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 Irigaray1,2, Virginie Ogier1, Sandrine Jacquenet1, Veronique Notet1, Pierre Sibille3, ´ Luc Mejean1,2, Bernard E Bihain1 and Frances T Yen1,2 ` JE2482 Lipidomix – Institut National Polytechnique de Lorraine, Vandoeuvre-les-Nancy, France ` Laboratoire de Sciences Animales, ENSAIA, Vandoeuvre-les-Nancy, France Polyclinique de Gentilly, Nancy, France Keywords adipocytes; lipolysis; obesity; polycyclic aromatic hydrocarbons; triglycerides Correspondence F.T Yen, JE2482 Lipidomix, Laboratoire de ´ ´ ´ Medecine et Therapeutique Moleculaire, 15 rue du Bois de la Champelle, ` 54500 Vandoeuvre-les-Nancy, France Fax: +33 83 67 89 99 Tel: +33 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 inhibition was obtained with a B[a]P concentration of 0.9 mgỈL)1 (3.5 lm) Lipolysis induced by b1-, b2- and b3-adrenoreceptor-specific agonists, as well as ACTH, were also significantly inhibited by B[a]P, whereas forskolininduced lipolysis was not B[a]P-sensitive Similar inhibition of catecholamine-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 concentration of 0.02 mgỈL)1 (0.08 lm) In vivo treatment of C57Bl ⁄ 6J mice with 0.4 mgỈkg)1 B[a]P inhibited epinephrine-induced release of free fatty acids by 70% Chronic exposure of mice to B[a]P (0.5 mgỈkg)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 disorders, such as obesity 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 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 P Irigaray et al 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 liposoluble 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 pollutant released was positively associated with a lower relative 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, environmental 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 environmental pollutants exert a toxic effect on the adipocyte 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 organochlorines, 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 compounds 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 hydrocarbons (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, originating from incomplete combustion or pyrolysis of organic matter Under normal conditions, the diet is Benzo[a]pyrene inhibition of lipolysis the main source of B[a]P exposure [15] B[a]P contamination of food results from either specific food processing, e.g open flame cooking, or contamination of food by B[a]P released into the environment For example, the B[a]P content of fast-food hamburgers reaches levels of 200 ng per serving [15] In Asia, very high levels of B[a]P are produced in cooking oil (> 400 ngỈg)1) [16] B[a]P is also a well-known carcinogen and consumption 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 transported in the plasma via the lipoprotein system [5,6] Tissue-distribution studies have shown that B[a]P accumulates in lipid-storing tissues including the mammary glands and adipose tissue The carcinogenicity of B[a]P has been well-documented This pollutant is metabolized via the cytochrome P450 system into reactive dihydrodiol epoxide derivatives (e.g B[a]P-7,8dihydrodiol-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 various 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 adipocyte function, one of the main sites of storage We therefore decided to address the question of B[a]P toxicity 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 liberated was mediated by hormone sensitive lipase (HSL) However, recent studies using mice with inactivation of the HSL gene suggest that another adipose triglycerides lipase also participates in this process [20] Activation 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 signalling proceed via stimulatory and inhibitory G-proteins 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 FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS 1363 Benzo[a]pyrene inhibition of lipolysis P Irigaray et al tions of mgỈL)1 (Fig 1A) The estimated Kd value for B[a]P inhibition of lipolysis was 0.9 mgỈL)1 (3.5 lm), i.e in the same range as that measured for the known b blocker atenolol (Kd ẳ 0.4 mgặL)1 or 1.5 lm for b1-adrenoreceptor and 2.3 mgỈL)1 or 8.6 lm for b2-adrenoreceptor) [25] The inhibitory effect of 1.8 mgỈL)1 B[a]P on epinephrine-induced lipolysis was detectable within (Fig 1B), suggesting that B[a]P directly affected the cellular signal transduction 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 ⁄ norepinephrine-induced lipolytic cascade includes activation of the b-adrenergic receptor system We thus determined the effect of B[a]P on lipolysis induced by the phosphorylation of cAMP-dependent protein kinase 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 significant 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 measurement of the FFA released in the media A significant inhibitory effect on epinephrine-induced FFA release (P < 0.01) was achieved with B[a]P concentra- 1.6 1.2 0.8 0.4 10 15 B[a]P (mg/L) 20 25 1.6 1.2 0.8 0.4 25 10 15 20 30 Incubation time in the presence of B[a]P (min) 4.5 FFA release (mM/mg prot) 1.6 1.2 0.8 0.4 0 10 15 B[a]P (mg/L) 20 25 E D ns 3.5 2.5 * * * ** 1.5 in sk ol fo r 37 34 L 0 ACTH (nM) 10 BR sa lb ut a m ol in e m ut a b ep in ep hr in e 0.5 ACTH-induced FFA release (mM/mg prot) C Norepinephrine-induced FFA release (mM/mg prot) B Epinephrine-induced FFA release (mM/mg prot) Epinephrine-induced FFA release (mM/mg prot) A Fig Effect of B[a]P on the release of FFA from mice adipocytes Freshly isolated mice adipocytes were incubated (A) for 45 with the indicated concentrations of B[a]P or (B) for the indicated times with 2.5 mgỈL)1 (10 lM) B[a]P After this, epinephrine (1.8 mgỈL)1 or 10 lM) was added and the incubation continued for 45 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 with the indicated