TOXICOLOGICAL SCIENCES 84, 319–327 (2005)
doi:10.1093/toxsci/kfi088
Advance Access publication January 19, 2005
Bisphenol A Accelerates Terminal Differentiation of 3T3-L1 Cells into
Adipocytes through the Phosphatidylinositol 3-Kinase Pathway
Hiroshi Masuno,*,1 Jun Iwanami,† Teruki Kidani,‡ Kenshi Sakayama,‡ and Katsuhisa Honda†
*Department of Medical Technology, Faculty of Health Sciences, Ehime Prefectural University of Health Sciences, Takooda, Tobe-cho, Iyo-gun,
Ehime 791-2101, Japan; †Department of Environmental Science for Industry, Ehime University, 3-5-7 Tarumi, Matsuyama, Ekime 790-8566, Japan; and
‡Department of Orthopaedic Surgery, Ehime University School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan
Received October 8, 2004; accepted January 11, 2005
In order to identify whether bisphenol A (BPA) acts as an
adipogenic agent, following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for six days with
BPA alone. Treatment with BPA increased the triacylglycerol
(TG) content of the cultures, increased the percentage of Oil Red
O-staining cells in the cultures, and increased the levels of
lipoprotein lipase (LPL) and adipocyte-specific fatty acid binding
protein (aP2) mRNAs. These findings indicate that BPA was able
to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. LY294002, a chemical inhibitor of phosphatidylinositol 3kinase (PI 3-kinase), blocked completely the increasing effect of
BPA on TG accumulation and expression of LPL and aP2
mRNAs. Western blot analysis revealed that BPA increased the
level of phosphorylated Akt kinase. Based on these findings, we
concluded that BPA acted through the PI 3-kinase and Akt kinase
pathway, resulting in increased TG accumulation and expression
of adipocyte genes. The structure-activity relationship for BPArelated chemicals was examined. Eight derivatives of BPA (three
diphenylalkanes with different substituents at the central carbon
atom, three diphenylalkanes with ester bonds on hydroxyl groups
in the phenolic rings, one bisphenol consisting of a sulphur atom
at the central position, one chemical with cyanic groups, instead of
hydroxyl groups, in the phenolic rings) accelerated terminal
adipocyte differentiation and their potencies to increase TG
accumulation were 73–97% of that of BPA. Two diphenylalkanes
with ether bonds on hydroxyl groups and two alkylphenols (4nonylphenol and 4-tert-octylphenol) did not have the ability to
accelerate terminal adipocyte differentiation.
Key Words: bisphenols; 3T3-L1 cells; terminal adipocyte
differentiation; LY294002; Akt kinase.
Bisphenol A (BPA) is an environmental endocrine disrupting
chemical that is present ubiquitously in the environment. For
example, BPA is used commercially in products containing
polycarbonate plastics such as baby bottles and leaches from
them when subjected to simulated use by dishwashing, boiling,
and brushing (Brede et al., 2003). This chemical also leaches
1
To whom correspondence should be addressed. Fax: þ81-89-958-2177.
E-mail: [email protected].
from polycarbonate culture flasks when subjected to autoclaving (Krishnan et al., 1993). Microgram amounts of BPA
are found in the liquid of preserved vegetables in cans (Brotons
et al., 1995) and in the saliva of patients treated with dental
sealants (Olea et al., 1996). A small amount of BPA also is
found in tap water and in human serum (Inoue et al., 2000).
BPA mimics the actions of estrogens (Korach, 1993). Most
research to date on BPA has focused on the growth of
reproductive organs and tumor cells. For example, administration of BPA to ovariectomized rodents increases uterine
weight, stimulates cell proliferation in the uterus and
vagina, and induces hypertrophy of the luminal epithelium
(Goloubkova et al., 2000; Papaconstantinou et al., 2000;
Steinmetz et al., 1998). In cultures, BPA stimulates proliferation of estrogen-responsive human breast cancer cells,
MCF-7 cells (Rivas et al., 2002). Recently, BPA was reported
to affect non-reproductive organs as well as reproductive
organs. In ovariectomized rats, BPA stimulated the growth of
the anterior pituitary gland (Goloubkova et al., 2000) and
increased the expression of progesterone receptor mRNA in
the preoptic area (Funabashi et al., 2001). Moreover, twogeneration studies in rats (Rubin et al., 2001) and mice
(Howdeshell et al., 1999) showed that perinatal exposure to
BPA caused an increase in body weight after birth. However,
whether this increased body weight was due to an increased
adipose tissue mass remains unclear.
