Báo cáo khoa học: 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 doc

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
Philippe Irigaray
1,2
, Virginie Ogier
1
, Sandrine Jacquenet
1
,Ve
´
ronique Notet
1
, Pierre Sibille
3
,
Luc Me
´
jean
1,2
, Bernard E. Bihain
1
and Frances T. Yen
1,2
1 JE2482 Lipidomix – Institut National Polytechnique de Lorraine, Vandoeuvre-le
`
s-Nancy, France
2 Laboratoire de Sciences Animales, ENSAIA, Vandoeuvre-le
`
s-Nancy, France

3 Polyclinique de Gentilly, Nancy, France
Obesity is a multifactorial disease that occurs because
of an imbalance between food intake and energy
expenditure. Several molecular mechanisms acting on
either neurological centres controlling eating behaviour
or peripheral systems regulating energy expenditure
have been identified as contributing to this imbalance
[1,2]. The current view of the recent dramatic
increase in the incidence of the obesity implicates the
Keywords
adipocytes; lipolysis; obesity; polycyclic
aromatic hydrocarbons; triglycerides
Correspondence
F.T. Yen, JE2482 Lipidomix, Laboratoire de
Me
´
decine et The
´
rapeutique Mole
´
culaire,
15 rue du Bois de la Champelle,
54500 Vandoeuvre-le
`
s-Nancy, France
Fax: +33 3 83 67 89 99
Tel: +33 3 83 67 89 98
E-mail:
inserm.fr
(Received 26 October 2005, revised 24

January 2006, accepted 31 January 2006)
doi:10.1111/j.1742-4658.2006.05159.x
Benzo[a]pyrene (B[a]P) is a common food pollutant that causes DNA
adduct formation and is carcinogenic. The report of a positive correlation
between human plasma B[a]P levels and body mass index, together with
B[a]P’s lipophilicity, led us to test for possible adverse effects of B[a]P on
adipose tissue. In ex vivo experiments using primary murine adipocytes,
B[a]P rapidly (within minutes) and directly inhibited epinephrine-induced
lipolysis (up to 75%) in a dose-dependent manner. Half-maximum inhibi-
tion was obtained with a B[a]P concentration of 0.9 mgÆL
)1
(3.5 lm). Lipo-
lysis induced by b
1
-, b
2
- and b
3
-adrenoreceptor-specific agonists, as well as
ACTH, were also significantly inhibited by B[a]P, whereas forskolin-
induced lipolysis was not B[a]P-sensitive. Similar inhibition of catecholam-
ine-induced lipolysis by B[a]P was also seen in isolated human adipocytes;
half-maximum inhibition of lipolysis was achieved with a B[a]P concentra-
tion of 0.02 mgÆL
)1
(0.08 lm). In vivo treatment of C57Bl ⁄ 6J mice with
0.4 mgÆkg
)1
B[a]P inhibited epinephrine-induced release of free fatty acids
by 70%. Chronic exposure of mice to B[a]P (0.5 mgÆkg

)1
injected i.p. every
48 h) for 15 days also decreased lipolytic response to epinephrine and
induced a 43% higher weight gain compared with controls (B[a]P:
2.23 ± 0.12 g versus control: 1.56 ± 0.18 g, P < 0.01) due to increased
fat mass. The weight gain occurred consistently without detectable changes
in food intake. These results reveal a novel molecular mechanism of
toxicity for the environmental pollutant B[a]P and introduce the notion
that chronic exposure of human population to B[a]P and possibly other
polycyclic aromatic hydrocarbons could have an impact on metabolic dis-
orders, such as obesity.
Abbreviations
ANP, atrial natriuretic peptide; B[a]P, benzo[a]pyrene; BMI, body mass index; BSA, bovine serum albumin; FFA, free fatty acids; HSL,
hormone sensitive lipase; KRBB, Krebs Ringer bicarbonate buffer; PAH, polycyclic aromatic hydrocarbons.
1362 FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS
conjunction of increased consumption of energy-rich
food with decreased physical activity and common
genetic variants [3].
Obesity results from a large increase in fat mass or
adipose tissue, which serves as the storage site for free
fatty acids (FFA) in the form of triglycerides. This
lipophilic tissue can also serve as a reservoir for lipo-
soluble pollutants such as organochlorines [4] and
polycyclic aromatic hydrocarbons [5,6]. The potential
impact of the accumulation of toxins in adipose tissue
was recently demonstrated by the observed increase in
plasma levels of organochlorines released from adipose
tissue during weight loss [7–9]. The amount of pollut-
ant released was positively associated with a lower rel-
ative metabolic rate as well as decreased oxidative

