Accumulation of polychlorinated biphenyls in adipocytes: selective targeting to lipid droplets and role of caveolin-1.
ABSTRACT Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that preferentially accumulate in lipid-rich tissues of contaminated organisms. Although the adipose tissue constitutes a major intern reservoir of PCBs and recent epidemiological studies associate PCBs to the development of obesity and its related disorders, little is known about the mechanisms involved in their uptake by the adipose tissue and their intracellular localization in fat cells.
We have examined the intracellular distribution of PCBs in mouse cultured adipocytes and tested the potential involvement of caveolin-1, an abundant adipocyte membrane protein, in the uptake of these compounds by fat cells. We show that 2,4,4'-trichlorobiphenyl (PCB-28), 2,3',4,4',5-pentachlorobiphenyl (PCB-118) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) congeners rapidly and extensively accumulate in 3T3-L1 or mouse embryonic fibroblast (MEF) derived cultured adipocytes. The dynamics of accumulation differed between the 3 congeners tested. By subcellular fractionation of primary adipocytes, we demonstrate that these pollutants were almost exclusively recovered within the lipid droplet fraction and practically not associated to cell membranes. The absence of caveolin-1 expression in primary adipocytes from cav-1 deficient mice did not modify lipid droplet selective targeting of PCBs. In cav-1 KO MEF differentiated adipocytes, PCB accumulation was decreased, which correlated with reduced cell triglyceride content. Conversely, adenoviral mediated cav-1 overexpressing in 3T3-L1 cells, which had no impact on total cell lipid content, did not change PCB accumulation.
Our data indicate that caveolin-1 per se is not required for selective PCB accumulation, but rather point out a primary dependence on adipocyte triglyceride content. If the crucial role of lipid droplets in energy homeostasis is considered, the almost exclusive accumulation of PCBs in these organelles warrants future attention as the impairment of their function could be linked to the worldwide obesity epidemic.
- SourceAvailable from: cas.cz[show abstract] [hide abstract]
ABSTRACT: White adipose tissue (WAT) represents a reservoir of lipophilic environmental pollutants, especially of those which are resistant to biological and chemical degradation - so-called persistent organic pollutants (POPs). Large amounts of different congeners and isomers of these compounds exhibit a variety of adverse biological effects. Interactions among different classes of compounds, frequently with opposing effects, complicate hazard evaluation and risk assessment. WAT is the key organ for energy homeostasis and it also releases metabolites into the circulation and adipokines with systemic effects on insulin sensitivity and fuel partitioning in muscles and other tissues. Its beneficial role is lost in obesity when excessive accumulation of WAT contributes to severe diseases, such as diabetes. POPs may crossroad or modulate the effect of endogenous ligands of nuclear transcription factors, participating in differentiation, metabolism and the secretory function of adipocytes. These mechanisms include, most importantly: i) endocrine disrupting potency of POPs mixtures on androgen, estrogen or thyroid hormone metabolism/functions in WAT, ii) interference of dioxin-like chemicals with retinoic acid homeostasis, where impact on retinoid receptors is expected, and iii) interaction with transcriptional activity of peroxisome proliferator-activated receptors is likely. Thus, the accumulation and action of POPs in WAT represents a unitary mechanism explaining, at least in part, the effects of POPs in the whole organism. By modulating WAT differentiation, metabolism and function, the POPs could affect not only the physiological role of WAT, but they may also influence the development of obesity-associated diseases.Physiological research / Academia Scientiarum Bohemoslovaca 02/2007; 56(4):375-81. · 1.53 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The polychlorinated biphenyls (PCBs) are synthetic organochlorine chemicals that were useful industrial products in the past, but their production was ended because they persist in both the environment and living organisms. The PCBs are mixtures of up to 209 different components (congeners), depending on the number and position of chlorines around the biphenyl ring. The PCBs are fat-soluble substances to which everyone is exposed through ingesting animal fats, inhalation, or dermal contact. Exposure to PCBs suppresses the immune system, thereby increasing the risk of acquiring several human diseases. Both ortho-substituted and coplanar (dioxin-like) congeners are tumor promoters that enhance the effects of other carcinogenic substances. PCB exposure, especially during fetal and early life, reduces IQ and alters behavior. The PCBs alter thyroid and reproductive function in both males and females and increase the risk of developing cardiovascular and liver disease and diabetes. Women are at high risk of giving birth to infants of low birth weight, who are at high lifetime risk for several diseases. As knowledge of their toxic effects has grown faster than environmental levels have declined, PCBs remain dangerous contaminants.Reviews on environmental health 21(1):1-23.
- [show abstract] [hide abstract]
ABSTRACT: Persistent organic pollutants (POPs) are endocrine-disrupting chemicals associated with the development of the metabolic syndrome and type 2 diabetes. In humans, little is known about their role in the potential origin of obesity. This study aims to assess the associations between serum levels of POPs and the prevalence of obesity in a cohort of obese and lean adult men and women. POP serum samples were investigated cross-sectionally in 98 obese and 47 lean participants, aged ≥18 years. Serum samples were analyzed for the presence of polychlorinated biphenyl (PCB) congeners 153, 138, 180, and 170 and for the organochlorine pesticides, dichloro-diphenyl-dichloroethylene (pp-DDE), and β-hexachlorocyclohexane (βHCH). We established a significant negative correlation between BMI, waist, fat mass percentage, total and subcutaneous abdominal adipose tissue, and serum levels of PCB 153, 180, 170, and the sumPCBs. For βHCH, we demonstrated a positive correlation with BMI, waist, fat mass percentage, and total and subcutaneous abdominal adipose tissue. PCBs 180, 170, and the sum of PCBs correlated significantly negative with homeostasis model assessment for insulin resistance (HOMA(IR)). βHCH correlated significantly positively with HOMA(IR). A strong correlation was established between all POP serum levels and age. We established a positive relationship between high serum levels of βHCH and BMI and HOMA(IR), whereas serum PCB levels were inversely correlated with BMI and HOMA(IR). Combined, these results suggest that the diabetogenic effect of low-dose exposure to POPs might be more complicated than a simple obesogenic effect.Obesity 04/2011; 19(4):709-14. · 3.92 Impact Factor
Accumulation of Polychlorinated Biphenyls in
Adipocytes: Selective Targeting to Lipid Droplets and
Role of Caveolin-1
Sophie Bourez1, Soazig Le Lay2, Carine Van den Daelen1, Caroline Louis1, Yvan Larondelle1, Jean-Pierre
Thome ´3, Yves-Jacques Schneider1, Isabelle Dugail2, Cathy Debier1*
1Institut des Sciences de la Vie, UCLouvain, Louvain-la-Neuve, Belgium, 2Universite ´ Pierre et Marie Curie – Paris 6, UMR S 872, Paris, France, 3Laboratoire d’Ecologie
animale et d’Ecotoxicologie, Universite ´ de Lie `ge, Lie `ge, Belgium
Background: Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that preferentially accumulate in
lipid-rich tissues of contaminated organisms. Although the adipose tissue constitutes a major intern reservoir of PCBs and
recent epidemiological studies associate PCBs to the development of obesity and its related disorders, little is known about
the mechanisms involved in their uptake by the adipose tissue and their intracellular localization in fat cells.
