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.

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 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 Mechanism of insulin exocytosis in pancreatic B cells by analyzing single insulin granule motion with TIRF imaging system

2003 ◽  
Vol 43 (supplement) ◽  
pp. S5
Author(s):  
M. Ohara-Imaizumi ◽  
Y. Nakamichi ◽  
C. Nishiwaki ◽  
T. Kikuta ◽  
S. Nagai ◽  
...  
Keyword(s):  
B Cells ◽  
Imaging System ◽  
Pancreatic B Cells ◽  
Insulin Granule ◽  
Granule Motion ◽  
Insulin Exocytosis

scholarly journals Sub-chronic stimulation of glucocorticoid receptor impairs and mineralocorticoid receptor protects cytosolic Ca2+ responses to glucose in pancreatic β-cells

2008 ◽  
Vol 197 (2) ◽  
pp. 221-229 ◽  
Author(s):  
Masaru Koizumi ◽  
Toshihiko Yada
Keyword(s):  
Glucocorticoid Receptor ◽  
Mineralocorticoid Receptor ◽  
Term Treatment ◽  
Β Cells ◽  
Β Cell ◽  
Chronic Stimulation ◽  
Glucose Signaling ◽  
Ru 486 ◽  
Long Term Treatment

The development of diabetes associated with stress, obesity, and metabolic syndrome involves elevated plasma glucocorticoid levels. It has been shown that short-term (<1 day) exposure to glucocorticoids reduces insulin secretion from pancreatic islets by affecting several steps of glucose signaling in β-cells. However, longer term direct effects of glucocorticoids on β-cells remain to be established. In this study, single β-cells isolated from rat islets were treated with glucocorticoids, mineralocorticoids, and their receptor agonists/antagonists for 3 days in culture, followed by assessment of the β-cell responsiveness to glucose by measuring cytosolic Ca2+ concentration ([Ca2+]i) using fura-2. Following treatment with corticosterone at 10–500 ng/ml for 3 days, the first-phase [Ca2+]i response to 8.3 mM glucose in β-cells was suppressed. Simultaneous administration of RU-486, a glucocorticoid receptor (GR) antagonist, prevented this suppression. RU-486 by itself promoted the β-cell [Ca2+]i response to glucose. Conversely, dexamethasone (1000 ng/ml), a highly selective GR agonist, impaired β-cell [Ca2+]i responses to glucose. A mineralocorticoid receptor (MR) antagonist spironolactone, co-administered with corticosterone, further depressed [Ca2+]i responses to glucose, while an MR ligand aldosterone attenuated the corticosterone inhibition of [Ca2+]i responses. Neither spironolactone nor aldosterone by itself affected [Ca2+]i responses. These results indicate that long-term treatment with corticosterone impairs β-cell [Ca2+]i responses to glucose. This effect is mediated by GR and attenuated partially by simultaneous MR stimulation by corticosterone. The results show a novel function of MR to protect islet β-cells against deteriorating glucocorticoid action via GR.

scholarly journals Morphology of pancreatic endocrine cells in rats with type 2 diabetes after the long-term treatment with sulphonylureas

2020 ◽  
Vol 4 (6) ◽  
pp. 329-333
Author(s):  
T.P. Tuchina ◽  
◽  
K.P. Skotnikova ◽  
A.A. Vtorushina ◽  
O.V. Rogoza ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Endocrine Cells ◽  
Diabetic Rats ◽  
Term Treatment ◽  
Β Cells ◽  
Ki 67 ◽  
Long Term Treatment ◽  
Pancreatic Endocrine Cells

Aim: to study the morphology of pancreatic islet cells in rats aged more than 1 year with experimental type 2 diabetes (T2D) that receive sulphonylureas for a long time.Patients and Methods: albino laboratory rats aged more than 1 year (equal to 40 years in humans) were enrolled in this experimental study. Streptozotocin and nicotinamide were administered to induce T2D (streptozotocin-nicotinamide-induced diabetic rats). The animals were divided into four groups, i.e., healthy controls, T2D with no treatment, T2D treated with glibenclamide, and T2D treated with gliclazide. After the experimental study, immunohistochemistry with the antibodies against insulin, glucagon, and Ki-67 was performed. Morphometric analysis was performed using microphotographs. Insulin-, glucagon- and Ki-67-positive cells in islets were calculated.Results: by the 24th week of the study, the ratio of the total volume of α-cells to islet area in rats receiving glibenclamide was similar compared to diabetic rats receiving no treatment (p=0.75). Meanwhile, the significant reduction in the number of α-cells was reported in rats receiving gliclazide compared to diabetic rats receiving no treatment (p=0.000004). Moreover, the number of α-cells was equal to healthy controls. The total volume of β-cells remained unchanged in rats receiving glibenclamide or gliclazide as compared with diabetic rats with no treatment.Conclusion: our findings demonstrate that older animals (>1 year of age) are characterized by the changes in the ratio of α- and β-cells that are similar to the adults with T2D, i.e., the reduction in the percentage of β-cells and the increase in the percentage of α-cells. Long-term treatment with sulphonylureas does not result in the additional changes in the number and total volume of β-cells (rats receiving sulphonylureas for 24 weeks are similar to T2D rats with no treatment in terms of the volume of β-cells). However, 24-week treatment with gliclazide results in the normalization of the total volume of α-cells, and their number is comparable to healthy controls.KEYWORDS: sulphonylureas, α-cells, β-cells, pancreas, type 2 diabetes. FOR CITATION: Tuchina T.P., Skotnikova K.P., Vtorushina A.A. et al. Morphology of pancreatic endocrine cells in rats with type 2 diabetes after the long-term treatment with sulphonylureas. Russian Medical Inquiry. 2020;4(6):329–333. DOI: 10.32364/2587-6821-2020-4-6-329-333.

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.

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