SIGNALING AND CELL PHYSIOLOGY
Does NAD(P)H oxidase-derived H
2
O
2
participate
in hypotonicity-induced insulin release
by activating VRAC in
β-cells?
R. Crutzen&V. Shlyonsky&K. Louchami&M. Virreira& E. Hupkens&A. Boom&A. Sener&W. J. Malaisse& R. Beauwens
Received: 28 February 2011 / Revised: 14 October 2011 / Accepted: 17 October 2011 / Published online: 18 November 2011 # Springer-Verlag 2011
Abstract NAD(P)H oxidase (NOX)-derived H2O2 was
recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion chan-nels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 and rat β-cells. Exogenous H2O2(100 to 200μM) at basal glucose
concentration (1.1 to 2.8 mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H2O2. In patch clamp experiments, exogenous
H2O2 was observed to stimulate NPPB-sensitive anion
channel activity, which induced cell membrane depolariza-tion. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenyleneiodonium chloride (DPI), a universal NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by
expo-sure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl-L-cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by H2O2. Taken
together, these data suggest that NOX-derived H2O2plays a
key role in the insulin secretory response of BRIN-BD11 and nativeβ-cells to extracellular hypotonicity.
Keywords BRIN-BD11 cells .β-Cells . Rat pancreatic islet . Hypotonicity . H2O2. NAD(P)H oxidase . NOX .
Insulin release
Introduction
An acute reduction in the osmolality of the medium bathing isolated pancreatic islets has been recognized as early as 1975 as a stimulus for insulin release and was found to reproduce the first phase of glucose-induced insulin release [7]. As for glucose-induced insulin release, hypoosmolarity-induced insulin release critically depends on extracellular calcium. Subsequently, studies performed by the group of Drews [8, 11] and of Best [2,3] have demonstrated that hypotonicity not only rapidly induces the fast swelling of theβ-cells but also depolarizes them by opening of a volume-regulated anion channel (VRAC) sensitive to NPPB and DIDS. Further studies by the latter group [4–6,27] further advocated that
R. Crutzen and V. Shlyonsky contributed equally to this study. R. Crutzen
:
M. Virreira:
A. Boom:
R. Beauwens (*) Laboratory of Cell and Molecular Physiology, Université Libre de Bruxelles,Campus Erasme, CP 611. Room E1.6.214, Route de Lennik, 808, 1070 Bruxelles, Belgium
e-mail: renbeau@ulb.ac.be V. Shlyonsky
Laboratory of Pathophysiology, Université Libre de Bruxelles, Brussels, Belgium
K. Louchami
:
E. Hupkens:
A. Sener:
W. J. Malaisse Laboratory of Experimental Hormonology,raising extracellular glucose concentration also leads to β-cell swelling that results from a relative intraβ-cellular hypertonicity induced by glucose metabolism (producing bicarbonate, lactate and possibly other organic anions), since 3-O-methylglucose failed to reproduce a similar cell volume increase. The present study aimed at clarifying the mecha-nism of hypotonicity-induced insulin release, which poten-tially also contributes to glucose-induced insulin release in addition to the well-demonstrated KATPclosure. Mostβ-cell
derived cell lines exhibit strongly reduced insulinotropic response to glucose but they keep responding to hypotonic-ity. We turned to the BRIN-BD11 cell line, as its insulinotropic response to hypotonicity is one of the largest and as it was also shown to depend critically on extracellular calcium [1].
It has been recently proposed in several cell lines that the activation of VRAC under hypotonic conditions (and the ensuing volume regulatory decrease) results from NAD(P) H oxidase (NOX)-derived H2O2that is recognized to act as
a signaling molecule [9,15,19,38]. The present study aims at investigating whether a comparable situation prevails in insulin-producing BRIN-BD11 cells, i.e., whether an increase in intracellular H2O2is instrumental in the opening
of VRAC in the process of hypotoncity-induced insulin release. The effects of H2O2on VRAC activity and insulin
release were also studied in rat dispersed β-cells and isolated pancreatic islets in vitro.
Materials and methods Materials
All inhibitors were purchased from Sigma (St. Louis, MO, USA). The oxidation-sensitive dye, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) was purchased from Molecular Probes
(Eugene, OR, USA). Compounds of the buffers were purchased from Merck (Darmstadt, Germany). Tissue culture materials were purchased from Sarstedt (Nümbrecht, Germany). Culture media were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).
Preparation of rat pancreatic islets
Pancreatic islets were isolated by the collagenase technique [25]. Briefly, Wistar rats were sacrificed and dissected. Bile duct was clamped at its entrance in the duodenum and a canula was inserted to inject Hank's glucose solution (15 ml) containing 5.8 mg collagenenase P (68–130 kDa; Roche, GmbH, Mannheim, Germany) in order to distend the pancreas and digest the exocrine gland. The inflated pancreas was extracted and incubated for 15 min at 37°C
for further digestion. Digestion was stopped by addition of ice-cold Hank's glucose solution and the preparation was washed three times with Hank's glucose by aspiration to remove collagenase and floating exocrine acini. Aliquots of 3 ml were successively transferred in a Petri dish. The islets were collected individually under a microscope using a siliconized Pasteur pipette and laid on a Petri dish containing 4 ml isotonic NaCl medium FBS at room temperature. Four hundred to 600 pancreatic islets could be so obtained from a single rat pancreas. The composition of Hank's glucose solution was (in mM): 137 NaCl, 5.4 KCl, 1.2 CaCl2, 0.8 MgSO4, 0.3 Na2HPO4, 0.4 KH2PO4,
4.2 NaHCO3, 11.1 glucose; pH 7.4 and that of isotonic
NaCl medium FBS (in mM): 111 NaCl, 10 HEPES, 24 NaHCO3, 5 KCl, 1 MgCl2, 1 CaCl2, 2.8 glucose, 10% heat
inactivated fetal bovine serum (#10500-064 GIBCO); pH 7.4.
