scholarly journals Polarization of chlorotetracycline fluorescence in pancreatic islet cells and its response to calcium ions and D-glucose

1979 ◽  
Vol 178 (1) ◽  
pp. 187-193 ◽  
Author(s):  
I B Täljedal
Keyword(s):  
Beta Cell ◽  
Fluorescence Intensity ◽  
Calcium Ions ◽  
Pancreatic Beta Cells ◽  
Plasma Membranes ◽  
Islet Cells ◽  
Pancreatic Islet Cells ◽  
Cell Plasma ◽  
Polarization Of Fluorescence ◽  
Parametric Testing

Suspensions rich in pancreatic beta-cells were prepared from non-inbred ob/ob-mice, incubated with 10 micrometer-chlorotetracycline, and analysed for fluorescence polarization in a microscope. Throughout the temperature range 16–38 degrees C, fluorescence was enhanced by 5 mM-Ca2+ in the incubation medium; 20 mM-D-glucose decreased the fluorescence measured in the presence of Ca2+. Fluorescence showed a curvilinear negative regression on temperature. The curves were rectified to a virtually ideal degree by Arrhenius transformations of data. Non-parametric testing of differences between linearized regression lines forms the basis for the following conclusions. The temperature-dependence of fluorescence intensity appeared to be smaller for Ca2+-specific signals than for the background fluorescence of chlorotetracycline in Ca2+-deficient cells. D-Glucose significantly diminished the polarization of fluorescence in cells incubated with Ca2+. It is suggested that D-glucose increases the mobility of Ca2+ in beta-cell plasma membranes; this mobility increase may help to explain previously reported effects of D-glucose on 45Ca2+ fluxes and membrane electric potential.

scholarly journals Chlorotetracycline as a fluorescent Ca2+ probe in pancreatic islet cells.

1978 ◽  
Vol 76 (3) ◽  
pp. 652-674 ◽  
Author(s):  
I B Täljedal
Keyword(s):  
Plasma Membranes ◽  
Islet Cells ◽  
Dependent Manner ◽  
Cell Surfaces ◽  
Noticeable Effect ◽  
Cell Plasma ◽  
Glass Capillaries ◽  
Asymmetrically Distributed ◽  
Dose Dependent ◽  
Dispersed Cells

Pancreatic islets, or suspensions of islet cells, from noninbred ob/ob-mice were incubated with chlorotetracycline and analyzed for Ca2+-dependent fluorescence in a microscope. Unless logarithmically transformed, signals from islets were asymmetrically distributed with unstable variance. Signals from cells pelleted in glass capillaries were more homogeneous and depended linearly on the thickness of the sample. The effect of sample thickness and a significant enhancement of fluorescence by alloxan suggest that beta-cells were involved in producing the signal from whole islets. The signal from dispersed cells was probably diagnostic of Ca2+ in beta-cell plasma membranes because it was suppressed by La3+ and had a spectrum indicative of an apolar micromilieu; fluorescent staining of cell surfaces was directly seen at high magnification. Fluorescence from cells was enhanced by 0.5-10 mM Ca2+ in a dose-dependent manner, whereas less than 0.5 mM Ca2+ saturated the probe alone in methanol. The signal from islets or dispersed cells was suppressed by 5 mM theophylline; that from cells was also suppressed by 0.5 mM 3-isobutyl-1-methylxanthine, 1.2 or 15 mM Mg2+, 3-20 mM D-glucose, and, to a lesser extent, 20 mM 3-O-methyl-D-glucose. D-glucose was more inhibitory in the absence than in the presence of Mg2+, as if Mg2+ and D-glucose influenced the same Ca2+ pool. L-glucose, D-mannopheptulose, or diazoxide had no noticeable effect and 20 mM bicarbonate was stimulatory. The results suggest that microscopy of chlorotetracycline-stained cells can aid in characterizing calcium pools of importance for secretion. Initiation of insulin release may be associated with an increas

scholarly journals Video image analysis of labile zinc in viable pancreatic islet cells using a specific fluorescent probe for zinc.