capacity in muscle. We also reported that mobilization
of FFA from adipocytes leads to the release of dioxins
stored in adipose tissue [10]. An environmental study
in 1983 reported that higher plasma benzo[a]pyrene
(B[a]P) concentrations were associated with a higher
body mass index (BMI) in human subjects living in the
New York area [11]. These studies unambiguously
established that, because of their lipophilicity, environ-
mental pollutants accumulate in the fat of organisms
and their concentrations increase up the food chain, a
phenomenon called ‘bioaccumulation’ [4]. It is also
clear that body weight reduction leads to increased
plasma concentrations of potentially toxic compounds
that can affect different targets, e.g. thyroid hormone
metabolism [8]. It is currently unknown whether envi-
ronmental pollutants exert a toxic effect on the adipo-
cyte itself. This led us to question whether the
pollutant had a negative impact on one of the key
functions of the adipocyte, i.e. its capacity to release
stored FFA.
Most attention has thus far been paid to organo-
chlorines, which are endocrine disrupters and enzyme
inducers and are associated with breast cancer [12] and
impairment of thyroid function [13,14]. Because of
this, organochlorine insecticides are prohibited in
North America and Europe, but still are in use in
developing countries. Therefore, residues of these com-
pounds are still found in all organisms on the planet.
Persistent organic pollutants with strong lipophilic
properties are not limited to organochlorines and ⁄ or

pesticides, but also include polycyclic aromatic hydro-
carbons (PAH) that are by-products of industrial
activity. Although protocols intended to reduce the
emission rate of persistent organic pollutants are in
place, the emission rate of PAH remains elevated.
B[a]P is a widely studied representative of PAH, ori-
ginating from incomplete combustion or pyrolysis of
organic matter. Under normal conditions, the diet is
the main source of B[a]P exposure [15]. B[a]P contam-
ination of food results from either specific food pro-
cessing, e.g. open flame cooking, or contamination of
food by B[a]P released into the environment. For exam-
ple, the B[a]P content of fast-food hamburgers reaches
levels of 200 ng per serving [15]. In Asia, very high lev-
els of B[a]P are produced in cooking oil (> 400 ngÆg
)1
)
[16]. B[a]P is also a well-known carcinogen and con-
sumption of B[a]P-rich foods contributes to the overall
cancer burden affecting human populations [15]. Upon
ingestion, B[a]P is rapidly absorbed by the intestine
and, because of its highly lipophilic nature, is transpor-
ted in the plasma via the lipoprotein system [5,6].
Tissue-distribution studies have shown that B[a]P accu-
mulates in lipid-storing tissues including the mammary
glands and adipose tissue. The carcinogenicity of B[a]P
has been well-documented. This pollutant is metabo-
lized via the cytochrome P450 system into reactive
dihydrodiol epoxide derivatives (e.g. B[a]P-7,8-
dihydrodiol-9,10-epoxide or BPDE). These metabolites

bind covalently to DNA resulting in the formation of
adducts, which leads to mutations, uncontrolled cell
growth and consequently tumour formation in var-
ious tissues (lung adenocarcinoma, lymphoreticular
tumours, hepatomas, mammary adenocarcinomas) [17].
B[a]P has also been shown to display immunotoxic
properties that affect macrophage function [18] and
increase local inflammatory response leading to
increased atherosclerotic lesion size [19]. However, little
has been documented on the effect of B[a]P on adipo-
cyte function, one of the main sites of storage. We
therefore decided to address the question of B[a]P tox-
icity on adipocytes, and specifically to test its effect on
the capacity of adipocytes to release FFA from stored
triglycerides.
It was initially thought that most degradation of
adipose tissue triglycerides into FFA that can be liber-
ated was mediated by hormone sensitive lipase (HSL).
However, recent studies using mice with inactivation
of the HSL gene suggest that another adipose triglycer-
ides lipase also participates in this process [20]. Activa-
tion of the lipolytic cascade is under tight hormonal
regulation [21]. The most potent lipolytic hormones
are the catecholamines, which act via b-adrenergic
receptors. In humans, the atrial natriuretic peptides
have been shown to exert potent lipolytic effects [22].
FFA release is inhibited by a
2
-adrenergic and insulin
receptors. The mechanisms of adrenergic receptor sig-

nalling proceed via stimulatory and inhibitory G-pro-
teins that control adenylate cyclase activity and thus
cAMP formation. Insulin signalling is mediated by
type IIIB phosphodiesterase that inactivates cAMP by
its conversion to 5¢AMP [23,24]. cAMP levels regulate
P. Irigaray et al. Benzo[a]pyrene inhibition of lipolysis
FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS 1363
the phosphorylation of cAMP-dependent protein kin-
ase A, which in its phosphorylated form activates the
HSL that hydrolyses triglycerides into FFA.
We report that acute B[a]P treatment profoundly
impaired catecholamine-induced lipolysis in both
murine and human adipocytes. Furthermore, a signifi-
cant weight gain, as well as increased fat mass, was
observed in mice treated 15 days with B[a]P.
Results
Primary adipocytes freshly isolated from murine white
adipose tissue were incubated with epinephrine and
increasing concentrations of B[a]P, followed by meas-
urement of the FFA released in the media. A signifi-
cant inhibitory effect on epinephrine-induced FFA
release (P<0.01) was achieved with B[a]P concentra-
tions of 1 mgÆL
)1
(Fig. 1A). The estimated K
d
value
for B[a]P inhibition of lipolysis was 0.9 mgÆL
)1
(3.5 lm), i.e. in the same range as that measured

