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 a
Trang 1tissue lipolysis and causes weight gain in mice
A novel molecular mechanism of toxicity for a common food
pollutant
Philippe Irigaray1,2, Virginie Ogier1, Sandrine Jacquenet1, Ve´ronique Notet1, Pierre Sibille3,
Luc Me´jean1,2, Bernard E Bihain1 and Frances T Yen1,2
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: frances.yenpotin@mtm.nancy.
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 concentrainhibi-tion of 0.9 mgÆL)1(3.5 lm) Lipo-lysis induced by b1-, b2- and b3-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)1injected 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.
Trang 2conjunction 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 oxidrel-ative
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 mediliber-ated 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 a2-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
Trang 3the 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 Kd 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 (Kd¼ 0.4 mgÆL)1 or 1.5 lm for b1-adrenoreceptor and 2.3 mgÆL)1 or 8.6 lm for b2-adrenoreceptor) [25] The inhibitory effect of 1.8 mgÆL)1B[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
B[a]P (mg/L)
2
1.6
1.2
0.8
0.4
0
B[a]P (mg/L)
2 1.6 1.2 0.8 0.4 0
A
FFA release (m M
0 0.5 1.5 2.5 3.5 4.5
epinephrine dobutamine salbutamol BRL 37344 forskolin
*
ns
D
4 3 2 1 0
E
Incubation time in the presence of B[a]P (min)
2 1.6 1.2 0.8 0.4 0
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
duplicate (C) Freshly isolated mice adipocytes were incubated for 15 min with the indicated concentrations of B[a]P, after which
vehi-cle (s) were incubated with the indicated concentrations of ACTH In six separate experiments, the mean FFA concentration measured in
Trang 4several 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 b1(dobutamine; P < 0.01),
b2(salbutamol; P < 0.01) and b3(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)1B[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, b1- and b2-adrenergic receptors are predominant in human adipocytes, whereas b3 -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
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) of B[a]P significantly reduced
0
2
4
6
8
10
A
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
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
Trang 5(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)1B[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
B[a]P (mg/kg)
A
0.8
0.6
0.4
0.2
0 1
100
Time (min)
B
injection of epinephrine or saline, the animals were anaesthetized, blood samples were collected, and plasma FFA levels determined In (A)
Trang 6rine-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,
decrease in the expression of b1- and b2-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
mice on normal chow diet Mice (20–22 g,
11 weeks of age) maintained on normal
chow diet were injected every 48 h with
(A–F), basal plasma FFA levels were
control mice were subjected to epinephrine
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
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Time (days) Fig 5 Effect of withdrawal of B[a]P treatment on weight gain in
age) maintained on normal chow diet were injected every 48 h with
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.
Trang 7stems 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 b1-, b2-, b3-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
‘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 n103580), 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 b2, b3 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
Trang 8interpret-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 (20C), 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 37C in the presence of collage-nase (2 mgÆg)1tissue 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 37C 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, 4C), 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
