) to dense-core secretory granules (diameter 200–1000 nm), identified by co-immunolocalization with anti-(pro-)insulin antibodies in INS-1 cells and dopamine β-hydroxylase in PC12 cells. Using laser-scanning confocal microscopy and digital image analysis, we have used this chimaera to monitor the effects of secretagogues on the dynamics of secretory granules in single living cells. In unstimulated INS-1 β-cells, granule movement was confined to oscillatory movement (dithering) with period of oscillation 5–10 s and mean displacement < 1 µm. Both elevated glucose concentrations (30 mM), and depolarization of the plasma membrane with K+, provoked large (5–10 µm) saltatory excursions of granules across the cell, which were never observed in cells maintained at low glucose concentration. By contrast, long excursions of granules occurred in PC12 cells without stimulation, and occurred predominantly from the cell body towards the cell periphery and neurite extensions. Purinergic-receptor activation with ATP provoked granule movement towards the membrane of PC12 cells, resulting in the transfer of fluorescence to the plasma membrane consistent with fusion of the granule and diffusion of the chimaera in the plasma membrane. These results illustrate the potential use of phogrin–EGFP chimeras in the study of secretory-granule dynamics, the regulation of granule–cytoskeletal interactions and the trafficking of a granule-specific transmembrane protein during the cycle of exocytosis and endocytosis.
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LU, JINLING, NATALIA GUSTAVSSON, QIMING LI, GEORGE K. RADDA, THOMAS C. SÜDHOF, and WEIPING HAN. "GENERATION OF TRANSGENIC MICE FOR IN VIVO DETECTION OF INSULIN-CONTAINING GRANULE EXOCYTOSIS AND QUANTIFICATION OF INSULIN SECRETION." Journal of Innovative Optical Health Sciences 02, no. 04 (October 2009): 397–405. http://dx.doi.org/10.1142/s1793545809000711.
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Insulin secretion is a complex and highly regulated process. Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation, it remains unclear how conclusions from these studies apply to living animals. That few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in vivo. When combined with genetically encoded biosensor, optical imaging is a powerful tool for visualization of molecular events in vivo. In this study, we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins, a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the cytosol. Upon exocytosis of secretory granules, the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH, while mCherry fluorescence remained constant in the process, thus allowing ratiometric quantification of insulin secretory events. Furthermore, mCherry fluorescence enabled tracking the movement of secretory granules in living cells. We validated this approach in insulin-secreting cells, and generated a transgenic mouse line expressing the optical sensor specifically in pancreatic β-cells. The transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.
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Kuliawat, Regina, Daniel Prabakaran, and Peter Arvan. "Proinsulin Endoproteolysis Confers Enhanced Targeting of Processed Insulin to the Regulated Secretory Pathway." Molecular Biology of the Cell 11, no. 6 (June 2000): 1959–72. http://dx.doi.org/10.1091/mbc.11.6.1959.
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Recently, two different prohormone-processing enzymes, prohormone convertase 1 (PC1) and carboxypeptidase E, have been implicated in enhancing the storage of peptide hormones in endocrine secretory granules. It is important to know the extent to which such molecules may act as “sorting receptors” to allow the selective trafficking of cargo proteins from the trans-Golgi network into forming granules, versus acting as enzymes that may indirectly facilitate intraluminal storage of processed hormones within maturing granules. GH4C1 cells primarily store prolactin in granules; they lack PC1 and are defective for intragranular storage of transfected proinsulin. However, proinsulin readily enters the immature granules of these cells. Interestingly, GH4C1 clones that stably express modest levels of PC1 store more proinsulin-derived protein in granules. Even in the presence of PC1, a sizable portion of the proinsulin that enters granules goes unprocessed, and this portion largely escapes granule storage. Indeed, all of the increased granule storage can be accounted for by the modest portion converted to insulin. These results are not unique to GH4C1 cells; similar results are obtained upon PC1 expression in PC12 cells as well as in AtT20 cells (in which PC1 is expressed endogenously at higher levels). An in vitro assay of protein solubility indicates a difference in the biophysical behavior of proinsulin and insulin in the PC1 transfectants. We conclude that processing to insulin, facilitated by the catalytic activities of granule proteolytic enzymes, assists in the targeting (storage) of the hormone.
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Rizzo, Mark A., and David W. Piston. "Regulation of β cell glucokinase by S-nitrosylation and association with nitric oxide synthase." Journal of Cell Biology 161, no. 2 (April 21, 2003): 243–48. http://dx.doi.org/10.1083/jcb.200301063.
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Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic β cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured β cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic β cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.
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Norris, Nicholas, Belinda Yau, and Melkam Alamerew Kebede. "Isolation and Proteomics of the Insulin Secretory Granule." Metabolites 11, no. 5 (April 30, 2021): 288. http://dx.doi.org/10.3390/metabo11050288.
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Insulin, a vital hormone for glucose homeostasis is produced by pancreatic beta-cells and when secreted, stimulates the uptake and storage of glucose from the blood. In the pancreas, insulin is stored in vesicles termed insulin secretory granules (ISGs). In Type 2 diabetes (T2D), defects in insulin action results in peripheral insulin resistance and beta-cell compensation, ultimately leading to dysfunctional ISG production and secretion. ISGs are functionally dynamic and many proteins present either on the membrane or in the lumen of the ISG may modulate and affect different stages of ISG trafficking and secretion. Previously, studies have identified few ISG proteins and more recently, proteomics analyses of purified ISGs have uncovered potential novel ISG proteins. This review summarizes the proteins identified in the current ISG proteomes from rat insulinoma INS-1 and INS-1E cell lines. Here, we also discuss techniques of ISG isolation and purification, its challenges and potential future directions.