for the known b blocker atenolol (K
d
¼ 0.4 mgÆL
)1
or
1.5 lm for b
1
-adrenoreceptor and 2.3 mgÆL
)1
or
8.6 lm for b
2
-adrenoreceptor) [25]. The inhibitory
effect of 1.8 mgÆL
)1
B[a]P on epinephrine-induced lipo-
lysis was detectable within 5 min (Fig. 1B), suggesting
that B[a]P directly affected the cellular signal trans-
duction pathway, rather than gene expression at the
transcriptional or translational levels. Stimulation of
adipocyte lipolysis by low doses of norepinephrine
exerted a significant inhibition on lipoysis (Fig. 1C).
The initial step involved in the epinephrine ⁄ nor-
epinephrine-induced lipolytic cascade includes activa-
tion of the b-adrenergic receptor system. We thus
determined the effect of B[a]P on lipolysis induced by
0 5 10 15 20 25
B[a]P (mg/L)
2
1.6

1.2
0.8
0.4
0
Epinephrine-induced FFA release
(m
M
/mg prot)
0
51015
20
25
B[a]P (mg/L)
2
1.6
1.2
0.8
0.4
0
Norepinephrine-induced FFA release
(m
M
/mg prot)
A
FFA release
(m
M
/mg prot)
0
0.5

1.5
2.5
3.5
4.5
epinephrine
dobutamine
salbutamol
BRL 37344
forskolin
*
*
***
ns
D
4
3
2
1
0
0246810
ACTH (n
M
)
ACTH-induced FFA release
(m
M
/mg prot)
E
0
5

10 15 20
25
30
Incubation time in the presence of B[a]P (min)
2
1.6
1.2
0.8
0.4
0
Epinephrine-induced FFA release
(m
M
/mg prot)
BC
Fig. 1. Effect of B[a]P on the release of FFA from mice adipocytes. Freshly isolated mice adipocytes were incubated (A) for 45 min with the
indicated concentrations of B[a]P or (B) for the indicated times with 2.5 mgÆL
)1
(10 lM)B[a]P. After this, epinephrine (1.8 mgÆL
)1
or 10 lM)
was added and the incubation continued for 45 min. Each point represents the mean ± SEM (n ¼ 4) in which FFA levels were measured in
duplicate. (C) Freshly isolated mice adipocytes were incubated for 15 min with the indicated concentrations of B[a]P, after which norepineph-
rine (1.7 mgÆL
)1
or 10 lM) was added and the incubation continued for 45 min. Each point represents the mean ± SEM (n ¼ 6) in which
FFA levels were measured in duplicate. (D) Effect of 15 min preincubation of isolated adipocytes without (h) or with 12.6 mg L
)1
(50 lM)
B[a]P (n) followed by 45 min incubation with the indicated pharmacological agent: epinephrine (1.8 mgÆL

)1
or 10 lM), dobutamine (8.4 mgÆL
)1
or 25 lM), salbutamol (23.9 mgÆL
)1
or 100 lM), BRL 37344 (9.6 mgÆL
)1
or 25 lM) and forskolin (41 mgÆ L
)1
or 100 lM). Significance is noted
as the following: *P<0.01, **P<0.03. (E) Isolated adipocytes preincubated for 15 min with 12.6 mgÆL
)1
(50 lM)B[a]P (d) or with the vehi-
cle (s) were incubated with the indicated concentrations of ACTH. In six separate experiments, the mean FFA concentration measured in
the incubation media in absence of any pharmacological agents (basal lipolysis) was 0.096 ± 0.016 m
MÆmg
)1
cell protein. For both (D) and
(E), each bar or point represents the mean ± SEM (n ¼ 3) of triplicate samples with FFA concentrations measured in duplicate.
Benzo[a]pyrene inhibition of lipolysis P. Irigaray et al.
1364 FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS
several b-adrenergic agonists. Prior to performing these
experiments, we defined the concentration required for
each agonist to induce maximal stimulation of lipolysis.
Our goal was to determine if, even under maximal sti-
mulation, B[a]P exerted significant inhibitory effects.
The results in Fig. 1D show that B[a]P significantly
inhibited lipolysis induced by b
1
(dobutamine; P < 0.01),

