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Original Paper

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Quercetin Stimulates Insulin Secretion and Reduces the Viability of Rat INS-1 Beta-Cells

Kittl M.a · Beyreis M.a · Tumurkhuu M.a, b · Fürst J.c · Helm K.a · Pitschmann A.d · Gaisberger M.a,e,f · Glasl S.d · Ritter M.a,e,f · Jakab M.a

Author affiliations

aInstitute of Physiology and Pathophysiology, Paracelsus Medical University, Salzburg, Austria; bSchool of Pharmacy and Bio-Medicine, Mongolian National University of Medical Sciences, Ulaanbaatar, Mongolia; cDepartment of Physiology and Medical Physics, Division of Physiology, Medical University of Innsbruck, Innsbruck, Austria; dDepartment of Pharmacognosy, University of Vienna, Vienna, Austria; eDepartment for Radon Therapy Research, Ludwig Boltzmann Cluster for Arthritis and Rehabilitation, Salzburg, Austria; fGastein Research Institute, Paracelsus Medical University, Salzburg, Austria

Corresponding Author

Martin Jakab

Paracelsus Medical University, Institute of Physiology and Pathophysiology, Strubergasse

22, 5020 Salzburg, (Austria)

Tel. +43-662-242080504, E-Mail martin.jakab@pmu.ac.at

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Cell Physiol Biochem 2016;39:278-293

Abstract

Background/Aims: Previously we described insulinotropic effects of Leonurus sibiricus L. plant extracts used for diabetes mellitus treatment in Traditional Mongolian Medicine. The flavonoid quercetin and its glycoside rutin, which exert anti-diabetic properties in vivo by interfering with insulin signaling in peripheral target tissues, are constituents of these extracts. This study was performed to better understand short- and long-term effects of quercetin and rutin on beta-cells. Methods: Cell viability, apoptosis, phospho-protein abundance and insulin release were determined using resazurin, annexin-V binding assays, Western blot and ELISA, respectively. Membrane potentials (Vmem), whole-cell Ca2+ (ICa)- and ATP-sensitive K+ (IKATP) currents were measured by patch clamp. Intracellular Ca2+ (Cai) levels were measured by time-lapse imaging using the ratiometric Ca2+ indicator Fura-2. Results: Rutin, quercetin and the phosphoinositide-3-kinase (PI3K) inhibitor LY294002 caused a dose-dependent reduction in cell viability with IC50 values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively. Quercetin (50 µM) significantly increased the percentage of Annexin-V+ cells within 48 hrs. The mean cell volume (MCV) of quercetin-treated cells was significantly lower. Within 2 hrs, quercetin significantly decreased basal- and insulin-stimulated Akt(T308) phosphorylation and increased Erk1/2 phosphorylation, without affecting P-Akt(S473) abundance. Basal- and glucose-stimulated insulin release were significantly stimulated by quercetin. Quercetin significantly depolarized Vmem by ∼25 mV which was prevented by the KATP-channel opener diazoxide, but not by the L-type ICa inhibitor nifedipine. Quercetin significantly stimulated ICa and caused a 50% inhibition of IKATP. The effects on Vmem, ICa and IKATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute. With a similar time-response quercetin induced an elevation in Cai which was completely abolished in the absence of Ca2+ in the bath solution. Rutin (50 µM) did not significantly alter the percentage of Annexin-V+ cells, MCV, Akt or Erk1/2 phosphorylation, insulin secretion, or the electrophysiological behavior of INS-1 cells. Conclusion: We conclude that quercetin acutely stimulates insulin release, presumably by transient KATP channel inhibition and ICa stimulation. Long term application of quercetin inhibits cell proliferation and induces apoptosis, most likely by inhibition of PI3K/Akt signaling.

© 2016 The Author(s) Published by S. Karger AG, Basel


Introduction

In a previous study we described the insulinotropic effects of Leonurus sibiricus L. (LS) plant extracts used in Traditional Mongolian Medicine (TMM) for the treatment of diabetes mellitus (DM) and DM-related symptoms [1]. We have demonstrated that insulin release from INS-1E cells was significantly increased in presence of aqueous as well as methanolic LS extracts. Acute application of water extract resulted in a persisting depolarization of the cell membrane potential (Vmem) paralleled by an initial increase and subsequent silencing of action potential frequency, by KATP channel inhibition and an increase in intracellular calcium (Cai). Furthermore, all LS extracts stimulated INS-1E cell proliferation. The present study was performed to identify LS extract compounds accounting for the observed effects on beta-cell viability, insulin release and electrophysiological behavior. We focused on the flavonoid quercetin and its glycoside rutin, two phytochemicals which could be identified as constituents of LS extracts by HPLC analyses [1,2] and which are known to exert comparable effects in different cell systems.

Quercetin is a naturally occurring flavonoid commonly found in plants. It is the aglycone of a number of flavonoid glycosides including rutin [3]. In the gut quercetin is formed through hydrolysis of rutin by intestinal microorganisms [4]. The pharmacological actions of rutin and quercetin include inhibition of lipopolysaccharide-induced nitric oxide production, antioxidant, anti-hyperglycemic, anti-inflammatory, cytoprotective, hepatoprotective and chemopreventive activities [5,6,7,8,9,10,11,12]. In several cell types including pancreatic beta-cells it suppresses NF-κβ, decreases pro-inflammatory cytokines and suppresses the production of oxidized LDL and TNF-α [13,14,15,16]. Both rutin and quercetin show insulinotropic properties in vivo and in vitro, have beneficial effects in metabolic syndrome and exert anti-diabetic properties. Studies in animals and humans have suggested its potential in the treatment of metabolic syndrome and diabetes [13,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33].