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Grimaldi, K. A., K. Siddle, and J. C. Hutton. "Biosynthesis of insulin secretory granule membrane proteins. Control by glucose." Biochemical Journal 245, no. 2 (July 15, 1987): 567–73. http://dx.doi.org/10.1042/bj2450567.
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The biosynthesis of a component SGM 110, specifically localized to the membrane of insulin secretory granules, was studied in rat insulinoma cells and in normal islets of Langerhans. Cells or islets were labelled with [35S]methionine or [3H]mannose and SGM 110 was immunoprecipitated by using a monoclonal antibody. Pulse-chase experiments demonstrated that the nascent polypeptide was cotranslationally glycosylated to form a 97,000 Da peptide which in turn was processed to the mature 110,000 Da form. A 50,000 Da form detected by immunoblotting with the same antibody was not conspicuously labelled even after a 20 h chase incubation, suggesting that it represented late processing of SGM 110 in lysosomes. With insulinoma cells, an increase in medium glucose concentration from 3 mM to 20 mM was without effect on the secretion of insulin or on the biosynthesis of (pro)insulin or SGM 110. In normal islets, however, 20 mM-glucose produced a 17-fold increase in (pro)insulin biosynthesis and a 13-fold increase in SGM 110 biosynthesis, compared with only a 2-fold increase in total protein synthesis, as judged by incorporation of [35S]methionine during a 1 h incubation. The effect of glucose on both (pro)insulin and SGM 110 biosynthesis was blocked by the addition of mannoheptulose, but not by the removal of extracellular calcium, both of which conditions inhibit insulin secretion. In contrast tolbutamide, an agent which stimulates insulin secretion, did not enhance the biosynthesis of (pro)insulin or SGM 110. It is concluded that at least one protein component of the insulin secretory granule membrane is synthesized co-ordinately with proinsulin and is subject to similar regulatory mechanisms. Factors which acutely control insulin secretion may also control granule biogenesis, although the two processes are not coupled in an obligatory fashion.
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Min, Le, Yuk M. Leung, Alejandra Tomas, Robert T. Watson, Herbert Y. Gaisano, Philippe A. Halban, Jeffrey E. Pessin, and June Chunqiu Hou. "Dynamin Is Functionally Coupled to Insulin Granule Exocytosis." Journal of Biological Chemistry 282, no. 46 (September 11, 2007): 33530–36. http://dx.doi.org/10.1074/jbc.m703402200.
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The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 β-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of β-cell insulin secretion.
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Bailyes, Eiaine M., Deborah L. Bennett, and John C. Hutton. "Proprotein-Processing Endopeptidases of the Insulin Secretory Granule." Enzyme 45, no. 5-6 (1991): 301–13. http://dx.doi.org/10.1159/000468903.
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Suckale, Jakob, and Michele Solimena. "The insulin secretory granule as a signaling hub." Trends in Endocrinology & Metabolism 21, no. 10 (October 2010): 599–609. http://dx.doi.org/10.1016/j.tem.2010.06.003.
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Schvartz, Domitille, Yannick Brunner, Yohann Couté, Michelangelo Foti, Claes B. Wollheim, and Jean-Charles Sanchez. "Improved characterization of the insulin secretory granule proteomes." Journal of Proteomics 75, no. 15 (August 2012): 4620–31. http://dx.doi.org/10.1016/j.jprot.2012.04.023.
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OHARA-IMAIZUMI, Mica, Yoko NAKAMICHI, Toshiaki TANAKA, Hidenori KATSUTA, Hitoshi ISHIDA, and Shinya NAGAMATSU. "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." Biochemical Journal 363, no. 1 (March 22, 2002): 73–80. http://dx.doi.org/10.1042/bj3630073.
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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.
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Mizuno, Kouichi, José S. Ramalho, and Tetsuro Izumi. "Exophilin8 transiently clusters insulin granules at the actin-rich cell cortex prior to exocytosis." Molecular Biology of the Cell 22, no. 10 (May 15, 2011): 1716–26. http://dx.doi.org/10.1091/mbc.e10-05-0404.
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Exophilin8/MyRIP/Slac2-c is an effector protein of the small GTPase Rab27a and is specifically localized on retinal melanosomes and secretory granules. We investigated the role of exophilin8 in insulin granule trafficking. Exogenous expression of exophilin8 in pancreatic β cells or their cell line, MIN6, polarized (exophilin8-positive) insulin granules at the cell corners, where both cortical actin and the microtubule plus-end–binding protein, EB1, were present. Mutation analyses indicated that the ability of exophilin8 to act as a linker between Rab27a and myosin Va is essential for its granule-clustering activity. Moreover, exophilin8 and exophilin8-associated insulin granules were markedly stable and immobile. Total internal reflection fluorescence microscopy indicated that exophilin8 restricts the motion of insulin granules at a region deeper than that where another Rab27a effector, granuphilin, accumulates docked granules directly attached to the plasma membrane. However, the exophilin8-induced immobility of insulin granules was eliminated upon secretagogue stimulation and did not inhibit evoked exocytosis. Furthermore, exophilin8 depletion prevents insulin granules from being transported close to the plasma membrane and inhibits their fusion. These findings indicate that exophilin8 transiently traps insulin granules into the cortical actin network close to the microtubule plus-ends and supplies them for release during the stimulation.
