Pharmacology & Pharmacy, 2013, 4, 451-460 Published Online August 2013 ( 451
Impact of Bisphenol A (BPA) and Free Fatty Acids (FFA)
on Th2 Cytokine Secretion from INS-1 Cells
Johan N. van Oppen, Eugen J. Verspohl*
Department of Pharmacology, Institute of Medicinal Chemistry, Münster, Germany.
Received June 17th, 2013; revised July 15th, 2013; accepted August 1st, 2013
Copyright © 2013 Johan N. van Oppen, Eugen J. Verspohl. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Bisphenol A (BPA) is used in huge amounts for many plastic products and is a hormone (estrogen) disrupting agent.
BPA as well as FFAs may be deleterious for the immune system. The aim was to identify Th2 cytokines and some of
their signal transduction mechanisms in INS-1 cells, an insulin secreting cell line. Screening using a proteome profile
indicated an increase of IL-1, IL-2, IL-4, IL-6, IL-10, IL-13 and IL-17 by BPA. Also FFAs (in combination with LPS)
were positive. In detailed qu antitative measurements, these results were con firmedly indicating a co mplex array of pro-
and anti-inflammatory potential. The interaction of BPA with 17β-estradiol was non-additive with respect to IL-4 and
IL-6 release and additive with respect to FFA interaction indicating same and different mechanisms of action, respect-
tively. As signal transduction PI3K (Wortmannin-sensitive) and STAT-3/6 (Tofacitinib-sensitive) are involved in va-
rious effects, INS-1 cells release several cytokines due to BPA and FFA attack which may be involved in disturbance of
glucose homoeostasis and type 1 diabetes.
Keywords: Bisphenol A; Th2 Cytokines; INS-1 Cells
1. Introduction
Bisphenol A (BPA) is the building block of polycarbo-
nate plastic used to make numerous consumer products
including baby bottles, beverage containers, food cans
(inner layer), Tetra-PakTM, dental materials, thermal
paper and impact-resistant safety equipment. BPA is also
a part of epoxy resins. It even shows up in cell culture
media during cell research when plastic materials are
used. 800,000 tons are produced worldw ide.
BPA is an endocrine disrupting chemical (EDC) with a
toxic influence on reproduction [1]. BPA interacts with
G-protein coupled receptors, e.g. GPR-30 and binds to
both types of estrogen receptors and various cytokine
receptors with low affinity (genomic effect). BPA
imitates 17β-estradiol effects [2] which is not surprising
because of the similarity of phenol groups of either
compound resulting e.g. in effects on blood glucose
homeostasis. BPA disrupts pancreatic beta-cell function
[3]. Even low levels can detrimentally affect the glucose
metabolism mediated by the glucose-regulated-protein
(grp) [4]. BPA is linked to several other diseases such as
allergy, cardiovascular diseases [5] chronic inflammation
and e.g. asthma [6]. It modulates immunological pro-
cesses with respect to differentiation of Th0 cells to Th1
or Th2 cells [7]. BPA increases body weight [8], and
since obesity is linked to inflamma tion [9 ] ( i.e. mor e t han
100 hormones and cytokines are secreted by fat cells),
this process may be linked to T cells.
BPA is completely (95% - 100%) absorbed [10] and
was found in the urine of nearly 100% of investigated
children [1,11-13]. Average urine concentration is 3 µg/L.
Plasma concentrations are higher in children th an in adu-
lts [14] which may partly be due to a slower glucuro-
nidation [10,15,16 ] making children high ly susceptib le to
damage. In the European Union BPA is not allowed in
baby bottles since 2011.
Plasma FFAs play important physiological roles in
skeletal muscle, heart, liver and pancreas. FFAs (e.g.
palmitate as used in this study) supply energy, build cell
membranes and are precursors for intracellular signal
molecules such as prostaglandins and leucotriens. Nor-
mal concentrations of 200 - 600 µM are elevated more
than three-fold in pathophysiological situations such as
obesity and diabetes mellitus [17]. Increased FFAs have
*Corresponding a uthor.
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Impact of Bisphenol A (BPA) and Free Fatty Acids (FFA) on Th2 Cytokine Secretion from INS-1 Cells
an immunological potential [18], e.g. inhibiting the
T-cell activity. The induction of various proinflammato ry
effects was described for β-cells [19]. The pancreatic
β-cell function and the insulin receptor signaling cascade
are inhibited (insulin resistance) [20]. FFA-induced
hepatic insulin resistance is associated with increased
hepatic diacylglycerol content, increased activities of
protein kinase C (PKC), of pro-inflammatory NFκB
pathway and increased inflammatory cytokine expression
[21]. FFAs are a major link between obesity and insulin
resistance (inhibition of insulin-stimulated glucose up-
take), activate PI3K [22] and lead to a chronic inflam-
matory status [23].