b
2
(salbutamol; P <0.01)andb
3
(BRL37344; P <0.03)
adrenoreceptor-specific agonists. Similar experiments
were also conducted in the presence of forskolin, which
bypasses b-adrenergic signalling and directly increases
cellular cAMP levels [26]. Interestingly, B[a]P had
no inhibitory effect on forskolin-induced lipolysis
(Fig. 1D). Taken together, these results indicate that
B[a]P acts as a potent and nonspecific antagonist of the
early b-adrenoreceptor signalling step. The lack of effect
on forskolin-induced lipolysis indicates that, at least
under these acute conditions, adipocytes retain their
capacity to hydrolyse stored triglycerides and release
FFA. In rodents, lipolysis is also activated via other
G-coupled receptors, e.g. the ACTH receptor. This
alternate process of lipolysis stimulation was found to
be significantly inhibited by acute exposure of adipo-
cytes to 12.6 mgÆL
)1
B[a]P (Fig. 1E) (P < 0.02).
Other experiments using adipocytes isolated from
murine brown adipose tissue incubated with 1 lm epi-
nephrine showed FFA release of 1.05 ± 0.3 and
0.65 ± 0.3 mm FFA per mg protein (P<0.05) in the
absence and presence of 12.6 mgÆL
)1
B[a]P, respect-

ively. Thus, B[a]P exerted an inhibitory effect on epi-
nephrine-induced lipolysis both in murine white and
brown adipocytes.
The relative importance of the various receptors that
participate in the regulation of lipolysis is species speci-
fic [21]. Indeed, b
1
- and b
2
-adrenergic receptors are
predominant in human adipocytes, whereas b
3
-adrener-
gic receptor activity is predominant in rodent brown
and white adipose tissue. We therefore sought to deter-
mine if B[a]P also exerted an inhibitory effect on lipo-
lysis in human adipocytes. Figure 2A shows that in
human adipocytes freshly isolated from abdominal
subcutaneous tissue, B[a]P inhibited epinephrine-
induced lipolysis in a dose-dependent manner. Most
importantly, in these cells, maximal inhibition was
achieved with 0.05 mgÆL
)1
(0.2 lm)B[a]P, i.e. at con-
centrations 40–50-fold lower than those required to
inhibit lipolysis in murine adipocytes (2.5 mgÆL
)1
,
10 lm B[a]P for murine adipocytes, Fig. 1A). Interest-
ingly, lipolysis induced in human adipocytes by the

atrial natriuretic peptide (ANP) was not inhibited by
exposure to B[a]P (Fig. 2B). This lack of effect of
B[a]P on ANP receptor signalling might result from
the fact that, unlike b-adrenergic and ACTH receptors
that contain seven transmembrane spanning domains,
the ANP receptor (NPR-A) is a guanyl cyclase that
contains only a single transmembrane spanning
domain [27].
We next tested in vivo whether acute exposure of
mice to B[a]P had an effect on the adipocyte lipolytic
process. C57Bl ⁄ 6J mice were injected with increasing
concentrations of B[a]P followed by epinephrine. After
45 min, the mice were bled and plasma FFA levels
were immediately measured. Figure 3A shows that a
single dose (0.1 mgÆkg
)1
)ofB[a]P significantly reduced
0
2
4
6
8
10
0 0.05 0.2 0.250.15
0.1
Epinephrine-induced FFA release
(mM/mg prot)
A
ANP-induced FFA release
(mM mg/prot)

0
1
2
3
control
B
B[a]P
B[a]P (mg/L)
Fig. 2. Effect of B[a]P on the release of FFA from human adipocytes. Human adipocytes freshly isolated from abdominal subcutaneous tis-
sue were first incubated for 15 min with the indicated concentrations of B[a]P. After this, 4.6 mgÆL
)1
(25 lM) epinephrine or saline were
added for 45 min. Results are represented as the differences in FFA concentrations between epinephrine and saline incubations (A). (B)
Effect of 15 min preincubation of human adipocytes with either B[a]P (j) or saline (h) followed by 45 min incubation with ANP. In the
absence of pharmacological agents (basal lipolysis), FFA concentrations in the incubation media were 0.087 ± 0.010 and
0.077 ± 0.03 m
MÆmg
)1
cell protein for the epinephrine and ANP experiments, respectively. For (A) and (B), each point is the mean ± SEM
(n ¼ 3) with FFA concentrations measured in duplicate.
P. Irigaray et al. Benzo[a]pyrene inhibition of lipolysis
FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS 1365
(P<0.02) the increase in plasma FFA levels that fol-
lows epinephrine injections. Maximal B[a]P inhibition
corresponding to 70% inhibition of the epinephrine
lipolytic effect was achieved with a B[a]P concentration
of 0.4 mgÆkg
)1
. Injection of B[a]P had no effect on
basal FFA levels measured in the absence of epineph-

