Expert Answer:WCU Bisphenol-A In Diabetes Pathophysiology Discus


Solved by verified expert:Go to the WCU Library and search the database for the following article. Read the article, and then answer the questions that follow: (ARTICLE ATTACHED)Soriano S., Alonso-Magdalena P., Garcıa-Arevalo M., Novials A., & Muhammed S. J. (2012). Rapid insulinotropic action of low doses of Bisphenol-A on mouse and human islets of langerhans: Role of estrogen receptor b. PLoS ONE 7(2): e31109. doi:10.1371/journal.pone.0031109What are the causes of type 2 diabetes?What is the relationship between insulin resistance and hyperinsulinemia?What are endocrine disrupters and what are their functions in our body? Provide some examples.How does concentration of glucose effect insulin secretion?Explain how data from article support or refute the hypothesis that endocrine disrupters can change insulin secretion. Be sure to cite the article in text. Paraphrase key points from the article that support your answers.

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Rapid Insulinotropic Action of Low Doses of Bisphenol-A
on Mouse and Human Islets of Langerhans: Role of
Estrogen Receptor b
Sergi Soriano1, Paloma Alonso-Magdalena1, Marta Garcı́a-Arévalo1, Anna Novials2, Sarheed J.
Muhammed3, Albert Salehi3, Jan-Ake Gustafsson4, Ivan Quesada1, Angel Nadal1*
1 Instituto Bioingenierı́a and CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Universidad Miguel Hernández de Elche, Elche, Alicante, Spain,
2 Institut D’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and CIBERDEM, Barcelona, Spain, 3 Department of Clinical Sciences, Lund University, Malmö, Sweden,
4 Department of Cell Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, Texas, United States of America
Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical (EDC) used as the base compound in the manufacture of
polycarbonate plastics. It alters pancreatic b-cell function and can be considered a risk factor for type 2 diabetes in rodents.
Here we used ERb2/2 mice to study whether ERb is involved in the rapid regulation of KATP channel activity, calcium
signals and insulin release elicited by environmentally relevant doses of BPA (1 nM). We also investigated these effects of
BPA in b-cells and whole islets of Langerhans from humans. 1 nM BPA rapidly decreased KATP channel activity, increased
glucose-induced [Ca2+]i signals and insulin release in b-cells from WT mice but not in cells from ERb2/2 mice. The rapid
reduction in the KATP channel activity and the insulinotropic effect was seen in human cells and islets. BPA actions were
stronger in human islets compared to mouse islets when the same BPA concentration was used. Our findings suggest that
BPA behaves as a strong estrogen via nuclear ERb and indicate that results obtained with BPA in mouse b-cells may be
extrapolated to humans. This supports that BPA should be considered as a risk factor for metabolic disorders in humans.
Citation: Soriano S, Alonso-Magdalena P, Garcı́a-Arévalo M, Novials A, Muhammed SJ, et al. (2012) Rapid Insulinotropic Action of Low Doses of Bisphenol-A on
Mouse and Human Islets of Langerhans: Role of Estrogen Receptor b. PLoS ONE 7(2): e31109. doi:10.1371/journal.pone.0031109
Editor: Kathrin Maedler, University of Bremen, Germany
Received September 5, 2011; Accepted January 2, 2012; Published February 8, 2012
Copyright: ß 2012 Soriano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by grants from Ministerio de Ciencia e Innovación grants BFU2008-01492, BFU2011-28358 and BFU2010-21773,
Generalitat Valenciana grants Prometeo/2011/080 and ACOMP/2010/113, the European Commission (Program PEOPLE). The ‘‘Centro de Investigación Biomédica
en Red de Diabetes y Enfermedades Metabólicas Asociadas’’ is an initiative of the Instituto de Salud Carlos III. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail:
on their electrical activity. This consists of oscillations of the
membrane potential that range from electrically silent periods to
depolarized plateaus on which Ca2+-action potential originate
[12]. Classically, stimulus-secretion coupling involves the closure
of KATP channels after an increase in the ATP/ADP ratio because
of the glucose metabolism [13]. KATP channels are responsible for
the resting membrane potential of b-cells and its closure elicits a
depolarization that opens voltage dependent calcium channels and
induces Ca2+ influx [14]. As a consequence of the oscillatory
membrane potential, a [Ca2+]i oscillatory pattern originates [15–
19], which triggers a pulsatile insulin secretion [17–19]. In
addition to the KATP dependent process of insulin secretion there
is a KATP independent process which involves cAMP dependent
phosphorylation [20,21].
