scholarly journals Imaging analysis reveals mechanistic differences between first- and second-phase insulin exocytosis

2007 ◽  
Vol 177 (4) ◽  
pp. 695-705 ◽  
Author(s):  
Mica Ohara-Imaizumi ◽  
Tomonori Fujiwara ◽  
Yoko Nakamichi ◽  
Tadashi Okamura ◽  
Yoshihiro Akimoto ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Second Phase ◽  
Imaging Analysis ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Biphasic Insulin ◽  
Insulin Granules ◽  
Biphasic Insulin Release ◽  
Two Phases ◽  
Insulin Exocytosis

The mechanism of glucose-induced biphasic insulin release is unknown. We used total internal reflection fluorescence (TIRF) imaging analysis to reveal the process of first- and second-phase insulin exocytosis in pancreatic β cells. This analysis showed that previously docked insulin granules fused at the site of syntaxin (Synt)1A clusters during the first phase; however, the newcomers fused during the second phase external to the Synt1A clusters. To reveal the function of Synt1A in phasic insulin exocytosis, we generated Synt1A-knockout (Synt1A−/−) mice. Synt1A−/− β cells showed fewer previously docked granules with no fusion during the first phase; second-phase fusion from newcomers was preserved. Rescue experiments restoring Synt1A expression demonstrated restoration of granule docking status and fusion events. Inhibition of other syntaxins, Synt3 and Synt4, did not affect second-phase insulin exocytosis. We conclude that the first phase is Synt1A dependent but the second phase is not. This indicates that the two phases of insulin exocytosis differ spatially and mechanistically.

scholarly journals ELKS, a Protein Structurally Related to the Active Zone-associated Protein CAST, Is Expressed in Pancreatic β Cells and Functions in Insulin Exocytosis: Interaction of ELKS with Exocytotic Machinery Analyzed by Total Internal Reflection Fluorescence Microscopy

2005 ◽  
Vol 16 (7) ◽  
pp. 3289-3300 ◽  
Author(s):  
Mica Ohara-Imaizumi ◽  
Toshihisa Ohtsuka ◽  
Satsuki Matsushima ◽  
Yoshihiro Akimoto ◽  
Chiyono Nishiwaki ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Active Zone ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Insulin Granules ◽  
Insulin Exocytosis

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic β cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic β cells.

scholarly journals The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma membrane

2013 ◽  
Vol 24 (3) ◽  
pp. 319-330 ◽  
Author(s):  
Hao Wang ◽  
Ray Ishizaki ◽  
Jun Xu ◽  
Kazuo Kasai ◽  
Eri Kobayashi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Knockout Mice ◽  
Secretory Granules ◽  
Small Gtpase ◽  
Membrane Phospholipids ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Insulin Granules ◽  
Insulin Exocytosis

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.

scholarly journals Exocytosis from pancreatic β-cells: mathematical modelling of the exit of low-molecular-weight granule content

2010 ◽  
Vol 1 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Juris Galvanovskis ◽  
Matthias Braun ◽  
Patrik Rorsman
Keyword(s):  
Molecular Weight ◽  
Membrane Receptors ◽  
Fusion Pore ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Release Process ◽  
Cell Plasma ◽  
Insulin Granules ◽  
Large Dense Core Vesicles ◽  
Insulin Exocytosis

Pancreatic β-cells use Ca 2+ -dependent exocytosis of large dense core vesicles to release insulin. Exocytosis in β-cells has been studied biochemically, biophysically and optically. We have previously developed a biophysical method to monitor release of endogenous intragranular constituents that are co-released with insulin. This technique involves the expression of ionotropic membrane receptors in the β-cell plasma membrane and enables measurements of exocytosis of individual vesicles with sub-millisecond resolution. Like carbon fibre amperometry, this method allows fine details of the release process, like the expansion of the fusion pore (the narrow connection between the granule lumen and the extracellular space), to be monitored. Here, we discuss experimental data obtained with this method within the framework of a simple mathematical model that describes the release of low-molecular constituents during exocytosis of the insulin granules. Our findings suggest that the fusion pore functions as a molecular sieve, allowing differential release of low- and high-molecular-weight granule constituents.

scholarly journals TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic β-cells: different behaviour of granule motion between normal and Goto–Kakizaki diabetic rat β-cells

2004 ◽  
Vol 381 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Mica OHARA-IMAIZUMI ◽  
Chiyono NISHIWAKI ◽  
Toshiteru KIKUTA ◽  
Shintaro NAGAI ◽  
Yoko NAKAMICHI ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Release ◽  
Secretory Granules ◽  
Dynamic Change ◽  
Second Phase ◽  
Β Cells ◽  
Gk Rat ◽  
Pancreatic Β Cells ◽  
Insulin Granules ◽  
Granule Motion