1994 ◽  
Vol 42 (7) ◽  
pp. 877-884 ◽  
Author(s):  
P D Zalewski ◽  
S H Millard ◽  
I J Forbes ◽  
O Kapaniris ◽  
A Slavotinek ◽  
...  
Keyword(s):  
Image Analysis ◽  
Pancreatic Islet ◽  
Fluorescence Intensity ◽  
Islet Cells ◽  
Video Image ◽  
Zinc Homeostasis ◽  
Pancreatic Acinar Cells ◽  
Pancreatic Islet Cells ◽  
Video Image Analysis ◽  
Insulinoma Cells

We used an intracellular zinc-specific fluorophore, Zinquin, in conjunction with fluorescence video image analysis, to reveal labile zinc in pancreatic islet cells, which concentrate this metal for use in synthesis, storage, and secretion of insulin. Zinquin vividly demonstrated zinc in the islet cell secretory granules, which formed a brightly labeled crescent in the cytoplasm between one side of the nucleus and the plasma membrane. Lower but still appreciable amounts of zinc were detected in the remaining cytoplasm, but there was little labeling in the nucleus. Fluorescence intensity varied among islet cells, suggesting differences in zinc content. Their average fluorescence intensity greatly surpassed that of the surrounding pancreatic acinar cells in frozen sections of pancreas and in all other types of cell studied, including lymphocytes, neutrophils, fibroblasts, and erythrocytes. Less labile zinc was detected in cells of the mouse insulinoma cell line NIT-1, regardless of whether they were maintained in long-term culture in the presence or absence of exogenous extracellular zinc. Exposure of islet or insulinoma cells to a high concentration of glucose or other secretagogue decreased the content of labile zinc. Zinquin should be a useful probe for revealing changes in zinc homeostasis in islet B-cells that may be important in their dysfunction and death during diabetes.

scholarly journals Pancreatic Duct Cells Isolated From Canines Differentiate Into Beta-Like Pancreatic Islet Cells

2022 ◽  
Vol 8 ◽  
Author(s):  
Yuhua Gao ◽  
Weijun Guan ◽  
Chunyu Bai
Keyword(s):  
Pancreatic Islet ◽  
Pancreatic Beta Cells ◽  
Islet Cells ◽  
Pancreatic Islet Cells ◽  
Cell Therapies ◽  
Ductal Cells ◽  
Pancreatic Ductal Cells ◽  
Insulin Granules ◽  
Glucose Stimulated Insulin Secretion

In this study, we isolated and cultured pancreatic ductal cells from canines and revealed the possibility for using them to differentiate into functional pancreatic beta cells in vitro. Passaged pancreatic ductal cells were induced to differentiate into beta-like pancreatic islet cells using a mixture of induced factors. Differentiated pancreatic ductal cells were analyzed based on intracellular insulin granules using transmission electron microscopy, the expression of insulin and glucagon using immunofluorescence, and glucose-stimulated insulin secretion using ELISA. Our data revealed that differentiated pancreatic ductal cells not only expressed insulin and glucagon but also synthesized insulin granules and secreted insulin at different glucose concentrations. Our study might assist in the development of effective cell therapies for the treatment of type 1 diabetes mellitus in dogs.

scholarly journals The β-cell glibenclamide receptor is an ADP-binding protein

1990 ◽  
Vol 268 (3) ◽  
pp. 713-718 ◽  
Author(s):  
I Niki ◽  
J L Nicks ◽  
S J H Ashcroft
Keyword(s):  
Beta Cell ◽  
Insulin Release ◽  
Plasma Membranes ◽  
Pancreatic Beta Cell ◽  
Atp Depletion ◽  
K Channel ◽  
Whole Cells ◽  
Cell Plasma ◽  
Beta Cell Line ◽  
Extracellular Side