The major cellular component of adipose tissue is adipocytes. 3T3-L1 adipocytes have been widely used to understand
adipocyte physiology. Insulin is well known to be a potent
adipogenic agent in cultures of 3T3-L1 adipocytes (Cornelius
et al., 1994; Gregoire et al., 1998; Rubin et al., 1978; Spooner
et al., 1979). The adipogenic actions of insulin are mediated by
the insulin signaling pathway (White and Kahn, 1994; Xia and
Serrero, 1999). The binding of insulin to its specific receptor at
the cell surface causes autophosphorylation and activation of
the receptor and this activated receptor phosphorylates insulin
receptor substrate-1 (IRS-1). Phosphorylated IRS-1 activates
phosphatidylinositol 3-kinase (PI 3-kinase). The function of PI
3-kinase is implicated in terminal differentiation of 3T3-L1
Toxicological Sciences vol. 84 no. 2 Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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cells into adipocytes. For example, inhibition of PI 3-kinase
by a chemical inhibitor LY294002 blocked insulin-induced
lipid accumulation in 3T3-L1 cells (Xia and Serrero 1999).
In the present study, we examined whether BPA and BPArelated chemicals had the ability to accelerate terminal
differentiation of 3T3-L1 cells into adipocytes. We used three
phenotypic markers for adipocytes as follows: (1) triacylglycerol (TG) accumulation in cells, (2) expression of lipoprotein
lipase (LPL) and adipocyte-specific fatty acid binding protein
(aP2) genes, and (3) catalytic activity of LPL. We also explored
whether BPA acted through the PI 3-kinase pathway.
MATERIALS AND METHODS
Materials. Bisphenol A (BPA), bisphenol B (BPB), bisphenol A dicyanate
(BPA-DC), bisphenol A bis(chloroformate) (BPA-BCF), bisphenol A diacetate
(BPA-DA), bisphenol A O,O-diacetic acid (BPA-DAA), bisphenol A
dimethacrylate (BPA-DM) and bisphenol A diglycidyl ether (BPA-DGE), 4nonylphenol (NP, mixture of compounds with branched sidechain), 4-tertoctylphenol (OP) were obtained from Tokyo Kasei Co., Tokyo, Japan.
Bisphenol E (BPE), bisphenol F (BPF), bisphenol S (BPS), and a kit for TG
were obtained from Wako Pure Chemicals Co., Osaka, Japan. A GenElute
Mammalian Total RNA kit and LY294002 hydrochloride were obtained from
Sigma. An AlkPhos direct labeling kit, an ECL Plus Western Blotting Starter
kit, hybridization buffer, blocking reagent, Hyperfilm MP, Hybond-Nþ, and
Hybond-P were obtained from Amersham Pharmacia Biotech, U.K. A mouse
monoclonal antibody to Akt1 (B-1) and a rabbit polyclonal antibody to p-Akt1/
2/3 (Ser 473) were obtained from Santa Cruz Biotechnology, Inc. A DC Protein
Assay was obtained from Bio-Rad Laboratories.
Cell culture and measurements of DNA and TG contents and LPL
activity. 3T3-L1 cells were grown to confluence in a standard medium, which
consisted of 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 lg/ml
streptomycin, and 0.25 lg/ml amphotericin B in Dulbecco’s modified Eagle’s
medium, on a 60-mm plate. Confluent cultures were induced to differentiate
into adipocytes by treating them for two days in a standard medium supplemented with 10 lg/ml insulin, 1 lM dexamethasone (DEX), and 0.5 mM
3-isobutyl-1-methylxanthine (MIX). The medium was then replaced with
a standard medium supplemented with either 80 lM BPA alone or a combination of 80 lM BPA and LY294002 at the indicated concentrations and changed
every two days. Six days later, cells were harvested in 1.2 ml of 50 mM NH4Cl/
NH4OH buffer (pH 8.2) containing 20 lg/ml heparin and 2% (w/v) bovine
serum albumin and sonicated briefly at 0°C. Aliquots of the homogenate were
used for DNA and TG measurements. DNA was measured fluorometrically by
the method of Hinegardner (1971) and TG was measured using a kit for TG.