Rutin and quercetin act on cellular signaling mechanisms on many levels and affect e.g. glucose transport, metabolism, cell proliferation and survival. In rat skeletal muscle rutin stimulates Ca2+ uptake from the extracellular space through voltage-gated Ca2+ channels and glucose uptake is increased as a consequence of calcium-calmodulin-dependent protein kinase II (CaMKII)-stimulated glucose transporter (GLUT)-4 translocation to the plasma membrane [34]. Rutin has further been shown to enhance insulin-dependent GLUT-4 translocation by insulin-receptor (IR) tyrosin kinase activation [35]. Quercetin and its metabolite quercetin-3-O-methylglucose were shown to protect the umbilical vein endothelium against oxidative stress-induced inflammation and insulin resistance. Both compounds facilitated phosphoinositide-3-kinase (PI3K) signaling by positive regulation of serine/tyrosine phosphorylation of IR substrate (IRS)-1 and restoration of downstream Akt/eNOS activation [36]. In INS-1 insulinoma cells quercetin potentiates glucose- and glibenclamide-induced insulin release and Erk1/2 phosphorylation and protects the cells against H2O2-induced oxidative damage [19]. In the same cells as well as in isolated rat pancreatic islets quercetin has been found to enhance insulin secretion by direct activation of L-type Ca2+ channels [17]. In INS-1E insulinoma cells quercetin enhances glucose-induced insulin secretion under hyperglycemic and glycotoxic conditions and it modulates gene expression profiles - including GLUT-2, glucokinase, insulin receptor substrate (IRS-1), Akt1/2, Bcl2 and Bax among others [18] - to improve beta-cell survival and function during glucotoxicity. Quercetin affects cell proliferation and cell death. Recently it was shown that in mouse MC3T3-G2/PA6 cells differentiated into mature adipose cells quercetin inhibits insulin-stimulated glucose uptake and IR-beta subunit phosphorylation, while rutin was without effect. Moreover, quercetin appears to inhibit insulin-stimulated activation of Akt and suppresses insulin-dependent GLUT-4 translocation to the plasma membrane [37]. In MDA-MB-231 breast cancer cells quercetin suppresses insulin-induced IR dimerization by interfering with ligand-receptor interactions, which reduces the phosphorylation of IR and Akt. This further inhibits insulin stimulated glucose uptake due to impaired translocation of GLUT-4 to the cell membrane leading to impaired cancer cell proliferation [38]. By inhibiting the PI3K/Akt pathway quercetin also inhibits proliferation of HeLa cells and breast carcinoma cell lines and induces apoptosis in promyelocytic HL-60 cells [39,40,41]. In HepG2 hepatocellular carcinoma cells quercetin was found to mediate autophagy by release of Ca2+ from intracellular stores [42] and to enhance apoptosis by induction of a positive feedback loop consisting of p53, miR-34a miRNA and SIRT1 [43].

The present study was performed to investigate short and long-term effects of quercetin and rutin on beta-cells [1,2]. While most studies which describe insulinotropic influences of flavonoids on beta-cells focus on short term effects, data on chronic exposure are lacking. We designed this study to investigate both long- and short term effects on insulin secreting cells using rat INS-1 cells - a beta-cell model well described with respect to proliferation, pro-and anti-apoptotic signal transduction pathways, insulin release and electrophysiological behavior [19,44,45,46,47,48,49]. Cell viability, apoptosis, phospho(P)-Akt and P-Erk1/2 protein abundance and insulin release were determined and cell membrane potentials (Vmem), whole-cell Ca2+ (ICa)- and ATP-sensitive K+ (IKATP) currents and intracellular Ca2+ (Cai) concentrations were measured.

Materials and Methods

Chemicals and reagents

All salts and chemicals used for the preparation of experimental solutions and cell culture media were p.a. grade. Tolbutamide, LY294002 and porcine insulin were purchased from Sigma-Aldrich. Rutin (Quercetin-3-rutoside) and quercetin were from Carl Roth. Stock solutions of rutin, quercetin and LY294002 (50 or 100, and 10 mM, respectively) were prepared in dimethyl sulfoxide (DMSO). Tolbutamide was dissolved in ethanol at a stock concentration of 100 mM. Resazurin was obtained from Sigma Aldrich and dissolved in phosphate buffered saline (PBS) to give a 2.5 mM stock solution. Stocks were stored in aliquots at -20°C until use.

Cell culture

For cell viability measurements, Western blot experiments, flow cytometry and electrophysiological recordings, INS-1 cells [48] were grown in RPMI 1640 medium containing 11.1 mM D-glucose, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, 2 mM glutamine, 10 mM HEPES, 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin B at 37°C, humidified 5% CO2 and 95% air (standard conditions). Subcultures were established once a week by trypsin/EDTA treatment. Cells within passages 100 and 120 were used for the experiments.

Cell viability

For cell viability measurements INS-1 cells were seeded in black, clear bottom, tissue culture treated 96-well microplates at a density of 12,500 cells/well. After over-night incubation under standard conditions cells were either left untreated, incubated in culture medium containing rutin or quercetin (1.5, 3.1, 6.25, 12.5, 25, 50 and 100 µM), LY294002 (1, 10 or 100 µM), or 0.1% (v/v) DMSO (solvent control) for further 48 hrs. Insulin (10 µM) was added to the culture medium as indicated. Triton X-100 (1% v/v) causing maximum cell damage was used as positive control. Experimental conditions were run in 5-6 replicates per plate. For quantification of cell viability a resazurin (alamar blue) assay was used [50]. This assay is a fluorescence-based assay to determine the number of viable, metabolically active cells, given that only viable cells are able to metabolize resazurin to the fluorescent resorufin. The reduction of resazurin to resorufin is irreversible and fluorescence intensity is proportional to the amount of viable cells. Resazurin was used at a final concentration of 0.5 mM. Fluorescence measurements were performed on a Zenyth 3100 multimode reader (Anthos Labtec Instruments GmbH, Austria). Excitation and emission wavelength were 535 and 595 nm, respectively. For background subtraction background fluorescence was determined for each experimental treatment from blank, cell-free wells containing incubation medium with drugs only. Data are presented as relative fluorescence units (RFU). As the reduction of resazurin depends on cell metabolism, changes in fluorescence might be caused by effects on the cells' metabolic state rather than the absolute number of viable cells. To validate the resazurin assay we therefore performed a parallel series of experiments based on live-cell protease activity assessment using a fluorogenic, cell-permeant, peptide substrate (CellTiter Fluor™ cell viability assay; Promega). Intracellular cleavage of the substrate by a conserved and constitutively active protease generates a fluorescent signal proportional to the number of living cells. The results obtained with this assay were identical to those of the resazurin assay (n = 3; data not shown).