The effects of ADP on [3H]glibenclamide binding to membranes and whole cells, the activity of the ATP-sensitive K+ channel (K-ATP channel), intracellular Ca2+ concentration and insulin secretion were studied in a hamster pancreatic beta-cell line, HIT T15. ADP dose-dependently inhibited [3H]glibenclamide binding to membranes and to whole cells in a competitive manner. ADP-agarose also inhibited the binding to whole cells. The activity of the K-ATP channel was assayed by measuring 86Rb efflux from whole cells. ADP inhibited the 86Rb efflux elicited either by diazoxide or by ATP depletion. In the presence, but not in the absence, of extracellular Ca2+, ADP evoked a rapid and sustained increase in intracellular Ca2+ concentration as estimated with the fluorescent dye quin 2. Insulin release from HIT cells was also increased by 0.5-2 mM-ADP in the presence of 0.5 mM-glucose. These effects of ADP on glibenclamide binding, K-ATP channel activity and insulin release were specific for ADP, and were not reproduced by any other nucleotide so far tested. The present findings strongly suggest that ADP and sulphonylureas have common binding sites on the extracellular side of beta-cell plasma membranes, where they inhibit the activity of the K-ATP channel, resulting in an increase in intracellular Ca2+ concentration and insulin release.

scholarly journals Oxidative stress induces p21 expression in pancreatic islet cells: possible implication in beta-cell dysfunction

Diabetologia ◽  
1999 ◽  
Vol 42 (9) ◽  
pp. 1093-1097 ◽  
Author(s):  
H. Kaneto ◽  
Y. Kajimoto ◽  
Y. Fujitani ◽  
T. Matsuoka ◽  
K. Sakamoto ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Beta Cell ◽  
Pancreatic Islet ◽  
Islet Cells ◽  
Beta Cell Dysfunction ◽  
Pancreatic Islet Cells ◽  
Cell Dysfunction

scholarly journals Exogenous Ghrelin Increases Plasma Insulin Level in Diabetic Rats

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 633 ◽  
Author(s):  
Haba Elabadlah ◽  
Rasheed Hameed ◽  
Crystal D’Souza ◽  
Sahar Mohsin ◽  
Ernest A. Adeghate
Keyword(s):  
Body Weight ◽  
Insulin Secretion ◽  
Amino Acid ◽  
Beta Cell ◽  
Beta Cells ◽  
Islet Cells ◽  
Insulin Level ◽  
Diabetic Rats ◽  
Pancreatic Islet Cells ◽  
Beta Cell Line

Ghrelin, a 28-amino acid peptide, is a strong growth hormone secretagogue and a regulator of food intake. In addition, ghrelin is thought to play a role in insulin secretion and in glucose homeostasis. A lot of contradictory data have been reported in the literature regarding the co-localization of ghrelin with other hormones in the islet of Langerhans, its role in insulin secretion and attenuation of type 2 diabetes mellitus. In this study, we investigate the effect of chronic ghrelin treatment on glucose, body weight and insulin level in normal and streptozotocin-induced diabetic male Wistar rats. We have also examined the distribution pattern and co-localization of ghrelin with insulin in pancreatic islet cells using immunohistochemistry and immune-electron microscopy and the ability of ghrelin to stimulate insulin release from the CRL11065 beta cell line. Control, non-diabetic groups received intraperitoneal injection of normal saline, while treated groups received intraperitoneal injection of 5 µg/kg body weight of ghrelin (amino acid chain 24–51) on a daily basis for a duration of four weeks. Our results show that the administration of ghrelin increases the number of insulin-secreting beta cells and serum insulin level in both normal and diabetic rats. We also demonstrated that ghrelin co-localizes with insulin in pancreatic islet cells and that the pattern of ghrelin distribution is altered after the onset of diabetes. Moreover, ghrelin at a dose of 10−6 M and 10−12 M increased insulin release from the CRL11065 beta cell line. In summary, ghrelin co-localizes with insulin in the secretory granules of pancreatic beta cells and enhances insulin production.