Another aliquot of the homogenate was used for preparing an acetone/ether
powder for measurement of LPL activity (Masuno et al., 1990). LPL activity in
the extract of an acetone/ether powder was measured using phosphatidylcholine-stabilized triolein as a substrate (Masuno et al., 2002). One unit of lipolytic
activity was defined as that releasing 1 lmol of fatty acid/min at 37°C.
Lipid staining in cells. The cultures were fixed with 10% (v/v) formalin in
phosphate-buffered saline, and then stained with Oil Red O as described by
Kuri-Harcuch and Green (1978). Cells were considered as lipid-positive when
droplets were stained red. Four different microscopic fields (3100 magnification) per culture were photographed. The percentage of lipid-positive cells,
which was calculated by dividing the number of lipid-positive cells by the
number of total cells (at least 250 cells), in each photograph was determined.
A value was obtained as an average value for four fields per culture. The results
were expressed as the average value for two cultures.
Northern blot. Total RNA was isolated from the cultures using a
GenElute Mammalian Total RNA kit. RNA samples from two plates were
combined for each treatment. RNA samples (20 lg/lane) were denatured
with formamide and formaldehyde and electrophoresed on a 1.5% agarose
gel containing 6.3% formaldehyde. The RNAs were blotted onto a nylon
membrane (Hybond-Nþ) and crosslinked with 0.05 N NaOH. The blotted
membrane was prehybridized for 30 min at 55°C in hybridization buffer
containing 0.5 M NaCl and 4% (w/v) blocking reagent. A probe labeled
using an AlkPhos direct labeling kit was then hybridized for 15–20 h at 55°C.
After hybridization, the membrane was washed twice for 10 min at 55°C in
the primary wash buffer (50 mM NaH2PO4, 2 M urea, 0.1% sodium dodecyl
sulfate, 150 mM NaCl, 1 mM MgCl2, 0.2% blocking reagent, pH 7.0) and
washed twice for 5 min at room temperature in the secondary wash buffer
(50 mM Tris, 2 mM MgCl2, pH 10.0). The blot was left in CDP-Star
detection reagent (30–40 ll/cm2) for 3 min and excess detection reagent was
drained off. The membrane was exposed to a Hyperfilm MP with an
intensifying screen for approximately 60 min. Relative densitometric units
were determined using the analysis software Diversity Database.
The probes used for detection of mRNAs of LPL and aP2 were
a 542bp EcoRI fragment (nucleotide 194–735) of mouse LPL cDNA clone
and a 600bp BamHI fragment of mouse aP2 cDNA clone. The probe for
aP2 was kindly donated by Dr. S. A. Kliewer (GlaxoSmithKline, North
Carolina).
Western blot. Cells were lysed in a lysis buffer (20 mM HEPES, 3 mM
MgCl2, 2 mM EDTA, 100 mM NaF, 1% Triton X-100, 1 mM PMSF, 100 lg/ml
leupeptin, 10 lg/ml aprotinin, pH 7.4) for 20 min at 4°C and centrifuged to
remove insoluble materials. The protein concentrations in the cell lysates were
measured using a DC Protein Assay. The same amount (10 lg of protein/lane)
of proteins were denatured by boiling in Laemmli sample buffer containing
10% 2-mercaptoethanol, separated by SDS-PAGE, and transferred electrophoretically to a PVDF membrane (Hybond-P). The blotted membranes were
incubated with indicated primary antibodies (1:500) and horseradish peroxidase-conjugated secondary antibody (1:25,000). Blots were visualized with an
ELC Plus Western Blotting Starter kit according to the manufacturer’s
instructions. The membrane was exposed to a Hyperfilm MP with an
intensifying screen for approximately 5–15 min. Relative densitometric units
were determined using the analysis software Diversity Database.
Statistical analysis. Statistical analysis of group means was conducted by
ANOVA followed by post hoc comparisons using Fisher’s protected least
significant difference test. Statistical analysis was conducted using Student’s
t-test for unpaired samples. For all the statistical analyses, the criterion of
significance was p < 0.05. All values were expressed as means ± SD.