Beta cells express estrogen receptor a (ERa), estrogen receptor b
(ERb) and the G-protein coupled receptor (GPR30), also named
GPER1 [22,23]. The use of genetically modified mice has revealed
the role of these estrogen receptors [23]. ERa is involved in the
regulation of pancreatic insulin biosynthesis in response to both E2
and BPA. Remarkably, nanomolar concentrations of either BPA
or E2 act via extranuclear ERa to activate ERK1/2 and regulate
insulin content [24]. This action involves the activation of the
transcription factor NeuroD1 [25]. In addition, activation of
extranuclear ERb by physiological concentrations of E2 rapidly
Bisphenol-A (BPA) is a widespread endocrine disruptor that
produces insulin resistance and alterations in pancreatic b-cell
function [1]. It has been suggested that together with other
endocrine disrupting chemicals (EDCs), BPA constitutes a risk
factor for type 2 diabetes and other metabolic disorders [2–4].
Recent work shows the association between increasing urinary
BPA levels and Diabetes Mellitus. A 12.8% of diabetic patients
show high BPA levels in urine samples (4.20 ng/ml or 18 nM) [5].
Moreover an association between urinary levels of BPA, obesity
and insulin resistance in middle-aged and elderly chinese adults
has been recently described [6]. The range of BPA levels found in
humans is from 0.7 to 20 nM [7,8]. To support the evidence that
BPA may be a risk for the development of diabetes it is critical to
study its effect on human tissues involved in glucose and lipid
metabolism, including the endocrine pancreas which is key in
glucose homeostasis.
The islet of Langerhans is the physiological unit of the
endocrine pancreas; it is a group of 1500–3000 cells of five
different types [9–11], and the most abundant are b-cells. The
main function of b-cells is the biosynthesis and release of insulin in
response to neurotransmitters, hormones and nutrients, the most
important being glucose. The secretory response of b-cells depends
February 2012 | Volume 7 | Issue 2 | e31109
Rapid Insulinotropic Action of Bisphenol-A
RPMI 1640 without phenol red containing 11 mM glucose
supplemented with 10% charcoal dextran-treated fetal bovine
serum (HyClone Laboratories, Logan, UT), 2 mM L-glutamine,
200 U/mL penicillin and 0.2 mg/mL streptomycin and plated on
glass coverslips. Cells were kept at 37uC, in a humidified
atmosphere of 95% O2 and 5% CO2, and used within 1–2 days
of culture.
regulates KATP channel activity, increases glucose stimulated
[Ca2+]i signals and insulin release. It is important to clarify that
action of E2 on KATP channel activity was mimicked by specific
agonist of ERb 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN) but
not by the ERa specific agonist propylpyrazole-triol (PPT).
Moreover KATP channel activity was not modified in ERa2/2
[26]. These results indicated that ERb plays an important role in
rapid regulation of insulin secretion in pancreatic b-cells. Bisphenol-A imitated rapid estradiol regulation of KATP channel and
calcium signaling [1], however, whether ERb acting out of the
nucleus is able to mediate BPA actions is still unknown.
In the present work we use b-cells and islets of Langerhans from
wild type (WT) and ERb2/2 mice to show that ERb is involved
in the BPA-mediated rapid regulation of KATP channel activity,
potentiation of glucose induced-[Ca2+]i signals and insulin release.