Flow cytometry

For assessment of phosphatidylserine exposure at the cell surface by Annexin-V binding and cell membrane integrity by 7-actinomycin D (7-AAD) staining, cells were seeded in 30 mm diameter petri dishes at a density of 600,000 cells/dish. Following over night culture under standard conditions, cells were either left untreated, or incubated with 50 µM rutin, 50 µM quercetin, or 0.1% (v/v) DMSO (solvent control) in absence or presence of 10 µM insulin for 48 hrs. Thereafter cells were harvested by trypsin/EDTA treatment, washed twice in PBS and resuspended in the binding buffer to a concentration of 1 × 106 cells/ml. 100 µl of the respective cell suspensions were subjected to staining for 15 minutes in the dark with FITC (fluorescein isothiocyanate)-conjugated Annexin-V and 7-AAD (BioLegend, Inc., USA) according to the manufacturer's protocol. After addition of 400 µl binding buffer to each sample, cells were analyzed by flow cytometry. Fluorescence emissions of FITC-Annexin-V on FL-1 (525 nm band pass filter) and 7-AAD on FL-3 (670 nm long pass filter) were measured upon excitation with a 488-nm argon laser using a Cell Lab Quanta™ SC flow cytometer (Beckman-Coulter). Unstained and single-stained samples were used for setting the electronic volume (EV) gain, FL-1 and FL-3 PMT-voltages and for compensation of FITC-spill over into the 7-AAD channel. Debris (particles diameter <7 µm) and cell aggregates (>20 µm) were excluded from analysis. 30,000-5 0,000 single cells (diameter 7-20 µm) were analyzed in each sample. Parameter dividers were set to segregate Annexin-V- and Annexin-V+ cells. Cell diameter/volume was directly measured with the Cell Lab Quanta™ employing the Coulter principle for volume assessment, which is based on measuring changes in electrical resistance produced by nonconductive particles suspended in an electrolyte solution. The electronic volume channel was calibrated using 10 µm Flow-Check fluorospheres (Beckman-Coulter) by positioning this size bead in channel 200 on the volume scale. The mean cell volume (MCV) is given in femtoliters (fl).

Western blot

For analysis of phospho-protein abundance, cells were seeded in 30 mm petri dishes at a density of 106 cells/dish and grown for 48 hrs under standard conditions to 70-80% confluence. Subconfluent cells were incubated for 2 hrs in culture medium containing 50 µM rutin, 50 µM quercetin, 0.1% (v/v) DMSO (solvent control) in absence or presence of 10 µM insulin, or left untreated. After the incubation period culture media were aspirated and cells were washed with ice cold PBS. Cells were scraped off with lysis buffer composed of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin and 1 mM phenylmethanesulfonyl fluoride (PMSF). Cell lysates were sonicated (5×1 second) and centrifuged at 13,000×g for 10 minutes. Samples were mixed with 2× sample buffer containing 125 mM TRIS-HCl, 4% (w/v) SDS, 0.7% (v/v) β-mercaptoethanol, 20% (v/v) glycerol and 0.004% (w/v) bromphenol blue, and 25 µl/sample were separated by SDS-PAGE on 4-20% Mini-PROTEAN® TGX™ gels (Bio-Rad, Germany). Proteins were transferred onto 0.2 µm nitrocellulose membranes using a Bio-Rad Trans-Blot® Turbo™ system. Membranes were incubated over night at 4°C with rabbit monoclonal antibodies against total Akt (pan), phospho-Akt (Thr308), phospho-Akt (Ser473), total Erk1/2 (p44/42 MAPK), or phospho-Erk1/2 (Thr202/Tyr204) at dilutions of 1:1000 or 1:500. The secondary antibody was a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2000). All antibodies were from CellSignaling Technology, USA. Immunodetection, imaging and data analysis was performed using SignalFire™ enhanced chemiluminescent (ECL) substrate (CellSignaling Technology, USA), a ChemiDoc™ MP imaging system and Image Lab™ software (Bio-Rad, Germany). Data are presented as phospho-protein/total protein ratios.

Glucose stimulated insulin secretion (GSIS) assay

On day 1 of the experiment, INS-1 cells were detached by Trypsin/EDTA treatment, counted using a CASY TT cell counter (Schärfe, Germany), seeded into 35 mm cell culture dishes (Sarstedt, Austria) at a density of ∼400,000 cells per dish, and cultured in RPMI1640 (Sigma Aldrich) supplemented with 1 mM sodium pyruvate (Sigma Aldrich), 23.8 mM sodium bicarbonate (AppliChem), 84.4 µM 2-mercaptoethanol (Sigma Aldrich), 10 mM HEPES (AppliChem) and 5% FBS Superior (Biochrom) at 37°C, 5% CO2 and 95% air. On day 3, INS-1 cells were gently washed with PBS and then preconditioned for 30 minutes in 2 ml per cell culture dish of glucose-free Krebs Ringer HEPES buffer (KRH 0 Gluc) containing either 0.1% DMSO (KRH 0 Gluc + D; solvent control) or 50 µM of quercetin (KRH 0 Gluc + Q), or rutin (KRH 0 Gluc + R). Preconditioning was followed by the treatment of the cells for one hour with 1 ml per cell culture dish of either KRH 0 Gluc + D, KRH 0 Gluc + Q, KRH 0 Gluc + R, or KRH containing 20 mM glucose (KRH 20 Gluc) and 0.1% DMSO (KRH 20 Gluc + D; solvent control), or 50 µM quercetin (KRH 20 Gluc + Q), or rutin (KRH 20 Gluc + R). Preconditioning and treatment of cells was carried out at 37°C in humidified air. At the end of the treatment period, 200 µl of each cell culture supernatant was collected, centrifuged (800×g, 5 minutes at 4°C) and stored at -20°C. Cells were then detached from each cell culture dish by Trypsin/EDTA treatment and the number of cells per dish determined as described above. The composition of KRH 0 Gluc was (in mM): 145 NaCl, 3.6 KCl, 1.5 CaCl2, 0.5 MgCl2, 10 HEPES, 0.5 NaH2PO4, 5 NaHCO3 with 0.1% bovine serum albumin (BSA; fraction V, fatty acid free), pH 7.4 (adjusted with NaOH). KRH 20 Gluc had the same composition as KRH 0 Gluc, except that NaCl was reduced to 132 mM and 20 mM glucose added.

ELISA, insulin release

Insulin concentration in the cell culture supernatants was determined by a rat insulin ELISA (Mercodia, Sweden) according to the protocol suggested by the supplier. The insulin concentration in the cell culture supernatants was normalized to the number of cells (determined at the end of the treatment period (see above)) in each of the corresponding cell culture dishes, and expressed as µg/l per 105 cells.