Whereas the influence of BPA on Th1-cytokines is
well investigated, not so much is known about Th2,
especially not with respect to INS-1 cells, an insulib
secreting cell line. The aim of this study is to investigate
the impact of pathophysiologically important factors
such as BPA and FFA and their complex interplay with
17β-estradiol with respect to various Th2 cytokines using
INS-1 cells. Cytokines which are released by Th2 cells
are IL-4, IL-6, IL-10 and IL-13. Also the signal
transduction system of cytokines, STAT-3, STAT-6 and
Akt, is of interest. The basic question is which pro- and
anti-inflammatory cytokines are released from INS-1
cells and which secondary mechanisms are involved.
INS-1 cells are a valuable to ol since they show physiolo-
gical and immunological similarities to human pancreas
cells [24].
We altogether focus on pathophysiological effects of
BPA and FFA on Th2 cytokine secretion from INS-1
cells and some signal tran sduction mechanisms inclu ding
interactions with 17β-estradiol.
2. Materials and Methods
2.1. Chemicals
The proteome profiler was from R&D systemsTM
(Minneapolis, USA). Photo paper was from Kodak®
BioMaxTM Light 1, AGFA Rodinal as developer from
A&O imaging solutions GmbH, Koblenz, and Tetenal
Superfix Plus for fixation from Tetenal AG, Norderstedt,
Germany. 24-well plates Cellstar were from Greiner
bio-one, Frickenhausen, Germany, and 24-well plates
TPP were from Techno Plastic Products AG, Trasadin-
gen, Switzerland. IL assays were from Quantikine® rat
ELISA (IL-4, IL-6, IL-10), R&D systemsTM (Minnea-
polis) and RayBiotechTM (Norcross, USA) (rat IL-13,
STAT-3 and STAT-6 both phoshphorylated and not
phosphorylated). Tofacitinib (JAK3 inhibition) was from
Pfizer (Berlin, Germany), Wortmannin and palmitate
were from Sigma-Aldrich (Hamburg, Germany). All
chemicals not mentioned here were of analytical grade
and were from PAA, Colbe, or Sigma-Aldrich, Steinheim,
2.2. Cell Culture of INS-1 Cells
Asfari et al. established in 1992 a X-ray-induced rat
transplantable insulin secreting cell line (INS-1). The
continuous growth of the INS-1 cells, kindly supplied by
Dr. C. Wollheim, Geneva, Switzerland, was found to be
dependent on the reducing agent 2-mercaptoethanol to
increase the total cellular glutathione levels (Asfari et al.,
1992). INS-1 cells with the passage numbers 39-70 were
used. They were subcultivated once weekly and the
growth medium was changed four days after the subcul-
A problem induced by antibiotics added to culture
media thus releasing endotoxins from hypothetically
present bacteria was circumvented by doing once a while
experiments lacking the presence of antibiotics. In fact
when the production of endotoxins is decreased by
omitting penicillin an d streptomycin in culture media, th e
IL-1 secretion for example was reduced. This has to be
kept in mind when the influence of cytokines on
initiation of type 1 diabetes is discussed to those experi-
2.3. Proteome Profiler (ARY 008)
Selected capture antibodies have been spotted in dupli-
cate on nitrocellulose membranes. Cell culture superna-
tant mixed with a cocktail of biotinylated detection
antibodies. The sample/antibody mixture is then incu-
bated. Any cytokine/detection antibody complex present
is bound by its cognate immobilized capture antibody on
the membrane. Streptavidin-Horseradish Peroxidase and
chemiluminescent detection reagents are added, and a
signal is produced in proportion to the amount of cyto-
kine bound. The darkening of the photo was scanned and
calculated in relative terms (semiquantitative measure-
2.4. ELISA for IL
The basis is a quantitative sandwich enzyme immunoas-
say technique (ELISA Quantikine®). A monoclonal anti-
body specific for various rat interleukins has been
precoated onto a microplate. Standards, control (DMSO
1%) and samples are added and any specific cytokine
present is bound by the immobilized antibody. After
washing away unbound substances, an enzyme-linked
polyclonal antibody specific for the interleukin antibody
of interest is added to the wells. The enzyme reaction
yields a coloured product. Its colour intensity is in
proportion to the amount of interleukin bound in the
initial step to the first antibody. The sample values were
then read off the standard curve which was linear
between 31.25 and 2000 pg/mL of either cytokine.