RESULTS
Can BPA Accelerate Terminal Differentiation of 3T3-L1
Cells into Adipocytes?
Following the hormonal induction of differentiation, 3T3L1 cells were treated for six days with BPA alone. The
untreated cultures, in which BPA was absent during the
treatment period, contained 30.7 lg/plate of DNA and 1.97
lg/lg DNA of TG. The addition of BPA to the cultures did
not cause changes in the DNA content (Fig. 1A) but caused
a dose-dependent increase in the TG content (Fig. 1B). BPA
at 40 and 80 lM increased the TG content by 110 and
310%, respectively. However, in cultures treated with 160 lM
BPA for more than two days, cells began to detach from
the bottom of the plates. Therefore, the following
BISPHENOLS AND TERMINAL DIFFERENTIATION
321
FIG. 1. Effect of BPA on the DNA and TG contents and expression of LPL and aP2 mRNAs. (A and B) Following the hormonal induction of
differentiation, 3T3-L1 cells were treated for six days with BPA at the indicated concentrations. Cells were harvested and sonicated briefly at
0°C. The DNA and TG contents in aliquots of the homogenate were measured. Values given are the mean ± SD for four plates. ap < 0.01
(compared with the value obtained from the untreated cultures). (C) Following the hormonal induction of differentiation, 3T3-L1 cells were
treated for six days without or with 80 lM BPA. Total RNA was extracted from cells. RNA samples from two plates were combined for each
treatment. Twenty lg of RNA was loaded to each lane. Expression of LPL and aP2 mRNAs was analyzed by Northern blot. Relative
densitometric units were determined using the analysis software. Values given for LPL mRNA are the mean ± SD for four experiments. Values
given for aP2 mRNA are the mean ± SD for seven experiments. 28S shows ethidium bromide-stained rRNA.
experiments were performed using cultures treated with 80 lM
BPA, termed the BPA-treated cultures.
Oil Red O staining showed that approximately 25% of
cells in the untreated cultures contained discrete lipid
droplets (Fig. 2A). The presence of BPA caused an
increase in the percentage of lipid-positive cells to approximately 85% (Fig. 2B). These results suggest that BPA may
accelerate terminal differentiation of 3T3-L1 cells into adipocytes.
To confirm this, the expression of LPL and aP2 genes
was analyzed by Northern blot and the catalytic activity of
LPL in the extract of acetone/ether powder of cells was
measured. BPA increased the expression of these two genes
(Fig. 1C). Densitometrical analysis revealed that BPA increased the levels of LPL and aP2 mRNAs by 293 and
438%, respectively. The catalytic activity of LPL was also
higher in the BPA-treated cultures than in the untreated cultures [the former cultures (n ¼ 4), 1.71 ± 0.06 units/mg
DNA; the latter cultures (n ¼ 4), 1.29 ± 0.11 units/mg DNA;
p < 0.01].
Does BPA Act through the PI 3-Kinase and Akt Kinase
Pathway?
Since the insulin signaling pathway plays a pivotal role in
terminal adipocyte differentiation (Kohn et al., 1996; Xia and
Serrero, 1999), we examined using LY294002 whether BPA
acted through the PI 3-kinase pathway. Following the hormonal
induction of differentiation, 3T3-L1 cells were treated with
either BPA alone or a combination of BPA and LY294002. The
DNA content of the cultures decreased with an increase in
LY294002 concentrations (Fig. 3A). The DNA content of the
cultures treated with a combination of BPA and 8 lM
LY294002 was 76% of that of the cultures treated with BPA
alone. Thus, LY294002 inhibited proliferation of 3T3-L1 cells.
This result is compatible with the findings of others that PI
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MASUNO ET AL.
FIG. 2. Oil Red O staining of lipid droplets. Following the hormonal induction of differentiation, 3T3-L1 cells were treated for six days
without or with 80 lM BPA in the absence or presence of 8 lM LY294002. The cells were fixed with 10% formalin and then stained with Oil Red
O. Arrow, lipid droplets. (A) No additive; (B) 80 lM BPA alone; (C) 80 lM BPA plus 8 lM LY294002.
3-kinase plays an important role in mediating the action of
insulin on cell growth (Backer et al., 1992; Kapeller et al.,
1991). Of course, there is a concern that this inhibition could
be due to a toxic effect of LY294002. However, this does
not appear to be the case, because cells had not been
detached from the bottom of the plates during the experiment
period. Moreover, cells attached to the bottom of the plates
excluded trypan blue (data not shown).