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Geng, Xuehui, Haiyan Lou, Jian Wang, Lehong Li, Alexandra L. Swanson, Ming Sun, Donna Beers-Stolz, Simon Watkins, Ruth G. Perez, and Peter Drain. "α-Synuclein binds the KATP channel at insulin-secretory granules and inhibits insulin secretion." American Journal of Physiology-Endocrinology and Metabolism 300, no. 2 (February 2011): E276—E286. http://dx.doi.org/10.1152/ajpendo.00262.2010.
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α-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic β-cells, α-synuclein might therefore play a similar role by interacting with organelles involved in insulin secretion. We tested for α-synuclein localizing to insulin-secretory granules and characterized its role in glucose-stimulated insulin secretion. Immunohistochemistry and fluorescent sulfonylureas were used to test for α-synuclein localization to insulin granules in β-cells, immunoprecipitation with Western blot analysis for interaction between α-synuclein and KATP channels, and ELISA assays for the effect of altering α-synuclein expression up or down on insulin secretion in INS1 cells or mouse islets, respectively. Differences in cellular phenotype between α-synuclein knockout and wild-type β-cells were found by using confocal microscopy to image the fluorescent insulin biosensor Ins-C-emGFP and by using transmission electron microscopy. The results show that anti-α-synuclein antibodies labeled secretory organelles within β-cells. Anti-α-synuclein antibodies colocalized with KATP channel, anti-insulin, and anti-C-peptide antibodies. α-Synuclein coimmunoprecipitated in complexes with KATP channels. Expression of α-synuclein downregulated insulin secretion at 2.8 mM glucose with little effect following 16.7 mM glucose stimulation. α-Synuclein knockout islets upregulated insulin secretion at 2.8 and 8.4 mM but not 16.7 mM glucose, consistent with the depleted insulin granule density at the β-cell surface membranes observed in these islets. These findings demonstrate that α-synuclein interacts with KATP channels and insulin-secretory granules and functionally acts as a brake on secretion that glucose stimulation can override. α-Synuclein might play similar roles in diabetes as it does in other degenerative diseases, including Alzheimer's and Parkinson's diseases.
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Kowluru, A., and S. A. Metz. "Stimulation by prostaglandin E2 of a high-affinity GTPase in the secretory granules of normal rat and human pancreatic islets." Biochemical Journal 297, no. 2 (January 15, 1994): 399–406. http://dx.doi.org/10.1042/bj2970399.
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Recent reports of a pertussis-toxin (Ptx)-sensitive inhibition of glucose-induced insulin release by prostaglandin E2 (PGE2) in transformed beta-cells prompted us to look for the presence of prostaglandin-regulatable GTP-binding proteins (G-proteins) on the secretory granules of normal pancreatic islets. PGE2 (but not PGF2 alpha, PGA2, PGB2 or PGD2) stimulated in a concentration-dependent manner a high-affinity GTPase activity in the secretory-granule-enriched fractions of both normal rat and human islets. Similar results were found after sucrose-density-gradient-centrifugation-based isolation of secretory granules to those after a differential-centrifugation procedure. Half-maximal stimulation occurred at 800 nM PGE2, a concentration known to inhibit both phases of glucose-induced insulin secretion from pure beta-cell lines. The GTPase stimulatory effect of PGE2 was blocked virtually totally by Ptx pretreatment; it was not due to an effect on substrate binding since no measurable effect of PGE2 on binding of guanosine 5′-[gamma-[35S]thio]triphosphate was observed in cognate fractions. Other Ptx-sensitive inhibitors of insulin secretion (such as adrenaline or clonidine) also stimulated GTPase activity, suggesting that one (or more) inhibitory exocytotic G-proteins (i.e. a putative GEi) is located on the secretory granules. These studies demonstrate, for the first time in an endocrine gland, the presence of a regulatable G-protein, strategically located on the secretory granules where it might regulate the exocytotic cascade distal to both plasma-membrane events and the generation of soluble mediators of insulin secretion.
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JAIKARAN, Emma T. A. S., Melanie R. NILSSON, and Anne CLARK. "Pancreatic beta-cell granule peptides form heteromolecular complexes which inhibit islet amyloid polypeptide fibril formation." Biochemical Journal 377, no. 3 (February 1, 2004): 709–16. http://dx.doi.org/10.1042/bj20030852.
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Islet amyloid polypeptide (IAPP), or ‘amylin’, is co-stored with insulin in secretory granules of pancreatic islet β-cells. In Type 2 diabetes, IAPP converts into a β-sheet conformation and oligomerizes to form amyloid fibrils and islet deposits. Granule components, including insulin, inhibit spontaneous IAPP fibril formation in vitro. To determine the mechanism of this inhibition, molecular interactions of insulin with human IAPP (hIAPP), rat IAPP (rIAPP) and other peptides were examined using surface plasmon resonance (BIAcore), CD and transmission electron microscopy (EM). hIAPP and rIAPP complexed with insulin, and this reaction was concentration-dependent. rIAPP and insulin, but not pro-insulin, bound to hIAPP. Insulin with a truncated B-chain, to prevent dimerization, also bound hIAPP. In the presence of insulin, hIAPP did not spontaneously develop β-sheet secondary structure or form fibrils. Insulin interacted with pre-formed IAPP fibrils in a regular repeating pattern, as demonstrated by immunoEM, suggesting that the binding sites for insulin remain exposed in hIAPP fibrils. Since rIAPP and hIAPP form complexes with insulin (and each other), this could explain the lack of amyloid fibrils in transgenic mice expressing hIAPP. It is likely that IAPP fibrillogenesis is inhibited in secretory granules (where the hIAPP concentration is in the millimolar range) by heteromolecular complex formation with insulin. Alterations in the proportions of insulin and IAPP in granules could disrupt the stability of the peptide. The increase in the proportion of unprocessed pro-insulin produced in Type 2 diabetes could be a major factor in destabilization of hIAPP and induction of fibril formation.