Preparation of dispersed rat islet cells
Freshly isolated pancreatic islets were incubated at room temperature for 30 min in 1 ml calcium-free Earle-HEPES buffer containing 1 mM EGTA. After incubation, cells were mechanically separated by succion-pushing with a pipette. β-Cells (representing 90% of the cells) were easily identified by their large size and their characteristic granular appearance [6]. Calcium-free Earle-HEPES buffer had the following composition (in mM): 124 NaCl, 5.4 KCl, 0.8 MgSO4, 1 Na2HPO4, 10 HEPES, 14.3 NaHCO3, 2.8
glucose, 1 EGTA, pH 7.4. Cell culture
BRIN-BD11 cells was a kind gift from Prof. A. Herchuelz (Laboratoire de Pharmacodynamie et de Thérapeutique, Université Libre de Bruxelles, Brussels, Belgium). Cells were grown at 37°C in a humidified incubator gassed with 5% CO2 in air, and cultured in a RPMI 1640 medium
(#21875-034 GIBCO), complemented with 10% heat inactivated fetal bovine serum (#10500-064 GIBCO), 50 IU/ml penicillin and 50μg/ml streptomycin.
37°C in a humidified incubator gassed with 5% CO2in air
at least overnight before preincubation or incubation in specified media.
Insulin release BRIN-BD11 cells
Half a million cells were subcultured onto 6-well plate (10 cm²) 48 h before insulin release experiments. Cells were first washed twice with 2 ml isotonic NaCl medium before preincubating for 30 min in 1 ml isotonic NaCl medium containing, when so required, the inhibitor to be tested. Next, cells were incubated in 1 ml isotonic or hypotonic NaCl medium for 30 min containing, when so required, the inhibitor to be tested and/or 100 μM H2O2.
Isotonic NaCl medium had the following composition (in mM): 111 NaCl, 10 HEPES, 24 NaHCO3, 5 KCl, 1 MgCl2,
1 CaCl2, 1.1 glucose, 0.5 mg/ml BSA; pH 7.4, osmolarity
300 mOsmol/l. The composition of the hypotonic medium was the same except for NaCl concentration: 61 mM, osmolarity 200 mOsmol/l.
Dispersed rat islet cells
Dispersed cells from groups of 100 islets were subcultured onto three wells of a 6-well plate (10 cm²) precoated with poly-L-lysine (PLL, #P6282 Sigma) 24 h before insulin release experiments. The coating procedure consists of covering the wells with 1 ml 0.3 mg/ml PLL solution in water for 1 h at room temperature before removing by suction followed by three washings with 2 ml sterile water. The plates were dried overnight at 4°C wrapped in aluminum foil before cell culture. Twenty-four-hour cul-tured cells were washed three times with 2 ml isotonic NaCl medium (composition (in mM): 111 NaCl, 10 HEPES, 24 NaHCO3, 5 KCl, 1 MgCl2, 1 CaCl2, 2.8
glucose, 1 mg/ml BSA; pH 7.4, osmolarity 300 mOsmol/l) before a first preincubation for 1 h in 2 ml isotonic NaCl medium FBS (same composition except for BSA, replaced by 10% heat inactivated fetal bovine serum (#10500-064 GIBCO)). The medium was discarded and cells were washed again with 2 ml isotonic NaCl medium before a second preincubation for 20 min in 1 ml isotonic NaCl medium. The medium was collected and replaced by 1 ml of the same medium containing or not H2O2100 μM with
or without NPPB 100 μM for 20-min incubation. The medium was again collected. The second preincubation and the incubation collected media were centrifuged for 10 min (150 g at 4°C) to avoid potential cell contamination of the samples and 500μl of the upper side of each supernatant were harvested and frozen for determination of insulin content. Cells were trypsinized with 1 ml isotonic 0.05%
Trypsin–EDTA (#25300-054 GIBCO) and counted with a coulter-type handheld cell counter (ScepterTM, Millipore, Bedford, MA). Preincubations and incubation were per-formed at 37°C in a humidified incubator gassed with 5% CO2in air.
Rat pancreatic islets
Groups of 35 islets were placed in cell strainers 40 μm (#352340 BD Falcon, Franklin Lakes, NJ, USA) deposed in 6-well plates, each well containing 6 ml isotonic NaCl medium FBS (composition: see“Dispersed rat islet cells”). Islets were preincubated for 1 h before transfer of each cell strainer in a new well containing 6 ml of the same medium (to get rid of the remaining dispersed cells suspended out of the strainer) for 1 h. Strainers were then washed twice by transfer in new wells containing 7 ml isotonic NaCl medium (composition: see “Dispersed rat islet cells”) before a new preincubation of 20 min in 5 ml isotonic NaCl medium. Strainers were transferred again for 20-min incubation in 5 ml isotonic NaCl medium containing or not 200 μM H2O2. The strainers were discarded and the last
preincubation and the incubation media were individually collected and frozen for determination of insulin content. Preincubations and incubation were performed at 37°C in a humidified incubator gassed with 5% CO2in air.
The insulin content of each preincubation or incubation medium was measured by radioimmmunoassay [20] or by ELISA (#10-1250-01 Insulin, rat ELISA, Mercodia, Uppsala, Sweden).
Patch clamp experiments Voltage measurements
Nystatin-perforated whole cell configuration patch clamp experiments were conducted as described previously [35]. BRIN-BD11 cells: 2,500 cells were subcultured directly onto 1.3-cm diameter glass coverslips (0.25 cm² covered with cells) 48 h before patch clamp experiments. Dispersed ratβ-cells: 10,000 cells were cultured onto 1.3-cm diameter glass coverslips precoated with poly-L-lysine (PLL, #P6282 Sigma). The coating procedure consists of bathing the coverslips for 5 min in 0.1 mg/ml PLL solution in water before drying overnight at room temperature. Cells were used in patch clamp experiments between day culture +1 and day culture +4.