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Impact of Bisphenol A (BPA) and Free Fatty Acids (FFA) on Th2 Cytokine Secretion from INS-1 Cells 453
Blanks were between 19.1% and 24.4% of maximum
2.5. Dot Blot of IL-17 Receptor
Washed cells (5 ml cold PBS), lysed in the presen ce of a
protease inhibitor and pelleted (12000 rpm). They were
spotted on a nitrocellulose membrane and incubated for
12 hours. After washing the membrane was incubated for
10 min with a primary antibody directed against the
IL-17 receotor. Thereafter a secondary antibody linked to
horse radish peroxidase, an enzyme which induces the
degradation of luminol in the presence of H2O2. The
luminescence was detected using a photo paper Kodak®
2.6. Statistics
Results are shown as means + SEM of n independent
results. Each of these n results consists of 7 - 9 replicates.
Statistical significance was determined using a nonpara-
metric Mann-Whitney test (BBN Software Products
Corp.) followed by a post-hoc test (t-test for individual
results). P < 0.05 was considered significan t.
3. Results
In an a priori screening using a Proteome Profiler®, IL-1,
IL-2, IL-4, IL-6, IL-10, Il-13 and IL-17 were highly
increased in INS-1 cells by 40 µg/mL BPA (data of 5
µg/mL are not shown, but were rather the same), whereas
the effects of FFA [40 µg/mL] and LPS [100 ng/mL;
control] were smaller when given alone, albeit higher
when both were added in combination (Table 1). IL-17
was not increased by FFA and LPS alone (Table 1).
Many other compounds that are not indicated were
positively tested, but only effects on distinct cytokines
are shown and only these were evaluated later in more
BPA increased IL-4 secretion in a concentration
dependent manner and LPS had the same tendency
(Figures 1(a) and (b)). The effect was time-dependent
when data of 6 h (Figure 1(a)), 12 h (data not shown)
and 24 h incubation (Figure 1(b)) were compared. For
all further experiments 24 h incubations were preferred.
The IL-4 secretion induced by BPA at three different
concentrations was superimposed by 17β-estradiol except
at high, possibly maximally effective BPA concentration
(Figure 2(a)) indicating a right shift of the BPA curved
by 17β-estradiol.
The IL-4 secretion induced by FFA at three different
concentrations was superimposed by 17β-estradiol (Fig-
ure 2(b)).
BPA increased the release of IL-6 (Figure 3(a)) and
17β-estradiol [100 ng/mL] did not increase this effect at a
high BPA concentration (Figure 3(a)). 17β-Estradiol
Table 1. Secretion of IL-1, IL-2, IL-4, IL-6, IL-10, IL-13
and IL-17 from INS-1 cells being incubated with LPS, FFA,
their combination and BPA. Cells were incubated for 24 h;
secretion was determined in a semiquantitative way using a
proteome profiler (biotinylated antibodies, nitrocellulose
membrane, detection system). Semiquantitative data are
indicated: – no secretion, + weak, ++ average, +++ strong
secretion. Three replicates with rather identical results.
LPS 100
ng/mL FFA 40
µg/mL FFA 40 µg/mL and
LPS 100 ng/mL BPA 40
IL-1 + + ++ +++
IL-1α - - + +
IL-1β - - - -
IL-1ra + - - -
IL-2 + + ++ ++
IL-4 + ++ +++ +++
IL-6 - + + ++
IL-10 ++ + ++ +++
IL-13 + +++ + ++
IL-17 - - + ++
BPA [10 0ng/ml]
BPA [250ng/ml]
BPA [1µg/ml]
LPS [100ng/ ml]
LPS [500ng/ml]
Positive Control IL-4
IL-4 [pg/ml]
BP A [100ng/ml]
BPA [250ng/ml]
BPA [1µg /ml]
LPS [100ng/ml]
LPS [500ng/ml]
Positive Control IL-4
IL-4 [pg/ml]
Figure 1. (a) and (b) Effect of BPA and LPS on IL-4
secretion from INS-1 cells. Various concentrations of LPS
and BPA were used for a 6 (a) and 24 hour (b) incubation.