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Matz, Magnus, Kirstin Schumacher, Kathrin Hatlapatka, Dirk Lorenz, Knut Baumann, and Ingo Rustenbeck. "Observer-Independent Quantification of Insulin Granule Exocytosis and Pre-Exocytotic Mobility by TIRF Microscopy." Microscopy and Microanalysis 20, no. 1 (November 13, 2013): 206–18. http://dx.doi.org/10.1017/s1431927613013767.
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AbstractTotal internal reflection fluorescence microscopy of fluorescently labeled secretory granules permits monitoring of exocytosis and the preceding granule behavior in one experiment. While observer-dependent evaluation may be sufficient to quantify exocytosis, most of the other information contained in the video files cannot be accessed this way. The present program performs observer-independent detection of exocytosis and tracking of the entire submembrane population of insulin granules. A precondition is the exact localization of the peak of the granule fluorescence. Tracking is based on the peak base radius, peak intensity, and the precrossing itineraries. Robustness of the tracking was shown by simulated tracks of original granule patterns. Mobility in the X–Y dimension is described by the caging diameter which in contrast to the widely used mean square displacement has an inherent time resolution. Observer-independent detection of exocytosis in MIN6 cells labeled with insulin-EGFP is based on the maximal decrease in fluorescence intensity and position of the centroid of the dissipating cloud of released material. Combining the quantification of KCl-induced insulin exocytosis with the analysis of prefusion mobility showed that during the last 3 s pre-exocytotic granules had a smaller caging diameter than control granules and that it increased significantly immediately before fusion.
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Huang, X. F., and P. Arvan. "Formation of the insulin-containing secretory granule core occurs within immature beta-granules." Journal of Biological Chemistry 269, no. 33 (August 1994): 20838–44. http://dx.doi.org/10.1016/s0021-9258(17)31898-7.
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Yin, Peng, Nikhil R. Gandasi, Swati Arora, Muhmmad Omar-Hmeadi, Jan Saras, and Sebastian Barg. "Syntaxin clusters at secretory granules in a munc18-bound conformation." Molecular Biology of the Cell 29, no. 22 (November 2018): 2700–2708. http://dx.doi.org/10.1091/mbc.e17-09-0541.
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Syntaxin (stx)-1 is an integral plasma membrane protein that is crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the plasma membrane and membrane fusion. During docking, stx1 clusters at the granule docking site, together with the S/M protein munc18. Here we determined features of stx1 that contribute to its clustering at granules. In live insulin-secreting cells, stx1 and stx3 (but not stx4 or stx11) accumulated at docked granules, and stx1 (but not stx4) rescued docking in cells expressing botulinum neurotoxin-C. Using a series of stx1 deletion mutants and stx1/4 chimeras, we found that all four helical domains (Ha, Hb, Hc, SNARE) and the short N-terminal peptide contribute to recruitment to granules. However, only the Hc domain confers specificity, and it must be derived from stx1 for recruitment to occur. Point mutations in the Hc or the N-terminal peptide designed to interfere with binding to munc18-1 prevent stx1 from clustering at granules, and a mutant munc18 deficient in binding to stx1 does not cluster at granules. We conclude that stx1 is recruited to the docking site in a munc18-1–bound conformation, providing a rationale for the requirement for both proteins for granule docking.
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WASMEIER, Christina, and John C. HUTTON. "Secretagogue-dependent phosphorylation of phogrin, an insulin granule membrane protein tyrosine phosphatase homologue." Biochemical Journal 341, no. 3 (July 26, 1999): 563–69. http://dx.doi.org/10.1042/bj3410563.
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Phogrin, a 60/64 kDa integral membrane protein localized to dense-core secretory granules of neuroendocrine cells, was found to be reversibly phosphorylated in intact pancreatic β-cells. Phosphorylation occurred in response to a variety of secretory stimuli, including glucose and depolarizing concentrations of K+. In MIN6 cells, the glucose dose-response and time course of phogrin phosphorylation paralleled that of insulin secretion. Like secretion, glucose- or K+-stimulated phosphorylation required the presence of Ca2+. The calmodulin antagonist W-7 and the Ca2+/calmodulin-dependent kinase II inhibitor KN-93 dose-dependently inhibited both phosphorylation and secretion, while the ‘inactive’ analogue KN-92 was effective only at significantly higher concentrations. Phosphorylation of phogrin was also stimulated in cells exposed to forskolin, an effect presumably mediated by protein kinase A (cAMP-dependent protein kinase). Under these conditions, phogrin phosphorylation could be dissociated from the secretory response. In MIN6 cells, as in pancreatic islets, cAMP potentiates rather than initiates insulin release. Thus our observations are consistent with a role for phogrin phosphorylation in the signal-transduction pathway at a site proximal to the exocytic event itself, possibly regulating secretory-granule mobilization and recruitment to the exocytic site.
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Mosedale, Merrie, Sonya Egodage, Rei C. Calma, Nai-Wen Chi, and Steven D. Chessler. "Neurexin-1α Contributes to Insulin-containing Secretory Granule Docking." Journal of Biological Chemistry 287, no. 9 (January 10, 2012): 6350–61. http://dx.doi.org/10.1074/jbc.m111.299081.