Coverslips were placed in the patch clamp chamber and continuously perfused (1 ml/min) with a physiological solution containing (in mM): 140 NaCl, 4 KCl, 10 HEPES, 1 CaCl2, 1 MgCl2, 5D-glucose (BRIN-BD11 cells) or 4D
was omitted and replaced by 56 mM KCl. The pipette solution contained (in mM): 10 NaCl, 20 KCl, 60 K2SO4
(BRIN-BD11 cells) or 75 K2SO4(dispersed ratβ-cells), 10
HEPES, 300μg/ml nystatin (BRIN-BD11 cells) or 350 μg/ ml nystatin (dispersed rat β-cells), pH 7.2, osmolarity 252 mOsmol/l (BRIN-BD11 cells) or 297 mOsmol/l (dis-persed rat β-cells). Slightly hypotonic pipette solution prevents spontaneous activation of VRAC channels in nystatin-perforated whole cell recordings in BRIN-BD11 cells [6]. Stock solution of nystatin (30 or 35 mg/ml in DMSO) was prepared daily. The patch pipettes were double-step pulled from borosilicate glass capillaries (Hilgenberg, Malsfeld, Germany) using a vertical puller (PC-10, Narishige International, London, UK). Filled pipettes had resistances of 4–6 MΩ. Liquid junction potentials were balanced after formation of a gigaseal contact. Whole cell patch clamp configuration induced by nystatin permeabilization was achieved within 15 to 20 min at room temperature. Access resistance (Rs<35 MΩ) was constant for at least 20 min after stabilization and it was compensated by 70%. Cells were used within 60 min after being taken from the incubator. Zero-current whole cell voltages were continuously recorded using PC-501A amplifier (Warner Instruments, Hamden, CT, USA) and WinEDR software (John Dempster, Strathclyde Institute of Pharmacy and Biomedical Sciences, UK).
Current measurements
In single-channel cell-attached experiments, pipette and bath solutions were high potassium solution as above. To meet the electrophysiological convention, inward chloride channel currents (pipette to cell) were inverted in single-channel records to be represented as upward transitions. Single-channel records were analyzed using pCLAMP software, where all event lists of single-channel records were generated by Fetchan program (Molecular Devices, Sunnyvale, CA). NPo, the product of the number of
channels in a patch (N) by the open probability (Po), which
reflects channel activity within a patch, was calculated using the equation
NPo¼
XN i¼1
i»ti=T
where T is the total recording time, i is the number of open channels, ti is the recording time during which i channels
were open, and N is the apparent number of channels within the patch determined as the highest observable level. Therefore, NPo can be calculated without making any
assumption about the total number of channels in a patch or the open probability of single channels. All NPo values
were calculated for every 60 s of recording.
Cell volume measurement
BRIN-BD11 cells (1.25 to 2.5×106) were subcultured onto 75-cm² culture flasks 24 to 48 h before experiments, preincubated or not for 30 min, 24 h or 48 h with the inhibitor, and then rinsed with isotonic HBSS without Ca2+ and Mg2+ (Invitrogen #14170) and Trypsin 0.05%–EDTA (Invitrogen #25300). After 3 min in the incubator (37°C, 5% CO2 in air), cells were suspended in 20 ml isotonic
RPMI 1640 (Invitrogen #11879) supplemented with 5 mM glucose, containing or not the inhibitor.
Hypotonic condition was obtained by diluting 33% of the isotonic medium containing the cell suspension (20 ml) with water (10 ml), containing or not the inhibitor. Cell diameters were measured by an electronic sizing technique with a coulter-type handheld cell counter (ScepterTM, Millipore, Bedford, MA). The diameter cell histogram distribution was fitted using the program Microcal Origin 6.0. The center of the Gaussian represents the average diameter of the cell for each condition. The volume of the cell was then calculated as the volume of a sphere (suspended cells).
Detection of intracellular H2O2
BRIN-BD11 cells (2.5 × 104) were subcultured onto 1.8 cm×1.8 cm glass coverslips (1 cm2covered with cells) 48 h before experiments, and then rinsed with isotonic RPMI 1640 (Invitrogen #11879) supplemented with 5 mM glucose (isotonic RPMI). The coverslips were loaded 30 min at 37°C in the dark in isotonic RPMI containing 50 μM of the oxidation-sensitive probe CM-H2DCFDA
(Invitrogen Molecular Probes #C6827; λexc=492–495 nm;
λem=517–527 nm) and the inhibitor if specified. The
coverslips were rinsed three times in 100 ml isotonic RPMI (37°C). Cells were then incubated in the dark for 30 min at 37°C in isotonic or 33% diluted with water hypotonic RPMI containing or not the inhibitor. Positive control: isotonic RPMI containing 100 μM H2O2. Cells were then
in the cell by surface unit of cell (or pixel in the cell), we used the function Threshold of the program, adjusted to the same value for each picture of a set and permitting to vizualize entirely the less fluorescent cells. These amounts (Mean fluorescence/pixel of cell) are reported to the amount present in the mean of the controls of the same set to express the relative content of fluorescence. Four percent PAF isotonic phosphate buffer contained (in mM): 98.3 NaH2PO4, 44.4 PAF, pH adjusted to 7.4 with NaOH,
osmolarity 300 mOsm/l. Hypotonic buffer had the same composition except for NaH2PO4: 60 mM, osmolarity
200 mOsm/l. The composition of Mowiol solution was: 140 mM NaCl, 20 mM KH2PO4, pH 7.2 adjusted with
NaOH, 25% Mowiol 4–88 (#475904 Calbiochem). This solution was further diluted 2/3 with pure glycerol and centrifuged.
Reverse transcriptase for NOX4
RT-PCR analysis was carried out on cDNA prepared from mRNA extracted from BRIN-BD11 cells cultured in the presence of betulinic acid for 24 and 48 h as well as in its absence. Primers used for NOX4 were: forward GTTAAACACCTCTGTTCGCTTG-3′ and reverse 5′-CACCTGTCAGGCCCGGAACA-3′. Thermocycling con-ditions were: denaturation at 95°C for 90 s, followed by 40 cycles (95°C for 30 s, 60°C for 30 s, 72°C for 1 min) terminated by a final elongation at 72°C of 5 min. Amplicons were separated on a 1.2% agarose gel contain-ing 0.5μg/ml ethidium bromide.
Statistics
All results are presented as mean values (±SEM) together with the number of individual experiments (n). The statistical significance of differences between mean values was assessed by use of paired or independent Student'st-test when appropriate.