rine. Time-course experiments (Fig. 3B) revealed that
the inhibitory effect on the lipolytic response after a
single injection of B[a]P (0.5 mgÆkg
)1
) was detectable
within 2 h and maximal at 24 h. FFA release after
injection of epinephrine returned to normal levels 72 h
after a single B[a]P injection.
Because exposure to pollutants typically occurs in a
chronic manner, we examined the effect of repeated
B[a]P injections (0.5 mgÆkg
)1
) in C57BL ⁄ 6J male mice
every 48 h over a two-week period. At the end of chro-
nic B[a]P exposure, plasma FFA levels were not signifi-
cantly different from controls (Fig. 4A), consistent
with the observed lack of effect of B[a]P on basal lipo-
lysis (Fig. 3A). However, FFA release in response to
epinephrine was significantly lower in the B[a]P-treated
group (Fig. 4B). Most strikingly, chronic B[a]P expo-
sure caused a 43% higher weight gain compared
with controls (B[a]P: 2.23 ± 0.12 g versus control:
1.56 ± 0.18 g, P < 0.01; Fig. 4C). This experiment
was repeated three times with similar results. Dose–
response experiments showed that the lowest B[a]P
dose to cause a statistically significant weight gain was
0.1 mgÆkg
)1
injected every 48 h (data not shown). In
this study, mice were kept on a normal diet and in

none of these experiments did we observe detectable
changes in food consumption (Fig. 4D). Fifteen-day
chronic B[a]P exposure did not change plasma trigly-
ceride or total cholesterol levels significantly (Fig. 4E).
However, plasma leptin levels tended to be lower
(control: 2.84 ± 0.376 ngÆmL
)1
versus B[a]P: 2.28 ±
0.188 ngÆmL
)1
; mean ± SEM), but not significantly
different. By normalizing leptin values to body weight,
it was observed that the ratio of leptin to body weight
was significantly decreased in the B[a]P-treated group
(Fig. 4F). This is in contrast to the reported inhibitory
action of beta-agonists on leptin secretion and expres-
sion [28–31]. Examination of body composition after
2 weeks chronic exposure of mice to 0.5 mgÆkg
)1
B[a]P
every 48 h revealed a significant increase in fat mass.
Fat represented 15.9 ± 0.7 and 17.5 ± 1.2% (P<
0.03) of total body weight in control and B[a]P-treated
groups, respectively. We next examined whether the
weight gain caused by chronic B[a]P exposure contin-
ued after withdrawal of the PAH. Figure 5 shows that
there was no significant change in body weight 3 days
after the end of chronic B[a]P exposure, indicating that
upon withdrawal of the compound, the animal was
unable to immediately reduce its fat mass.

Discussion
Our results show that micromolar concentrations of
a common food pollutant, B[a]P, caused a rapid,
direct and profound inhibition of adipose tissue lipo-
lysis stimulated by epinephrine, dobutamine, salbuta-
mol, BRL37344 and ACTH. This inhibitory effect
was first observed in ex vivo experiments using iso-
lated mouse white and brown adipocytes, as well as
human adipocytes. Acute exposure of mice to B[a]P
also significantly inhibited epinephrine ⁄ norepineph-
0.8
0.6
0.4
0.2
0
1
0 0.5 1 1.5
2
Plasma FFA (mM)
B[a]P (mg/kg)
A
0.8
0.6
0.4
0.2
0
1
100020406080
Time (min)
FFA (mM)

B
Fig. 3. Acute effect of B[a]P on FFA levels in C57Bl ⁄ 6J mice. Between 08.00 and 09.00, ad libitum-fed male mice were injected i.p. with (A)
the indicated concentrations of B[a]P 2 h prior to receiving i.p. injections of either 250 lgÆkg
)1
epinephrine (d) or saline (s), or (B)
0.5 mgÆkg
)1
B[a]P for the indicated times prior to receiving injections of either saline or 0.25 mgÆkg
)1
epinephrine. Forty-five minutes after
injection of epinephrine or saline, the animals were anaesthetized, blood samples were collected, and plasma FFA levels determined. In (A)
each point represents the mean ± SEM (n ¼ 6) of FFA concentrations measured in duplicate. In (B) each point represents the mean ± SEM
(saline-treated, n ¼ 3, epinephrine-treated, n ¼ 6) of FFA concentrations measured in duplicate.
Benzo[a]pyrene inhibition of lipolysis P. Irigaray et al.
1366 FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS
rine-induced lipolysis. Chronic (15 day) B[ a]P expo-
sure caused a significant weight gain and increased
fat mass without detectable changes in food intake.
Upon withdrawal of this PAH, the excess weight
gain was not corrected.
Our data indicate that inhibition of lipolysis by
B[a]P proceeds via direct inhibition of the early step of
b-adrenergic receptor and ACTH receptor signalling to
their respective G-coupled proteins. Indeed, the inhibi-
tory effect of B[a]P occurred within minutes, which is
consistent with the notion that the principle action
does not proceed via alterations of gene expression or
by interference with translation processes. However,
this does not imply that changes in gene expression
does not occur upon chronic B[a]P exposure. Indeed,