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Knoch, Klaus-Peter, Hendrik Bergert, Barbara Borgonovo, Hans-Detlev Saeger, Anke Altkrüger, Paul Verkade, and Michele Solimena. "Polypyrimidine tract-binding protein promotes insulin secretory granule biogenesis." Nature Cell Biology 6, no. 3 (February 22, 2004): 207–14. http://dx.doi.org/10.1038/ncb1099.
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Hutton, J. C. "Insulin secretory granule biogenesis and the proinsulin-processing endopeptidases." Diabetologia 37, S2 (September 1994): S48—S56. http://dx.doi.org/10.1007/bf00400826.
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Kuliawat, R., and P. Arvan. "Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells." Journal of Cell Biology 126, no. 1 (July 1, 1994): 77–86. http://dx.doi.org/10.1083/jcb.126.1.77.
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In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C-peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus-dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.
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Fan, Fan, Yumei Wu, Manami Hara, Adam Rizk, Chen Ji, Dan Nerad, Natalia Tamarina, and Xuelin Lou. "Dynamin deficiency causes insulin secretion failure and hyperglycemia." Proceedings of the National Academy of Sciences 118, no. 32 (August 6, 2021): e2021764118. http://dx.doi.org/10.1073/pnas.2021764118.
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Pancreatic β cells operate with a high rate of membrane recycling for insulin secretion, yet endocytosis in these cells is not fully understood. We investigate this process in mature mouse β cells by genetically deleting dynamin GTPase, the membrane fission machinery essential for clathrin-mediated endocytosis. Unexpectedly, the mice lacking all three dynamin genes (DNM1, DNM2, DNM3) in their β cells are viable, and their β cells still contain numerous insulin granules. Endocytosis in these β cells is severely impaired, resulting in abnormal endocytic intermediates on the plasma membrane. Although insulin granules are abundant, their release upon glucose stimulation is blunted in both the first and second phases, leading to hyperglycemia and glucose intolerance in mice. Dynamin triple deletion impairs insulin granule exocytosis and decreases intracellular Ca2+ responses and granule docking. The docking defect is correlated with reduced expression of Munc13-1 and RIM1 and reorganization of cortical F-actin in β cells. Collectively, these findings uncover the role of dynamin in dense-core vesicle endocytosis and secretory capacity. Insulin secretion deficiency in the absence of dynamin-mediated endocytosis highlights the risk of impaired membrane trafficking in endocrine failure and diabetes pathogenesis.
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Davidson, H. W., and J. C. Hutton. "The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing." Biochemical Journal 245, no. 2 (July 15, 1987): 575–82. http://dx.doi.org/10.1042/bj2450575.
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A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.
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Barg, Sebastian, Ping Huang, Lena Eliasson, Deborah J. Nelson, Stefanie Obermüller, Patrik Rorsman, Frank Thévenod, and Erik Renström. "Priming of insulin granules for exocytosis by granular Cl− uptake and acidification." Journal of Cell Science 114, no. 11 (June 1, 2001): 2145–54. http://dx.doi.org/10.1242/jcs.114.11.2145.
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ATP-dependent priming of the secretory granules precedes Ca2+-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H+-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl− uptake through granular ClC-3 Cl− channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl− fluxes, such as 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca2+-dependent exocytosis that is also likely to be operational in other secretory cell types.
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POULI, E. Aristea, Nedim KARAGENC, Christina WASMEIER, C. John HUTTON, Nick BRIGHT, Sue ARDEN, J. George SCHOFIELD, and A. Guy RUTTER. "A phogrin–aequorin chimaera to image free Ca2+ in the vicinity of secretory granules." Biochemical Journal 330, no. 3 (March 15, 1998): 1399–404. http://dx.doi.org/10.1042/bj3301399.
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Microdomains of high Ca2+ concentration ([Ca2+]) may be critical to the control of intracellular processes such as secretion and metabolism without compromising other cell functions. To explore changes in [Ca2+] in the outer mantle (< 30 nm deep) that surrounds the surface of dense-core secretory granules, we have designed a recombinant chimaera between the granule protein phogrin and aequorin. When expressed in populations of insulin-secreting MIN6 or phaeochromocytoma PC12 cells, the chimaera was targeted to secretory granules as expected. The recombinant protein reported a similar [Ca2+] at the granule surface to that in the bulk cytosol, measured with untargeted aequorin. This was the case both at rest ([Ca2+] = 80-120 nM) and after stimulation with agents that provoke Ca2+ entry or Ca2+ mobilization from intracellular pools, and during activated secretion. Thus depolarization of MIN6 cell populations with high K+ increased [Ca2+] both in the bulk cytosol and close to the granules to approx. 4 μM, with near-identical kinetics of increase and recovery. Similarly, stimulation of PC12 cells with ATP provoked an increase in [Ca2+] in either domain to 1.3 μM. These data argue that, in MIN6 and PC12 neuroendocrine cells (i) significant mobilization of Ca2+ from most secretory granules probably does not occur during activated Ca2+ influx or mobilization of internal Ca2+ stores, and (ii) agonist-stimulated Ca2+-dependent secretion can occur without development of a large gradient of [Ca2+] between the surface of most secretory vesicles and the rest of the cytosol.
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Kuliawat, R., and P. Arvan. "Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment." Journal of Cell Biology 118, no. 3 (August 1, 1992): 521–29. http://dx.doi.org/10.1083/jcb.118.3.521.
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We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C-peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Arvan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. J. Biol. Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (t1/2 approximately equal to 1.5 h) compared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20 degrees C temperature block reveals a t1/2 less than or equal to 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway.