Blanks (no addition) were subtracted. DMSO (solvent; 1%)
and a high concentration of IL-4 [3000 pg/mL] were used as
negative and positive controls. Mean + SEM, n = 3; *p <
0.05 vs. DMSO control.
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Impact of Bisphenol A (BPA) and Free Fatty Acids (FFA) on Th2 Cytokine Secretion from INS-1 Cells
[100 ng/m]l
BPA [100ng/ml ]+Estr a d io l [100ng/ml]
[250n g /ml]
BPA [250ng/ml]+Estradiol [250ng/ml]
BPA [1µg/ml]
[1µg/ml]+ Estradiol [1µg/ml]
Posit ive Control IL-4
IL-4 pg/ml
FFA [100n g /ml]
FFA [100ng /ml]+Estrad iol [1µg/ml]
FFA [500n g/ml]
FFA [500ng/ml]+Estr ad i ol [1µg/ml]
FFA [g/ml]+Estrad i ol [1µg/ml]
Positive C ontrol IL-4
IL-4 [pg/ml]
Figure 2. (a) and (b) Modulatory effect of 17β-Esradiol on
BPA-induced (a) and FFA-induced (b) IL-4 secretion from
INS-1 cells. Blanks (no addition) were subtracted. DMSO
(solvent; 1%) and a high concentration of IL-4 [3000 pg/mL]
were used as negative and positive co ntrols. Mean + SEM, n
= 3: *p < 0.05 vs. DMSO control; +p < 0.05 vs. absence of
[100 ng/mL] increased the release of IL-6 (Figure 3(b))
which is superimposed by increasing concentrations of
BPA until a maximum BPA concentration of 1.0 µg/mL
indicating the same type of mechanism.
Table 2 shows the effect of various concentrations of
FFA and LPS on IL-6 secretion. Both compounds increa-
sed IL-6 release in a concentration-dependent manner.
17β-Estradiol [100 ng/mL] was not able to increase the
effect of FFA at 1 µg/mL.
BPA, LPS and FFA increased IL-10 secretion (Figure
4(a)). This effect of BPA at 1 µg/mL was further
BPA [250ng/ml ]
BPA [500ng/ml]
BPA [g/ml]
BPA [2,5µg/ml]
BPA [1µg/ml]+Estradiol [100ng/ ml]
Positive Control IL-6
IL-6 [pg/ml]
Estra di ol [100ng/ml ]
BPA [250ng/ml]+Estradiol [100ng/ml]
BPA [500ng/ml]+Estradiol [100ng/ml]
BP A [1µg/ml]+ Es tr adi ol [100ng/ml]
BPA [2.5µg/ml]+Estr adiol [100ng/ml]
Positive Control IL-6
I L -6 [p g /ml ]
Figure 3. Effect of various concentrations of BPA and a
combination with 17β-estradiol on IL-6 secretion from
INS-1 cells. 17β-estradiol [100 ng/mL] was combined with
various concentrations of BPA [0 to 2.5 µg/mL]. Blanks (no
addition) were subtracted. DMSO (solvent; 1%) and a high
concentration of IL-6 [650 pg/mL] were used as negative
and positive controls. Mean + SEM, n = 4; *p = 0.05 vs.
DMSO control.
Table 2. Effect of FFA and LPS on IL-6 release from INS-1
cells (24 h incubation). Details see in the legend of Figure 3.
DMSO (0.1%) 14.3 ± 0.4
FFA [0.5 µg/mL] 99.7 ± 8 .3*
FFA (1 µg/mL) 197.2 ± 22.4*
FFA [2.5 µg/mL] 276.4 ± 35.6*
FFA [2.5 µg/mL]
+ 17β-Estradiol [1 00 ng /mL] 262.6 ± 33.7*
(N.S. vs. absence of 17β-estradiol)
FFA [5.0 µg/ml] 296.9 ± 18.7*
LPS [50 ng/mL] 186.3 ± 24.6*
LPS [100 ng/ml ] 298.3 ± 16.7*
Positive control: IL-6 [650 pg/mL] 639.2 ± 24.6
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Impact of Bisphenol A (BPA) and Free Fatty Acids (FFA) on Th2 Cytokine Secretion from INS-1 Cells 455
BPA [500ng/ml]
BPA [g/ml]
LPS [50n g/ml]
LPS [100ng/ml]
FFA [1µg/ml]
Positive Control IL-10
IL-10 [p g/ ml]
BPA [1µg/ml]
BPA [1µg/ml]+IL-4 [1000ng/ml]
BPA [1µg/ml]+IL-13 [100ng/ml]
BPA [1 µg/ m l]+ I L-10 [50ng/ml]
BPA [1µg/ml]+IL-10 [1 00n g/ml]
Positive Control IL-10
IL-10 [pg/ml]
Figure 4. (a) and (b) Effect of BPA, LP S and FFA on IL-10
secretion from INS-1 cells. Two concentrations of LPS [50
and 100 ng/mL], BPA [500 and 1000 ng/mL] and FFA [1000
ng/mL] were used. Blanks (no addition) were subtracted.