LY294002 suppressed dose-dependently the increasing
effect of BPA on the TG content (Fig. 3B). In the cultures
treated with a combination of BPA and 8 lM LY294002, the
TG content was decreased to a level similar to that of the
untreated cultures. The percentage of lipid-positive cells in
these cultures was also similar to that in the untreated cultures
(Fig. 2C). In addition, LY294002 suppressed dose-dependently
the increasing effect of BPA on expression of LPL and aP2
FIG. 3. Effect of LY294002 on the DNA and TG contents and expression of LPL and aP2 mRNAs. (A and B) Following the hormonal
induction of differentiation, 3T3-L1 cells were treated for six days with 80 lM BPA in the presence of LY294002 at the indicated concentrations.
Cells were harvested and sonicated briefly at 0°C. The DNA and TG contents in aliquots of the homogenate were measured. Values given are the
mean ± SD for four plates. ap < 0.05; bp < 0.01 (compared with the value obtained from the untreated cultures). cp < 0.05; dp < 0.01 (compared
with the value obtained from the cultures treated with BPA alone). (C) Total RNA was extracted from cells. RNA samples from two plates were
combined for each treatment. Twenty lg of RNA was loaded to each lane. Expression of LPL and aP2 mRNAs was analyzed by Northern blot.
Relative densitometric units were determined using the analysis software. Values given are the mean ± SD for three experiments. ap < 0.05; bp <
0.01 (compared with the value obtained from the cultures treated with BPA alone). 28S shows ethidium bromide-stained rRNA.
BISPHENOLS AND TERMINAL DIFFERENTIATION
323
FIG. 4. Effect of BPA on phosphorylation of Akt. Following the hormonal induction of differentiation, 3T3-L1 cells were treated for six days
without or with 80 lM BPA in the absence or presence of 8 lM LY294002. The proteins (10 lg of protein/lane) in the cell lysates were separated
by SDS-PAGE. Expression of Akt (A) and phospho-Akt (B) was analyzed by Western blot. Relative densitometric units were determined using
the analysis software. Values given are the mean ± SD for three experiments. ap < 0.05 (compared with the value obtained from the cultures
treated with BPA alone).
genes (Fig. 3C). The levels of LPL and aP2 mRNAs in the
cultures treated with a combination of BPA and 8 lM
LY294002 were similar to those in the untreated cultures.
Since Akt kinase functions downstream of PI 3-kinase
(Burgering and Coffer, 1995; Franke et al., 1995), we
examined the effect of BPA on the levels of Akt and
phosphorylated Akt (phospho-Akt) by Western blot. Treatment
with BPA alone decreased the level of Akt by 53% (Fig. 4A)
and increased the level of phospho-Akt by 97% (Fig. 4B),
compared with those of the untreated cultures. Treatment
with LY294002 alone did not affect the level of Akt but
decreased the level of phospho-Akt by 26%. In the cultures
treated with a combination of BPA and LY294002, the levels of
Akt and phospho-Akt were 88 and 106%, respectively, of
those of the untreated cultures. Thus, LY294002 reversed
the decreasing effect of BPA on the level of Akt and suppressed
the increasing effect of BPA on the level of phospho-Akt.
Relationship between the Structures of BPA-Related
Chemicals and Terminal Adipocyte Differentiation
The effects of chemicals with different substituents at the
central carbon atom on the TG content and the expression of
aP2 gene were examined. The chemical structures are shown
in Figure 5A. BPA, BPB, BPE, and BPF are chemicals
with different lengths of alkyl substituents at the central
carbon atom. Like BPA, BPB, BPE, and BPF increased the
TG content (Fig. 5B) and the level of aP2 mRNA (Fig. 5C).
The potency of BPB to increase TG accumulation was similar
to that of BPA, while the potencies of BPE and BPF were 93
and 89%, respectively, of that of BPA. BPS is a bisphenol
consisting of a sulphur atom, instead of a carbon atom, at the
central position; the whole chemical structure of BPS is shown
in Figure 5A. This chemical also increased the TG content and
the level of aP2 mRNA. Its potency to increase TG accumulation was 92% of that of BPA.