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Ito, Seiki, Toshimitsu Suzuki, Tooru Izumi, Takeshi Momotsu, Satoko Isemura, Eiichi Saitoh, Kazuo Sanada, and Akira Shibata. "Intracellular localization of salivary peptide P-C-like immunoreactivity in the human pancreatic B-cells." Acta Endocrinologica 108, no. 1 (January 1985): 119–29. http://dx.doi.org/10.1530/acta.0.1080119.
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Abstract. In order to clarify the intracellular localization of salivary peptide P-C-like immunoreactivity in human pancreatic B-cells, an immunohistochemical study at electron microscopic levels was carried out by the protein A-gold technique using antisera against insulin and salivary peptide P-C. Both salivary peptide P-C-like immunoreactivity and insulin-like immunoreactivity were present only in the insulin secretory granules of the pancreatic B-cells. However, the former immunoreactivity was lacking in many insulin secretory granules of foetal pancreatic B-cells while the latter immunoreactivity was seen in all insulin secretory granules. Salivary peptide P-C-like immunoreactivity was not found in the other kinds of cells in the islets. In a previous immunohistochemical study at light microscopic level, salivary peptide P-C-like immunoreactivity appeared in a few pancreatic B-cells at about the 16th week of gestation, in an increasing number during gestation, and was seen in all pancreatic B-cells a few months after birth. The present finding together with the above results suggest that absence of salivary peptide P-C-like immunoreactivity in some foetal pancreatic B-cells may be due to the underdevelopment of salivary peptide P-C-like immunoreactivity in each insulin secretory granule. From the examination of cross-reactivity of antisera against salivary peptide P-C to other kinds of salivary peptides and salivary Protein C, and from the results of an indirect immunofluorescence technique using three kinds of antisera including antisera against salivary peptide P-C, salivary peptide P-B and salivary Protein C, it was thought that salivary peptide P-C-like immunoreactivity in human pancreatic B-cells belongs neither to salivary Protein C nor to salivary peptide P-B nor to salivary peptide P-E, but either to salivary peptide P-C itself or to an unknown substance which has common antigenic determinants with salivary peptide P-C, salivary peptide P-B and salivary Protein C. Salivary peptide P-C-like immunoreactivity was not found in the pancreatic B-cells of other mammals. Thus, although a new substance other than insulin is present in the insulin secretory granules of the human pancreatic B-cells, its pathophysiological function remains unclear.
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Rhodes, C. J., B. A. Thorne, B. Lincoln, E. Nielsen, J. C. Hutton, and G. Thomas. "Processing of proopiomelanocortin by insulin secretory granule proinsulin processing endopeptidases." Journal of Biological Chemistry 268, no. 6 (February 1993): 4267–75. http://dx.doi.org/10.1016/s0021-9258(18)53605-x.
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Chang, Jennie C. C., Louie G. Linarelli, Julie A. Laxer, Karen J. Froning, Lisa L. Caralli, Steven W. Brostoff, and Dennis J. Carlo. "Insulin-Secretory-Granule Specific T Cell Clones in Human IDDM." Journal of Autoimmunity 8, no. 2 (April 1995): 221–34. http://dx.doi.org/10.1006/jaut.1995.0017.
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Pedersen, Morten Gram, Alessia Tagliavini, and Jean-Claude Henquin. "Calcium signaling and secretory granule pool dynamics underlie biphasic insulin secretion and its amplification by glucose: experiments and modeling." American Journal of Physiology-Endocrinology and Metabolism 316, no. 3 (March 1, 2019): E475—E486. http://dx.doi.org/10.1152/ajpendo.00380.2018.
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Glucose-stimulated insulin secretion from pancreatic β-cells is controlled by a triggering pathway that culminates in calcium influx and regulated exocytosis of secretory granules, and by a less understood amplifying pathway that augments calcium-induced exocytosis. In response to an abrupt increase in glucose concentration, insulin secretion exhibits a first peak followed by a lower sustained second phase. This biphasic secretion pattern is disturbed in diabetes. It has been attributed to depletion and subsequent refilling of a readily releasable pool of granules or to the phasic cytosolic calcium dynamics induced by glucose. Here, we apply mathematical modeling to experimental data from mouse islets to investigate how calcium and granule pool dynamics interact to control dynamic insulin secretion. Experimental calcium traces are used as inputs in three increasingly complex models of pool dynamics, which are fitted to insulin secretory patterns obtained using a set of protocols of glucose and tolbutamide stimulation. New calcium and secretion data for so-called staircase protocols, in which the glucose concentration is progressively increased, are presented. These data can be reproduced without assuming any heterogeneity in the model, in contrast to previous modeling, because of nontrivial calcium dynamics. We find that amplification by glucose can be explained by increased mobilization and priming of granules. Overall, our results indicate that calcium dynamics contribute substantially to shaping insulin secretion kinetics, which implies that better insight into the events creating phasic calcium changes in human β-cells is needed to understand the cellular mechanisms that disturb biphasic insulin secretion in diabetes.
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Thévenod, Frank. "Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins." American Journal of Physiology-Cell Physiology 283, no. 3 (September 1, 2002): C651—C672. http://dx.doi.org/10.1152/ajpcell.00600.2001.