Results
Secretory response to H2O2and hypotonicity
The insulin secretory response of BRIN-BD11 cells to increasing concentrations of H2O2is illustrated in the inset
of Fig. 1. In this set of three experiments, the reference basal value for insulin output averaged 61.3±3.2 μU/2· 106 cells·30 min and the maximal output at 100–250 μM H2O2206.3±38.0μU/2·106cells·30 min. The concentration–
response relationship suggested a threshold concentration for the insulinotropic action of H2O2close to 38μM and a
maximal stimulation of insulin release at about 100 μM
H2O2. In the light of these results, a concentration of
100 μM H2O2 was used in all further experiments. In a
larger set of five experiments (Fig.1, left panel), 100μM H2O2 augmented insulin release from a basal value that
averaged 49.9 ±3.9 μU/2·106 cells · 30 min to a value of 125.1 ±16.7μU/2·106cells · 30 min (p < 0.01). Thus, in the presence of H2O2, the output of insulin represented 250.7 ±
41.3% of the basal value. Likewise, hypotonicity (Fig. 1, right panel), as achieved by a decrease of extracellular NaCl concentration by 50 mM, increased insulin secretion from a basal value of 49.9 ±3.9μU/2·106cells · 30 min to 134.4 ±5.5 μU/2·106 cells ·30 min, the latter value repre-senting 269.3 ±18.8% (p < 0.001) of the basal value. The effect of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) upon the secretory response to H2O2 (Fig. 1)
was observed to be qualitatively similar to that previously
0 25 50 75 100 250 100 150 200 250 300 350 400 450 0 20 40 60 80 100 120 140 °°° *** ° ** NPPB H2O 2 + NPPB HO2 2 Hypo + NPPB NPPB Hypo Iso Iso
Insulin output (µU / 2 . 10
6 cells . 30 min)
Insulin output (% of control)
H2O2 (µM)
Fig. 1 Effect of NPPB (100μM) upon insulin output from BRIN-BD11 cells incubated in an isotonic medium, a hypotonic medium or an isotonic medium containing 100μM H2O2either in the absence or
presence of NPPB, a general inhibitor of VRAC. n=5, two asterisks p<0.01 and three asterisks p<0.001 vs. control; one open circle p< 0.05 and three open circles p<0.001 vs. H2O2- or
hypotonicity-stimulated insulin release (independent Student'st-tests). The inset shows a sigmoidal concentration–dependence relationship between H2O2 and insulin release in BRIN-BD11 cells (p<0.05, one-way
ANOVA test). The maximal response to H2O2is reached at 100μM.
reported for hypotonicity. In isotonic medium and in the absence of H2O2, NPPB (100 μM) failed to affect insulin
output significantly (p >0.2) while in the presence of H2O2,
NPPB inhibited the increment of insulin output by 62.5% (p < 0.05), thus to a level of insulin output remaining still significantly above the basal value of insulin release (p < 0.05). By comparison, NPPB totally abolished this insulinotropic response to hypotonicity.
Niflumic acid (100 μM), another inhibitor of volume-sensitive anion channels, also inhibited insulin release induced by either H2O2 or hypotonicity but to a
signifi-cantly lesser extent, at most by 50% (n=3, data not shown) and it was therefore not further studied.
Effect of NAD(P)H oxidase inhibitors
The similarity between the effect of hypotonicity and of exogenous H2O2 and their sensitivity to VRAC inhibitors
raises the question whether hypotonicity increases intracel-lular H2O2 in BRIN-BD11 cells, possibly by activating a
NAD(P)H oxidase (NOX). NOX inhibitors were, therefore, tested on insulin release during hypotonic conditions.
At 10μM concentration, diphenylene iodonium chloride (DPI), a blocker of all NOX enzymes, did not affect basal insulin release in isotonic conditions. During exposure to hypotonicity, the secretion of insulin increased to 309.8±
62.5% of the basal secretion (n=6, p<0.001) but this increase was abolished (p<0.01) in the presence of DPI (Fig. 2).
A second NOX inhibitor, plumbagin, was tested because of its claimed specificity [10] for NOX4. Plumbagin, 30μM, inhibited again hypotonicity-induced insulin release (n=5, p<0.001) but slightly augmented basal insulin output in isotonic medium (Fig.2).
The antioxidant N-acetyl-L-cysteine (NAC), a precursor of glutathione, was preincubated for 24 h to allow sufficient cell accumulation. It drastically inhibited both basal and hypotonicity-induced insulin release (Fig.2). Preincubation with betulinic acid for 48 h was observed to reduce NOX4 expression in BRIN-BD11 cells (Fig.2, inset) as reported in endothelial cells [36]. This treatment also drastically inhibited both basal and hypotonicity-induced insulin release (Fig.2).
Effect of hypotonicity on cell H2O2production
BRIN-BD11 cells were loaded for 30 min with the oxidation-sensitive dye CM-H2DCF-DA that is
deacety-lated by intracellular esterase, trapping within the cytosol CM-H2DCF that can then be visualized by confocal
fluorescence microscopy when oxidized to CM-DCF. Thus, this method does not distinguish between intracellular
0 20 40 60 80 100 120 140 160 °°° °°° °°° °° *** *** *** * *** *** Hypo + bet Iso + bet Hypo + NAC Iso + NAC Hypo Hypo
Hypo Iso Iso
Iso
Hypo + Plumb Iso + Plumb
Insulin output (µU / 2 . 10
6 cells . 30 min) Ct neg. bet 48h bet 24h CTRL MWM 454 bp Hypo + DPI Iso + DPI Hypo Iso
Fig. 2 Effect of NAD(P)H oxidase inhibitors DPI, 10μM (n=6) and plumbagin, 30μM (n=5) upon insulin output from BRIN-BD11 cells incubated in an isotonic medium or hypotonic medium either in the absence or presence of one of these drugs. Similar experiments were performed on cells preincubated with N-acetyl cysteine (NAC), 5 mM for 24 h (n=4) or betulinic acid (bet) 10μM for 48 h (n=4). One asterisk p<0.05 and three asterisks p<0.001 vs. control; two open circles p<0.01 and three open circles p<0.001 vs.
reactive oxygen species (ROS): superoxide anion or hydrogen peroxide. The intracellular ROS produced by BRIN-BD11 cells exposed to various conditions is illus-trated in Fig.3. Following 30-min stimulation with H2O2,
100μM, the amount of intracellular ROS was increased to 191.5±4.5% of control (n=50, p<0.001) while following exposure to a hypotonic medium, it amounted to 147.6± 3.8% of the control value (n=50, p<0.001). This latter increase was almost completely abolished after preincuba-tion and incubapreincuba-tion with the NAD(P)H oxidase inhibitor DPI (the amount was reduced to 112.0±3.6% of the control value, n = 50, p < 0.001 vs. hypotonic condition). The increase in intracellular ROS was already apparent within 1 min of hypotonicity (data not shown).