We imaged and analysed the motion of single insulin secretory granules near the plasma membrane in live pancreatic β-cells, from normal and diabetic Goto–Kakizaki (GK) rats, using total internal reflection fluorescence microscopy (TIRFM). In normal rat primary β-cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from ‘newcomers’. In diabetic GK rat β-cells, the number of fusion events from previously docked granules were markedly reduced, and, in contrast, the fusion from newcomers was still preserved. The dynamic change in the number of docked insulin granules showed that, in GK rat β-cells, the total number of docked insulin granules was markedly decreased to 35% of the initial number after glucose stimulation. Immunohistochemistry with anti-insulin antibody observed by TIRFM showed that GK rat β-cells had a marked decline of endogenous insulin granules docked to the plasma membrane. Thus our results indicate that the decreased number of docked insulin granules accounts for the impaired insulin release during the first phase of insulin release in diabetic GK rat β-cells.

scholarly journals Glinide, but Not Sulfonylurea, Can Evoke Insulin Exocytosis by Repetitive Stimulation: Imaging Analysis of Insulin Exocytosis by Secretagogue-Induced Repetitive Stimulations

2009 ◽  
Vol 2009 ◽  
pp. 1-6 ◽  
Author(s):  
Kyota Aoyagi ◽  
Mica Ohara-Imaizumi ◽  
Chiyono Nishiwaki ◽  
Yoko Nakamichi ◽  
Shinya Nagamatsu
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
Total Internal Reflection ◽  
Repetitive Stimulation ◽  
Internal Reflection ◽  
Imaging Analysis ◽  
Tirf Microscopy ◽  
Insulin Granules ◽  
Insulin Exocytosis ◽  
Secondary Stimulation

To investigate the different effects between sulfonylurea (SU) and glinide drugs in insulin secretion, pancreaticβ-cells were repeatedly stimulated with SU (glimepiride) or glinide (mitiglinide). Total internal reflection fluorescent (TIRF) microscopy revealed that secondary stimulation with glimepiride, but not glucose and mitiglinide, failed to evoke fusions of insulin granules although primary stimulation with glucose, glimepiride, and mitiglinide induced equivalent numbers of exocytotic responses. Glimepiride, but not glucose and mitiglinide, induced abnormally sustained[Ca2+]ielevations and reductions of docked insulin granules on the plasma membrane. Our data suggest that the effect of glinide on insulin secretory mechanisms is similar to that of glucose.

Conditional expression of the FTO gene product in rat INS-1 cells reveals its rapid turnover and a role in the profile of glucose-induced insulin secretion

2011 ◽  
Vol 120 (9) ◽  
pp. 403-413 ◽  
Author(s):  
Mark A. Russell ◽  
Noel G. Morgan
Keyword(s):  
Insulin Secretion ◽  
Expression System ◽  
Proteasome Inhibitors ◽  
Inducible Promoter ◽  
Insulin Biosynthesis ◽  
Second Phase ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Peripheral Tissues ◽  
Conditional Expression

Common polymorphisms within the FTO (fat mass and obesity-associated) gene correlate with increased BMI (body mass index) and a rising risk of Type 2 diabetes. FTO is highly expressed in the brain but has also been detected in peripheral tissues, including the endocrine pancreas, although its function there is unclear. The aim of the present study was to investigate the role of FTO protein in pancreatic β-cells using a conditional expression system developed in INS-1 cells. INS-1 cells were stably transfected with FTO–HA (haemagluttinin) incorporated under the control of a tetracycline-inducible promoter. Induction of FTO protein resulted in localization of the tagged protein to the nucleus. The level of FTO–HA protein achieved in transfected cells was tightly regulated, and experiments with selective inhibitors revealed that FTO–HA is rapidly degraded via the ubiquitin/proteasome pathway. The nuclear localization was not altered by proteasome inhibitors, although following treatment with PYR-41, an inhibitor of ubiquitination, some of the protein adopted a perinuclear localization. Unexpectedly, modestly increased expression of FTO–HA selectively enhanced the first phase of insulin secretion when INS-1 monolayers or pseudoislets were stimulated with 20 mM glucose, whereas the second phase remained unchanged. The mechanism responsible for the potentiation of glucose-induced insulin secretion is unclear; however, further experiments revealed that it did not involve an increase in insulin biosynthesis or any changes in STAT3 (signal transducer and activator of transcription 3) expression. Taken together, these results suggest that the FTO protein may play a hitherto unrecognized role in the control of first-phase insulin secretion in pancreatic β-cells.