DMSO (solvent; 1%) and a high concentration of IL-10
[1250 pg/mL] were used as negative and positive controls.
When IL-10 was added, this concentration was subtracted
from data shown in column 6 of Figure 4(b). Mean + SEM,
n = 3; *p < 0.05 vs. DMSO control, + < 0.05 vs. BPA effect
increased by IL-4, IL-13 and IL-10 (Figure 4(B)) (Note:
The added IL-10 has been subtracted from data).
BPA increased IL-13 secretion which was further
increased by IL-4 (Figure 5).
FFA and LPS increased IL-13 secretion (Table 3). The
effect of FFA, but not of LPS was inhibited by 1 ng/mL
The IL-17 receptor is expressed by INS-1 cells which
was verified by a dot-blot experiment shown in Figure 6.
BPA and FFA increased p-Akt in a concentration-
dependent manner (Figure 7). The effect of FFA was
inhibited by Wortmannin (PI3K inhibitor) (Figure 7).
Figure 8 shows the effect of BPA on both STAT-3
and phosphorylated STAT-3. Both factors are increased
by BPA. Tofacitinib (JAK3 inhibition, interferes with
JAK-STAT signaling pathway) and wortmannin (PI3K
BPA [250ng/ml ]
BPA [500ng/ml]
BPA [1µg/ml]
BPA [10µg/ml]
BPA [250ng/ ml ]+IL-4 [1n g/ml]
Positi ve Control IL-13
I L-13 [p g / ml ]
Figure 5. Modulation of the effect of BPA on IL-10 secre-
tion from INS-1 cells; modulators are IL-4, IL-10, IL-13.
BPA effect (1 µg/mL) was modulated by various interleu-
kins, mostly at 100 ng/mL. Blanks (no addition) were
subtracted. DMSO (solvent; 1%) and a high concentration
of IL-10 (1250 pg/mL) were used as negative and positive
controls. When IL-4 was added, this concentration was
subtracted from data shown in column 6 of Figure 5. Mean
+ SEM, n = 3; *p < 0.05 vs. DMSO control.
Table 3. Release of IL-13 by FFA and LPS and the
modulation of this effect by IL-4. Details see in the legend of
Figure 5.
Compound IL-13 [PG/ML]
DMSO (0.1 %) 11.2 ± 0.3
FFA (250 ng/mL) 76.3 ± 2.6*
FFA (500 ng/mL) 109.5 ± 5.3*
FFA [1000 ng/mL] 623.7 ± 27.1*
FFA [1000 ng/mL] + IL-4 [1 ng/mL] 103.4 ± 8.7*
(significant vs. absence of IL-4)
FFA (10 µg/mL) 911.6 ± 19.4*
LPS [10 ng/mL] 203.5 ± 11.3*
LPS [5.0 ng/mL] 433.9 ± 9.7*
LPS [100 ng/mL] 834.3 ± 21.7*
LPS [100 ng/mL] + IL-4 [1 ng/mL] 1087 ± 41.7*
LPS [1000 ng/mL] 838.9 ± 19.6*
Positive control: IL-13 [1250 pg/mL] 1241 ± 28.3
inhibitor) inhibited this BPA effect.
In Table 4 the effects of various concentrations of
BPA, FFA and LPS on STAT-6 expression (phosphory-
lated and non-phosphorylated) and their inhibition by
Tofacitinib (JAK3 inhibitor) and Wortmannin (PI3K in-
hibitor) is shown.
4. Discussion
4.1. General Comments
The immunological answer to compounds of patho-
physiological impact such as the chemical BPA or the
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Impact of Bisphenol A (BPA) and Free Fatty Acids (FFA) on Th2 Cytokine Secretion from INS-1 Cells
Figure 6. Dot Blot of IL-17 receptor (IL-17R) of INS-1 cells.