quantitative PCR analysis showed a significant
decrease in the expression of b
1
- and b
2
-adrenergic
receptors, lipoprotein lipase and diacylglycerol
acyltransferase in adipose tissue of mice exposed to
B[a]P for 2 weeks (unpublished data). These changes
in gene expression most likely occurred as secondary
effect of B[a]P chronic exposure and require more
detailed analysis that will be reported elsewhere. The
differences in adipose tissue gene expression profile
observed after chronic B[a]P exposure contrasted with
the lack of changes in muscle gene expression profile
observed in the same animals, suggesting that the toxic
effect of B[a]P is to some extent tissue specific.
The observed inhibition of lipolysis by B[a]P most
likely results from physical perturbation of the plasma
membrane phospholipid bilayer. This interpretation
Fig. 4. Chronic effect of B[a]P on C57Bl ⁄ 6J
mice on normal chow diet. Mice (20–22 g,
11 weeks of age) maintained on normal
chow diet were injected every 48 h with
0.5 mgÆkg
)1
B[a]P (n ¼ 16) (n) or the vehicle
alone (n ¼ 14) (h). After 2 weeks treatment
(A–F), basal plasma FFA levels were deter-
mined (A) and subsets (n ¼ 6) of B[a]P and

control mice were subjected to epinephrine
(0.25 mgÆkg
)1
) injections: plasma FFA levels
were measured 15 min after epinephrine
injections (B). Body weight and food intake
of animals housed in pairs were measured
daily at 09.00, i.e. immediately after the dark
cycle. (C) and (D) show the average weight
gain ± SEM and the average food consump-
tion ± SEM, respectively. Plasma triglycer-
ides and total cholesterol levels were
determined enzymatically (E). Leptin levels
were measured using ELISA and are pre-
sented here as a ratio to body weight (F).
Results for (A–F) are represented as mean ±
SEM.
B[a]P exposure
0
1.6
1.2
0.8
0.4
01234567891011121314151617
Weight gain (g)
Time (da
y
s)
Fig. 5. Effect of withdrawal of B[a]P treatment on weight gain in
C57Bl ⁄ 6J mice on normal chow diet. Mice (20–22 g, 11 weeks of

age) maintained on normal chow diet were injected every 48 h with
0.5 mgÆkg
)1
B[a]P (n ¼ 6) (d) or the vehicle alone (n ¼ 6) (s).
B[a]P treatment was stopped after 14 days of treatment. The body
weight of animals housed in pairs was monitored on a daily basis
at 09.00, i.e. immediately after the dark cycle. Results are shown
as the average weight gain every 2 days ± SEM.
P. Irigaray et al. Benzo[a]pyrene inhibition of lipolysis
FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS 1367
stems from the observation that, in acute ex vivo
experiments, B[a]P strongly and rapidly inhibited the
signalling capacity of at least four distinct receptors:
the b
1
-, b
2
-, b
3
-adrenergic and ACTH receptors. These
receptors share common features: all contain seven
transmembrane spanning domains and are coupled to
G-proteins themselves anchored to the inner leaflet of
the plasma membrane. In contrast ANP-induced lipo-
lysis via stimulation of NPR-A, which contains a sin-
gle transmembrane-spanning domain, was not affected
by B[a]P (Fig. 2B) [27].
Physicochemical studies using differential scanning
calorimetry, infrared spectroscopy and small-angle
X-ray diffraction have shown that B[a]P incorporated

into phospholipid bilayers localizes in the most apolar
region of the phospholipid matrix, resulting in an
expanded and swollen membrane [32]. We, therefore,
propose that distortion of the physiochemical proper-
ties of the adipocyte plasma membrane by B[a]P
decreases the signalling capacity of G-coupled recep-
tors intimately linked to the phospholipid bilayer via
their seven transmembrane domains. This provides a
novel mechanism for B[a ]P toxicity. Indeed, thus far,
B[a]P toxicity has been attributed to its ability to
induce DNA adduct formation [33]. In the ex vivo
experiments reported here, the concentrations needed
to achieve the B[a]P toxic effect on adipocytes were
2000-fold lower than those causing carcinogenesis in
rodents and 20-fold lower than those causing altera-
tions of the EGF receptor in cultured human uterine
cells, RL95-2 [34]. In vivo the maximal inhibitory effect
on epinephrine-induced lipolysis was achieved with
B[a]P at a dose of 0.4 mgÆkg
)1
, i.e. 100-fold lower than
those used to induce a tumorogenic response in mice
(typically 50 mgÆkg
)1
) [35].
Chronic B[a]P exposure of mice on a normal diet
caused weight gain that was not immediately correc-
ted upon withdrawal of the pollutant. In acute in vivo
experiments catecholamine-induced release of FFA
returned to baseline values between 48 and 72 h. This