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Regulated secretion in exocrine and neuroendocrine cells occurs through exocytosis of secretory granules and the subsequent release of stored small molecules and proteins. The introduction of biophysical techniques with high temporal and spatial resolution, and the identification of Ca2+-dependent and -independent “docking” and “fusion” proteins, has greatly enhanced our understanding of exocytosis. The cloning of families of ion channel proteins, including intracellular ion channels, has also revived interest in the role of secretory granule ion channels in exocytotic secretion. Thus secretory granules of pancreatic acinar cell express a ClC-2 Cl−channel, a HCO[Formula: see text]-permeable member of the CLCA Ca2+-dependent anion channel family, and a KCNQ1 K+channel. Evidence suggests that these channels may facilitate the release of digestive enzymes and/or prevent exocytosed granules from collapsing during “kiss and run” recycling. In pancreatic β-cells, a granular ClC-3 Cl−channel provides a shunt pathway for a vacuolar-type H+-ATPase. Acidification “primes” the granules for Ca2+-dependent exocytosis and release of insulin. In summary, secretory granules are equipped with specific sets of ion channels, which modulate regulated exocytosis and the release of macromolecules. These channels could represent excellent targets for therapeutic interventions to control exocytotic secretion in relevant diseases, such as pancreatitis, cystic fibrosis, or diabetes mellitus.
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Saccomanno, K., G. Taccagni, E. Bosi, P. Preti, N. Dozio, and A. Cantaboni. "Immunoelectron microscopy: a new method for detection of insulin antibodies." Journal of Histochemistry & Cytochemistry 41, no. 8 (August 1993): 1233–39. http://dx.doi.org/10.1177/41.8.8331287.
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The aim of the present study was to set up a sensitive technical alternative to the classical procedures for detection of human insulin antibodies. We developed a method of post-embedding immunoelectron microscopy (IEM) using as the substrate fresh human pancreas, embedded in acrylic resin to maintain its antigenic structure. The antigen was insulin within the mature secretory granule. Serum samples obtained from 10 patients with insulin antibodies detected at various titers by either radio binding assay (RBA) or enzymatic immunoassay (EIA) were incubated for 2 hr at 37 degrees C at dilutions of 1:25, 1:100, 1:400, 1:1600, 1:6400, 1:25,600, and 1:102,400. The electron microscope photographs were analyzed by computerized morphometry and the number of protein A-gold-IgG complexes was calculated per micron2 of insulin granule. IEM results were compared with those obtained with EIA. The specificity of both techniques towards insulin was assessed as the difference between the signals (number of gold particles per micron2 of insulin granule in IEM or optical density > or = 0.193 in EIA) with and without excess insulin. Sensitivity was defined as the detection limit of the assay. In all the 10 sera investigated, IEM was more sensitive, with a 12- to 40-fold lower detection limit than EIA. IEM, with native insulin granules as substrate, is a specific, reproducible, and sensitive method for detection of human serum insulin antibodies. These findings also suggest IEM as a procedure potentially suitable for identifying antigen specificity of autoantibodies circulating at low concentration.
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Binger, Katrina J., Martin Neukam, Sudhir Gopal Tattikota, Fatimunnisa Qadri, Dmytro Puchkov, Diana M. Willmes, Sabrina Wurmsee, et al. "Atp6ap2 deletion causes extensive vacuolation that consumes the insulin content of pancreatic β cells." Proceedings of the National Academy of Sciences 116, no. 40 (September 16, 2019): 19983–88. http://dx.doi.org/10.1073/pnas.1903678116.
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Pancreatic β cells store insulin within secretory granules which undergo exocytosis upon elevation of blood glucose levels. Crinophagy and autophagy are instead responsible to deliver damaged or old granules to acidic lysosomes for intracellular degradation. However, excessive consumption of insulin granules can impair β cell function and cause diabetes. Atp6ap2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy. Here, we show that Cre recombinase-mediated conditional deletion of Atp6ap2 in mouse β cells causes a dramatic accumulation of large, multigranular vacuoles in the cytoplasm, with reduction of insulin content and compromised glucose homeostasis. Loss of insulin stores and gigantic vacuoles were also observed in cultured insulinoma INS-1 cells upon CRISPR/Cas9-mediated removal of Atp6ap2. Remarkably, these phenotypic alterations could not be attributed to a deficiency in autophagy or acidification of lysosomes. Together, these data indicate that Atp6ap2 is critical for regulating the stored insulin pool and that a balanced regulation of granule turnover is key to maintaining β cell function and diabetes prevention.
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Torrejón-Escribano, Benjamín, Jessica Escoriza, Eduard Montanya, and Juan Blasi. "Glucose-Dependent Changes in SNARE Protein Levels in Pancreatic β-Cells." Endocrinology 152, no. 4 (February 1, 2011): 1290–99. http://dx.doi.org/10.1210/en.2010-0898.
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Abstract Prolonged exposure to high glucose concentration alters the expression of a set of proteins in pancreatic β-cells and impairs their capacity to secrete insulin. The cellular and molecular mechanisms that lie behind this effect are poorly understood. In this study, three either in vitro or in vivo models (cultured rat pancreatic islets incubated in high glucose media, partially pancreatectomized rats, and islets transplanted to streptozotozin-induced diabetic mice) were used to evaluate the dependence of the biological model and the treatment, together with the cell location (insulin granule or plasma membrane) of the affected proteins and the possible effect of sustained insulin secretion, on the glucose-induced changes in protein expression. In all three models, islets exposed to high glucose concentrations showed a reduced expression of secretory granule-associated vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins synaptobrevin/vesicle-associated membrane protein 2 and cellubrevin but minor or no significant changes in the expression of the membrane-associated target-SNARE proteins syntaxin1 and synaptosomal-associated protein-25 and a marked increase in the expression of synaptosomal-associated protein-23 protein. The inhibition of insulin secretion by the L-type voltage-dependent calcium channel nifedipine or the potassium channel activator diazoxide prevented the glucose-induced reduction in islet insulin content but not in vesicle-SNARE proteins, indicating that the granule depletion due to sustained exocytosis was not involved in the changes of protein expression induced by high glucose concentration. Altogether, the results suggest that high glucose has a direct toxic effect on the secretory pathway by decreasing the expression of insulin granule SNARE-associated proteins.