Effect of H2O2on BRIN-BD11 cell membrane potential
and single chloride channel currents
Representative zero-current nystatin-perforated whole cell configuration patch clamp experiments are shown in Fig.4a and b. Exogenous addition of H2O2 (100 μM) induced
within 7–9 min a depolarization of about 33 mV (triggered within 5 min). Cell membrane rapidly repolarized towards control values upon addition of NPPB (100μM). The mean values of six such experiments are reported in Fig.4c. As seen in the Fig. 4a, exposure to 60 mM potassium bath solution completely annulated the membrane potential. This experimental condition was, therefore, selected to record
single-channel cell-attached currents in order to prevent effect of voltage changes due to cell depolarization on single-channel chloride channel activity. Fig.5a and b show typical single-channel recordings in the cell-attached configuration. With a kinetic similar to that observed in whole cell voltage measurements, exogenous addition of 100 μM H2O2 induced the activation of single-channel
openings (Fig.5a), while in the presence of 100μM NPPB in the pipette solution, no significant increase in single-channel activity was anymore observed after addition of H2O2(Fig.5b). Mean values of NPo, product of the number
of channels in a patch (N) by the open probability (Po),
calculated for every 60 s of a recording are presented in Fig. 5c. Thus, as previously reported for hypotonicity [1], the exogenous addition of H2O2 activates a
NPPB-inhibitable volume-regulated anion channel that will lead to cell depolarization, hence insulin secretion.
Effect of H2O2on cell volume
Using the Scepter device, we observed that hypotonicity induced a rapid swelling of BRIN-BD11 cells secondarily followed by regulatory volume decrease (RVD) that was abolished in the presence of NPPB (Fig.6a) as previously reported [1]. NOX inhibitors likewise blocked the RVD (Fig. 6b); preincubation with the antioxidant N-Acetyl-L-cysteine (Fig.6c) or with betulinic acid (Fig.6d) reduced it. On the other hand, addition of H2O2in isotonic conditions
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 Iso H2O2 Hypo DPI Hypo Iso °°° *** *** H2O 2 Hypo +DPI Hypo Iso Relative DCF fluorescence
Fig. 3 Effects of hypotonicity and exogenous H2O2on intracellular
ROS content in BRIN-BD11 cells preincubated for 30 min in isotonic medium with the oxidation-sensitive dye, CM-H2DCFDA. The left
panel shows the mean amount (±SEM) of oxidized CM-DCF normalized to the mean control value in isotonic solution after 30 min in cells exposed to a hypotonic medium, in cells preincubated with DPI (10μM) before exposure to a hypotonic medium and in cells
incubated in isotonic conditions with the addition of H2O2(100μM).
failed to modify cell volume, in the presence as well as in the absence of NPPB (Fig.6a).
Effect of H2O2on rat pancreatic dispersedβ-cell membrane
potential
The concentration-dependant response to exogenous hydro-gen peroxide on the membrane potential of rat dispersed
β-cells is presented in Fig. 7. Panel a shows four represen-tative zero-current nystatin-perforated patch clamp voltage recordings at rising doses of H2O2.
The control value of membrane potential averaged −60.32±1.85 mV (Fig. 7b, n=12). Addition of 50 to 100 μM exogenous H2O2 triggered a membrane
depolar-ization within 6 min with a maximal depolardepolar-ization reached within 7–10 min (23.26±4.18 mV for 50 μM (n=5) and
0 3 6 9 12 15 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 2 s 1 min 2pA 2pA 2pA 2pA -Vp= +50 mV, NPPB in the pipette -Vp= +50 mV
c
b
H2O2 °° °° 2 s 2 s 2 s 1 min 2pA 2pA H2O2a
° °°° H2O2 NPPB NPo Time, minFig. 5 Effect of hydrogen peroxide on single channel currents. Representative cell-attached single-channel recordings in the absence (a) and in the presence (b) of 100μM NPPB in the pipette solution. Expanded time scale portions of the recordings are also shown. Dotted lines represent zero-current level. c Time course of single-channel activity changes before and during exposure to 100μM H2O2in the
absence (n=5) and in the presence (n=4) of 100μM NPPB in the
pipette solution. Mean (±SEM) NPovalues, i.e., the product of the
number of channels in a patch (N) by the open probability (Po),
calculated for each minute of the recordings are shown. NPPB completely prevents stimulation of single-channel activity induced by H2O2. Open circle p<0.05, two open circles p<0.01 and three open
circles p<0.001 vs. H2O2alone (independent Student's t-tests)
H2O2 H2O2 NPPB Control Membrane voltage, mV
c
NPPB KCl NPPB H2O2 H2O2 20 mV 3 minb
a
3 min 20 mV -50 -40 -30 -20 -10 0 °°° **Fig. 4 Effect of hydrogen peroxide on the membrane potential in BRIN-BD11 cells. a and b Two representative zero-current nystatin-perforated whole cell patch clamp voltage recordings. Dotted lines represent zero-voltage level. In a, depolarizing effect of high potassium solution (60 mM KCl) is also shown. c Mean (±SEM)
values of whole cell voltage in control condition after 8 to 10 min in the presence of 100μM H2O2and after inhibition of VRAC by 100μM
NPPB in the presence of H2O2. n=6, two asterisks p<0.01 vs. control;
26.82±9.05 mV for 100μM H2O2(n=5). These
depolari-zations were significant (p<0.01 vs. the corresponding control, paired t-test), but the effects of the two H2O2
concentrations (50 and 100 μM) were not significantly different from one another (p=0.65, independent t-test).