scholarly journals Intersection of the ATF6 and XBP1 ER stress pathways in mouse islet cells

2020 ◽  
Vol 295 (41) ◽  
pp. 14164-14177 ◽  
Author(s):  
Rohit B. Sharma ◽  
Christine Darko ◽  
Laura C. Alonso
Keyword(s):  
Beta Cell ◽  
Er Stress ◽  
Pancreatic Beta Cells ◽  
Islet Cells ◽  
Transcriptional Response ◽  
Similar Response ◽  
Response Dynamics ◽  
Transcriptional Responses ◽  
Mouse Islet ◽  
Primary Mouse

Success or failure of pancreatic beta cell adaptation to ER stress is a determinant of diabetes susceptibility. The ATF6 and IRE1/XBP1 pathways are separate ER stress-response effectors important to beta cell health and function. ATF6α. and XBP1 direct overlapping transcriptional responses in some cell types. However, the signaling dynamics and interdependence of ATF6α and XBP1 in pancreatic beta cells have not been explored. To assess pathway-specific signal onset, we performed timed exposures of primary mouse islet cells to ER stressors and measured the early transcriptional response. Comparing the time course of induction of ATF6 and XBP1 targets suggested that the two pathways have similar response dynamics. The role of ATF6α in target induction was assessed by acute knockdown using islet cells from Atf6αflox/flox mice transduced with adenovirus expressing Cre recombinase. Surprisingly, given the mild impact of chronic deletion in mice, acute ATF6α knockdown markedly reduced ATF6-pathway target gene expression under both basal and stressed conditions. Intriguingly, although ATF6α knockdown did not alter Xbp1 splicing dynamics or intensity, it did reduce induction of XBP1 targets. Inhibition of Xbp1 splicing did not decrease induction of ATF6α targets. Taken together, these data suggest that the XBP1 and ATF6 pathways are simultaneously activated in islet cells in response to acute stress and that ATF6α is required for full activation of XBP1 targets, but XBP1 is not required for activation of ATF6α targets. These observations improve understanding of the ER stress transcriptional response in pancreatic islets.

scholarly journals Long Noncoding RNA Meg3 Regulates Mafa Expression in Mouse Beta Cells by Inactivating Rad21, Smc3 or Sin3α

2018 ◽  
Vol 45 (5) ◽  
pp. 2031-2043 ◽  
Author(s):  
Ning Wang ◽  
Yanan Zhu ◽  
Min Xie ◽  
Lintao Wang ◽  
Feiyan Jin ◽  
...  
Keyword(s):  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Noncoding Rna ◽  
Islet Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Insulin Biosynthesis ◽  
Expression Levels ◽  
Beta Cell Line

Background/Aims: The main pathogenic mechanism of diabetes is a decrease in the number of islet beta cells or a decline in their function. Recent studies have shown that pancreatic long noncoding RNAs (lncRNAs) have a high degree of tissue specificity and may be involved in the maintenance of islet cells function and the development of diabetes. The aim of this study was to investigate the molecular regulatory mechanism of mouse maternal expressed gene 3 (Meg3) in insulin biosynthesis in pancreatic islets. Methods: Chromatin immunoprecipitation–quantitative polymerase chain reaction (qPCR) and RNA immunoprecipitation–qPCR were used to investigate the molecular mechanism of lncRNA Meg3 in insulin biosynthesis by regulating v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), a mature beta cell marker in the MIN6 beta cell line. Further, the expression levels of Meg3, Ezh2, MafA, Rad21, Smc3, and Sin3α were analyzed in vivo and in vitro by RT-PCR and western blotting. Results: Intranuclear lncRNA Meg3 can bind EZH2, a methyltransferase belonging to the Polycomb repressive complex-2, in pancreatic islet cells. In addition, knockdown of Ezh2 can also inhibit the expression of MafA and Ins2, while expression levels of Rad21, Smc3, and Sin3α are upregulated, by interfering with Ezh2 or Meg3 in pancreatic beta cells. Knockdown of Meg3 resulted in the loss of EZH2 binding and H3K27 trimethylation occupancy of Rad21, Smc3, and Sin3α promoter regions. The inhibition of Rad21, Smc3, or Sin3α, which directly act on the MafA promoter, leads to upregulated expression of MafA in both MIN6 cells and mouse islets. Moreover, the synthesis and secretion of insulin were increased by inhibition of these transcription factors. Conclusions: Pancreatic lncRNA Meg3 can epigenetically regulate the expression of Rad21, Smc3, and Sin3α via EZH2-driven H3K27 methylation. By inhibiting the expression of Rad21, Smc3, or Sin3α, Meg3 promotes the expression of MafA and affects the production of insulin.