Trang 9abdomen 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)
References
1 Friedman JM (2000) Obesity in the new millennium Nature 404, 632–634
2 Ravussin E, Lillioja S, Knowler WC, Christin L, Frey-mond D, Abbott WG, Boyce V, Howard BV & Bogar-dus C (1988) Reduced rate of energy expenditure as a risk factor for body-weight gain N Engl J Med 318, 467–472
3 Barsh GS, Farooqi IS & O’Rahilly S (2000) Genetics of body-weight regulation Nature 404, 644–651
4 Pelletier C, Imbeault P & Tremblay A (2003) Energy balance and pollution by organochlorines and poly-chlorinated biphenyls Obes Rev 4, 17–24
5 Laher JM, Rigler MW, Vetter RD, Barrowman JA & Patton JS (1984) Similar bioavailability and lymphatic transport of benzo(a)pyrene when administered to rats
in different amounts of dietary fat J Lipid Res 25, 1337–1342
6 Shu HP & Nichols AV (1979) Benzo(a)pyrene uptake
by human plasma lipoproteins in vitro Cancer Res 39, 1224–1230
7 Pelletier C, Despres JP & Tremblay A (2002) Plasma organochlorine concentrations in endurance athletes and obese individuals Med Sci Sports Exerc 34, 1971–1975
8 Pelletier C, Doucet E, Imbeault P & Tremblay A (2002) Associations between weight loss-induced changes in plasma organochlorine concentrations, serum T(3) con-centration and resting metabolic rate Toxicol Sci 67, 46–51
9 Tremblay A, Pelletier C, Doucet E & Imbeault P (2004) Thermogenesis and weight loss in obese individuals: a primary association with organochlorine pollution Int J Obes Relat Metab Disord 28, 936–939
10 Irigaray P, Mejean L & Laurent F (2005) Behaviour of dioxin in pig adipocytes Food Chem Toxicol 43, 457–460
11 Hutcheon DE, Kantrowitz J, Van Gelder RN & Flynn
E (1983) Factors affecting plasma benzo[a]pyrene levels
in environmental studies Environ Res 32, 104–110
12 Romieu I, Hernandez-Avila M, Lazcano-Ponce E, Weber JP & Dewailly E (2000) Breast cancer, lactation history, and serum organochlorines Am J Epidemiol
152, 363–370
13 Hagmar L, Rylander L, Dyremark E, Klasson-Wehler
E & Erfurth EM (2001) Plasma concentrations of persis-tent organochlorines in relation to thyrotropin and thyr-oid hormone levels in women Int Arch Occup Environ Health 74, 184–188
14 Sala M, Sunyer J, Herrero C, To-Figueras J & Gri-malt J (2001) Association between serum concentra-tions of hexachlorobenzene and polychlorobiphenyls with thyroid hormone and liver enzymes in a sample
of the general population Occup Environ Med 58, 172–177
Trang 1015 Kazerouni N, Sinha R, Hsu CH, Greenberg A &
Rothman N (2001) Analysis of 200 food items for
benzo[a]pyrene and estimation of its intake in an
epi-demiologic study Food Chem Toxicol 39, 423–436
16 Li S, Pan D & Wang G (1994) Analysis of polycyclic
aromatic hydrocarbons in cooking oil fumes Arch
Environ Health 49, 119–122
17 IARC (1983) IARC Monographs on the Evaluation of
Carcinogenic Risk of Chemicals to Humans – Polynuclear
Aromatic Compounds, Vol 32 (33-91), pp 211–224
International Agency for Research into Cancer, Lyon
18 van Grevenynghe J, Rion S, Le Ferrec E, Le Vee M,
Amiot L, Fauchet R & Fardel O (2003) Polycyclic
aro-matic hydrocarbons inhibit differentiation of human
monocytes into macrophages J Immunol 170, 2374–
23781
19 Curfs DM, Lutgens E, Gijbels MJ, Kockx MM, Daemen
MJ & van Schooten FJ (2004) Chronic exposure to the
carcinogenic compound benzo[a]pyrene induces larger
and phenotypically different atherosclerotic plaques in
ApoE-knockout mice Am J Pathol 164, 101–108
20 Zechner R, Strauss JG, Haemmerle G, Lass A &
Zim-mermann R (2005) Lipolysis: pathway under
construc-tion Curr Opin Lipidol 16, 333–340
21 Lafontan M & Berlan M (1993) Fat cell adrenergic
receptors and the control of white and brown fat cell
function J Lipid Res 34, 1057–1091
22 Lafontan M, Moro C, Sengenes C, Galitzky J, Crampes