Moreover, we have used human islets of Langerhans to
demonstrate that 1 nM BPA blocked KATP channels and
produced a potent enhancement of insulin secretion in response
to glucose.
Insulin secretion measurements in mouse islets
Freshly isolated islets of Langerhans from mice were left to
recover in the isolation medium for 2 h in the incubator. After
recovery, groups of 5 were transferred to 400 ml of a buffer
solution containing 140 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl2,
1 mM MgCl2, 20 mM HEPES and the glucose concentration
corresponding to each experimental condition with final pH at 7.4.
Afterwards, 100 ml of the corresponding buffer solution with 5%
Bovine Serum Albumin (BSA) was added, incubated at room
temperature for 3 min and let to cool down for 15 min on ice.
Then, the medium was collected and insulin was measured in
duplicate samples by radioimmunoassay using a Coat-a-Count kit
(Siemens, Los Angeles, CA, USA).
Materials and Methods
Recording intracellular calcium concentration ([Ca2+]i)
Adult C57 female mice were used. All animals were kept under
standard housing conditions. The ethical committee of Miguel
Hernandez University ‘‘Comisión de Ética en la Investigación
Experimental’’ reviewed and approved the method used. ERb2/2
mice were generated as described prior [27] and supplied by JanAke Gustafsson’s laboratory. All genetically modified animals and
wild types were from the same supplier and the same colony. Islets
of Langerhans from ERb2/2 mice were treated as described
below for islets from C57 mice. Animals were kept in new
polycarbonate cages and polycarbonate water bottles were avoided
to minimize contamination of mice with free BPA.
Freshly isolated islets of Langerhans from mice were loaded with
5 mM Fura-2 AM for at least 1 h at room temperature. Calcium
recordings in islets were obtained by imaging intracellular calcium
under an inverted epifluorescence microscope (Zeiss, Axiovert
200). Images were acquired every 2 s with an extended
Hamamatsu Digital Camera C4742-95 (Hamamatsu Photonics,
Barcelona, Spain) using a dual filter wheel (Sutter Instrument CO,
CA, USA) equipped with 340 nm and 380 nm, 10 nm bandpass
filters (Omega optics, Madrid, Spain). Data were acquired using
Aquacosmos software from Hamamatsu (Hamamatsu Photonics,
Barcelona, Spain). Results were plotted and analyzed using
commercially available software (Sigmaplot, Jandel Scientific).
Data were represented as a frequency of calcium oscillations
(min21) comparing BPA responses with their respective control.
Islet of Langerhans isolation from mice
Adult mice were killed by cervical dislocation and islets were
isolated with a collagenase technique previously described [26]
and used according to the kind of experiment to be performed.
Patch Clamp recordings
KATP channel activity was recorded using standard patch clamp
recording procedures from isolated b-cells [29]. 80–90% of the
single cells were identified as b-cells by their large size
(capacitance: 8–12 pF) and by their electrophysiological properties, such as presence of KATP channel activity in absence of
glucose and their response to glucose by the appearance of action
currents in cell-attached configuration. Currents were recorded
using an Axopatch 200B patch-clamp amplifier (Axon Instruments
Co. CA, USA). Patch pipettes were pulled from borosilicate
capillaries (Sutter Instruments Co. CA, USA) using a flaming/
brown micropipette puller P-97 (Sutter Instruments Co. CA, USA)
with resistance between 3–5 MV when filled with the pipette
solutions as specified below. Bath solution contained (in mM): 5
KCl, 135 NaCl, 2.5 CaCl2, 10 Hepes and 1.1 MgCl2 (pH 7.4) and
supplemented with glucose as indicated. The pipette solution
contained (in mM): 140 KCl, 1 MgCl2, 10 Hepes and 1 EGTA
(pH 7.2). The pipette potential was held at 0 mV throughout
recording. KATP channel activity was quantified by digitising 60 s
sections of the current record, filtered at 1 kHz, sampled at
10 kHz by a Digidata 1322A (Axon Instruments Co. CA, USA),
and calculating the mean open probability of the channel (NP o)
during the sweep. Channel activity was defined as the product of
N, the number of functional channels, and Po, the open-state
probability. Po was determined by dividing the total time channels
spent in the open state by the total sample time. Data were
Human islets isolation for electrophysiological
Human pancreases were obtained from 3 cadaveric organ
donors, after written consent of their families and approval of
‘‘Comité Ético de Investigación Clı́nica’’, and ‘‘Comité de
Investigación’’, Hospital Clı́nic of Barcelona (ID: 2009/5157).