The effect of NPPB on the membrane potential of rat β-cell stimulated by 50 and 100 μM H2O2 is presented in
Fig. 8. Two representative zero-current nystatin-perforated patch clamp voltage recordings are shown with 50 μM H2O2(Fig. 8a) and 100 μM H2O2 (Fig. 8b), followed by
addition of 100μM NPPB. The control value of membrane potential averaged−57.05±4.03 mV (Fig.8c, n=4) and it depolarized by 27.88±8.22 mV upon addition of exogenous H2O2 (50 or 100 μM) yielding an average membrane
potential of−29±4.19 mV (n=4, p=0.02 vs. control, paired t-test). Addition of NPPB still in the presence of H2O2
immediately repolarized the cell membrane to an average potential of −54.32±6.18 mV (n=4, p=0.002 vs. H2O2
alone, p=0.72 vs control, paired t-test) (Fig.8c). Effect of H2O2on insulin output from dispersed rat islet
cells and from rat pancreatic islets
The insulin secretory response of 24-h cultured dispersed rat islet cells [39] and of freshly isolated rat pancreatic islets
to H2O2 in isotonic NaCl medium containing 2.8 mM
glucose is illustrated Fig. 9.
Panel a shows the stimulatory effect of 100μM H2O2on
insulin secretion by 15·103dispersed ratβ-cells cultured for 24 h and the abolition of this stimulation in the presence of 100μM NPPB. In a set of eight experiments, 100 μM H2O2
augmented insulin release from a basal value averaging 47.6±4.8 μU/15·103 cells·20 min to a value of 70.5± 3.5 μU/15·103cells·20 min (p=0.002 vs. control, indepen-dent t-test). In the presence of 100 μM H2O2, the insulin
release represented 148.1±7.4% of the basal value. NPPB (100 μM) completely abolished the increase in secretion induced by H2O2 (p=0.0003 vs. H2O2, independent t-test),
decreasing insulin secretion back to the basal value of 47.7± 3.3 μU/15·103 cells·20 min (representing 100.2±6.9% of control value; p=0.989 vs. control, independent t-test.
Panel b shows the stimulatory effect of 200μM H2O2on
insulin secretion by freshly isolated rat pancreatic islets. In a set of seven experiments performed on groups of 35 islets, 200μM H2O2increased the insulin secretion from an
average basal value of 2.6±0.3μU/islet·20 min to a value of 4.6±0.6 μU/islet·20 min, the latter value representing 178.7 ± 22.8% of control value (p = 0.010 vs. control, independent t-test). This set of experiments confirms the observations published by Maechler et al. [22].
0 5 10 15 20 0,8 1,0 1,2 1,4 1,6 1,8 2,0 0 5 10 15 20 0,8 1,0 1,2 1,4 1,6 1,8 2,0 0 5 10 15 20 0,8 1,0 1,2 1,4 1,6 1,8 2,0 0 5 10 15 20 0,8 1,0 1,2 1,4 1,6 1,8 2,0
b
d
a
NPPB °° °°c
Reative cell volume
Time (min)
NOX inhibitors
°°
Relative cell volume
Time (min)
NAC
°
Relative cell volume
Time (min)
betulinic acid
°
Relative cell volume
Time (min)
Fig. 6 Effects of hypotonicity and hydrogen peroxide on cell volume regulation for 20 min. Cell volume was measured by an electronic sizing technique using a handheld coulter-type counter (Scepter, Millipore, Billerica, MA, USA). The volume of the BRIN-BD11 cells in isotonic condition averaged 1.60±0.03 pl (n=41). Dashed lines represent treated conditions. Open circles represent isotonic condition and filled black triangles hypotonic condition. a Effect of NPPB, 100μM (filled gray squares cells incubated with 100 μM H2O2); b
effect of the NADPH oxidase inhibitors DPI, 10μM and plumagin,
Discussion
In 2003, Oliveira et al. [32] documented by RT-PCR, Western blotting and immunohistochemistry, the presence of NOX2, a phagocytelike NOX, in rat pancreatic insulin-producing β-cells. By RT-PCR, the expression of three NOX2 subunits (gp91PHOX, p22PHOX and p47PHOX) was documented in rat pancreatic islets. The kinetics of superoxide production in pancreatic islets was slower than in HIT-T15 cells, neutrophils or macrophages. The time
course for nitroblue tetrazolium (NBT) reduction by rat pancreatic islets was linear with time over 120-min incubation. D-Glucose (5.6 mM or more) increased by 75% NBT reduction when compared to control islets incubated in the absence of the hexose. Incubation of freshly isolated islet cells in the presence of 5.6 mMD -glucose promoted a translocation of p47PHOX from cytosol to plasma membrane. Phorbol myristate acetate, an activa-tor of protein kinase C (PKC), enhanced NBT reduction by the islets and potentiated the effect of glucose, while either
-60 -40 -20 0 -60 -40 -20 0 -60 -40 -20 0 -80 -60 -40 -20 0 H2O2 100 µM 3 min H2O2 50 µM 3 min H2O2 10 µM 3 min 3 min
b
a
mV H2O2 25 µM mV mV mV -70 -60 -50 -40 -30 -20 -10 0 H2O2 100 µM H2O2 50 µM H2O2 10 µM H2O2 25 µM**
**
CTRL Membrane voltage, mV Fig. 7 Concentration– dependence of the response to exogenous hydrogen peroxide on the membrane potential in rat dispersedβ-cells. a Four representative zero-current nystatin-perforated whole cell configuration voltage recordings at rising doses of H2O2(control,n=12; 10μM, n=1; 25 μM, n=1; 50μM, n=5 and 100 μM, n=5). Dotted lines represent zero-voltage level. b Mean (±SEM) values of whole cell voltage in control condition and after exposure to rising concentrations of H2O2
(1 min measurement at maximal stimulation). Two asterisks p<0.01 vs. respective controls (paired Student's t-tests). Stimulations at maximal level with 50 and 100μM H2O2
bysindoylmaleinide, a PKC-specific inhibitor, or dipheny-lene iodonium (DPI), a universal NOX inhibitor, opposed
the effect of both D-glucose and phorbol myristate acetate on NBT reduction.