Electrophysiology

INS-1 cells were seeded on poly-D-lysine-coated glass coverslips, cultured under standard conditions and used for patch clamp experiments after 24-48 hrs. The coverslips were transferred to a recording chamber and mounted on an Olympus IMT-2 inverted microscope. All experiments were performed at room temperature in the conventional ‘ruptured' patch clamp mode. Patch electrode resistances were 3-5 MΩ. Data were acquired and analyzed using an EPC-10 amplifier and Pulse/FitMaster software (HEKA, Germany). Cell membrane potential recordings were performed in the zero current clamp mode. The intracellular (pipette) solution contained (in mM): 120 potassium D-gluconate, 5 NaCl, 10 KCl, 2 CaCl2, 4 MgCl2, 2 ATP (Mg2+ salt), 5 HEPES free acid (FA), 10 EGTA, 5 raffinose (pH 7.2 adjusted with KOH, 300 mOsm/kg). The extracellular solution contained (in mM): 140 NaCl, 5.6 KCl, 2.5 CaCl2, 1.5 MgCl2, 10 HEPES FA, 4.5 D-glucose, 5 mannitol (pH 7.4 adjusted with NaOH, 300 mOsm/kg). Whole cell KATP currents were recorded using same solutions at a holding potential of -70 mV and during 500-ms pulses to -80 and -60 mV at 10-s intervals as previously described [51,52]. In this range of potentials, the membrane conductance is predominately determined by KATP currents [53]. The pipette solution for whole cell Ba2+ current recordings contained (in mM): 100 CsCl, 5 MgCl2, 2 ATP (Mg2+ salt), 10 HEPES FA, 11 EGTA, 65 raffinose (pH 7.2 adjusted with CsOH, 310 mOsm/kg). The extracellular solution consisted of (in mM): 80 tetraethylammonium (TEA)-Cl, 10 BaCl2, 10 HEPES FA, 5 D-glucose, 120 mannitol (pH 7.2 adjusted with TEA-OH, 303 mOsm/kg). Ba2+ currents were elicited by voltage ramps from -100 to +100 mV (500 ms duration, 10-s intervals) from a holding potential of -70 mV [52]. Osmolalities were measured with a vapor pressure osmometer (Wescor, USA). A gravity-driven perfusion system (ALA Scientific Instruments, Inc., USA) with a flow-rate of 3-5 ml/min was used for extracellular solution exchange. Drugs were added to the extracellular solutions as indicated.

Calcium imaging

Intracellular Ca2+ was monitored by time-lapse fluorescence imaging. Cells grown for 24-48 hrs in glass-bottom petri dishes (3-cm diameter; MatTek Corporation, USA) under standard cell culture conditions were loaded with 2 µM Fura-2/AM plus 0.08% Pluronic F-127 (Molecular Probes-LifeTechnologies) for 20 minutes at 37°C in the dark in serum-free medium. Cells were washed once and incubated for further 20 minutes in the dark to allow for complete de-esterification of the dye. Dishes were placed on the microscope (iMIC; TILL Photonics-FEI, Germany), which was equipped with a filter set consisting of a 395 nm cleanup filter, a 409 nm beamsplitter and a 510/84 nm bandpass filter (AHF Analysentechnik, Germany) compatible with the Polychrome V monochromator (TILL Photonics) used for dye excitation. Cells were alternately illuminated at 340 and 380 nm for 100-200 ms at 10-second intervals. The emitted light was passed through the clean-up filter, the beamsplitter and the bandpass emission filter before detection by a PCO Sensicam QE CCD camera (PCO AG, Germany). Life Acquisition and Offline Analysis Software (TILL Photonics) was used for microscope, camera and monochromator control, data acquisition and analysis. Cells were continuously perfused with a solution containing (in mM): 140 NaCl, 5.6 KCl, 2.5 CaCl2, 1.5 MgCl2, 10 HEPES FA, 4.5 D-glucose, 5 mannitol (pH 7.2 adjusted with NaOH, 296 mOsm/kg) at a flow rate of 2-3 ml/minute. The Ca2+-free extracellular solution was prepared by omission of CaCl2 and addition of 2 mM EGTA. Drugs were added to the solution as indicated in the figure legend (Fig. 6). The image background was recorded from cell free areas and subtracted from the respective signals. Data are given as 340/380 nm excitation ratios detected at 510 nm.

Fig. 6

Quercetin induces a transient elevation in intracellular Ca2+ (Cai) which requires extracellular Ca2+. Cells were loaded with Fura-2/AM and fluorescence ratios (F340/F380) were recorded in the absence or presence of quercetin (Q; 50 µM) or tolbutamide (TOL; 50 µM). (A) and (C) Responses of single cells (grey tracings) and mean responses (black tracings) of 13 and 39 cells, respectively, of individual experiments. In experiments as shown in (A) Ca2+ was present in the extracellular solution during the whole experiment. (B) Bar graph showing the average of the mean responses of 4 individual experiments (13-34 cells per experiment) as shown in (A). Q(max), maximal effect of Q; Q(trans), fluorescence ratios after ∼6 min of Q application. (D) Average of mean responses of 4 individual experiments (30-41 cells per experiment) as shown in (C). Ca2+ free+Q, fluorescence ratios after ∼7 min of Q application in Ca2+ free extracellular solution. Asterisks indicate significant differences to solvent (DMSO) control (CTL). # indicates a significant difference to Ca2+ free conditions.

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Statistics and data presentation

Data are expressed as mean ± SEM of at least three biological replicates (n≥3). In all experimental series, solvent control samples were included and compared to untreated controls. In neither series a statistically significant difference between untreated- and solvent (DMSO)-treated cells was observed. Statistical analysis was carried out using Student's t-test or one-way ANOVA with Dunnett's, or Bonferroni's post-test, as applicable. Means were considered significantly different at p-values <0.05. * /#, **, ***, **** denotes p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively. ns = not significant. n refers to the number of independent experiments. Data were analyzed and plotted using GraphPad Prism 6 (GraphPad Software, USA) or Igor Pro 6 (WaveMetrics, Inc., USA).

Results

Using a resazurin-based assay, we found that incubation with rutin, quercetin, or the PI3K inhibitor LY294002 for 48 hrs dose-dependently reduced INS-1 cell viability with IC50 values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively (Fig. 1A). Addition of 10 µM insulin to the culture medium over 48 hrs stimulated cell proliferation, but had no influence on the inhibiting effect of quercetin on cell viability (Fig. 1B). To test if the effects of rutin and quercetin on cell viability were related to induction of apoptosis, we performed Annexin-V binding assays to quantify phosphatidylserine (PS) exposure and measurements of the mean cell volume (MCV) using flow cytometry. Quercetin (50 µM) significantly increased the percentage of Annexin-V binding (Annexin-V+) cells from 16.2 ± 0.9% to 36.9 ± 8.9% within 48 hrs (Fig. 1C and E) and the MCV of Annexin-V+ cells was significantly lower in quercetin-treated samples compared to control cells (1340.0 ± 202.8 fl vs. 1622.0 ± 135.2 fl, respectively; Fig. 1D). Rutin (50 µM) had no effect on the percentage of Annexin-V+ cells or the MCV.

