Cavβ3 regulates Ca2+-signalling and insulin expression in pancreatic β-cells in a cell-autonomous manner

10.2337/figshare.15173556 ◽

2021 ◽

Author(s):

Alexander Becker ◽

Barbara Wardas ◽

Houssein Salah ◽

Maryam Amini ◽

Claudia Fecher‐Trost ◽

...

Keyword(s):

Protein Profiling ◽

Dependent Manner ◽

Ip3 Receptor ◽

Β Cells ◽

Pancreatic Β Cells ◽

Concentration Dependent Manner ◽

Insulin Expression ◽

Low Concentrations ◽

A Cell ◽

Nitric Oxide Synthase 1

Voltage-gated Ca2+ (Cav) channels consist of a pore-forming Cavα1 subunit and auxiliary Cavα2-δ and Cavβ subunits. In fibroblasts, Cavβ3, independent of its role as a Cav subunit, reduces the sensitivity to low concentrations of inositol-1,4,5-trisphosphate (IP3). Similarly, Cavβ3 could affect cytosolic [Ca2+] in pancreatic β-cells. Here, we deleted the Cavβ3-encoding gene Cacnb3 in insulin-secreting rat β-(Ins-1) cells using CRISPR/Cas9. These cells were used as controls to investigate the role of Cavβ3 on Ca2+-signalling, glucose-induced insulin secretion (GIIS), Cav-channel activity and gene expression in wild-type cells in which Cavβ3 and the IP3-receptor were co-immunoprecipitated. Transcript and protein profiling revealed significantly increased levels of insulin transcription factor Mafa, CaMKIV, neuroendocrine convertase1 (Pcsk1) and nitric oxide synthase-1 (NOS-1) in Cavβ3-KO cells. In the absence of Cavβ3, Cav-currents were not altered. In contrast, CREB activity, the amount of MAFA protein and GIIS, the extent of IP3-dependent Ca2+ release and the frequency of Ca2+-oscillations were increased. These processes were decreased by the Cavβ3 protein in a concentration-dependent manner. Our study shows that Cavβ3 interacts with the IP3-receptor in isolated β-cells, controls IP3-dependent Ca2+-signalling independently of Cav channel functions, and thereby regulates insulin expression and its glucose-dependent release in a cell-autonomous manner.

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Evidence for voltage-dependent Cl− permeability in mouse pancreatic β-cells

Bioscience Reports ◽

10.1007/bf01122729 ◽

1987 ◽

Vol 7 (1) ◽

pp. 67-72 ◽

Cited By ~ 4

Author(s):

Janove Sehlin

Keyword(s):

Dependent Manner ◽

Β Cells ◽

Efflux Rate ◽

Β Cell ◽

Pancreatic Β Cells ◽

Concentration Dependent Manner ◽

Cell Plasma ◽

Voltage Dependent ◽

The Mean ◽

Cl Permeability

Microdissected β-cell-rich pancreatic islets from ob/ob-mice were used in studies of transmembrane36Cl− efflux. The mean rate coefficient for36Cl− efflux was stable at 0.158 min−1 during the initial 10 min. Depolarization of the β-cell plasma membrane by acute increases in extracellular K+ (5–130mM) stimulated the36Cl− efflux in a concentration-dependent manner. Glucose-induced (20mM) and K+-induced increases in36Cl− efflux were largely overlapping, but even at 135.9 mM K+, glucose slightly further enhanced the36Cl− efflux rate. The data suggest (1) that pancreatic β-cells are equipped with a voltage-dependent Cl− permeability, (2) that glucose-induced increase in Cl− permeability may, at least partly, be mediated by primary membrane depolarization, and (3) that glucose in addition may activate other mechanisms for β-cell Cl− transport.