Methodology/Principal Findings: We have examined the intracellular distribution of PCBs in mouse cultured adipocytes
and tested the potential involvement of caveolin-1, an abundant adipocyte membrane protein, in the uptake of these
compounds by fat cells. We show that 2,4,49-trichlorobiphenyl (PCB-28), 2,39,4,49,5-pentachlorobiphenyl (PCB-118) and
2,29,4,49,5,59-hexachlorobiphenyl (PCB-153) congeners rapidly and extensively accumulate in 3T3-L1 or mouse embryonic
fibroblast (MEF) derived cultured adipocytes. The dynamics of accumulation differed between the 3 congeners tested. By
subcellular fractionation of primary adipocytes, we demonstrate that these pollutants were almost exclusively recovered
within the lipid droplet fraction and practically not associated to cell membranes. The absence of caveolin-1 expression in
primary adipocytes from cav-1 deficient mice did not modify lipid droplet selective targeting of PCBs. In cav-1 KO MEF
differentiated adipocytes, PCB accumulation was decreased, which correlated with reduced cell triglyceride content.
Conversely, adenoviral mediated cav-1 overexpressing in 3T3-L1 cells, which had no impact on total cell lipid content, did
not change PCB accumulation.
Conclusion/Significance: Our data indicate that caveolin-1 per se is not required for selective PCB accumulation, but rather
point out a primary dependence on adipocyte triglyceride content. If the crucial role of lipid droplets in energy homeostasis
is considered, the almost exclusive accumulation of PCBs in these organelles warrants future attention as the impairment of
their function could be linked to the worldwide obesity epidemic.
Citation: Bourez S, Le Lay S, Van den Daelen C, Louis C, Larondelle Y, et al. (2012) Accumulation of Polychlorinated Biphenyls in Adipocytes: Selective Targeting to
Lipid Droplets and Role of Caveolin-1. PLoS ONE 7(2): e31834. doi:10.1371/journal.pone.0031834
Editor: Partha Mukhopadhyay, National Institutes of Health, United States of America
Received October 24, 2011; Accepted January 12, 2012; Published February 20, 2012
Copyright: ? 2012 Bourez 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: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Polychlorinated biphenyls (PCBs) are persistent environmental
pollutants that are found at elevated concentrations in the adipose
tissue of contaminated organisms . These organic contaminants
have shown numerous harmful effects on animal and human health
[2,3]. Moreover, there is increasing information in the literature
correlating the presence of PCBs in contaminated organisms and
the development of obesity related disorders such as type II diabetes
and cardiovascular diseases [4–6], and suggesting a putative role for
these chemicals in the fundamental mechanisms controlling the
adipose tissue metabolism and obesity pandemy. A recent study
demonstrated increased persistent organic pollutant (POP) total
body burden in obese subjects and increased POP serum
concentrations after bariatric surgery leading to drastic weight loss
. Conversely, how these compounds interfere with metabolic
regulation remains poorly understood [4,7,8].
It is however clearly established that adipose tissues are
preferential sites for PCB accumulation [1,9]. Due to their
lipophilic properties, at least two preferential cellular compart-
ments are likely candidates for their accumulation: polar lipids of
biological membranes or the lipid droplet, which contains the most
important part of adipocyte triglyceride stores.
Although very little is known about mechanisms that lead to the
preferential targeting of PCBs towards adipose tissues, some recent
experimental data might suggest a role for caveolae in this process.
Caveolae are 50–100 nm invaginations in the plasma membrane
that are particularly abundant in endothelial cells, muscle cells and
adipocytes . The caveolar membrane is invaginated by a
major coat constituent, caveolin, and its composition is closely
related to that of lipid rafts, enriched in cholesterol and
sphingolipids. The caveolin family comprises three related
members of intramembrane proteins, caveolin-1, -2 and -3, which
mostly differ in their tissue distribution. Caveolin-1 and -2 are
PLoS ONE | www.plosone.org1February 2012 | Volume 7 | Issue 2 | e31834
coexpressed and are especially abundant in the adipose tissue and
vascular endothelium, whereas caveolin-3 is muscle restricted .
Several lines of evidence indicate that caveolae/caveolins might
be critical adipose cell components leading to an intracellular
accumulation of PCBs. Firstly, caveolae formation was shown to
be induced by some PCB congeners, suggesting that caveolae
could be involved in the uptake of toxic, lipophilic xenobiotics by
endothelial cells [12,13]. Secondly, specific receptors for albumin
and certain lipoproteins (high-density lipoproteins and oxidized
low-density lipoproteins), which participate to PCB transport in
plasma [14,15], are localized in caveolae . Thirdly, endothelial
cell exposure to 3,39,4,49-tetrachlorobiphenyl (PCB-77), induced
specific accumulation of this congener in the caveolae-rich fraction
of cellular membranes . Fourthly, fat cells, in which PCBs
preferentially accumulate in vivo, contain numerous caveolae
[10,17]. Fifthly, caveolin-1 is dually distributed between the cell
surface and the lipid droplet organelle in adipocytes, and
exogenous lipid addition to cultured adipocytes directs caveolin-
1 to the lipid droplet pool, suggesting a role for lipophilic
molecules in the stimulation of caveolar endocytosis [18–20].
Thus, caveolin trafficking between the plasma membrane and lipid
droplet pools  might regulate PCB intracellular distribution.