Fig. 1

(A) Dose-dependent inhibition of INS-1 cell viability by rutin (R; n = 4), quercetin (Q; n = 5) and LY294002 (LY; n = 9) measured after 48 hrs. (B) Effects of insulin (10 µM) and insulin (I) plus Q (50 µM) on cell viability within 48 hrs (n = 6). (C) and (D) Phosphatidylserine (PS) exposure (percentage of Annexin-V+ cells) and mean cell volume (MCV), respectively, of INS-1 cells cultured for 48 hrs in absence or presence of R and Q (50 µM each; n = 5) measured by flow cytometry. (E) Data of a single experiment shown as histograms (Annexin-V- and Annexin-V+ cell populations) of cells cultured for 48 hrs in absence or presence of R or Q. Asterisks indicate significant differences to solvent (DMSO) control (CTL); RFU, relative fluorescence units.

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Given the effects of quercetin and rutin on cell viability, we next tested if the compounds interfere with signaling pathways that regulate cell survival and proliferation. Using Western blot we quantified the abundance of phosphorylated Akt (PKB) and extracellular signal-regulated kinase 1 and 2 (Erk1/2, or p44/42) protein in cells incubated for 2 hrs in absence or presence of rutin or quercetin. We found that quercetin (50 µM) caused a significant decrease in basal- and insulin-stimulated Akt(T308) phosphorylation by ∼40% and 50%, respectively, without affecting basal-, or stimulated phosphor (P)-Akt(S473) abundance. Further Erk1/2 phosphorylation was significantly increased by ∼50% in quercetin-treated cells compared to control cells. At the same concentration rutin (50 µM) had no significant effect on (P)-Akt(T308/S473), or P-Erk1/2 protein abundances (Fig. 2).

Fig. 2

(A) Western blot analysis of the effect of 50 µM rutin (R) and quercetin (Q) on Akt- and Erk1/2 phosphorylation. Akt(T308) and Erk1/2 (n = 5), Akt(S473) (n = 4). Akt(T308/S473) phosphorylation was stimulated by addition of 10 µM insulin (I) to the culture medium during the 2 hrs incubation period with R and Q. Data are shown as ratios of phosphorylated protein to total protein under the given experimental conditions relative to solvent (DMSO)-treated cells (CTL). Asterisks indicate significant differences to CTL. # indicates a significant difference between I and I+Q for Akt(T308) phosphorylation. (B) Western blot images (cropped) of total Akt protein (upper bands) and Akt(T308)-phosphorylated protein (lower bands) under the indicated experimental conditions.

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By performing insulin release measurements under basal- and glucose-stimulated conditions using ELISA, we next tested for short-term effects of rutin and quercetin on INS-1 cell function. Basal insulin secretion in glucose-free ringer was 1.3±0.1 µg/l per 100,000 cells (n = 3; Fig. 3). Under 20 mM glucose we observed a 17-fold increase to 22.7 ± 1.6 µg/l per 100,000 cells. Both basal- and glucose-stimulated secretion were significantly augmented 1.7- and 2.7-fold by 50 µM quercetin, respectively, whereas rutin did not significantly affect hormone release.

Fig. 3

Effects of quercetin (Q) and rutin (R) on insulin secretion measured by ELISA as cumulative release over 1 h (n = 3). CTL, solvent (DMSO) control. Asterisks indicate significant differences between groups as indicted.

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To test, if stimulated insulin secretion was due to an acute effect of quercetin on the electrophysiological behavior of INS-1 cells, we performed patch clamp recordings of the cell membrane potential (Vmem) before and after bath application of quercetin. As shown in Fig. 4A and B, quercetin (50 µM) rapidly depolarized Vmem from -65.7 ± 3.7 mV under control conditions to maximally -40.8 ± 7.5 mV (n = 8; Fig. 4B; Q(max)), which was associated with action potential firing. In the continued presence of quercetin, the action potential amplitudes gradually decreased and Vmem returned to control values within 57.5 ± 5.2 s (Fig. 4B; Q(trans)). The ATP-sensitive K+ current (IKATP) blocker tolbutamide (50 µM) caused a persistent, reversible depolarization to -27.5 ± 9.3 mV (n = 6). Rutin (50 µM) had no significant effect on Vmem (n = 5; p = 0.33). Therefore, we focused on quercetin in the following series of patch clamp experiments and did not further test rutin.

Fig. 4

Effect of quercetin (Q) on the cell membrane potential (Vmem) and electrical activity of INS-1 cells. (A) Current clamp recording in absence and presence of Q (50 µM), or tolbutamide (TOL; 50 µM). Sequences of action potentials elicited by Q or TOL are shown as insets at a higher temporal resolution (Q, full symbols; TOL, empty symbols). In contrast to the TOL-induced depolarization, the Q-induced effect is transient with Vmem returning to the control level within ∼1 min. Numbers 1-4 correspond to those in (B) and indicate time points at which Vmem was analyzed. (B) Average Vmem under the given conditions. Results of 5-8 individual experiments as shown in (A). Q(max), maximum Q effect; Q(trans), Vmem after ∼1 min of Q application; TOL, tolbutamide; R, rutin (50 µM). (C) Effects of Q and TOL on Vmem in presence of nifedipine (NIF; 10 µM), n = 7-8. (D) Effects of Q and TOL on Vmem in presence of diazoxide (DIA; 100 µM). Numbers 1-4 in (D) correspond to those in (E) indicating at which time points Vmem was analyzed. (E) Average Vmem under the given conditions. Results of 5-8 individual experiments as shown in (D). Asterisks indicate significant differences to solvent (DMSO) control (CTL).

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Vmem and electrical activity of beta-cells is mainly determined by ATP-sensitive K+ currents (IKATP) and voltage-dependent Ca2+ conductances (ICa). To find out, if effects on one or both of these conductances (i.e., inhibition of IKATP and/or activation of ICa) were underlying the quercetin-induced depolarization, we performed additional series of Vmem recordings applying the L-type Ca2+ current inhibitor nifedipine, or the KATP-channel opener diazoxide along with quercetin. In the presence of nifedipine (10 µM), which did not significantly affect Vmem when applied alone (p = 0.69; n = 8), both quercetin or tolbutamide caused depolarizations from -68.6 ± 3.6 (n = 8) to -53.9 ± 4.4 (n = 8) and -36.3±4.9 mV (n = 7), respectively (Fig. 4C). These changes in Vmem were smaller, but not significantly different to the depolarizations induced by quercetin or tolbutamide alone shown in Fig. 4B (p = 0.16 and 0.40, respectively). Application of diazoxide alone (100 µM) caused a significant hyperpolarization of Vmem from -65.9 ± 1.8 to -73.3 ± 1.5 mV (n = 8) (Fig. 4D and E). When quercetin was applied in addition of diazoxide, Vmem remained hyperpolarized at -72.8 ± 1.7 mV (n = 8), and also tolbutamide-induced electrical activity and depolarization were largely prevented reaching levels of Vmem as measured under control conditions (-63.7 ± 6.9 mV; n = 5; Fig. 4E).