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[Ca2+]i-reducing action of cAMP in rat pancreatic β-cells: involvement of thapsigargin-sensitive stores

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.2.c513 ◽

1998 ◽

Vol 274 (2) ◽

pp. C513-C521 ◽

Cited By ~ 24

Author(s):

Kazuro Yaekura ◽

Toshihiko Yada

Keyword(s):

Cell Metabolism ◽

Pyridine Nucleotide ◽

Dependent Manner ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Concentration Dependent Manner ◽

Elevated Glucose ◽

Glucagon Like Peptide 1 ◽

Pka Pathway

In the present study, we examined the ability of adenosine 3′,5′-cyclic monophosphate (cAMP) to reduce elevated levels of cytosolic Ca2+ concentration ([Ca2+]i) in pancreatic β-cells. [Ca2+]iand reduced pyridine nucleotide, NAD(P)H, were measured in rat single β-cells by fura 2 and autofluorescence microfluorometry. Sustained [Ca2+]ielevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activator forskolin (10 μM), and an incretin glucagon-like peptide-1-(7–36) amide (10−9 M), as well as by glucose (16.7 mM). The [Ca2+]i-reducing effects of cAMP were greater at elevated glucose (8.3–16.7 mM) than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA), H-89, counteracted [Ca2+]i-reducing effects of cAMP but not those of glucose. Okadaic acid, a phosphatase inhibitor, at 10–100 nM also reduced sustained [Ca2+]ielevation in a concentration-dependent manner. Glucose, but not DBcAMP, increased NAD(P)H in β-cells. [Ca2+]i-reducing effects of cAMP were inhibited by 0.3 μM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump. In contrast, [Ca2+]i-reducing effects of cAMP were not altered by ryanodine, an ER Ca2+-release inhibitor, Na+-free conditions, or diazoxide, an ATP-sensitive K+ channel opener. In conclusion, the cAMP-PKA pathway reduces [Ca2+]ielevation by sequestering Ca2+ in thapsigargin-sensitive stores. This process does not involve, but is potentiated by, activation of β-cell metabolism. Together with the known [Ca2+]i-increasing action of cAMP, our results reveal dual regulation of β-cell [Ca2+]iby the cAMP-signaling pathway and by a physiological incretin.

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Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646319 ◽

1992 ◽

Vol 68 (05) ◽

pp. 570-576 ◽

Cited By ~ 20

Author(s):

Mary A Selak

Keyword(s):

Neutrophil Elastase ◽

Cell Activation ◽

Thromboxane A2 ◽

Creatine Phosphate ◽

Dense Granule ◽

Cathepsin G ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Low Concentrations ◽

High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

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Peroxisome Proliferator-Activated Receptor α (PPARα) Potentiates, whereas PPARγ Attenuates, Glucose-Stimulated Insulin Secretion in Pancreatic β-Cells

Endocrinology ◽

10.1210/en.2004-1430 ◽

2005 ◽

Vol 146 (8) ◽

pp. 3266-3276 ◽

Cited By ~ 71

Author(s):

Kim Ravnskjaer ◽

Michael Boergesen ◽

Blanca Rubi ◽

Jan K. Larsen ◽

Tina Nielsen ◽

...

Keyword(s):