In the present study, we investigated the intracellular localiza-
tion of 3 PCB-congeners (PCB-28, -118, -153) in the adipose tissue
and the possible involvement of the caveolae and caveolin-1 in
their entry in fat cells. We used diverse adipocyte cell culture
systems to evaluate the dynamics of PCB uptake by adipocytes in
vitro. Our results show a massive accumulation of these pollutants
in fat cells and their almost exclusive association with the lipid
droplet compartment. The dynamics of entry however differed
between the 3 congeners investigated. In a second step, we
modulated the expression of caveolin-1 to examine the role of this
protein in both the uptake and the targeting of PCBs to lipid
droplets. Our data identify the adipocyte neutral lipids as major
determinants of PCB accumulation in fat cells, a process that does
not require caveolin-1.
Materials and Methods
Caveolin-1 KO mice and their wildtype littermates, maintained in
the animal facility, were described previously . All animal
experiments were approved by local authorities in accordance with
the criteria outlined by the French veterinary guidelines. The study
was approved by the Regional Ethics Committee for Animal
Cav-1 KO/WT Mouse Embryonic Fibroblasts (MEFs) were
obtained and cultured as described previously . To achieve
overexpression of caveolin-1 in adipocytes, fully differentiated
3T3-L1 cells (kind gift of Dr J. Pairault, Paris, FR), were infected
with an adenovirus encoding caveolin-1 (cav-1 3T3-L1) or the
Green Fluorescent Protein (GFP) as a control. The latter was also
used to monitor transfection efficiency. 3T3-L1 cells were
maintained and cultured into differentiated adipocytes as de-
scribed in  under 5% CO2atmosphere 237uC during 12 days.
Isolation of primary adipocytes
Cav-1 KO and WT mature adipocytes were isolated by
collagenase digestion of adipose tissue coming from cav-1 KO
and WT mice respectively, according to Rodbell . Briefly, the
excised fat pads were finely minced into small pieces and digested
with collagenase (0.15 U/ml) (Roche, Paris, FR) for 1 h at 37uC
under gentle shaking. The cell suspension was then filtered through
a 200 mm nylon mesh. Floating cells were collected and gently
rinsed twice with Dulbecco’s modified Eagle’s medium (DMEM) at
37uC and resuspended in the same medium for experiments.
Once differentiated into adipocytes, cav-1/control 3T3-L1 and
WT/cav-1 KO MEF adipocytes were incubated for 30 min,
90 min, 4 h and 8 h at 37uC with a cocktail of 3 PCB congeners:
phenyl (PCB-118) and 2,29,4,49,5,59-hexachlorobiphenyl (PCB-
153) (Dr Ehrenstorfer, Augsburg, DE). In other experiments,
differentiated adipocytes were incubated for 24 hours with the
same PCB cocktail and then submitted to a second dose for an
additional 8 hours of incubation. In all cultured adipocyte
experiments, PCBs were added to the medium as an ethanolic
solution at a final concentration of 500 nM for each congener,
which is in the range of concentrations found in humans in vivo as it
lies between the levels quantified in human serum after chronic or
acute exposures to PCBs [22,23]. Control cells received the
ethanol vehicle alone that did not represent more than 0.5% (v:v).
Primary adipocytes isolated from cav-1 KO and WT mice were
incubated with the same cocktail of PCBs for 2 h at 37uC under
gentle shaking. The lactate dehydrogenase assay (Roche), was used
to verify that PCB treatments were not cytotoxic (data not shown).
Isolation of lipid droplets
After incubation of primary adipocytes with PCBs, lipid droplets
were isolated as described previously . Briefly, adipocytes were
washedtwice with PBSand resuspended in3 mlofdisruptionbuffer
(25 mM Tris-HL, 100 mM KCl, 1 mM ethylenediaminetetraacetic
acid (EDTA), 5 ml ethyleneglycoltetraacetic acid (EGTA); pH 7.4,
further supplemented with a ‘Complete’ protease inhibitor cocktail-
one tablet per 25 ml, Roche). Cells were disrupted by nitrogen
cavitationat 800 psi for 10 min at 4uC. The lysatewascollected and
mixed with an equal volume of disruption buffer containing 1.08 M
sucrose.Itwasthen sequentially overlaidwith 2 mleachof270 mM
sucrose buffer, 135 mM sucrose buffer and Tris/EDTA/EGTA
buffer (25 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA; pH 7.4).
Following centrifugation at 150 000 g for 60 min, 7 fractions were
collected from the top of the gradient.
Total cell lysates and immunoblotting
Differentiated cav-1/control 3T3-L1 and WT/cav-1 KO MEFs
as well as primary adipocytes were lysed as described previously
. Cell lysates were then subjected to SDS-PAGE on 12% (w:v)
polyacrylamide gels and transferred onto nitrocellulose membranes
(Amersham Biosciences, Munich, DE), blocked for 2 h at room
temperature in 5% (w:v) skimmed milk/Tris buffered saline (TBS,
150 mM NaCl, 50 mM Tris-HCl; pH 7.4) supplemented with
0.1% (v:v) Tween 20 and probed with primary antibodies against
caveolin-1. Nitrocellulose membranes were washed three times in
TBS/0.1% (v:v) Tween-20 for 5 min prior to incubation with
secondary peroxidase conjugated antibodies. Protein signals were
visualized using enhanced chemiluminescence (Pierce-Perbio bio-
technology, Rockford, IL) by exposure to a Kodak X-Omat film.
Determination of triglyceride concentration
Cells were collected in 300 ml (for MEFs) or 450 ml (for 3T3-L1
adipocytes) of lysis buffer (35 mM sodium dodecyl sulfate, 10 mM
EDTA, 60 mM Tris buffer; pH=7.2). Aliquots of 200 ml were
transferred into microtubes followed by the addition of 200 ml of
0.1 M KOH in methanol (Labscan, Gliwice, PL) in a 70uC water
PCB Intracellular Targeting in Adipocytes
PLoS ONE | www.plosone.org2 February 2012 | Volume 7 | Issue 2 | e31834
bath with gentle shaking for 1 h. Samples were thoroughly vortexed
for 10 sec every 20 min. After saponification, the samples were
centrifuged at 17 000 g for 10 min. Total released glycerol was then
measured by using a sensitive clinical kit according to the
manufacturer’s recommendations (Free glycerol FS, DiaSys, Holz-
heim, DE). Glycerol being alsopresent in the backbone of membrane
each cell condition. The quantity of glycerol corresponding to the
phospholipids was then substracted from the total amount quantified
by the glycerol enzymatic kit after saponification.