delay in recovery from a longer ‘chronic’ treatment of
B[a]P suggests that constant exposure may lead to
significant changes in adipose tissue metabolism and
thus require a longer time to reverse the effects.
Indeed, this notion is supported by the significant
changes in mRNA as a result of 15 days of treatment
with B[a]P described earlier. This also suggests that
there may be other, as yet unexplained, interactions
between B[a]P and adipocytes resulting in delayed
recovery after withdrawal of B[a]P. Another possibil-
ity to be considered is that B[a]P may have a pro-
longed half-life as a result of chronic treatment and
thus requires a longer wash-out period compared with
those that received a single acute dose of B[a]P. It is
interesting to note that the obese mouse model, ob ⁄ ob
shows decreased levels of CYP1A1, a cytochrome
P450 enzyme essential for processing B[a]P [36]. A
study monitoring the kinetics of B[a]P and other
pollutants in both overweight and obese subjects
before and during weight loss would provide useful
information.
The molecular mechanisms directly responsible for
B[a]P-induced weight gain remain speculative. We do
know, however, that this increase in body weight was
not due to an increase in food intake despite a signi-
ficant decrease in leptin ⁄ weight ratio. Indeed, in three
separate experiments, significant weight gain com-
pared with controls was observed without any detect-
able changes in food intake. This weight gain was
most likely due to increased fat mass, as indicated by

results of body composition analysis. Our interpret-
ation of these data is that chronic inhibition by B[a]P
of physiological b-adrenergic and ACTH stimulation
caused a reduction of energy expenditure sufficient to
cause weight gain in rodents. It has been shown that
‘b-less’ mice that do not express any of the three b-
adrenergic receptors experience weight gain without
changes in food intake [37]. Furthermore, b-adrener-
gic-blocking medications acutely decrease energy
expenditure in normal human subjects. This leads to
an increase in fat mass if no subsequent alteration is
made in food consumption or activity pattern [38].
The precise mechanisms by which the b-adrenergic
system controls energy expenditure remain unclear.
However, the release of adipose tissue triglycerides as
FFA is the first step toward FFA oxidation that
causes partial uncoupling of respiratory chain
thereby physiologically increasing energy expenditure
[39,40].
A number of reports have shown impaired catechol-
amine-induced FFA release from adipose subcutaneous
tissue in obese subjects [41], a trait that tends to aggre-
gate in their families [42]. In severely obese adoles-
cents, induction of lipolysis by injections of small
doses of epinephrine is also decreased [43]. Genetic Gs
deficiency (Gs; OMIM n°103580), which leads to para-
thyroid hormone resistance, short stature, skeletal
defects (Albright’s hereditary syndrome) and obesity
was shown to cause decreased lipolytic response to epi-
nephrine [44]. In addition, defective lipolysis in obese

humans is associated with polymorphisms of the b
2
, b
3
adrenergic receptors and HSL genes [45]. Whether in
addition to these established genetics factors, exposure
to a common food and environmental pollutants con-
tribute to defective lipolysis observed in obese subjects
remains to be investigated. An alternative interpret-
Benzo[a]pyrene inhibition of lipolysis P. Irigaray et al.
1368 FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS
ation for the weight gain induced by B[a]P is that
it causes an as yet undefined toxicity on adipocyte
hormonal function leading to changes in energy
expenditure.
In this perspective, it is interesting to note that a
decrease in the leptin ⁄ body weight ratio was observed
despite the increase in fat mass. It is possible that this
may also contribute to the observed weight gain after
15 days of treatment with B[a]P. Indeed, leptin defici-
ency leads directly to increased fat mass and leptin
changes determine variations in energy expenditure
[46]. However, a characteristic of leptin deficiency is a
significant increase in food intake, caused by the lack
of a satiety signalling, whereas no change in food
intake was detected in the B[a]P-treated animals. In
addition, the absolute values of leptin in control and
B[a]P-treated animals were not significantly different
and remained within the normal range for mice.
B[a]P plasma levels were reported to correlate posi-

tively with the BMI of human subjects in an environ-
mental study [11]. This ‘correlation’ may simply result
from the increased reservoir capacity of fat mass for
this lipophilic pollutant. However, our results suggest
that the presence of B[a]P itself can have a deleterious
effect on adipocyte function. Interestingly, it has been
shown that the increased plasma concentration of
organochlorines in obese subjects observed during
weight loss is positively correlated with a decrease in
resting metabolic rate as well as oxidative capacity of
the muscle and the thyroid hormone T3 [4]. Taken
together, these results demonstrate the potential
importance of pollutants in obesity, with implications
for metabolism during both weight gain and weight
loss. Whether these elements form part of the cause of
metabolic dysfunction leading to the accumulation of
fat mass remains to be determined.
Available epidemiological data addressing the impli-
cation of PAH, in general, as a causal factor in the
pathogeny of metabolic disorders are currently too
few to draw any definitive conclusions. Nevertheless,
DNA adducts of PAH were significantly greater in a
population with increased atherosclerotic lesions and
higher BMI than in the control group with less severe
lesions [47]. Consistent with this are recent findings
that chronic exposure of apoE knockout mice to
B[a]P (5 mgÆkg
)1
, i.e. a dose tenfold greater than that
used in the studies reported here) induces larger