Insulin Secretion ◽

Mitochondrial Membrane ◽

Uncoupling Protein ◽

Peroxisome Proliferator ◽

Dependent Manner ◽

Β Cells ◽

Pancreatic Β Cells ◽

Peroxisome Proliferator Activated Receptor ◽

Glucose Stimulated Insulin Secretion

Abstract Fatty acids (FAs) are known to be important regulators of insulin secretion from pancreatic β-cells. FA-coenzyme A esters have been shown to directly stimulate the secretion process, whereas long-term exposure of β-cells to FAs compromises glucose-stimulated insulin secretion (GSIS) by mechanisms unknown to date. It has been speculated that some of these long-term effects are mediated by members of the peroxisome proliferator-activated receptor (PPAR) family via an induction of uncoupling protein-2 (UCP2). In this study we show that adenoviral coexpression of PPARα and retinoid X receptor α (RXRα) in INS-1E β-cells synergistically and in a dose- and ligand-dependent manner increases the expression of known PPARα target genes and enhances FA uptake and β-oxidation. In contrast, ectopic expression of PPARγ/RXRα increases FA uptake and deposition as triacylglycerides. Although the expression of PPARα/RXRα leads to the induction of UCP2 mRNA and protein, this is not accompanied by reduced hyperpolarization of the mitochondrial membrane, indicating that under these conditions, increased UCP2 expression is insufficient for dissipation of the mitochondrial proton gradient. Importantly, whereas expression of PPARγ/RXRα attenuates GSIS, the expression of PPARα/RXRα potentiates GSIS in rat islets and INS-1E cells without affecting the mitochondrial membrane potential. These results show a strong subtype specificity of the two PPAR subtypes α and γ on lipid partitioning and insulin secretion when systematically compared in a β-cell context.

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Metabolic inhibition impairs ATP-sensitive K+ channel block by sulfonylurea in pancreatic β-cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1998.274.1.e38 ◽

1998 ◽

Vol 274 (1) ◽

pp. E38-E44 ◽

Cited By ~ 5

Author(s):

Eri Mukai ◽

Hitoshi Ishida ◽

Seika Kato ◽

Yoshiyuki Tsuura ◽

Shimpei Fujimoto ◽

...

Keyword(s):

Metabolic Inhibition ◽

K Channel ◽

High Dose ◽

Dependent Manner ◽

Channel Activity ◽

Β Cells ◽

Pancreatic Β Cells ◽

Patch Clamp Technique ◽

Channel Inhibition ◽

Dose Dependent

The effect of metabolic inhibition on the blocking of β-cell ATP-sensitive K+ channels (KATP channels) by glibenclamide was investigated using a patch-clamp technique. Inhibition of KATP channels by glibenclamide was attenuated in the cell-attached mode under metabolic inhibition induced by 2,4-dinitrophenol. Under a low concentration (0.1 μM) of ATP applied in the inside-out mode, KATP channel activity was not fully abolished, even when a high dose of glibenclamide was applied, in contrast to the dose-dependent and complete KATP channel inhibition under 10 μM ATP. On the other hand, cibenzoline, a class Ia antiarrhythmic agent, inhibits KATP channel activity in a dose-dependent manner and completely blocks it, even under metabolic inhibition. In sulfonylurea receptor (SUR1)- and inward rectifier K+ channel (Kir6.2)-expressed proteins, cibenzoline binds directly to Kir6.2, unlike glibenclamide. Thus, KATPchannel inhibition by glibenclamide is impaired under the condition of decreased intracellular ATP in pancreatic β-cells, probably because of a defect in signal transmission between SUR1 and Kir6.2 downstream of the site of sulfonylurea binding to SUR1.

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Cu2+-induced modification of the kinetics of Aβ(1-42) channels

AJP Cell Physiology ◽

10.1152/ajpcell.00147.2003 ◽

2003 ◽

Vol 285 (4) ◽

pp. C873-C880 ◽

Cited By ~ 10

Author(s):

Randa Bahadi ◽

Peter V. Farrelly ◽

Bronwyn L. Kenna ◽

Cyril C. Curtain ◽

Colin L. Masters ◽

...