Non-functional role of syntaxin 2 in insulin exocytosis by pancreatic β cells

1997 ◽  
Vol 15 (4) ◽  
pp. 237-242 ◽  
Author(s):  
Shinya Nagamatsu ◽  
Hiroki Sawa ◽  
Yoko Nakamichi ◽  
Yoshinori Kondo ◽  
Satsuki Matsushima ◽  
...  
Keyword(s):  
Functional Role ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Insulin Exocytosis

scholarly journals Secretagogin affects insulin secretion in pancreatic β-cells by regulating actin dynamics and focal adhesion

2016 ◽  
Vol 473 (12) ◽  
pp. 1791-1803 ◽  
Author(s):  
Seo-Yun Yang ◽  
Jae-Jin Lee ◽  
Jin-Hee Lee ◽  
Kyungeun Lee ◽  
Seung Hoon Oh ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Focal Adhesion ◽  
Underlying Mechanism ◽  
Neuroendocrine Cells ◽  
Actin Dynamics ◽  
Second Phase ◽  
Dependent Manner ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Insulinoma Cells

Secretagogin (SCGN), a Ca2+-binding protein having six EF-hands, is selectively expressed in pancreatic β-cells and neuroendocrine cells. Previous studies suggested that SCGN enhances insulin secretion by functioning as a Ca2+-sensor protein, but the underlying mechanism has not been elucidated. The present study explored the mechanism by which SCGN enhances glucose-induced insulin secretion in NIT-1 insulinoma cells. To determine whether SCGN influences the first or second phase of insulin secretion, we examined how SCGN affects the kinetics of insulin secretion in NIT-1 cells. We found that silencing SCGN suppressed the second phase of insulin secretion induced by glucose and H2O2, but not the first phase induced by KCl stimulation. Recruitment of insulin granules in the second phase of insulin secretion was significantly impaired by knocking down SCGN in NIT-1 cells. In addition, we found that SCGN interacts with the actin cytoskeleton in the plasma membrane and regulates actin remodelling in a glucose-dependent manner. Since actin dynamics are known to regulate focal adhesion, a critical step in the second phase of insulin secretion, we examined the effect of silencing SCGN on focal adhesion molecules, including FAK (focal adhesion kinase) and paxillin, and the cell survival molecules ERK1/2 (extracellular-signal-regulated kinase 1/2) and Akt. We found that glucose- and H2O2-induced activation of FAK, paxillin, ERK1/2 and Akt was significantly blocked by silencing SCGN. We conclude that SCGN controls glucose-stimulated insulin secretion and thus may be useful in the therapy of Type 2 diabetes.

Insulin exocytosis in Goto-Kakizaki rat β-cells subjected to long-term glinide or sulfonylurea treatment

2008 ◽  
Vol 412 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Junko Kawai ◽  
Mica Ohara-Imaizumi ◽  
Yoko Nakamichi ◽  
Tadashi Okamura ◽  
Yoshihiro Akimoto ◽  
...  
Keyword(s):  
Insulin Release ◽  
Β Cells ◽  
Gk Rats ◽  
Insulin Granules ◽  
Long Term Treatment ◽  
Insulin Granule ◽  
Granule Motion ◽  
Insulin Exocytosis ◽  
First Phase Insulin Release

Sulfonylurea and glinide drugs display different effects on insulin granule motion in single β-cells in vitro. We therefore investigated the different effects that these drugs manifest towards insulin release in an in vivo long-term treatment model. Diabetic GK (Goto-Kakizaki) rats were treated with nateglinide, glibenclamide or insulin for 6 weeks. Insulin granule motion in single β-cells and the expression of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins were then analysed. Perifusion studies showed that decreased first-phase insulin release was partially recovered when GK rats were treated with nateglinide or insulin for 6 weeks, whereas no first-phase release occurred with glibenclamide treatment. In accord with the perifusion results, TIRF (total internal reflection fluorescence) imaging of insulin exocytosis showed restoration of the decreased number of docked insulin granules and the fusion events from them during first-phase release for nateglinide or insulin, but not glibenclamide, treatment; electron microscopy results confirmed the TIRF microscopy data. Relative to vehicle-treated GK β-cells, an increased number of SNARE clusters were evident in nateglinide- or insulin-treated cells; a lesser increase was observed in glibenclamide-treated cells. Immunostaining for insulin showed that nateglinide treatment better preserved pancreatic islet morphology than did glibenclamide treatment. However, direct exposure of GK β-cells to these drugs could not restore the decreased first-phase insulin release nor the reduced numbers of docked insulin granules. We conclude that treatment of GK rats with nateglinide and glibenclamide varies in long-term effects on β-cell functions; nateglinide treatment appears overall to be more beneficial.

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