Further work by the same research team as well as others drew attention to the view that NOX may not only play a key role in normal islet β-cell physiology but also, under specific conditions, contribute toβ-cell demise, e.g., in the phenomenon of glucolipotoxicity [28,30,31].
Several recent reports deal with the possible role of H2O2generation in the process of glucose-induced insulin
release. The hypothesis that H2O2generated from glucose
metabolism may represent one of the metabolic signals involved in glucose-stimulated insulin secretion is sup-ported by several findings resup-ported by Pi et al. [33]. First, in isolated mouse islets, glucose increases intracellular H2O2accumulation. Second, low concentrations of H2O2
(1–4 μM) increase insulin secretion from INS-1 cells exposed to 3 mM glucose. Last, both the insulinotropic action of glucose and that of H2O2are opposed by H2O2
scavengers such as N-Acetyl-L-cysteine and polyethylene glycol-coupled catalase. As a matter of fact, it was recently proposed that mitochondrial reactive oxygen species constitutes an obligatory signal for glucose-induced insulin secretion [21]. This view was mainly based on the findings that an increase in glucose concentration from 5.5 to 16.7 mM stimulated ROS generation in freshly isolated rat islets and that such an effect was reversed by the antioxidant trolox (1.0 nM to 1.0 mM), which also opposed glucose-stimulated insulin secretion. Furthermore, Maechler et al. [22] reported that exogenous H2O2 (200 μM) stimulated insulin secretion
from rat perifused islets exposed to 2.8 mM glucose
0 20 40 60 80 100 °°° ** H2O 2 + NPPB H2O 2 Iso
Insulin output (µU / 15 . 10
3 cells . 20 min) 0 1 2 3 4 5
b
a
* H2O 2 IsoInsulin output (µU / islet . 20 min)
Fig. 9 Effect of H2O2and NPPB on insulin output from dispersed
rat islet cells and rat pancreatic islets. a Effect of H2O2, 100μM in
the presence or absence of NPPB, 100μM on insulin secretion from 24 h cultured dispersed rat islet cells incubated in an isotonic medium (glucose 2.8 mM). n = 8, two asterisks p < 0.01 vs. control; three open circles p < 0.001 vs. H2O2-stimulated insulin release
(independent Student's t-tests). b Effect of H2O2, 200μM on insulin
secretion from rat pancreatic islets incubated in an isotonic medium (glucose 2.8 mM); n = 7, asterisk p < 0.05 vs. control (independent Student's t-test) 20 mV 20 mV NPPB H2O2 100µM 3 min NPPB H2O2 50µM 3 min -60 -50 -40 -30 -20 -10 0
c
b
a
H2O2 NPPB H2O2 Control°°
*
Membrane voltage, mVFig. 8 Effect of NPPB on the membrane potential in rat isolated β-cells after hydrogen peroxide exposure (50 and 100μM). a and b Two representative zero-current nystatin-perforated whole cell patch clamp voltage recordings. a Stimulation with 50μM H2O2, b stimulation
with 100μM H2O2; both are followed by treatment with 100 μM
NPPB. Dotted lines represent zero-voltage level. c Mean (±SEM)
values of whole cell voltage in control condition, after H2O2exposure
(50 to 100μM) and after inhibition of VRAC by 100 μM NPPB in the presence of H2O2 (1 min measurement at maximal stimulation/
although it blunted the secretory response to 16.7 mM glucose; it is possible that in the latter case, toxic levels of hydrogen peroxide were reached as a result of H2O2
accumulation derived from glucose on top of the exoge-nous H2O2addition (see infra).
In the perspective of exploring the role of NOX on insulin secretion, Uchizono et al. [37] reported the expression of transcripts of three different NOX isoforms in rat pancreatic islets and in the tumoral rat β-cell line RINm5F cells: NOX1, NOX2 and NOX4. Furthermore, the same group [16] reported that DPI, a compound identified as a potent, though not totally specific inhibitor of all NOX, did not affect insulin secretion from isolated rat islets exposed for 60 min to a low concentration of glucose (3.3 mM) but inhibited insulin release from islets exposed to 16.7 mM glucose, the insulinotropic action of the hexose being totally suppressed by 10μM DPI and this coinciding with a severe decrease of glucose oxidation and a lowering of the islet ATP content. A similar inhibition of glucose-induced insulin release by DPI was reported by Morgan et al. [29].
The present study aimed mainly at investigating (i) the possible participation of NOX-derived hydrogen peroxide in the secretory response of insulin-producing BRIN-BD11 cells to extracellular hypotonicity and (ii) whether H2O2
induced similar a effect on the membrane potential and insulin release in ratβ-cells.
Four sets of the present findings support the hypothesis that the effect of hypotonic shock in BRIN-BD11 cells is mediated by an intracellular H2O2 production. First,
exogenous H2O2 provoked a concentration-dependent
stimulation of insulin release, which was reduced or abolished by NPPB, a universal inhibitor of volume-regulated anion channels (VRAC) (Fig. 1). The fact that NPPB did not totally suppress H2O2-induced insulin release
in contrast to hypotonicity-induced insulin release, may merely be related to a lower intracellular concentration of H2O2 generated under the present hypotonic condition as
documented in Fig.3(see infra).
Second, when the tonicity of the extracellular medium was acutely reduced (hypotonic condition), a 50% increase in intracellular ROS production was observed in BRIN-BD11 cells and the latter was abolished by DPI. This is to be compared to a 100% increase induced by the exogenous addition of 100 μM H2O2 (Fig. 3).
Although the exact nature of the ROS produced is presently unknown, its rapid appearance (within 1 min) together with its inhibition by DPI highly suggests that it is either superoxide anion or hydrogen peroxide generated by some NOX enzyme.