Keyword(s):

Amyloid Β ◽

Chelating Agent ◽

Common Mechanism ◽

Amyloid Β Peptide ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Channel Inhibition ◽

Low Concentrations ◽

Redox Agents ◽

In Cis

We found that the amyloid β peptide Aβ(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu2+ or biological redox agents that have been reported to mediate Aβ(1-42) toxicity. The Aβ(1-42)-formed cation channel was inhibited by Cu2+ in cis solution ([Cu2+] cis) in a voltage- and concentration-dependent manner between 0 and 250 μM. The [Cu2+] cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 μM [Cu2+] cis and partially reversible at 250 μM [Cu2+] cis. The inhibitory effects of [Cu2+] cis between 50 and 250 μM on the channel could not be reversed with addition of Cu2+-chelating agent clioquinol (CQ) at concentrations between 64 and 384 μM applied to the cis chamber. The effects of 200-250 μM [Cu2+] cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 μM [CQ] cis. The kinetic analysis of the data indicate that Cu2+-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu2+ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu2+-binding site of the Aβ(1-42)-formed channels is modulated with Cu2+ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu2+ modulation of Aβ and PrP channel proteins linked to neurodegenerative diseases.

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

Biochemical Journal ◽

10.1042/bcj20160137 ◽

2016 ◽

Vol 473 (12) ◽

pp. 1791-1803 ◽

Cited By ~ 33

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.

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Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.1.f129 ◽

1997 ◽

Vol 273 (1) ◽

pp. F129-F135 ◽

Cited By ~ 9

Author(s):

J. M. Arthur ◽

G. P. Collinsworth ◽

T. W. Gettys ◽

L. D. Quarles ◽

J. R. Raymond

Keyword(s):

G Protein ◽

G Proteins ◽

Plasma Membranes ◽

Mdck Cells ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cellular Activation ◽

Protein Activation ◽

A Cell ◽

Cation Sensing

Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-32P]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members. Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1. Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells. We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1.

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A Minimum of Fuel Is Necessary for Tolbutamide to Mimic the Effects of Glucose on Electrical Activity in Pancreatic β-Cells*

Endocrinology ◽

10.1210/endo.139.3.5783 ◽

1998 ◽

Vol 139 (3) ◽

pp. 993-998 ◽

Cited By ~ 22

Author(s):

Jean-Claude Henquin

Keyword(s):

Membrane Potential ◽

Electrical Activity ◽

Slow Waves ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

K Channels ◽

Low Concentrations ◽

Low Glucose ◽

Intracellular Microelectrodes

Glucose stimulation of pancreatic β-cells triggers electrical activity (slow waves of membrane potential with superimposed spikes) that is best monitored with intracellular microelectrodes. Closure of ATP-sensitive K+ channels underlies the depolarization to the threshold potential and participates in the increase in electrical activity produced by suprathreshold (>7 mm) concentrations of glucose, but it is still unclear whether this is the sole mechanism of control. This was investigated by testing whether blockade of ATP-sensitive K+ channels by low concentrations of tolbutamide is able to mimic the effects of glucose on mouse β-cell electrical activity even in the absence of the sugar. The response to tolbutamide was influenced by the duration of the perifusion with the low glucose medium. Tolbutamide (25 μm) caused a rapid and sustained depolarization with continuous activity after 6 min of perifusion of the islet with 3 mm glucose, and a progressive depolarization with slow waves of the membrane potential after 20 min. In the absence of glucose, the β-cell response to tolbutamide was a transient phase of depolarization with rare slow waves (6 min) or a silent, small, but sustained, depolarization (20 min). Readministration of 3 mm glucose was sufficient to restore slow waves, whereas an increase in the glucose concentration to 5 and 7 mm was followed by a lengthening of the slow waves and a shortening of the intervals. In contrast, induction of slow waves by tolbutamide proved very difficult in the absence of glucose, because the β-cell membrane tended to depolarize from a silent level to the plateau level, at which electrical activity is continuous. Azide, a mitochondrial poison, abrogated the electrical activity induced by tolbutamide in the absence of glucose, which demonstrates the influence of the metabolism of endogenous fuels on the response to the sulfonylurea. The partial repolarization that azide also produced was reversed by increasing the concentration of tolbutamide, but reappearance of the spikes required the addition of glucose. It is concluded that inhibition of ATP-sensitive K+ channels is not the only mechanism by which glucose controls electrical activity inβ -cells.

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