The effects of chemicals with different substituents on
hydroxyl groups in the para position of the phenolic rings on
the TG content and the expression of aP2 gene were examined.
The chemical structures are shown in Figure 6A. BPA-DA,
BPA-BCF, and BPA-DM are ester derivatives of BPA. These
chemicals with ester bonds increased the TG content (Fig. 6B)
and the level of aP2 mRNA (Fig. 6C). The potencies of BPADA, BPA-BCF, and BPA-DM to increase TG accumulation
were 93, 85, and 73%, respectively, of that of BPA. BPA-DGE
and BPA-DAA are ether derivatives of BPA. These chemicals
with ether bonds did not have the ability to increase the TG
content and the level of aP2 mRNA. BPA-DC is a chemical
with cyanic groups, instead of hydroxyl groups, at the phenolic
rings. This chemical increased the TG content and the level of
aP2 mRNA. Its potency to increase TG accumulation was 80%
of that of BPA.
Effect of Alkylphenols on TG Accumulation and
LPL Activity
Following the hormonal induction of differentiation, 3T3-L1
cells were treated for six days with either 45 lM 4-nonylphenol
(NP) alone or 45 lM 4-tert-octylphenol (OP) alone. Treatment
of cells with either NP or OP caused a 28 or 35%, respectively,
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MASUNO ET AL.
(n ¼ 3), 1.75 ± 0.30 lg/lg DNA (p < 0.01, vs. the untreated
cultures)]. NP and OP did not affect the LPL activity of the
cultures (data not shown).
DISCUSSION
FIG. 5. Effect of BPA-related chemicals with different substituents at the central carbon atom on the TG content and expression of
aP2 mRNA. (A) Structures of substituents at the central carbon atom
and the whole structure of BPS are shown. (B) Following the hormonal
induction of differentiation, 3T3-L1 cells were treated for six days
with 80 lM BPA-related chemical. Cells were harvested and sonicated
briefly at 0°C. The DNA and TG contents in aliquots of the homogenate were measured. No chemicals caused changes in the DNA
content of the cultures (data not shown). Values given are the mean ±
SD for four plates. ap < 0.01 (compared with the value obtained
from the untreated cultures). bp < 0.05; cp < 0.01 (compared with
the value obtained from the cultures treated with BPA alone). (C) Total
RNA was extracted from cells. RNA samples from two plates were
combined for each treatment. Twenty lg of RNA was loaded to each
lane. Expression of aP2 mRNA was analyzed by Northern blot. 28S
shows ethidium bromide-stained rRNA. The representative of two
independent experiments is shown. BPA, bisphenol A; BPF, bisphenol
F; BPE, bisphenol E; BPB, bisphenol B; BPS, bisphenol S.
decrease in the TG content, compared with the untreated
cultures [the untreated cultures (n ¼ 3), 2.69 ± 0.08 lg/lg
DNA; the NP-treated cultures (n ¼ 3), 1.94 ± 0.20 lg/lg DNA
(p < 0.01, vs. the untreated cultures); the OP-treated cultures
Although 3T3-L1 cells are converted spontaneously to
adipocytes when maintained in culture with fetal bovine serum
alone, this process takes several weeks (Green and Meuth,
1974). A variety of agents that can retract the conversion
process by rapidly and irreversibly triggering 3T3-L1 cells to
differentiate into adipocytes have been reported. These triggers
include insulin (Russell and Ho, 1976; Spooner et al., 1979),
DEX (Rubin et al., 1978), MIX (Russell and Ho, 1976;
Spooner et al., 1979), and sodium butyrate (Toscani et al.,
1990). The most effective means to trigger the differentiation is
to treat confluent cultures of 3T3-L1 cells with a combination
of insulin, DEX, and MIX (Rubin et al., 1978). Previously, we
found that BPA had the ability to trigger the differentiation of
3T3-L1 cells into adipocytes (Masuno et al., 2002). Following
the induction of differentiation by BPA, treatment with
a combination of BPA and insulin increased markedly TG
accumulation in cells but treatment with BPA alone decreased
it. Thus, whether BPA by itself had the ability to accelerate
terminal differentiation of 3T3-L1 cells into adipocytes was
not clear. The present study was conducted to extend our
previous study on the effect of BPA on terminal adipocyte
differentiation. For this purpose, the confluent cultures of 3T3L1 cells were treated with a combination of insulin, DEX, and
MIX for two days and subsequently treated with BPA alone.