Modulation of Specific Beta Cell Gene (Re)Expression during In Vitro Expansion of Human Pancreatic Islet Cells

2003 ◽  
Vol 12 (7) ◽  
pp. 799-807 ◽  
Author(s):  
Thomas Bouckenooghe ◽  
Brigitte Vandewalle ◽  
Bruno Lukowiak ◽  
Julie Kerr-Conte ◽  
Sandrine Beläich ◽  
...  
Keyword(s):  
Beta Cell ◽  
Pancreatic Islet ◽  
Islet Cells ◽  
Pancreatic Islet Cells ◽  
Human Pancreatic Islet ◽  
In Vitro Expansion ◽  
Cell Gene

Abstract 483: Non-redundant and Opposing Roles of Group X and Group V Secretory Phospholipase A2s on Pancreatic Beta-cell Function

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Preetha Shridas ◽  
Victoria P Noffsinger ◽  
Nancy R Webb
Keyword(s):  
Beta Cell ◽  
Cell Lines ◽  
Pancreatic Islet ◽  
Cell Function ◽  
Islet Cells ◽  
Pancreatic Beta Cell ◽  
Pge2 Production ◽  
Pancreatic Islet Cells ◽  
Group V ◽  
Min6 Cells

Background: Group X and group V secretory phospholipase A2s (GX and GV sPLA2s) potently release arachidonic acid (AA) from the plasma membrane of intact cells. AA is an activator of glucose-stimulated insulin secretion (GSIS) by β-islet cells. However, the AA metabolite prostaglandin E2 (PGE2) is a known inhibitor of GSIS. Both GX and GV sPLA2s are expressed in mouse pancreatic islet cells. We previously demonstrated that GX sPLA2 negatively regulates GSIS by a PGE2-dependent mechanism. In this study we investigated whether GV sPLA2 similarly regulates GSIS. Methods and Results: GSIS was measured in pancreatic islet cells isolated from WT and GV sPLA2-deficient (GV KO) mice. To complement these studies, GSIS was also assessed in vitro using MIN6 pancreatic beta cell lines with or without GV sPLA2 overexpression or silencing. In marked contrast to our findings in GX KO mice, GSIS was significantly decreased in islets isolated from GV KO mice compared to WT mice. Similarly, there was a significant decrease in GSIS in MIN6 cells with siRNA-mediated GV sPLA2 suppression. Consistent with these findings, MIN6 cells overexpressing GV sPLA2 (MIN6-GV) showed a significant increase in GSIS compared to control cells. As expected, the amount of AA released into the media by MIN6-GV cells was significantly increased compared to control MIN6 cells. However, unlike MIN6 cells overexpressing GX sPLA2, MIN6-GV cells did not exhibit enhanced PGE2 production or decreased cAMP content compared to control MIN6 cells, despite similar amounts of sPLA2 activity produced by the two cell lines. Conclusions: We conclude that GX and GV sPLA2s play opposing and non-redundant roles in pancreatic β-cell function. Whereas GV sPLA2 activates GSIS, GX sPLA2 suppresses this process. This functional difference appears to be due to the extent to which AA generated by the respective sPLA2’s is coupled to PGE2 production.

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