Islets were isolated by collagenase digestion of the pancreas
(SERVA Electrophoresis, Heidelberg, Germany) and separated
from exocrine tissue by Biocoll density gradient (Biochrom,
Berlin,Germany), as previously described [28]. Islets were
transferred to RPMI-1640 medium (Gibco-BRL, Pisley, U.K.)
containing 11.1 mmol/l glucose and supplemented with 10%
FBS, 2 mmol/l L-glutamine, 100 units/ml penicillin, and 100 mg/
ml streptomycin and cultured overnight at 37uC with 5% CO2.
Islets were then picked and disaggregated to obtain beta cells for
electrophysiological studies as described below.
Obtaining b-cells from mouse and human islets
Once mouse or human islets were obtained as described above,
they were dispersed into single cells and cultured as previously
described [14]. The protocol of islet disaggregation into sinlge cells
was the same for both mouse and human islets. Once isolated,
islets were disaggregated into single cells in a low calcium medium.
Cells were then centrifuged, resuspended in culture medium
February 2012 | Volume 7 | Issue 2 | e31109
Rapid Insulinotropic Action of Bisphenol-A
represented as a percentage of activity with respect to resting
conditions (0 mM Glucose). Experiments were carried out at room
temperature (20–24uC).
Human pancreatic islets and measurement of human
islet insulin secretion
Isolated human pancreatic islets from non-diabetic males (age
58+6 years; Hb1Ac,6.1 and MBI: 27+2) were provided by the
Nordic network for clinical islet transplantation (Professor Olle
Korsgren, Uppsala University, Sweden). All procedures were
approved by the ethical committees at Uppsala and Lund
Universities. Prior to the experiments the islets had been cultured
at 37uC (5% CO2) for 2 days in CMRL 1066 (ICN Biomedicals,
Costa Mesa, CA) supplemented with 10 mmol/l HEPES,
2 mmol/l L-glutamine, 50 mg/ml gentamicin, 0.25 mg/ml Fungizone (Gibco, BRL, Gaithersburg, MD), 20 mg/ml ciprofloxacin
(Bayer Healthcare, Leverkusen, Germany) and 10 mmol/l nicotinamide. The islets had 70–90% purity when they arrived; the
islets were then hand-picked under stereomicroscope prior to use.
All islet preparations were treated in exactly the same way to avoid
results being due to handling differences. All procedures were
approved by the ethical committees at Uppsala and Lund
At the experiment day, the islets were preincubated for 30 min
at 37uC in Krebs-Ringer bicarbonate buffer, pH 7.4, supplemented with 10 mmol/l HEPES, 0.1% bovine serum albumin and
1.0 mmol/l glucose. After preincubation the buffer was changed
and the islets were incubated at 8.3 mmol/l glucose 6 different
test agents for 60 min at 37uC. Each incubation tube contained 12
islets in 1.0 ml of KRB solution and was gassed with 95% O2 5%
CO2 to obtain constant pH and oxygenation. All incubations were
performed in an incubation box at 30 cycles/min. immediately
after incubation an aliquot of the medium was removed and frozen
for subsequent assay of insulin. The secreted insulin was measured
using a radioimmunoassay kit (Millipore).
Figure 1. BPA increases insulin release in a dose dependent
manner in mouse islet of langerhans. The released insulin was
measured after one hour exposure of islets to increasing concentration
of BPA, in the presence of 8 mM glucose. All points contained same
amount of vehicle.