The results of TG measurement suggested that BPA might
increase TG accumulation in cells. This was confirmed by the
finding that there were many more Oil Red O-staining cells in
the BPA-treated cultures than in the untreated cultures.
Northern blot analysis revealed that BPA increased the
expression of LPL and aP2 genes. In addition, the catalytic
activity of LPL was higher in the BPA-treated cultures than in
the untreated cultures. These findings indicate that BPA by
itself had the ability to accelerate terminal differentiation of
3T3-L1 cells into adipocytes. This conclusion raises the
question of how BPA accelerates terminal adipocyte differentiation.
Expression of the constitutively active variant of Akt kinase
in 3T3-L1 cells resulted in spontaneous differentiation of
fibroblasts into adipocytes, associated with the accumulation
of increased lipid droplets (Kohn et al., 1996). A study with
LY294002 demonstrated that the function of PI 3-kinase was
indispensable for lipid accumulation in 3T3-L1 adipocytes
(Xia and Serrero, 1999). Thus, the PI 3-kinase and Akt
kinase pathway plays a pivotal role in terminal adipocyte
differentiation. Therefore, to explore the above-mentioned
question, we examined whether BPA increased TG accumulation and expression of adipocyte genes through this pathway.
BISPHENOLS AND TERMINAL DIFFERENTIATION
FIG. 6. Effect of BPA-related chemicals with different substituents at the phenolic hydroxyl groups on the TG content and expression
of aP2 mRNA. (A) Structures of substituents at the phenolic hydroxyl
groups are shown. (B) Following the hormonal induction of differentiation, 3T3-L1 cells were treated for six days with 80 lM BPA-related
chemical. Cells were harvested and sonicated briefly at 0°C. The DNA
and TG contents in aliquots of the homogenate were measured. No
chemicals caused changes in the DNA content of the cultures (data not
shown). Values given are the mean ± SD for four plates. ap < 0.01
(compared with the value obtained from the untreated cultures). bp <
0.05; cp < 0.01 (compared with the value obtained from the cultures
treated with BPA alone). (C) Total RNA was extracted from cells.
RNA samples from two plates were combined for each treatment.
Twenty lg of RNA was loaded to each lane. Expression of aP2 mRNA
was analyzed by Northern blot. 28S shows ethidium bromide-stained
rRNA. The representative of two independent experiments is shown.
BPA, bisphenol A; BPA-DA, bisphenol A diacetate; BPA-DGE,
bisphenol A diglycidyl ether; BPA-BCF, bisphenol A bis(chloroformate); BPA-DM, bisphenol A dimethacrylate; BPA-DAA, bisphenol
A O,O-diacetic acid; BPA-DC, bisphenol A dicyanate.
325
Since LY294002 blocked completely the increasing effect of
BPA on TG accumulation and expression of LPL and aP2
genes, this indicated that the function of PI 3-kinase was
required to accelerate terminal adipocyte differentiation.
Treatment with BPA alone increased the level of phosphoAkt, indicating that BPA activated Akt. Moreover, LY294002
blocked the increasing effect of BPA on the level of phosphoAkt. Based on these findings, we concluded that BPA acted
through the PI 3-kinase and Akt kinase pathway, resulting in
increased TG accumulation and expression of adipocyte genes.
Recently, Sakurai et al. (2004) reported that BPA affected
neither the expression of IRS-1 nor the insulin-induced
phosphorylation of IRS-1 in 3T3-F442A adipocytes. In addition, BPA alone was not able to phosphorylate IRS-1. Based on
these findings, they concluded that the action of BPA was not
mediated by the insulin signaling pathway in 3T3-F442
adipocytes. The discrepancy between our conclusion and theirs
may result from different treatment periods. We treated cells
with 80 lM BPA for six days but they treated cells with 100 lM
BPA for only 26 h. Of course, a possibility that this discrepancy
may be due to different cell lines cannot be excluded.