Determination of protein concentration
Cells were collected as described for lipid analyzes. Protein
concentration was determined using the Bicinchoninic Acid
Protein Assay kit (Sigma-Aldrich, St-Louis, MO), with a bovine
serum albumin (BSA) calibration curve.
Determination of PCB concentrations
Five milliliters of n-hexane (Burdick & Jackson Brand) were
added to 4 ml of media or to 300 ml (for MEFs)/450 ml (for 3T3-
L1 adipocytes) of cell suspension containing PCBs in an EPA vial
(Alltech, Lokeren, BE) for liquid-liquid extraction of the PCBs.
After a thorough 5 min shaking, the samples were stored at 4uC
until further analysis. All prepared samples (media and cells) were
then purified and analyzed as described in Debier et al. .
Quantification was performed by comparison with external
standards of the analyzed components in a certified calibration
mixture (Ultra Scientific and Dr Ehrenstorfer), using a linear
calibration curve for each PCB congener (concentration ranging
from 1 to 150 pg/ml). The PCB recovery was calculated on the
basis of the concentration of the surrogate standard (IUPAC 112,
Dr Ehrenstorfer) (50 pg/ml), which was added to the samples at the
beginning of the clean-ups.
The statistical analysis of the data was performed by SAS
Institute Inc. Software. Comparisons between cell groups were
made by two- and one-way ANOVA. Data were considered
statistically significant at p-values,0.05.
Kinetics of PCB accumulation and intracellular
distribution in adipocytes
Differentiated 3T3-L1 adipocytes, an extensively studied cell
model for adipocytes, were used to assess time-dependent intracel-
lular accumulation of PCBs in response to an ethanolic cocktail of
PCBs-28, -118 and -153, each congener at 500 nM. All congeners
were rapidly stored in adipocytes since 45 to 65% of the initially
added compounds were already recovered inside the cells after
90 min of incubation. After 24 h, 85% of PCB-28 as well as 98% of
PCBs-118 and -153 accumulated in fat cells (Fig. 1A). In all these
experiments, media and cells were systematically quantified for
residual and accumulated PCBs, respectively, and showed recoveries
between 90 and 110%. As a consequence, very little proportion of
the added PCB molecules remained in the medium after 24 h,
Figure 1. Efficient and differential accumulation patterns of PCBs -28, -118 and -153 in cultured adipocytes. A. 3T3-L1 adipocytes were
exposed for 24 h to a cocktail of PCBs-28, -118 and -153 (each congener at 500 nM). Cells were collected and quantified for PCB accumulation. Results
are expressed as the % of accumulated PCBs in cells as compared to the amounts initially added in the medium. Adipocytes stored nearly the entire
dose of PCBs within 24 h. B. After a first 24-hour incubation as described in A, 3T3-L1 adipocytes were incubated with a second dose of PCBs-28, 118
and 153 added at a final concentration of 500 nM. Cells were collected and quantified for PCB accumulation at each indicated time of incubation.
Results are expressed as pmoles of accumulated PCBs in cells. Although already contaminated by the first dose, adipocytes again stored up to 80% of
the second amount added in the medium. 3T3-L1 adipocytes (C) and differentiated adipocytes derived from mouse embryonic fibroblasts (MEFs)
obtained from WT mice (D) were independently incubated during 4 h with PCBs-28, -118 and -153 added at a final concentration of 500 nM, and
quantified for accumulated PCBs. Results are expressed as the % of accumulated PCBs in cells as compared to the amounts initially added in the
medium. Each PCB-congener entered the cells with its proper accumulation profile, governed by its own physico-chemical properties.
PCB Intracellular Targeting in Adipocytes
PLoS ONE | www.plosone.org3 February 2012 | Volume 7 | Issue 2 | e31834
illustrating the very high propensity of adipocytes to take up these
compounds and act like sinks to soak them from extracellular space.
In order to challenge adipocyte capacity to buffer PCBs, a second
dose of the PCB cocktail was added to cells that had been previously
exposed for 24 h. Within the following 8 h, up to 80% of each PCB
congener present in the second dose had accumulated in the
adipocytes (Fig. 1B), indicating an important capacity for PCBs
accumulation in cultured fat cells.
Since we observed rapid PCB uptake in 3T3-L1 adipocytes, we
a 4-hour period. PCB-28 accumulated more rapidly in the cells
followed by PCB-118 and then PCB-153 (Fig. 1C). MEF derived
adipocytes (Fig. 1D) were also tested for PCB accumulation.
Interestingly, they accumulated PCBs less extensively than 3T3-L1
cells, since only 30% of PCBs-28 and -118 and 10% of PCB-153
were quantified in cells after 4 h against approximately 70% of each
congener in 3T3-L1 adipocytes. Even more obvious differences in
accumulation profiles of each congener were observed in MEF
derived adipocytes when compared to 3T3-L1s, confirming a
marked slower rate of accumulation of PCB-153 in these cells. It is
well known that MEFs are less prone to adipocyte differentiation
than 3T3-L1 cells, and display lower lipid accretion upon
differentiation in culture (triglycerides expressed per unit of total
cell proteins were respectively 2461 ng/mg and 4463 ng/mg).
Considering that MEFs also concentrate less PCBs than 3T3-L1,
this might indicate that PCB accumulation by adipocytes is
dependent on triglyceride content. Indeed, when the total amounts
of accumulated PCBs after 4 hours were expressed per unit of
triglycerides in both MEF derived adipocytes and differentiated
3T3-L1 cells, levels were comparable between both cell conditions,
respectively 4366 ng/mg and 3861 ng/mg (P.0,05), pointing out
a possible correlation between the accumulation of PCBs and
triglyceride levels in cells. This suggests that these compounds
accumulate within the lipid droplet organelle, principally composed
ofa core ofesterified lipid,mainlytriglyceridesandcholesterylesters
 rather than in membranes or other cellular compartments.