Details in Methods. Top: sample, bottom: negative control
(no addition of primary antibody).
BPA [500ng/ml] p-Akt
BPA [1µg/ml] p-Akt
FFA [500ng/m l] p-Akt
FFA [1µg/ml] p-Akt
FFA [g/ml] p-Akt Wor tmannin
Positive Co n trol p-Akt
p-Akt [pg/ml]
Figure 7. Modulation of Akt (as pAkt) by BPA and FFA
and the modulation of FFA effect by Wortmannin in INS-1
cells. Blanks (no addition) were subtracted. DMSO (solvent;
1 %) and a high concentration of pAkt [2500 pg/mL] were
used as negative and positive contr ols. Mean + SEM, n = 3;
*p < 0.05 vs. DMSO control, + p < 0.05 vs. absence of
BPA [1µg/ml] STAT-3
BPA [1 µ g / ml] STAT-3+Tofacitinib
BPA [1µ g /m l] Phospho-S TAT-3
BPA [1 µ g / ml] Phospho-STAT- 3+Tofacitinib
BPA [1µ g /m l] S TAT-3
BPA [1µg/ml] STAT-3+Wortma nni n
BPA [1 µ g / ml] Phos ph o-STAT-3
BPA [g/ml] P hos pho-STAT-3+Wortmannin
STAT-3 [pg/ml]
Figure 8. Modulation of BPA-induced activation of STAT-3
(phospho-STAT-3) by Tofacitinib and Wortmannin. INS-1
cells were incubated for 24 hours. Blanks (no addition) w ere
subtracted. Blanks (no addition) were subtracted. DMSO
(solvent; 1 %) was used as a negative control. Mean + SEM,
n = 3; *p < 0.05 vs. DMSO control, p < 0.05 vs. absence of
either inhibitor.
Table 4. Effect of BPA and FFA on STAT-6 and p-STAT-6.
DMSO (0.1 %) 18.4 ± 0.6
BPA (1000 ng/mL) 11030 ± 54.9*
BPA (1000 ng/mL) + Tofacitinib9311 ± 22.5*+
BPA (1000 ng/mL) + Wortmannin 10760 ± 27.1*
FFA [1000 ng/mL] 8741 ± 51.4*
FFA [1000 ng/mL] + Tofacitinib7736 ± 39.4*+
FFA [1000 ng/mL] + Wortmannin 8576 ± 35.6*
LPS [1000 ng/mL] 7996 ± 44.6*
LPS [1000 ng/mL] + Tofacitinib10140 ± 87,6*+
LPS [1000 ng/mL] + Wortmannin7834 ± 43.6*+
pSTAT-6 [pg/mL]
BPA (1000 ng/mL) 12360 ± 36.4*
BPA (1000 ng/mL) + Tofacitinib9624 ± 22.6*+
BPA (1000 ng/mL) + Wortmannin 11120 44.5*+
FFA [1000 ng/mL] 10120 23.6*
FFA [1000 ng/mL] + Tofacitinib8224 25.4*+
FFA [1000 ng/mL] + Wortmannin 8651 27.9*+
LPS [1000 ng/mL] 9121 33.7*
LPS [1000 ng/mL] +Tofacitinib10730 76.2*+
LPS [1000 ng/mL] + Wortmannin8321 44.8*+
metabolic intermediate FFAs is strongly influenced by
various cytokines such as IL-4 promoting T helper type 2
(Th2) responses and IL-2 promoting T helper type 1 (Th1)
responses. Diabetes and β-cell death originating from
various cytokines and transcription factor were well
described [25,26]. The Th2 cytokines are not very well
investigated with respect to BPA and FFA. Cytokines
which are released by Th2 cells are IL-4, IL-6, IL-10 and
IL-13. A polarization into the direction of Th2 is partly
the answer to processes mediated by IL-4. BPA on a
long-term increases insulin levels thoug h not acutely [27];
but the focus here is on proinflammatory cytokines and
their balance to the anti-inflammatory cytokines IL-4,
IL-10 and IL-13, especially under the influence of BPA
and FFA.
17β-estradiol is of interest because it is known to
possess an IL-6 agonistic activ ity. Interesting ly BPA pos-
sesses only a low affinity to either estrogen receptor
subtype, nevertheless has a potent estrogen-like effect.
Estrogen receptors are present on β-cells and promote
insulin release [28].