Third, NOX inhibitors, i.e., DPI and plumbagin drasti-cally suppressed hypotonicity-stimulated insulin output (Fig. 2). Interestingly, plumbagin has been suggested to
specifically inhibit NOX4 [10]. Preincubation of BRIN-BD11 cells with betulinic acid for 48 h nearly totally suppressed NOX4 expression in these cells (Fig.2, inset) as it did in endothelial cells [36] and in the same conditions nearly suppressed hypotonicity-induced insulin release, thus suggesting that NOX4 might be one of the NAD(P)H oxidase involved in H2O2 generation under the present
setting. Also, 24-h preincubation with the general antiox-idant N-acetyl-L-cysteine, a precursor of intracellular glutathione (reduced status) drastically inhibited both basal and hypotonicity-induced insulin release. This further suggests that basal insulin release in BRIN-BD11 cells may also be due to some basal level of NOX4-derived H2O2production.
Last, in patch clamp experiments, exogenous addition of H2O2 (100 μM) to BRIN-BD11 cells induced cell
mem-brane depolarization that was reversible upon addition of NPPB (Fig. 4). As this depolarization (induced by VRAC opening) appears sufficient to activate L-type Ca channels, this also supports the paradigm that hypotonicity-induced H2O2production is instrumental in insulin release observed
under these conditions. The cell volume response was also studied in BRIN-BD11 cells following hypotonicity and exogenous H2O2 and it was observed to differ markedly
(Fig. 6). Reducing acutely the tonicity of the extracellular medium by 33% swelled the cells by about 50% within 2 min, hence activated a volume regulatory decrease mechanism (RVD) that was abolished in the presence of NPPB, indicating that VRAC is rate limiting for RVD. Presumably in this condition, anion (e.g., Cl−, HCO3−,
lactate, phosphates…) exit is accompanied by K+exit and osmotically obligated water, explaining the return towards initial volume. On the other hand, exogenous addition of H2O2 did not change cell volume significantly, possibly
because VRAC-mediated anion efflux was followed by their immediate recycling, e.g., via the Na–K–2Cl and/or NBCe1 cotransporters. Alternatively, the absence of changes in cell volume in BRIN-BD11 cells exposed to H2O2 could be attributable to a limited activation of K+
channels under this experimental condition. Quite impor-tantly, all NOX inhibitors (DPI, plumbagin) as well as preincubation with betulinic acid or N-Acetyl-L-cysteine strongly inhibited RVD further indicating that VRAC failed to open in these conditions despite exposure to hypotonicity and cell swelling. The experiments with betulinic acid and with plumbagin may further suggest that NOX4 might be one of the NADPH oxidase activated by hypotonicity but obviously this does not rule out the participation of other NOX, in particular NOX2 [32]. Thus, VRAC appears to be a target of H2O2
but this may well be quite indirect and the definition of the actual sensor of H2O2will now be a future challenge. The
genera-tion in BRIN-BD11 cells exposed to a hypotonic medium provides strong support to the notion that H2O2(or other
ROS) can serve as an intracellular signaling molecule [8], as observed in other cell types, e.g., in adipocytes or in A6 cells exposed to insulin [23,26]. This does not contradict the already alluded notion that a sustained production of high amount of ROS is implicated as a cause of many diseases including type 2 diabetes [30].
As documented here, low concentrations of H2O2(50–
100 μM) in the presence of basal glucose concentration (4.0 mM) also depolarized plasma membrane in rat dispersed β-cells (Figs. 7 and 8) and this effect was also reversed in the presence of NPPB (Fig. 8), further supporting the opening of VRAC by H2O2 not only in
BRIN-BD11 cells but in rat β-cells as well. At 2.8 mM glucose, 100 and 200 μM hydrogen peroxide also stimu-lated insulin release by respectively dispersed β-cells and isolated pancreatic islets in vitro. Given the low intracellu-lar level of antioxidant (enzyme glutathione, glutathione peroxidase, catalase, peroxiredoxin and thioredoxin) [16], H2O2 appears particularly well suited as a signaling
messenger molecule in isletβ-cells not being immediately blunted by ROS scavenging enzymes. However, these cells are more prone to oxidative stress [24] and in rodent β-cells, high concentrations of H2O2of the order of 1 mM
[15,16] or lower concentrations in the presence of 15 mM glucose (or more), which itself stimulates intracellular H2O2
production, hyperpolarized the β-cell membrane and blunted the increase in insulin secretion provoked by glucose alone [13,17,18,34]. This probably also explains the drastic reduction in insulin secretion observed by Maechler et al. [22] when 16.7 mM glucose was added after incubation with 200μM of exogenous H2O2, because
toxic H2O2 levels may then have been reached. This
reduction in insulin secretion in these conditions appears related to the opening KATPchannels [13] and is suggestive
of a more toxic effect reminiscent of the situation prevailing in diabetes [12].
In conclusion, the present findings strongly suggest that in BRIN-BD11 as well as in rat β-cells, the insulin secretory response to extracellular hypotonicity involves an increased intracellular H2O2 production derived from
some NOX activated upon cell swelling and that such an increase results in activation of VRAC, cell membrane depolarization, opening of L-type Ca2+channels and insulin release. Exogenous H2O2(50–100 μM) added to dispersed
rat pancreaticβ-cells exposed to basal glucose concentra-tion, induced a similar membrane depolarization by activation of NPPB-inhibitable VRAC. Finally, as a consequence of this depolarization, exogenous H2O2
(100–200 μM) also stimulated the release of insulin by both dispersed β-cells in culture and isolated pancreatic islets [22] exposed to basal glucose concentration.
Acknowledgments The present work was supported by grants from the Fonds Alphonse et Jean Forton, the Fonds de la Recherche Scientifique Médicale (FRSM), grant 3.4520.07 and the Fonds d' Encouragement a la Recherche (FER, ULB). We are grateful to Prof P. Lebrun, Laboratory of Pharmacology, Faculty of Medicine, ULB, for some measurements of insulin content, to Dr. Zhang, Laboratory of Experimental Hormonology, Faculty of Medicine, ULB for her help with RIA, and to C. Demesmaeker for secretarial help.
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