Intracellular PCB distribution was then investigated in mature
primary adipocytes that contain large unilocular lipid droplets that
can be easily purified by subcellular fractionation on sucrose
gradients, as described in Blouin et al . For each congener, up
to 98% of total intracellular PCBs were found to be localized in the
lipid droplet fractions (fraction 1 and to a much lesser extent
fraction 2) (Fig. 2A). No PCBs were found associated with fractions
3 to 6 but 1,5% of PCB-153 was recovered in fraction 7,
corresponding to total membranes. Western blot analysis of all
fractions (Fig. 2B) confirmed that the lipid droplet marker perilipin
was recovered on the top of the gradient. Results show that
caveolin-1 was essentially associated with the lipid droplet fraction
(fraction 1) and with the membranes (fraction 7).
Role of caveolin-1 in adipocyte accumulation and lipid
droplet targeting of PCBs
We tried to establish whether adipocytes devoid of caveolin
expression exhibit altered PCB uptake and/or intracellular
Figure 2. Selective PCB accumulation in lipid droplets of isolated primary adipocytes. Primary adipocytes from cav-1 KO and WT mice
were isolated by collagenase treatment and incubated during 2 h with a cocktail of PCB-s 28, -118 and -153 added at a concentration of 30 mM. Lipid
droplets were then isolated by sucrose gradient centrifugation. A. Quantification of PCBs in all collected fractions (1–7) derived from WT mice. Results
are expressed in nmoles of PCBs per fraction and show an almost exclusive association of PCBs with the first fraction corresponding to the lipid
droplets. B. Western blot analysis of all fractions for caveolin-1 and perilipin indicates the presence of caveolin-1 in both the first (lipid droplets) and
last (membranes) fractions. C. Association of PCBs with the lipid droplets of primary adipocytes isolated from cav-1 KO mice. Results represent PCB
accumulation in the membrane fraction (black bars) and in the lipid droplet fraction (grey bars). Results are expressed in nmoles of PCBs per fraction.
PCBs almost exclusively accumulate in the lipid droplets, even in cav-1 KO adipocytes. A small portion of PCB-153 (but not PCB-28 or -118) was found
associated to the membranes.
PCB Intracellular Targeting in Adipocytes
PLoS ONE | www.plosone.org4 February 2012 | Volume 7 | Issue 2 | e31834
distribution. We first treated primary adipocytes from epididymal
fat pads derived from cav-1 KO mice for 2 h with cocktail of PCBs
and observed that all three congeners almost exclusively distributed
in the lipid droplets (Fig. 2C), indicating that cav-1 deficiency does
not alter PCB targeting to the lipid storage organelle. In order to
examine whether the kinetics of PCB uptake was dependent on
caveolin-1, WT and cav-1 KO MEF derived adipocytes were
incubated for 8 hours with the PCB cocktail. Western blotting of
differentiated WT and cav-1 KO MEFs confirmed the absence of
caveolin-1 in the knock-out condition (Fig. 3A) and extensive
presence of lipid droplets reflected adipocyte differentiation of the
two cell lines (Fig. 3B). Within the 8 hour-incubation period,
differentiated WT MEFs accumulated approximately twice more
PCBs per unit of cell proteins than their cav-1 KO counterparts
(Fig. 3 C, E, G) suggesting a role for caveolin in PCB uptake by
adipocytes. However, adipocyte caveolin-1 deficiency has been
shown to reduce triglyceride accumulation in adipocytes, causing
lipoatrophy in rodents and humans [27,28]. Accordingly, we
observed reduced triglyceride contents in cav-1 deficient adipocytes
compared to controls (respectively 1161 ng/mg protein and
2461 ng/mg protein) (P,0,0001). Since PCB accumulation
occurred within the adipocyte lipid droplet (Fig. 2), we expressed
PCB accumulation per unit of triglycerides. This led to comparable
PCB uptakes in WT and Cav-1 KO adipocytes (Fig. 3 D, F, H)
(p.0.05), and indicates that reduced PCB accumulation in the
absence of caveolin might be related to its effect on lipid storage
rather than to caveolin-1 presence itself.
To assess a direct role of caveolin-1, independent of triglyceride
accumulation, we overexpressed cav-1 in 3T3-L1 adipocytes by
using an adenovirus. Transient cav-1 overexpression, confirmed
by western blotting (Fig. 4A), did not modify triglyceride levels
(Fig. 4B) (P.0,05) between controls and cav-1 3T3-L1 adipocytes
(5066 ng/mg protein and 4867 ng/mg protein, respectively).
When expressed per unit of total cell proteins or cell triglycerides,
no difference in PCB accumulation was observed upon caveolin
overexpression (Fig. 4 C–H) (p.0,05), clearly demonstrating that
caveolin had no direct effect on PCB uptake by adipocytes.
Although it is well known that PCBs accumulate in lipid-rich
tissues, and thus essentially in the adipose tissue, the mechanisms by
which they enter the fat cell and the final destination of the
pollutants once inside the cells had not yet been addressed. In the
present study, we used several culture models of adipocytes to
tissue. In both 3T3-L1 and MEF derived adipocytes, PCBs were
rapidly and efficiently transferred from the culture medium into the
cells, reaching concentrations (10 to 70 mg/g of lipids) that are
severalorders of magnitude higherthan in human adipose tissue (20
to 1300 ng/g of lipids) [29,30]. This substantial difference is quite
surprising as the PCB concentrations added to the culture medium
chronic or acute exposure. Several factors might explain this
phenomenon. Among others, the PCBs administered to 3T3-L1
and MEF adipocytes remained in close contact with the cell
monolayer during the entire experimental period, without any
circulation or flow in the system. To the contrary, in the in vivo
situation, PCBs present in the serum are continuously transported
through the blood circulation where they are tightly associated with
diverse lipoproteins or with plasma albumin [14,15,31]. In our
experiments, 10% of serum was present in the culture medium of
thecells,meaning that the concentrationsoflipoproteinsoralbumin
were at least 10 times less important than those found in the blood
circulation, probably leading to a lower retention of PCBs in the
extracellular compartment. In addition, in the in vivo situation, these
compounds have to cross the endothelium before being taken up by
the adipose tissue that is itself composed of several cell layers.
Adipocytes in vivo also undergo lipolysis, during periods of negative
energy balance, which induces the mobilization of PCBs from the
cells. Finally, PCBs may also be taken up by other organs in vivo,
such as the liver or the skin.