To directly assess possible effects of quercetin on voltage-dependent Ca2+ conductances, we measured whole-cell currents in response to voltage ramps from -100 to +100 mV from a holding potential of -70 mV using Ba2+ as charge carrier. As characteristic for L-type Ca2+ currents, the conductance reached peak amplitudes at high voltage (-5.54 ± 0.86 mV; n = 14) and were sensitive to nifedipine (data not shown). As shown in Fig. 5A and B, quercetin (50 µM) quickly and transiently increased the peak current amplitude from -255.8 ± 41.6 pA (CTL; n = 14) to -286.6±44.9 pA (Q(max); n = 14). In the continued presence of quercetin peak currents then gradually decreased back to control current levels within 66.7 ± 8.0 s (Q(trans); n = 7). While the increase in peak currents by quercetin was transient, a persistent broadening of the current-voltage curve was observed (Fig. 5B).

Fig. 5

Effects of quercetin (Q; 50 µM) on whole-cell Ca2+ (ICa) and ATP-dependent K+ currents (IKATP) in INS-1 cells. (A) Transient increase in peak ICa amplitudes by Q. CTL, solvent (DMSO) control (n = 14); Q(max), maximal effect of Q (n = 14); Q(trans), current amplitudes after ∼1 min in continued presence of Q (n = 7). (B) Original ICa tracings of an individual experiment in absence (CTL) and presence of Q. The inset shows the applied voltage-ramp protocol. (C) Inhibition of IKATP by Q and tolbutamide (TOL) (n = 6). Q(max), maximal effect of Q; Q(trans), current amplitudes after ∼1 min in continued presence of Q. (D) Original IKATP tracings of an individual experiment in absence (CTL) and presence of Q, or TOL. The inset shows the applied voltage-step protocol. Asterisks indicate significant differences to solvent (DMSO) control (CTL), or between groups as indicated.

http://www.karger.com/WebMaterial/ShowPic/511280

Given that the ATP-dependent K+ channel opener diazoxide efficiently prevented quercetin-induced depolarizations, we assumed that apart from ICa stimulation, IKATP played a major role in mediating the quercetin effect. To test this hypothesis, we measured IKATP by applying voltage steps to -60 and -80 mV from a holding potential of -70 mV before and after application of quercetin (50 µM). As shown in Fig. 5C and D, application of quercetin resulted in a maximally ∼50% inhibition of IKATP (Q(max)). Like observed for Vmem and ICa, this effect showed a rapid onset and then faded over ∼1 min (60.00 ± 10.33 s) until control current levels were re-obtained (Q(trans)). Current amplitudes at -80 and -60 mV were -137.4 ± 22.8 and 145.8 ± 23.1 pA under control conditions and -58.8 ± 7.2 and 58.1 ± 6.4 pA at Q(max) (n = 6). Tolbutamide at the same concentration (50 µM) caused a significantly stronger and persistent block to -16.6 ± 4.7 and 15.9 ± 4.6 pA (n = 6) at -80 and -60 mV, respectively.

To test if quercetin-induced cell membrane depolarization is reflected in changes in the intracellular Ca2+ (Cai) concentration, we performed time-lapse Ca2+ imaging. In the presence of extracellular Ca2+ quercetin (50 µM) elicits a similar rise in Cai as 50 µM tolbutamide (Fig. 6A and B). In line with Vmem recordings, the effect of quercetin on Cai was transient with peak responses after ∼1.5 min followed by a decline of the signal in the persisting presence of quercetin. Within ∼6 min Cai returned to control levels. In the absence of extracellular Ca2+ the response to quercetin was completely prevented (Fig. 6C and D). After removal of Ca2+, intracellular Ca2+ levels decreased and even dropped further upon application of quercetin.

Discussion

In this study we demonstrate that the acute application of quercetin leads to stimulation of both basal and glucose stimulated insulin secretion (GSIS) in rat insulinoma INS-1 cells (Fig. 3). This is in agreement with previous studies, showing potentiation of GSIS by quercetin in INS-1 cells [19] and INS-1E cells [18]. As underlying mechanisms, we could identify simultaneous transient inhibition of KATP channels and a transient activation of voltage-gated Ca2+ channels. While an effect of quercetin on KATP channels has not yet been described, activation of Ica has been shown in INS-1 cells and pancreatic islets [17]. In contrast to that study where quercetin only slightly affected Vmem and caused a permanent activation of Ca2+ channels, we observed a transient depolarization of Vmem and Ca2+ channel activation. The quercetin effects on Vmem, ICa and IKATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute (Fig. 4 and 5). However, the broadening of the Ca2+-current-voltage curve under quercetin, which persists after peak current amplitudes returned to control values (Fig. 5B), might indicate the activation of an additional, nifedipine-insensitive Ca2+ current component by quercetin. This might explain that, although nifedipine cannot prevent the quercetin-induced depolarization, the magnitude of the effect is slightly (though not significantly) smaller in the presence of nifedipine compared to the effects in its absence (15 mV vs. 25 mV depolarization by quercetin in presence vs. absence of nifedipine; Fig. 4B and C). The KATP channel opener diazoxide (100 µM) caused a significant hyperpolarization and completely prevented quercetin and tolbutamide-induced depolarization of Vmem and electrical activity (Fig. 4D and E), suggesting that KATP channels are a direct target of quercetin. Taken together we conclude that the stimulating effect of the aqueous extract of Leonurus sibiricus L. observed in INS-1E cells [1] can be attributed at least in part to its constituent quercetin. In other cell types, quercetin has also been reported to act on Ca2+- and K+- and channels. It was found to inhibit inward Ca2+ currents through L-type voltage-gated Ca2+ channels in coronary arterial smooth muscle cells and to enhance voltage-gated K+ channels leading to vasorelaxation [54]. In coronary artery smooth muscle cells quercetin was shown to promote relaxation by stimulation of large-conductance Ca2+-activated K+ currents [55]. In cultured murine intestinal cells of Cajal quercetin attenuates pacemaker activity by inhibiting TRPM7 channels and also Ca2+-activated Cl- channels (TMEM16A, ANO1) via opioid receptor signaling pathways [56]. In rat tail artery smooth muscle cells with intact endothelium quercetin leads to relaxation even though it acts as activator of L-type Ca2+ channels. In these cells it leads to a reversible increase in amplitude and a shift of the activation curve to more negative potentials while it slows the activation and deactivation kinetics as well as the rate of recovery from inactivation [57,58]. Rutin had no effect on L-type Ca2+ currents [58]. For the first time we show here that quercetin inhibits inwardly rectifying ATP-sensitive K+ currents. This might be highly relevant for KATP channels in other tissues and organs like vascular smooth muscle, skeletal muscle, cardiomyocytes or kidney cells. It needs to be tested in future studies, if this effect of quercetin is specific for the IKATP of insulin-secreting cells (Kir 6.2), or if other subtypes of inwardly-rectifying K+ channels are also affected.