concentrations of B[a]P, after which norepinephrine (1.7 mgỈL)1 or 10 lM) was added and the incubation continued for 45 Each point represents the mean ± SEM (n ¼ 6) in which FFA levels were measured in duplicate (D) Effect of 15 preincubation of isolated adipocytes without (h) or with 12.6 mg L)1 (50 lM) B[a]P (n) followed by 45 incubation with the indicated pharmacological agent: epinephrine (1.8 mgỈL)1 or 10 lM), dobutamine (8.4 mgỈL)1 or 25 lM), salbutamol (23.9 mgỈL)1 or 100 lM), BRL 37344 (9.6 mgỈL)1 or 25 lM) and forskolin (41 mgỈL)1 or 100 lM) Significance is noted as the following: *P < 0.01, **P < 0.03 (E) Isolated adipocytes preincubated for 15 with 12.6 mgỈL)1 (50 lM) B[a]P (d) or with the vehicle (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 mMỈmg)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 1364 FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS P Irigaray et al Benzo[a]pyrene inhibition of lipolysis 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 stimulation, 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 adipocytes to 12.6 mgỈL)1 B[a]P (Fig 1E) (P < 0.02) Other experiments using adipocytes isolated from murine brown adipose tissue incubated with lm epinephrine 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 mgỈL)1 B[a]P, respectively Thus, B[a]P exerted an inhibitory effect on epinephrine-induced lipolysis both in murine white and brown adipocytes B 10 ANP-induced FFA release (mM mg/prot) Epinephrine-induced FFA release (mM/mg prot) A The relative importance of the various receptors that participate in the regulation of lipolysis is species specific [21] Indeed, b1- and b2-adrenergic receptors are predominant in human adipocytes, whereas b3-adrenergic receptor activity is predominant in rodent brown and white adipose tissue We therefore sought to determine if B[a]P also exerted an inhibitory effect on lipolysis in human adipocytes Figure 2A shows that in human adipocytes freshly isolated from abdominal subcutaneous tissue, B[a]P inhibited epinephrineinduced lipolysis in a dose-dependent manner Most importantly, in these cells, maximal inhibition was achieved with 0.05 mgỈL)1 (0.2 lm) B[a]P, i.e at concentrations 40–50-fold lower than those required to inhibit lipolysis in murine adipocytes (2.5 mgỈL)1, 10 lm B[a]P for murine adipocytes, Fig 1A) Interestingly, 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 mgỈkg)1) of B[a]P significantly reduced 0 0.05 0.1 0.15 B[a]P (mg/L) 0.2 0.25 control B[a]P Fig Effect of B[a]P on the release of FFA from human adipocytes Human adipocytes freshly isolated from abdominal subcutaneous tissue were first incubated for 15 with the indicated concentrations of B[a]P After this, 4.6 mgỈL)1 (25 lM) epinephrine or saline were added for 45 Results are represented as the differences in FFA concentrations between epinephrine and saline incubations (A) (B) Effect of 15 preincubation of human adipocytes with either B[a]P (j) or saline (h) followed by 45 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 mMỈmg)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 FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS 1365 Benzo[a]pyrene inhibition of lipolysis P Irigaray et al B A 0.8 FFA (mM) 0.8 Plasma FFA (mM) 0.6 0.4 0.6 0.4 0.2 0.2 0 0.5 B[a]P (mg/kg) 1.5 20 40 60 Time (min) 80 100 Fig 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 h prior to receiving i.p injections of either 250 lgỈkg)1 epinephrine (d) or saline (s), or (B) 0.5 mgỈkg)1 B[a]P for the indicated times prior to receiving injections of either saline or 0.25 mgỈkg)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 (P < 0.02) the increase in plasma FFA levels that follows 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 mgỈkg)1 Injection of B[a]P had no effect on basal FFA levels measured in the absence of epinephrine Time-course experiments (Fig 3B) revealed that the inhibitory effect on the lipolytic response after a single injection of B[a]P (0.5 mgỈkg)1) was detectable within 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 mgỈkg)1) in C57BL ⁄ 6J male mice every 48 h over a two-week period At the end of chronic B[a]P exposure, plasma FFA levels were not significantly different from controls (Fig 4A), consistent with the observed lack of effect of B[a]P on basal lipolysis (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 exposure 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 mgỈkg)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 trigly1366 ceride or total cholesterol levels significantly (Fig 4E) However, plasma leptin levels tended to be lower (control: 2.84 ± 0.376 ngỈmL)1 versus B[a]P: 2.28 ± 0.188 ngỈmL)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 expression [28–31] Examination of body composition after weeks chronic exposure of mice to 0.5 mgỈkg)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 continued after withdrawal of the PAH Figure shows that there was no significant change in body weight 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 lipolysis stimulated by epinephrine, dobutamine, salbutamol, BRL37344 and ACTH This inhibitory effect was first observed in ex vivo experiments using isolated mouse white and brown adipocytes, as well as human adipocytes Acute exposure of mice to B[a]P also significantly inhibited epinephrine ⁄ norepineph- FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS P Irigaray et al Benzo[a]pyrene inhibition of lipolysis Fig 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 mgỈkg)1 B[a]P (n ¼ 16) (n) or the vehicle alone (n ¼ 14) (h) After weeks treatment (A–F), basal plasma FFA levels were determined (A) and subsets (n ¼ 6) of B[a]P and control mice were subjected to epinephrine (0.25 mgỈkg)1) injections: plasma FFA levels were measured 15 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 