Interestingly, in both culture systems used here, the dynamics of
accumulation differed quite importantly between the PCB conge-
ners tested. PCB-28, a mono-ortho tri-CB, entered the cells more
rapidly than PCB-118, a mono-ortho penta-CB, followed later on by
PCB-153, a di-ortho hexa-CB. These variations likely result from the
different molecular structures of the congeners, as the number and
the position of the chlorine atoms on the biphenyl structure
determine their physical, chemical and biological properties [3,31].
As far as mechanistic studies are concerned, each PCB congener
having its own physico-chemical properties, their behavior towards
cells should thus be considered independently from other
compounds, even from the same family of pollutants.
An important observation to be drawn from our study is that
although the three PCB congeners accumulated in adipocytes with
different kinetics, all of them had the lipid droplet as the major
final target. This underlines the cell lipid storage organelle as the
selective site for PCB intracellular accumulation. One can imagine
the lipid droplet as a detoxifying organelle in which organic
compounds could be sequestered and kept biologically inactive.
Inversely, our observation of massive and selective accumulation
of PCBs within lipid droplets could also provide a new basis to
understand how these compounds might act to worsen obesity-
related metabolic diseases in contaminated humans. Lipid droplets
have only recently been identified as highly regulated, complex
and dynamic organelles, controlling lipid storage and mobilization
[25,32]. However, the precise cellular interactions between all the
regulators involved in the signaling cascades of lipid storage and
release remain largely unknown to date. In this line, the possibility
that PCBs directly interfere with lipid droplet function, such as
fatty acid mobilization from this organelle, should warrant future
attention. So far, some in vivo and in vitro studies highlight a
disruption of lipid metabolism by PCBs and other common
lipophilic pollutants. Mechanisms involve interference with the
lipolysis pathway, impairment of triglyceride synthesis as well as
enhancement of adipocyte differentiation and increase in expres-
sion levels of diverse enzymes implicated in lipid metabolism or
transcription factors regulating energy homeostasis in fat cells [33–
35]. Taken together, these studies clearly prove that PCBs have an
impact on the mechanisms involved in the regulation of cell energy
homeostasis and could thus significantly contribute to the
development of obesity or obesity-related disorders. The identifi-
cation, in this work, of lipid droplets as the principal site of PCB
concentration in fat cells might thus help to understand the
diversity of the biological effects exerted by this family of POPs
and certainly deserves more consideration in the future.
It is however important to point out that a very small percentage
of PCB-153 (but not PCB-28 nor PCB-118) was localized in the
cell membranes. This finding is consistent and in accordance with
other studies investigating the potential toxic effects of PCBs
through the disruption of cellular membranes in cerebellar granule
cell neurons , skeletal and cardiac muscle cells , rat renal
tubular cells , mouse thymocytes and lipid bilayer vesicles
[39,40]. All these studies independently reported an increase in
cellular membrane fluidity following a treatment with di-ortho
substituted PCBs (either 2,29,5,59-tetrachlorobiphenyl (PCB-52) or
PCB-153), but not with other non-ortho tested PCB congeners.
PCB Intracellular Targeting in Adipocytes
PLoS ONE | www.plosone.org5 February 2012 | Volume 7 | Issue 2 | e31834
They show that di-ortho congeners dissolve in cell membranes,
causing important perturbations to the membrane lipids and
proteins and thereby exerting toxic effects on cells. Tan and co-
workers (2004) even showed membrane impairments at the
mitochondrial and endoplasmic reticulum level in thymocytes
and cerebellar granule cells. In our study, as the amounts of PCB-
153 associated to the membranes are very small as compared to
those found in the lipid droplets, we hypothesize that the pollutants
Figure 3. Accumulation of PCBs 28, 118 and 153 in WT and cav-1 KO MEFs. A. Western blot analysis for caveolin-1 in MEF differentiated into
adipocytes obtained from WT and cav-1 KO mice. Caveolin-1 was not expressed in the knock-out condition. B. Images of WT and cav1-KO MEF
derived adipocytes illustrating extensive lipid droplet accumulation in the two cell lines. Bar, 10 mm. C–H. Differentiated MEFs obtained from WT and
cav-1 KO mice were incubated with PCBs-28, -118 and -153 added at a concentration of 500 nM during 8 h, collected and quantified for accumulated
PCBs. Accumulated PCBs -28, -118 and -153 are respectively expressed per unit of total cell proteins (C, E, G) or per mg of triglycerides (D, F, H).
Although the amounts of accumulated PCBs were significantly different between both cell conditions when expressed per unit of proteins, they
became unsignificant when expressed per unit of triglycerides.
PCB Intracellular Targeting in Adipocytes
PLoS ONE | www.plosone.org6 February 2012 | Volume 7 | Issue 2 | e31834
Figure 4. Accumulation of PCBs-28, -118 and -153 in cav-1 overexpressing 3T3-L1 adipocytes and controls. A. Western blot analysis for
caveolin-1. Differentiated 3T3-L1 adipocytes were infected with an adenovirus encoding for caveolin-1 (Ad Cav-1) or with an adenovirus encoding for
the Green Fluorescent Protein (GFP) (Ctrl). Western blot confirms a higher expression of caveolin-1 in the transfected cells as compared to controls. B.
Cell triglyceride content was quantified in Ad Cav-1 and Ctrl adipocytes. Results are expressed as ng of triglycerides per mg of total cellular proteins
and show similar accumulation levels in both cell conditions. C–H. Cav-1 or control adipocytes were incubated with PCBs-28, -118 and -153 added at a
concentration of 500 nM during 8 h, collected and quantified for accumulated PCBs. Results represent the % of accumulated PCBs expressed per unit
of total cell proteins (C, E, G) or per unit of triglycerides (D, F, H). No significant differences were observed in the accumulation levels of PCBs in both
cell conditions, when expressed per unit of proteins or per unit of triglycerides.
PCB Intracellular Targeting in Adipocytes
PLoS ONE | www.plosone.org7 February 2012 | Volume 7 | Issue 2 | e31834
rapidly transferred from the membranes to the lipid droplet
triglyceride pools, which are significant in adipocytes as compared
to other cell types. However, the fact that a small percentage of
PCB-153, but none of the two other congeners, was found
associated to the membrane fraction supports the idea that at least
part of di-ortho-PCB mediated toxicity in the adipose tissue could
occur at the membrane level of the fat cell or even at the
membrane level of the lipid droplets.