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Wang, Hao, Ray Ishizaki, Jun Xu, Kazuo Kasai, Eri Kobayashi, Hiroshi Gomi, and Tetsuro Izumi. "The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma membrane." Molecular Biology of the Cell 24, no. 3 (February 2013): 319–30. http://dx.doi.org/10.1091/mbc.e12-04-0265.
Full textAbstract:
Granuphilin, an effector of the small GTPase Rab27a, mediates the stable attachment (docking) of insulin granules to the plasma membrane and inhibits subsequent fusion of docked granules, possibly through interaction with a fusion-inhibitory Munc18-1/syntaxin complex. However, phenotypes of insulin exocytosis differ considerably between Rab27a- and granuphilin-deficient pancreatic β cells, suggesting that other Rab27a effectors function in those cells. We found that one of the putative Rab27a effector family proteins, exophilin7/JFC1/Slp1, is expressed in β cells; however, unlike granuphilin, exophilin7 overexpressed in the β-cell line MIN6 failed to show granule-docking or fusion-inhibitory activity. Furthermore, exophilin7 has no affinities to either Munc18-1 or Munc18-1–interacting syntaxin-1a, in contrast to granuphilin. Although β cells of exophilin7-knockout mice show no apparent abnormalities in intracellular distribution or in ordinary glucose-induced exocytosis of insulin granules, they do show impaired fusion in response to some stronger stimuli, specifically from granules that have not been docked to the plasma membrane. Exophilin7 appears to mediate the fusion of undocked granules through the affinity of its C2A domain toward the plasma membrane phospholipids. These findings indicate that the two Rab27a effectors, granuphilin and exophilin7, differentially regulate the exocytosis of either stably or minimally docked granules, respectively.
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Guest, P. C., C. J. Rhodes, and J. C. Hutton. "Regulation of the biosynthesis of insulin-secretory-granule proteins. Co-ordinate translational control is exerted on some, but not all, granule matrix constituents." Biochemical Journal 257, no. 2 (January 15, 1989): 431–37. http://dx.doi.org/10.1042/bj2570431.
Full textAbstract:
The regulation of the biosynthesis of the insulin-secretory-granule matrix proteins insulin II, chromogranin A and carboxypeptidase H was studied in isolated rat islets of Langerhans. Islets were labelled with [35S]-methionine, and incorporation into total protein was determined by trichloroacetic acid precipitation and that into specific proteins by immunoprecipitation followed by polyacrylamide-gel electrophoresis and fluorography. Islets incubated in the presence of 16.7 mM-glucose incorporated 3 times as much [35S]-methionine into total protein as did islets incubated with 2.8 mM-glucose. The same conditions produced more than a 20-fold increase in incorporation into both proinsulin and chromogranin A, with no observable effect on carboxypeptidase H. The concentration-dependencies of the glucose-stimulated synthesis of chromogranin A and proinsulin were parallel, and in both cases the response to 16.7 mM-glucose was typified by an initial lag of 20 min, followed by a rapid activation to a new steady state over the ensuing 40 min. Synthesis of total protein, although activated to a lesser extent, responded with similar kinetics. Extracellular Ca2+ depletion did not affect the basal or glucose-stimulated biosynthesis of any of the proteins under investigation. Mannoheptulose (20 mM) abolished glucose-stimulated synthesis of insulin, chromogranin A and total protein, but had no effect on the synthesis of carboxypeptidase H. It is concluded that the biosynthesis of insulin and chromogranin A is regulated principally at the translational level by the same intracellular signal generated from the metabolism of glucose. Such regulation is not common to all insulin-secretory-granule proteins, since the synthesis of carboxypeptidase H was unaffected by the same stimulus.
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Iida, Yuji, Takao Senda, Yoshihisa Matsukawa, Koji Onoda, Jun-Ichi Miyazaki, Hiromi Sakaguchi, Yuji Nimura, Hiroyoshi Hidaka, and Ichiro Niki. "Myosin light-chain phosphorylation controls insulin secretion at a proximal step in the secretory cascade." American Journal of Physiology-Endocrinology and Metabolism 273, no. 4 (October 1, 1997): E782—E789. http://dx.doi.org/10.1152/ajpendo.1997.273.4.e782.
Full textAbstract:
The aim of this study was to investigate how insulin secretion is controlled by phosphorylation of the myosin light chain (MLC). Ca2+-evoked insulin release from pancreatic islets permeabilized with streptolysin O was inhibited by different monoclonal antibodies against myosin light-chain kinase (MLCK) to an extent parallel to their inhibition of purified MLCK. Anti-MLCK antibody also inhibited insulin release caused by the stable GTP analog guanosine 5′- O-(3-thiodiphosphate), even at a substimulatory concentration (0.1 μM) of Ca2+. Free Ca2+ increased MLC peptide phosphorylation by β-cell extracts in vitro. In contrast to the phosphorylation by purified MLCK or by calmodulin (CaM) kinase II, the activity partially remained with the β-cell under nonstimulatory Ca2+ (0.1 μM) conditions. The MLCK inhibitor ML-9 inhibited the activity in the β-cell with both substimulatory and stimulatory Ca2+, whereas KN-62, an inhibitor of CaM kinase II, only exerted an influence in the latter case. ML-9 decreased intracellular granule movement in MIN6 cells under basal and acetylcholine-stimulated conditions. We propose that MLC phosphorylation may modulate translocation of secretory granules, resulting in enhanced insulin secretion.