Perez et al. (1998) reported that in cultures of MCF-7 cells,
the longer the alkyl groups at the central carbon atom of
diphenylalkanes, the stronger the estrogenic potency estimated
from relative cell proliferation activity. They also found that
a lower polarity at the central carbon atoms enhanced estrogenicity. Thus, estrogenicity of diphenylalkanes was influenced
not only by the length of the substituents at the central carbon
atom but also by their nature. In the present study, we found
that in diphenylalkanes with free hydroxyl groups in the para
position of the phenolic rings, there was no relationship
between the length of alkyl groups at the central carbon atom
and the potency to increase TG accumulation. This raises the
question of whether the central carbon atom was required to
accelerate terminal adipocyte differentiation. To explore this
question, we used BPS, a bisphenol consisting of a sulphur
atom at the central position. Since this chemical increased the
expression of aP2 gene and its potency to increase TG
accumulation was 92% of that of BPA, this indicated that the
central carbon atom was not necessarily needed to accelerate
terminal adipocyte differentiation. This finding is consistent
with that of Hashimoto et al. (2001) that bisphenols consisting
of atoms and compounds other than the carbon atom at the
central position had a strong/moderate estrogenic potency in
cultures of MCF-7 cells.
Since NP and OP, alkylphenols consisting of a single
phenolic ring, also stimulated cell proliferation in cultures of
MCF-7 cells (Soto et al., 1991; White et al., 1994), we
examined whether they accelerated terminal differentiation
of 3T3-L1 cells into adipocytes. In contrast to diphenylalkanes,
these alkylphenols inhibited TG accumulation in cells. In
addition, they were not able to increase the LPL activity of
the cultures. Previously, we found that NP and OP both blocked
the increasing effect of insulin on TG accumulation and
326
MASUNO ET AL.
expression of adipocyte genes in culture of 3T3-L1 cells
(Masuno et al., 2003). Taken together, the present findings
indicate that both phenolic rings of diphenylalkanes are
required to accelerate terminal differentiation of 3T3-L1 cells
into adipocytes.
Leclercq (1992) reported that the existence of an oxygen
functional group in the para position of the phenolic rings
conferred estrogenicity. The replacement of the phenolic
hydroxyl groups with diethylaminoethoxy substituents reduced the proliferative activity in cultures of MCF-7 cells
(Gilbert et al., 1994). Perez et al. (1998) reported that ester
derivatives of BPA displayed a high proliferative activity in
cultures of MCF-7 cells, while ether derivatives displayed
a low or no proliferative activity. In our culture system, all
ester derivatives of BPA tested increased the expression of
aP2 gene and their potencies to increase TG accumulation
were 73–93% of that of BPA, whereas ether derivatives were
not able to increase TG accumulation and expression of
aP2 gene. Perez et al. (1998) found that bisphenol A ethoxylate
diacrylate (BPA-EDA) and BPA-DGE were estrogenic in the
proliferation bioassay even though they showed very low
(BPA-EDA) and no binding affinity (BPA-DGE) for the
estrogen receptor in in vitro competitive binding assay.
Based on this finding, they described that estrogenicity of
diphenylalkanes with ester and ether bonds on the para
hydroxyl groups could be related to the ability of cellular
enzymatic systems to break down these bonds and to generate molecules with free hydroxyl groups. Whether 3T3-L1 cells
were able to cleave ester bonds to release free hydroxyl groups
is unclear. It is likely that functional groups, which can form
the hydrogen bonds, were required to accelerate terminal
adipocyte differentiation, because BPA-DC, a chemical with
cyanic groups in the para position of the phenolic rings,
increased the expression of aP2 gene and its potency to
increase TG accumulation was 80% of that of BPA.
In conclusion, 9 of 11 bisphenols tested were able to
accelerate terminal differentiation of 3T3-L1 cells into adipocyteas. This action of BPA was mediated by the mechanism
that involved the PI 3-kinase and Akt kinase pathway.
ACKNOWLEDGMENTS
We thank Dr. Steven A. Kliewer, GlaxoSmithKline, North Carolina, U.S.A.,
for donating a mouse aP2 probe. This work was supported in part by a Grantin-Aid for Science Research (C) from the Japan Society for the Promotion of
Science (H.M.).
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