4.2. Screening by a Profiler
When the effects of BPA and FFA were tested using
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Impact of Bisphenol A (BPA) and Free Fatty Acids (FFA) on Th2 Cytokine Secretion from INS-1 Cells 457
screening by a Profiler, FFA and BPA induced increases
of IL-2, IL-4, IL-6, IL-10 and IL-13 (Table 1). The LPS
effects as used for control are those as described in the
literature except that IL-6 and IL-17 were not secreted.
We mainly concentrated on the screened ILs (Table 1)
except IL-17.
4.3. IL-4
IL-4 is known to influence the differentiation process of
Th0 and Th1 which is connected to pathogenesis of type
1 diabetes [29], possibly having a protective effect [30].
BPA increased IL-4 secretion in a concentration-and time
dependent manner (Figure 1). Thus IL-4, a regulatory
cytokine for the adaptive immune response, is regulated
by BPA.
17β-Estradiol had an additional effect to that of BPA
(Figure 2) which results in a rightward shift of the BPA
curve including no effect of 17β-estradiol at high BPA
concentration. This shift indi cates an estradiol-like effect
of BPA with probably no divergent mechanism of action.
There was no superadditive effect between both com-
pounds which would have indicated a different mode of
action of both compounds. This is not surprising since
BPA is an endocrine disrupting chemical possibly inter-
acting with estrogen receptors as already mentioned.
There was a superadditive effect of FFA and 17β-estra-
4.4. IL-6
IL-6 was released by BPA (Figure 3(a)) as well as by
LPS and FFA (Table 2). It is a pro-inflammatory Th2
cytokine being involved in acute phase responses to
infection and more important in immune responses.
Dysregulation of IL-6-type cytokine signalling is part of
inflammation, viral infections and contributes to the
onset and maintenance of several diseases, including type
1 diabetes. People suffering from type 1 diabetes show
elevated IL-6 levels [31,32]. On the other hand IL-6
protects β-cells from pro-inflammatory effect and func-
tional impairment.
17β-estradiol is already known to promote IL-6 secre-
tion and to modulate the IL-6 receptor [33]. 17β-Estra-
diol increases IL-6 secretion (Figure 3(b)); it also in-
creases the effect of BPA, at least at low BPA con-
centrations (Figures 3(a) and (b)) indicating the same
type of mechanism. This interaction was already obv ious
for IL-4 (Figure 2).
Our data corroborate data on release of IL-6 by FFA
from other tissues [34,35]. Interestingly, 17β-estradiol
did not modify the FFA effect on IL-6 (Table 2).
4.5. IL-10
IL-10 was secreted by BPA, FFA and LPS (Figure 4).
IL-10 is expressed by many cells of the adaptive immune
system, including Th2- and Treg-cells and it is well
characterized as an anti-inflammatory cytokine with
potent suppressive effects in autoimmune diseases [36].
IL-10 function is ambivalent: the absence of IL-10 leads
to a better clearance of pathogens with non-enhanced
immunopathology, during other infections the absence of
IL-10 can be accompanied by a dentrimental immuno-
pathology for the host. Permanently increased IL-10 can
induce an early type 1 diabetes manifestation [37] but
may also produce anti-inflammatory effects in this situ-
ation. Elevated IL-10 (e.g. when stimulated by LPS) is
correlated with diabetes. IL-10 is known to inhibit the
progression of Th1 cells.
Production of IL-10 is regulated in a complex manner:
e.g. by the activation of inhibitory pathways and secre-
tion of cytokines from macrophages or by blocking Toll-
like receptor mediated MAPK-activation or by IFN-γ in-
ducing the release of glycogen synthase kinase 3 (GSK3)
via antagonizing phosphoinositide 3-kinase (PI3K)-Akt
activation which leads to inhibition of IL-10 production
[38]. IL-10 also nega tively regulates p38 phospho rylation
and thus limits IL-10 secretion (negative feedback). In
INS-1 cells, IL-10 is triggered by IL-4, IL-10 or IL-13 in
the presence of BPA (Figure 4(b)) which hints at a
positive feedback.
4.6. IL-13
BPA, FFA and LPS induce IL-13 secretion from INS-1
cells (Figure 5, Table 3). IL-13 is a critical mediator of
(allergic) inflammation and shares many functional
properties with IL-4 which is obvious by sharing a com-
mon receptor unit and common signaling pathways.
During the development of type 1 diabetes IL-13 has an
inhibitory effect on IFN-γ secretion [39] which is known
to be important for diabetes [37]. The situation is com-
plex because IL-13 is a modulator: it inhibits IL-1, IL-6
or IL-12.