BPA closes KATP channels in human b-cells
In order to analyze whether the rapid action of BPA on KATP
channel activity occurred as well in human pancreatic b-cells, islets
from human donors were disaggregated into single cells and single
channel recordings were performed using the patch-clamp
technique as performed in the previous subheading. Figure 3A
shows a typical record in which 5 min after BPA application in the
absence of glucose, there is a dramatic decrease in KATP channel
activity by 83.4%, together with a depolarization of the membrane
noticed by the decrease in the amplitude of the channel opening
and the appearance of biphasic spikes (see * and ** in second and
third traces and figure 3B). These biphasic spikes (action currents)
were seen in all human cells exposed to either BPA or glucose and
are action potentials seen via the capacitance and resistance of the
patch and their recordings depend on the patch resistance [14,30].
Remarkably, the response to BPA in the average of experiments is
as high as that of 8 mM glucose (Fig. 3C). The action of BPA was
mimicked by the ERb agonist diaylpropionitrile (DPN) (Figure
BPA action on KATP channel activity is abolished in b-cells
from ERb2/2 mice
Bisphenol-A application in presence of 8 mM glucose during
1 hour increased insulin release from mouse islet of Langerhans in
a dose dependent manner (Figure 1A). For the rest of the work we
used 1 nM BPA because is within the range of BPA levels in
human serum and it is considered an environmentally relevant
dose [7,8].
Figure 2A shows, in a typical b-cell from wt mice, the basal
activity of KATP channels (upper trace). Channel openings are
represented by downward deflections, reflecting inward currents
due to the high potassium content of the pipette. The criterion
used for identification of KATP channels was their sensitivity to
glucose, which highly decreases channel activity (Fig. 2A, 8 mM
glucose trace) and diazoxide, a sulfonamide that opens KATP
channels (Fig. 2A, 100 uM diazoxide trace). Both glucose and
diazoxide sensitivity were tested in all records throughout this
work. Figure 2A (BPA trace) demonstrates the decrease in the
KATP channel activity elicited by BPA, 7 min after its application.
On average, BPA decreases KATP channel activity by 49.3%
(Fig. 2B).
To molecularly study the role of ERb in BPA-induced rapid
regulation of KATP channels, cell-attached patch recordings were
performed in b-cells from ERb2/2 mice. In these cells KATP
channel activity was not significantly altered after BPA application
(Fig. 2C and D).
BPA action on glucose induced [Ca2+]i oscillations and
insulin release is abolished in islets from ERb2/2 mice
It is well established that E2 rapidly induces Ca2+ signals and
insulin secretion via ERb in a process that depends on electrical
activity [26]. To investigate whether activation of ERb by BPA
potentiated glucose-induced [Ca2+]i signals and insulin secretion,
we performed the experiments described in Figures 4 and 5. Islets
loaded with the calcium fluorescence dye Fura-2 were imaged to
monitor the [Ca2+]i signal elicited by a change from a non
stimulatory glucose concentration (3 mM) to an insulin stimulatory
glucose concentration (8 mM). The response, as expected, was a
[Ca2+]i transient followed by a plateau with [Ca2+]i oscillations. In
the absence of BPA the frequency of [Ca2+]i oscillations was
similar in islets isolated from wt and ERb2/2 mice (Fig. 4A and
C). However the frequency of [Ca2+]i oscillations increased in islets
exposed to 1 nM BPA compared to control in wt islets (Fig. 4B
and C). It is of note that the BPA-induced increase in [Ca2+]i
oscillatory frequency is highly diminished in islets from ERb2/2
February 2012 | Volume 7 | Issue 2 | e31109
Rapid Insulinotropic Action of Bisphenol-A
Figure 2. KATP channel activity in pancreatic b-cells from WT and ERb2/2 mice in presence of 1 nM BPA. A, BPA at 1 nM decreased KATP
channel activity in pancreatic b-cells from WT mice. The records show channel activity before application of BPA (upper trace), 7 min after application
of BPA (1 nM BPA trace), 8 min after application of …
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