F & Berlan M (2005) An unsuspected metabolic role for
atrial natriuretic peptides: the control of lipolysis, lipid
mobilization, and systemic nonesterified fatty acids
levels in humans Arterioscler Thromb Vasc Biol 25,
2032–2042
23 Abumrad NA, Harmon CM & Barnela US & Whitesell
RR (1988) Insulin antagonism of catecholamine
stimula-tion of fatty acid transport in the adipocyte Studies on
its mechanism of action J Biol Chem 263, 14678–14683
24 Arner P (1999) Catecholamine-induced lipolysis in
obes-ity Int J Obes Relat Metab Disord 23 (Suppl 1), 10–13
25 Smith C & Teitler M (1999) Beta-blocker selectivity at
cloned human beta 1- and beta 2-adrenergic receptors
Cardiovasc Drugs Ther 13, 123–126
26 Litosch I, Hudson TH, Mills I, Li SY & Fain JN (1982)
Forskolin as an activator of cyclic AMP accumulation
and lipolysis in rat adipocytes Mol Pharmacol 22, 109–
115
27 Sengenes C, Berlan M, De Glisezinski I, Lafontan M &
Galitzky J (2000) Natriuretic peptides: a new lipolytic
pathway in human adipocytes FASEB J 14, 1345–1351
28 Berraondo B, Marti A, Duncan JS, Trayhurn P &
Mar-tinez JA (2000) Up-regulation of muscle UCP2 gene
expression by a new beta3-adrenoceptor agonist,
treca-drine, in obese (cafeteria) rodents, but down-regulation
in lean animals Int J Obes Relat Metab Disord 24, 156–
163
29 Li H, Matheny M & Scarpace PJ (1997) Beta 3-adrener-gic-mediated suppression of leptin gene expression in rats Am J Physiol 272, E1031–E1036
30 Mantzoros CS, Qu D, Frederich RC, Susulic VS, Lowell BB, Maratos-Flier E & Flier JS (1996) Activa-tion of beta(3) adrenergic receptors suppresses leptin expression and mediates a leptin-independent inhibition
of food intake in mice Diabetes 45, 909–914
31 Moreno-Aliaga MJ, Martinez JA, Stanhope KL, Fer-nandez-Otero MP & Havel PJ (2002) Effects of Treca-drine, a beta3-adrenergic agonist, on leptin secretion, glucose and lipid metabolism in isolated rat adipocytes Int J Obes Relat Metab Disord 26, 912–919
32 Jimenez M, Aranda FJ, Teruel JA & Ortiz A (2002) The chemical toxic benzo[a]pyrene perturbs the physical organization of phosphatidylcholine membranes Envi-ron Toxicol Chem 21, 787–793
33 Kwack SJ & Lee BM (2000) Correlation between DNA
or protein adducts and benzo[a]pyrene diol epoxide I– triglyceride adduct detected in vitro and in vivo Carcino-genesis 21, 629–632
34 McGarry MA, Charles GD, Medrano T, Bubb MR, Grant MB, Campbell-Thompson M & Shiverick KT (2002) Benzo(a)pyrene, but not 2,3,7,8-tetrachlorodi-benzo-p-dioxin, alters cell adhesion proteins in human uterine RL95-2 cells Biochem Biophys Res Commun
294, 101–107
35 Wu K, Shan YJ, Zhao Y, Yu JW & Liu BH (2001) Inhibitory effects of RRR-alpha-tocopheryl succinate on benzo(a)pyrene (B(a)P)-induced forestomach carcino-genesis in female mice World J Gastroenterol 7, 60–65
36 Roe AL, Howard G, Blouin R & Snawder JE (1999) Characterization of cytochrome P450 and glutathione S-transferase activity and expression in male and female
ob⁄ ob mice Int J Obes Relat Metab Disord 23, 48–53
37 Jimenez M, Leger B, Canola K, Lehr L, Arboit P, Sey-doux J, Russell AP, Giacobino JP, Muzzin P & Preitner
F (2002) Beta(1)⁄ beta(2) ⁄ beta(3)-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipo-lytic responses to fasting FEBS Lett 530, 37–40
38 Monroe MB, Seals DR, Shapiro LF, Bell C, Johnson D
& Parker Jones P (2001) Direct evidence for tonic sym-pathetic support of resting metabolic rate in healthy adult humans Am J Physiol Endocrinol Metab 280, E740–E744
39 Schrauwen P, Hesselink MK, Vaartjes I, Kornips E, Saris WH, Giacobino JP & Russell A (2002) Effect
of acute exercise on uncoupling protein 3 is a fat metabolism-mediated effect Am J Physiol Endocrinol Metab 282, E11–E17
40 Fleury C, Neverova M, Collins S, Raimbault S, Cham-pigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D et al (1997) Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia Nat Genet 15, 269–272