Quercetin-induced cell membrane depolarization and electrical activity is reflected by a transient elevation in the intracellular Ca2+ (Cai) concentration with peak responses similar to those of tolbutamide (Fig. 6). This is in line with the effect of aqueous Leonurus sibiricus L. extract we previously observed in INS-1E cells [1], but contrasts the findings of Bardy et al., who described a persistent effect of quercetin on Cai in INS-1 cells along with an activation of L-type Ca2+ channels [17]. However, in accordance with this study we find that in the absence of extracellular Ca2+ quercetin fails to elevate Cai, which clearly shows that the rise in Cai is due to Ca2+ influx and not due to release from intracellular stores. This supports the conclusion that quercetin interferes with stimulus-secretion-coupling [59] by inducing Vmem depolarization and electrical activity, which leads to a rise in Cai and insulin release.

In a number of studies flavonoids and other polyphenolic compounds have been shown to interfere with cell signaling pathways that regulate cell survival and proliferation by affecting key kinases such as PI3K, Akt (PKB) and extracellular signal-regulated kinase 1 and 2 (Erk1/2, or p44/42) [19,39,40,41,46,60]. As shown in the present study, rutin, quercetin and the PI3K inhibitor LY294002 cause a dose-dependent reduction in cell viability (Fig. 1). Within 2 hrs, quercetin causes a significant decrease in basal- and insulin-stimulated Akt(T308) phosphorylation without affecting P-Akt(S473) abundance and an increase in Erk1/2 phosphorylation (Fig. 2). The decreased Akt(T308) phosphorylation is therefore most likely due to inhibition of PI3K signaling by quercetin [61]. In a previous study we have shown that Akt phosphorylation is inhibited by resveratrol, another flavonoid, which was accompanied, however, by inhibition of insulin secretion [46]. Erk1/2 activation and stimulated insulin secretion by quercetin has already previously been described in INS-1 cells [19]. However, in this study quercetin had no effect on viability at a concentration of 20 µM where we find viability to be reduced by 50% (Fig. 1). 50 µM quercetin significantly increases the percentage of Annexin-V+ cells compared to control cells within 48 hrs and the MCV of quercetin-treated cells is significantly lower, indicating apoptotic cell shrinkage (apoptotic volume decrease, AVD [62]), while rutin does not exert any effect on Annexin-V binding, Erk1/2 or Akt phosphorylation (Fig. 1 and 2). Hence, quercetin but not rutin induces apoptosis in INS-1 cells. It is important to note that we observed a ∼20% increase in Annexin-V+ cells but a ∼90% reduction of resorufin fluorescence under 50 µM quercetin, which suggests that the quercetin effect might not only be caused by induction of apoptosis, but mainly by a stop of cell proliferation. The inhibitory effect of quercetin on cell viability contrasts the finding that the Leonurus sibiricus L. extracts enhances proliferation in INS-1E cells [1]. We assume that the effects of other constituents in Leonurus sibiricus L. extracts overrule the anti-proliferative/pro-apoptotic effects of quercetin and rutin shown here.

The concentrations of quercetin and rutin we tested (1.5-100 µM) are in the range as used in other studies on effects on insulin secretion and the electrophysiological behavior of insulinoma cells [17,18,19]. A significant increase in Cai has been shown at 2 µM quercetin in INS-1 cells as well as in dispersed rat pancreatic islets [17]. Glucose-stimulated insulin release was significantly increased at 1 µM in INS-1E cells [18]. Anti-proliferative, pro-apoptotic effects of quercetin on different cancer cell lines have been shown at concentrations from 10 µM up to 1 mM [39,40,41,42,43] and we show a significantly reduced cell viability and increased percentage of Annexin-V+ cells at 50 µM. Such concentrations are unlikely to be achieved in vivo upon oral or intravenous administration of the compound. In in vivo studies by Egert et al., in which quercetin has been shown to significantly reduce systolic blood pressure, oxidized LDL and TNF-α in overweight patients with high cardiovascular disease risk, mean fasting plasma quercetin concentrations of 269 nmol/l were measured after a daily intake of 150 mg quercetin [13,31]. Other bioavailability studies report plasma quercetin concentration from 50 nM up to 1.6 µM depending if subjects were consuming habitual diets, quercetin rich diets or the pure compound [3]. Quercetin is rapidly absorbed in the proximal parts of the gastrointestinal tract, whereas absorption of rutin occurs in the distal parts and is slow, since it must be hydrolyzed by the colonic microflora [3,4,63,64]. Like other flavonoids quercetin almost exclusively appears in the serum as glucuronide and sulfate conjugates, which have been shown to retain their antioxidant properties and which are slowly eliminated (half-lives from 11 to 28 hrs have been reported) so that repeated intake of rutin and quercetin-containing diet might lead to accumulation of metabolites in blood [3,63,64,65,66]. How far these metabolites can affect beta-cell function and viability as shown here needs to be investigated in further studies.

Insulinoma-derived INS-1 and INS-1E cells are reliable beta-cell surrogates displaying electrophysiological properties, secretagogue-induced electropysiological activity, Ca2+ signaling, stimulus-secretion-coupling and sulfonylurea and diazoxide-sensitivities similar to native islets [48,67]. Insulinoma cells have been used in numerous studies on drugs interfering with signal transduction pathways involved in regulating beta-cell mass, proliferation and apoptosis [19,44,45,46,47,49]. However, altered secretory responses and cell signaling processes regulating proliferation and apoptosis compared to native beta-cells must be considered. For instance differences among beta-cell lines and pancreatic islets in their insulin release responses to osmotic stimuli have been shown [68,69]. Therefore, future studies on native beta-cells need to be performed to validate the significance of our findings. Especially it needs to be clarified if the effect of quercetin on cell viability is specific for insulinoma cells or also have implications for native beta-cells.