atherosclerotic plaques [19]. Another environmental
study conducted on human subjects in the early 1980s
reports that higher plasma B[a]P levels correlated pos-
itively with BMI [11]. However, there are no current
epidemiological data that prospectively examines this
hypothesis.
Experimental procedures
Materials
B[a]P, epinephrine, dobutamine, salbutamol, BRL37344,
ACTH and ANP were purchased from Sigma (Lyon,
France).
Animals
C57Bl ⁄ 6J male mice weighing 20–22 g (11 weeks) were
obtained from Charles River Laboratories (L’Arbresle,
France) and housed in temperature-regulated (20 °C),
ventilated cabinets with a 12 h light, 12 h dark cycle
(09.00 to 21.00). Animals were acclimated in this con-
trolled environment for 1 week prior to any experiments
and allowed access to food and water ad libitum. All
experiments started between 08.00 and 09.00, i.e. at the
end of the dark period. Animals were anaesthetized using
isoflurane before blood sampling through either retro-
orbital sinus puncture or the carotid artery (in cases of
final bleeds). B[a]P was solubilized in physiological saline
containing 5% dimethylsulfoxide and 1% methyl carboxy
cellulose, and administered through i.p. injections.
Epinephrine in physiological saline was also injected i.p.
Animals were housed in an authorized specific pathogen-
free facility. Animal care protocols conducted were in
accordance with institutional guidelines and with Eur-

opean Communities Council Directive to minimize pain
and discomfort to animals.
Isolated adipocyte preparation
C57Bl ⁄ 6J male mice maintained under the environmental
conditions described were killed and epidydimal white adi-
pose tissue was rapidly dissected. Adipocytes were isolated
from adipose tissue using Rodbell’s method modified as des-
cribed below [48]. The samples were rinsed with Kreb’s
Ringer bicarbonate buffer (KRBB) supplemented with 4%
(w ⁄ v) bovine serum albumin (BSA) and 5 mm glucose and
then incubated 45 min at 37 °C in the presence of collage-
nase (2 mgÆg
)1
tissue in 1 mL sample) under gentle agitation
(80 r.p.m). After this, isolated cells were filtered through a
nylon mesh (pore size, 250 lm) and washed three times with
the same buffer. Cell suspensions were aliquoted in Eppen-
dorf tubes containing KRBB supplemented with 4% (w ⁄ v)
BSA and 1 mm glucose. After incubation at 37 °C under
gentle agitation (40 r.p.m) with the indicated pharmacologi-
cal agents, aliquots of the media were removed for enzymatic
determination of FFA. Cells were pelleted by centrifugation
(10 000 g, 20 min, 4 °C), washed twice in KRBB, and then
resuspended in Lowry reagent to determine cellular protein
content (80–120 lg protein per sample).
Human adipocytes were obtained from subcutaneous fat
dissected from a surgical specimen removed from the
P. Irigaray et al. Benzo[a]pyrene inhibition of lipolysis
FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS 1369
abdomen of patients undergoing plastic surgery, after

obtaining their informed consent. Cells were isolated from
fat lobules using the procedure described above.
Biochemical assays
Plasma triglycerides and total cholesterol were measured
enzymatically using reagents from Biome
´
rieux (Marcy
l’Etoile, France). FFA levels were determined within
60 min of completion of the experiments using enzymatic
determination kits from Roche Diagnostics (Meylan,
France). Mouse leptin levels were determined using an
ELISA method (R & D Systems, Minneapolis, MN).
Assessment of body fat mass
Body mass composition was analysed using the EM-Scan
model SA-3000 (EM-SCAN Inc, Springfield, MA). This
machine uses total body electrical conductivity to measure
a conductivity index, which is used to calculate body fat
and lean mass. This has been shown to detect a range of
5–30% body fat mass with a correlation of 0.98 to values
obtained by chemical analysis [49]. The machine was calib-
rated before performing measurements two times on mice
lightly anaesthetized with isoflurane. The fat-free mass was
then calculated to compensate for the body weight of each
mice as well as the length of the mouse. In pilot tests in
which two mice were measured twice daily three days in
a row, the coefficient of variation was measured to be
1.09 ± 0.96% (mean ± SD).
Statistical analysis
Statistical significance was determined by Fisher exact fol-
lowed by two-tailed Student’s t-test using statview (Palo

Alto, CA). Mann–Whitney test (statview) was used to
evaluate the significance of changes in percent of body
fat.
Acknowledgements
The authors gratefully acknowledge the scientific dis-
cussions with Professor Franc¸ ois Laurent. The excel-
lent technical work of Delphine Maurice and Erwan
Magueur is greatly appreciated. This work was suppor-
ted by grants from the Lorraine Region and Urban
Community of Grand Nancy (CUGN) and from the
French Ministry of Research and Higher Education.
PI is a recipient of a Research Fellowship from the
French Ministry of Research and Higher Education,
and FTY and BEB are Directors of Research at the
Institut National de la Sante
´
et de la Recherche Me
´
di-
cale (INSERM).
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