4.7. IL-17
IL-17 and its receptor (IL-17R) are basic members of a
newly described family of cytokines and receptors, whi-
ch are different from other cytokine families. IL-17, the
hallmark cytokine of the newly defined T helper 17 cell
subset, has an ambivalent role by on the one hand
protecting the host against extracellular pathogens, and
on the other hand in promoting inflammatory pathology
in autoimmune diseases such as type 1 diabetes. IL-17
levels are low in diabetes, but are increased by exercise.
The impact of IL-17 on the development of type 1
diabetes is discussed and not clear at present [40]. The
presence of the IL-17R was demonstrated for INS-1 cells
(Figure 6). A measurement of IL-17 was not possible at
Copyright © 2013 SciRes. PP
Impact of Bisphenol A (BPA) and Free Fatty Acids (FFA) on Th2 Cytokine Secretion from INS-1 Cells
present, but the profiler experiment has shown a BPA-
induced release (Table 1).
4.8. Akt/STAT Signal Transduction
Akt (protein kinase B) is known to modify insulin
activity on many enzymes and is activated by PI3K
which is involved e.g. in IL-10 action (see above). pAkt
stimulated by BPA and FFA may positively act via PI3K
which is demonstrated by the inhibitory effect of Wort-
mannin (Figure 7).
The important cytokines involved in the Th1 and Th2
cell response often trigger Janus kinase (Jak)-STAT
signalling pathways, whereas IL-17 family cytokines
being different (see above) mediate signalling through
the pro-inflammatory NFκB. Phosphorylated STATs are
found in rat pancreatic cells as an answer to released
cytokines [41]. STAT-3 is known to be induced by IL-6
and IL-10 (and insulin), STAT-6 by IL-4 and IL-13.
Gene knoc kout studies g av e information on STATs being
involved in development and function of the immune
system. Studies using STAT-6 deficient mice revealed
that IL-13 signalling uses the Jak/STAT-6 pathway [42].
Phosphorylated and non-phosphorylated STAT-3 can be
activated by a BPA- and FFA-incubatio n (Figure 8).
Also STAT-6 signal transduction is activated by BPA,
FFA and LPS also activate (Table 4). The inhibitory
effect of Wortmannin (Figure 7) shows that PI3K may
be involved in the BPA effect on Akt. Altogether these
signaling pathways are known to be involved in the de-
velopment of type 1 diabetes.
STAT-6 is an important regulator of inflammation in-
duced by Th2 cells. It is known to be activated by IL-4
and IL-13. STAT-6 is activated e.g. by IL-4 which firstly
is phosphorylated as a result. IL-4 also acts on PI3K;
STATs and PI3K are used by IL-6; PI3K acts on Akt.
Interestingly STAT-3 inhibition by Tofacitinib, a jak/
STAT inhibitor, is more pronounced than STAT-6 inhi-
bition (Figure 8 and Table 4).
With respect of INS-1 cells, activation releases a set of
primary inflammatory mediators known for acute-phase
response such as IL-1, IL-2 and IL-6. We only present
here a two-dimensional view which is in reality much
more complex: IL-1 for example is known to inhibit IL-6
induced acute-phase protein synthesis in hepatocytes [43].
NFκB was identified as a mediator for IL-1 dependent
suppression of IL-6 in liver cells [44]. Th e impact of IL-1
towards the secretion of IL-6 in INS-1 cells could not be
proven in this work (data not shown).
Data obtained from rodents may be relevant for hu-
mans although it has to be admitted that rats are less
sensitive to e.g. estrogens. The metabolisms of BPA is
similar albeit rats elimate the glucuronidated products via
faeces in contrast to humans (via urine) [45].
4.9. Summary and Conclusion
Altogether BPA, LPS and FFA can increase the release
of Th2-cytokines from INS-1 cells (IL-4, IL-6, IL-10 and
IL-13) and mediate pro- and anti-inflammatory effects.
Data underline the major pathophysiological relevance of
BPA and FFA. BPA induces the insulin-stimulated Akt
phosphoryation. FFA and BPA activate—probably by
one of the investigated released cytokines, STAT-3 and
STAT-6 as signal transduction pathways, and also the
PI3K/Akt signal transduc tion pathway. Thus it cannot be
excluded that these signals induced by BPA and FFAs
may influence development of type 1 diabetes or at least
disturb glucose homoeostasis. This involvement should
be a matter of further detailed investigation.
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