The present study also aimed at identifying specific cell
in the massive accumulation of PCBs in adipose tissue. Starting with
caveolin-1 as a candidate, we show here with cell models of genetic
loss and gain of function, that caveolin-1 is neither required for the
entry of PCBs into cells nor for their accumulation within the lipid
droplet organelle. Instead, it appears from our data that the extent of
PCB accumulation primarily depends on adipocyte triglyceride
content. Indeed, we clearly show that 3T3-L1 adipocytes, which are
characterized by higher triglyceride content, accumulated more
PCBs at each time of the incubation as compared to MEFs, although
the added amounts in the medium were the same. This result is
consistent with in vivo observations where total body burdens of PCBs
are higher in obese than in lean individuals [4,7]. If dilution
in the fat cell, the more PCBs it can accumulate.
To conclude, our study reveals a massive but differential
accumulation of PCBs in cultured adipocytes. The uptake of PCBs
was directly linked to the triglyceride content of adipocytes and
did not depend on the presence of caveolin-1. The distinct
accumulation patterns observed for each congener, as well as the
specific association of only PCB-153 to the membranes, points out
how important it is to consider a pollutant individually to describe
its mechanism of action before describing and understanding the
toxicity of mixtures. Eventually, an important conclusion to be
drawn from this study is that almost all PCBs were targeted to the
lipid droplets in fat cells, an observation suggesting that PCB intra-
adipocyte entry and trafficking is tightly linked to lipid metabolism
in the adipose tissue. Given the global rise of obesity and its related
disorders in humans worldwide, the understanding of the precise
impacts of lipophilic pollutants on energy homeostasis in fat cells
should be a matter of focus in the future.
We are very grateful to F. Lasnier, X. Le Liepvre and C. Prado from the
‘‘INSERM U872 team 8’’, Paris, France, for technical assistance in diverse
assays and for the cell cultures. We would also like to acknowledge M.
Louvet from the ‘‘Laboratoire d’Ecologie animale et d’Ecotoxicologie’’,
Universite ´ de Lie `ge, Belgium, for her help in PCB analyzes. We also thank
A. Joly and E. Mignolet from the ‘‘Institut des Sciences de la Vie’’,
UCLouvain, Belgium, for their help in the fatty acid analyses. We also
greatly appreciated the help and advice of Pr. B. Govaerts and C. Rasse
from the ‘‘Support en me ´thodologie et calcul statistique’’, Institut
multidisciplinaire pour la mode ´lisation et l’analyse quantitative, UCLou-
vain, Belgium, in the statistical analyzes of this study.
Conceived and designed the experiments: SB SLL CD ID. Performed the
experiments: SB CVDD SLL CL. Analyzed the data: SB CVDD.
Contributed reagents/materials/analysis tools: YL YJS JPT CD ID. Wrote
the paper: SB. Engaged in active discussions: SB CVDD CD SLL ID.
1. Mullerova D, Kopecky J (2007) White adipose tissue: storage and effector site for
environmental pollutants. Physiol Res 56: 375–381.
2. Ulbrich B, Stahlmann R (2004) Developmental toxicity of polychlorinated
biphenyls (PCBs): a systematic review of experimental data. Arch Toxicol 78:
3. Carpenter DO (2006) Polychlorinated biphenyls (PCBs): routes of exposure and
effects on human health. Rev Environ Health 21: 1–24.
4. Dirinck E, Jorens PG, Covaci A, Geens T, Roosens L, et al. (2011) Obesity and
persistent organic pollutants: possible obesogenic effect of organochlorine
pesticides and polychlorinated biphenyls. Obesity 19: 709–714.
5. Hectors T, Vanparys C, van der Ven K, Martens G, Jorens P, et al. (2011)
Environmental pollutants and type 2 diabetes: a review of mechanisms that can
disrupt beta cell function. Diabetologia 54: 1273–1290.
6. Lee D-H, Steffes MW, Sjodin A, Jones RS, Needham LL, et al. (2011) Low dose
organochlorine pesticides and polychlorinated biphenyls predict obesity,
dyslipidemia, and insulin resistance among people free of diabetes. PLoS One
7. Kim M-J, Marchand P, Henegar C, Antignac J-P, Alili R, et al. (2011) Fate and
complex pathogenic effects of dioxins and polychlorinated biphenyls in obese
subjects beforeand after drastic weight loss. Environ HealthPerspect 119: 377–383.
8. Pelletier C, Imbeault P, Tremblay A (2003) Energy balance and pollution by
organochlorines and polychlorinated biphenyls. Obes Rev 4: 17–24.
9. Yu GW, Laseter J, Mylander C (2011) Persistent organic pollutants in serum and
several different fat compartments in humans. J Environ Public Health 2011:
10. Thorn H, Stenkula KG, Karlsson M, Ortegren U, Nystrom FH, et al. (2003)
Cell surface orifices of caveolae and localization of caveolin to the necks of
caveolae in adipocytes. Mol Biol Cell 14: 3967–3976.
11. Couet J, Belanger MM, Roussel E, Drolet M-C (2001) Cell biology of caveolae
and caveolin. Adv Drug Delivery Rev 49: 223–235.
12. Lim EJ, Smart EJ, Toborek M, Hennig B (2007) The role of caveolin-1 in
PCB77-induced eNOS phosphorylation in human-derived endothelial cells.
Am J Physiol Heart Circ Physiol 293: 3340–3347.
13. Lim EJ, Ma ´jkova ´ Z, Xu S, Bachas L, Arzuaga X, et al. (2008) Coplanar
polychlorinated biphenyl-induced CYP1A1 is regulated through caveolae
signaling in vascular endothelial cells. Chem-Biol Interact 176: 71–78.
14. Becker MM, Gamble W (1982) Determination of the binding of 2,29,4,49,5,59-
hexachlorobiphenyl by low-density lipoprotein and bovine serum albumin.
J Toxicol Environ Health 9: 225–234.
15. Spindler-Vomachka M, Vodicnik MJ, Lech JJ (1984) Transport of 2,4,5,29,49,59-
hexachlorobiphenyl by lipoproteins in vivo. Toxicol Appl Pharmacol 74: 70–77.