We conclude that in INS-1 cells quercetin acutely stimulates insulin release, presumably by transient KATP channel inhibition and simultaneous transient stimulation of voltage-sensitive Ca2+ channels. Long-term application of quercetin and rutin for 48 hrs, however, inhibits cell proliferation and quercetin induces apoptosis, most likely by inhibition of PI3K/Akt signaling. Our data contribute to a better understanding of the mechanism of action of quercetin on beta-cell function and viability.

Acknowledgements

We thank Prof. Claes B. Wolheim for providing the INS-1 cells. We thank Leman Emin and Katharina Schuhbeck for their assistance. This project was supported by the PMU grants R-09/04/009-JAK and R-11/02/024-JAK to MJ.

Disclosure Statement

The authors declare that there are no conflicts of interest.


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  57. Saponara S, Sgaragli G, Fusi F: Quercetin as a novel activator of L-type Ca2+ channels in rat tail artery smooth muscle cells. Br J Pharmacol 2002;135:1819-1827.
  58. Fusi F, Saponara S, Pessina F, Gorelli B, Sgaragli G: Effects of quercetin and rutin on vascular preparations: a comparison between mechanical and electrophysiological phenomena. Eur J Nutr 2003;42:10-17.
  59. Ashcroft FM, Rorsman P: Electrophysiology of the pancreatic beta-cell. Prog Biophys Mol Biol 1989;54:87-143.
  60. Chan A, Dolinsky V, Soltys C, Viollet B, Baksh S, Light P, Dyck J: Resveratrol inhibits cardiac hypertrophy via AMP-activated protein kinase and Akt. J Biol Chem 2008;283:24194-24201.
  61. Vlahos CJ, Matter WF, Hui KY, Brown RF: A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994;269:5241-5248.
  62. Okada Y, Shimizu T, Maeno E, Tanabe S, Wang X, Takahashi N: Volume-sensitive chloride channels involved in apoptotic volume decrease and cell death. J Membr Biol 2006;209:21-29.
  63. Manach C, Morand C, Demigne C, Texier O, Regerat F, Remesy C: Bioavailability of rutin and quercetin in rats. FEBS Lett 1997;409:12-16.
  64. Manach C, Williamson G, Morand C, Scalbert A, Remesy C: Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 2005;81:230S-242S.
  65. Morand C, Crespy V, Manach C, Besson C, Demigne C, Remesy C: Plasma metabolites of quercetin and their antioxidant properties. Am J Physiol 1998;275:R212-219.
  66. Manach C, Morand C, Crespy V, Demigne C, Texier O, Regerat F, Remesy C: Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties. FEBS Lett 1998;426:331-336.
  67. Merglen A, Theander S, Rubi B, Chaffard G, Wollheim CB, Maechler P: Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells. Endocrinology 2004;145:667-678.
  68. Orecna M, Hafko R, Bacova Z, Podskocova J, Chorvat DJ, Strbak V: Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli. Physiol Res 2008;57:935-945.
  69. Orecna M, Hafko R, Toporcerova V, Strbak V, Bacova Z: Cell swelling-induced insulin secretion from INS-1E cells is inhibited by extracellular Ca2+ and is tetanus toxin resistant. Cell Physiol Biochem 2010;26:197-208.

Author Contacts

Martin Jakab

Paracelsus Medical University, Institute of Physiology and Pathophysiology, Strubergasse

22, 5020 Salzburg, (Austria)

Tel. +43-662-242080504, E-Mail martin.jakab@pmu.ac.at


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Abstract of Original Paper

Accepted: May 31, 2016
Published online: June 24, 2016
Issue release date: July 2016

Number of Print Pages: 16
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Number of Tables: 0

ISSN: 1015-8987 (Print)
eISSN: 1421-9778 (Online)

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  56. Gim H, Nam JH, Lee S, Shim JH, Kim HJ, Ha KT, Kim BJ: Quercetin Inhibits Pacemaker Potentials via Nitric Oxide/cGMP-Dependent Activation and TRPM7/ANO1 Channels in Cultured Interstitial Cells of Cajal from Mouse Small Intestine. Cell Physiol Biochem 2015;35:2422-2436.
  57. Saponara S, Sgaragli G, Fusi F: Quercetin as a novel activator of L-type Ca2+ channels in rat tail artery smooth muscle cells. Br J Pharmacol 2002;135:1819-1827.
  58. Fusi F, Saponara S, Pessina F, Gorelli B, Sgaragli G: Effects of quercetin and rutin on vascular preparations: a comparison between mechanical and electrophysiological phenomena. Eur J Nutr 2003;42:10-17.
  59. Ashcroft FM, Rorsman P: Electrophysiology of the pancreatic beta-cell. Prog Biophys Mol Biol 1989;54:87-143.
  60. Chan A, Dolinsky V, Soltys C, Viollet B, Baksh S, Light P, Dyck J: Resveratrol inhibits cardiac hypertrophy via AMP-activated protein kinase and Akt. J Biol Chem 2008;283:24194-24201.
  61. Vlahos CJ, Matter WF, Hui KY, Brown RF: A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994;269:5241-5248.
  62. Okada Y, Shimizu T, Maeno E, Tanabe S, Wang X, Takahashi N: Volume-sensitive chloride channels involved in apoptotic volume decrease and cell death. J Membr Biol 2006;209:21-29.
  63. Manach C, Morand C, Demigne C, Texier O, Regerat F, Remesy C: Bioavailability of rutin and quercetin in rats. FEBS Lett 1997;409:12-16.
  64. Manach C, Williamson G, Morand C, Scalbert A, Remesy C: Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 2005;81:230S-242S.
  65. Morand C, Crespy V, Manach C, Besson C, Demigne C, Remesy C: Plasma metabolites of quercetin and their antioxidant properties. Am J Physiol 1998;275:R212-219.
  66. Manach C, Morand C, Crespy V, Demigne C, Texier O, Regerat F, Remesy C: Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties. FEBS Lett 1998;426:331-336.
  67. Merglen A, Theander S, Rubi B, Chaffard G, Wollheim CB, Maechler P: Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells. Endocrinology 2004;145:667-678.
  68. Orecna M, Hafko R, Bacova Z, Podskocova J, Chorvat DJ, Strbak V: Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli. Physiol Res 2008;57:935-945.
  69. Orecna M, Hafko R, Toporcerova V, Strbak V, Bacova Z: Cell swelling-induced insulin secretion from INS-1E cells is inhibited by extracellular Ca2+ and is tetanus toxin resistant. Cell Physiol Biochem 2010;26:197-208.
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