16. Uittenbogaard A, Shaul PW, Yuhanna IS, Blair A, Smart EJ (2000) High density
lipoprotein prevents oxidized low density lipoprotein-induced inhibition of
endothelial nitric-oxide synthase localization and activation in caveolae. J Biol
Chem 275: 11278–11283.
17. Pilch PF, Liu L (2011) Fat caves: caveolae, lipid trafficking and lipid metabolism
in adipocytes. Trends Endocrinol Metab 22: 318–324.
18. Pol A, Martin S, Fernandez MA, Ingelmo-Torres M, Ferguson C, et al. (2005)
Cholesterol and fatty acids regulate dynamic caveolin trafficking through the
golgi complex and between the cell surface and lipid bodies. Mol Biol Cell 16:
19. Sharma DK, Brown JC, Choudhury A, Peterson TE, Holicky E, et al. (2004)
Selective stimulation of caveolar endocytosis by glycosphingolipids and
cholesterol. Mol Biol Cell 15: 3114–3122.
20. Le Lay S, Hajduch E, Lindsay MR, Le Lie `pvre X, Thiele C, et al. (2006)
Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: a role for
caveolar endocytosis. Traffic 7: 549–561.
21. Rodbell M (1964) The Metabolism of Isolated Fat Cells- I: effects of hormones
on glucose metabolism and lipolysis. J Biol Chem 239: 375–380.
22. Wassermann M, Wassermann D, Cucos S, Miller HJ (1979) World PCBs map:
storage and effects in man and his bio- logical environment in the 1970s. Ann
NY Acad Sci 320: 69–124.
23. Meeker JD, Maity A, Missmer SA, Williams PL, Mahalingaiah S, et al. (2011)
Serum concentrations of polychlorinated biphenyls in relation to in vitro
fertilization outcomes. Environ Health Perspect 119: 1010–1016.
24. Debier C, Pomeroy PP, Dupont C, Joiris C, Comblin V, et al. (2003)
Quantitative dynamics of PCB transfer from mother to pup during lactation in
UK grey seals Halichoerus grypus. Mar Ecol Prog Ser 247: 237–248.
25. Ducharme NA, Bickel PE (2008) Minireview: Lipid Droplets in Lipogenesis and
Lipolysis. Endocrinology 149: 942–949.
26. Blouin CM, Le Lay S, Eberl A, Ko ¨feler HC, Guerrera IC, et al. (2010) Lipid
droplet analysis in caveolin-deficient adipocytes: alterations in surface phospho-
lipid composition and maturation defects. J Lipid Res 51: 945–956.
27. Briand N, Le Lay S, Sessa WC, Ferre ´ P, Dugail I (2011) Distinct roles of
endothelial and adipocyte caveolin-1 in macrophage infiltration and adipose
tissue metabolic activity. Diabetes 60: 448–453.
28. Kim CA, Dele ´pine M, Boutet E, El Mourabit H, Le Lay S, et al. (2008)
Association of a homozygous nonsense caveolin-1 mutation with berardinelli-
seip congenital lipodystrophy. J Clin Endocrinol Metab 93: 1129–1134.
29. De Saeger S, Sergeant H, Piette M, Bruneel N, Van de Voorde W, et al. (2005)
Monitoring of polychlorinated biphenyls in Belgian human adipose tissue
samples. Chemosphere 58: 953–960.
PCB Intracellular Targeting in Adipocytes
PLoS ONE | www.plosone.org8February 2012 | Volume 7 | Issue 2 | e31834
30. Wang N, Kong D, Cai D, Shi L, Cao Y, et al. (2010) Levels of polychlorinated
biphenyls in human adipose tissue samples from southeast China. Environ Sci
Technol 44: 4334–4340.
31. Matthews HB, Surles JR, Carver JG, Anderson MW (1984) Halogenated
biphenyl transport by blood components. Fundam Appl Toxicol 4: 420–428.
32. Farese RV, Walther TC (2009) Lipid Droplets Finally Get a Little R-E-S-P-E-C-
T. Cell 139: 855–860.
33. Irigaray P, Ogier V, Jacquenet S, Notet V, Sibille P, et al. (2006) Benzo[a]pyrene
impairs b-adrenergic stimulation of adipose tissue lipolysis and causes weight
gain in mice - A novel molecular mechanism of toxicity for a common food
pollutant. FEBS J 273: 1362–1372.
34. Beranek SR, Becker MM, Kling D, Gamble W (1984) Phospholipid and
glyceride biosynthesis in 2, 4, 5, 29, 49, 59-hexachlorobiphenyl-treated human
skin fibroblasts. Environ Res 34: 103–109.
35. Arsenescu V, Arsenescu RI, King V, Swanson H, Cassis LA (2008)
Polychlorinated biphenyl-77 induces adipocyte differentiation and proinflam-
matory adipokines and promotes obesity and atherosclerosis. Environ Health
Perspect 116: 761–768.
36. Kodavanti PR, Shin DS, Tilson HA, Harry GJ (1993) Comparative effects of
two polychlorinated biphenyl congeners on calcium homeostasis in rat cerebellar
granule cells. Toxicol Appl Pharmacol 123: 97–106.
37. Wong PW, Pessah IN (1996) Ortho-substituted polychlorinated biphenyls alter
calcium regulation by a ryanodine receptor-mediated mechanism: structural
specificity toward skeletal- and cardiac-type microsomal calcium release
channels. Mol Pharmacol 49: 740–751.
38. Lopez-Aparicio P, Merino MJ, Sanchez E, Recio MN, Perez-Albarsanz MA
(1997) Effect of aroclor 1248 and two pure PCB congeners upon the membrane
fluidity of rat renal tubular cell cultures. Pestic Biochem Physiol 57: 54–62.
39. Yilmaz B, Sandal S, Chen C-H, Carpenter DO (2006) Effects of PCB 52 and
PCB 77 on cell viability, [Ca2+]i levels and membrane fluidity in mouse
thymocytes. Toxicology 217: 184–193.
40. Tan Y, Chen CH, David L, Carpenter DO (2004) Ortho-substituted PCBs kill
cells by altering membrane structure. Toxicol Sci 80: 54–59.
PCB Intracellular Targeting in Adipocytes
PLoS ONE | www.plosone.org9 February 2012 | Volume 7 | Issue 2 | e31834