consumption ± SEM, respectively Plasma triglycerides and total cholesterol levels were determined enzymatically (E) Leptin levels were measured using ELISA and are presented here as a ratio to body weight (F) Results for (A–F) are represented as mean ± SEM B[a]P exposure Weight gain (g) 1.6 1.2 0.8 0.4 0 10 11 12 13 14 15 16 17 Time (days) Fig 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 mgặkg)1 B[a]P (n ẳ 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 days ± SEM rine-induced lipolysis Chronic (15 day) B[a]P exposure 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 inhibitory 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 b1- and b2-adrenergic receptors, lipoprotein lipase and diacylglycerol acyltransferase in adipose tissue of mice exposed to B[a]P for 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 FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS 1367 Benzo[a]pyrene inhibition of lipolysis P Irigaray et al 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 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 lipolysis via stimulation of NPR-A, which contains a single 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 properties of the adipocyte plasma membrane by B[a]P decreases the signalling capacity of G-coupled receptors 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 alterations 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 mgỈkg)1, i.e 100-fold lower than those used to induce a tumorogenic response in mice (typically 50 mgỈkg)1) [35] Chronic B[a]P exposure of mice on a normal diet caused weight gain that was not immediately corrected 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 possibility to be considered is that B[a]P may have a prolonged half-life as a result of chronic treatment and thus requires a longer wash-out period compared with 1368 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 know, however, that this increase in body weight was not due to an increase in food intake despite a significant decrease in leptin ⁄ weight ratio Indeed, in three separate experiments, significant weight gain compared with controls was observed without any detectable changes in food intake This weight gain was most likely due to increased fat mass, as indicated by results of body composition analysis Our interpretation 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 not express any of the three badrenergic receptors experience weight gain without changes in food intake [37] Furthermore, b-adrenergic-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 catecholamine-induced FFA release from adipose subcutaneous tissue in obese subjects [41], a trait that tends to aggregate in their families [42] In severely obese adolescents, induction of lipolysis by injections of small doses of epinephrine is also decreased [43] Genetic Gs deficiency (Gs; OMIM n°103580), which leads to parathyroid hormone resistance, short stature, skeletal defects (Albright’s hereditary syndrome) and obesity was shown to cause decreased lipolytic response to epinephrine [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 contribute to defective lipolysis observed in obese subjects remains to be investigated An alternative interpret- FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS P Irigaray et al 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 deficiency 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 positively with the BMI of human subjects in an environmental 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 implication 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 mgỈkg)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 positively with BMI [11] However, there are no current epidemiological data that prospectively examines this hypothesis Benzo[a]pyrene inhibition of lipolysis 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 controlled environment for 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 retroorbital 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 pathogenfree facility Animal care protocols conducted were in accordance with institutional guidelines and with European 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 adipose tissue was rapidly dissected Adipocytes were isolated from adipose tissue using Rodbell’s method modified as described below [48] The samples were rinsed with Kreb’s Ringer bicarbonate buffer (KRBB) supplemented with 4% (w ⁄ v) bovine serum albumin (BSA) and mm glucose and then incubated 45 at 37 °C in the presence of collagenase (2 mgỈg)1 tissue in 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 Eppendorf tubes containing KRBB supplemented with 4% (w ⁄ v) BSA and mm glucose After incubation at 37 °C under gentle agitation (40 r.p.m) with the indicated pharmacological agents, aliquots of the media were removed for enzymatic determination of FFA Cells were pelleted by centrifugation (10 000 g, 20 min, °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 FEBS Journal 273 (2006) 1362–1372 ª 2006 The Authors Journal compilation ª 2006 FEBS 1369 Benzo[a]pyrene inhibition of lipolysis P Irigaray et al 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 Biomerieux (Marcy l’Etoile, France) FFA levels were determined within 60 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 calibrated 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 followed 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 discussions with Professor Francois Laurent The excel¸ lent technical work of Delphine Maurice and Erwan Magueur is greatly appreciated This work was supported 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 Medicale (INSERM) 1370 References 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Results are shown as the average weight gain every days ± SEM rine-induced lipolysis Chronic (15 day) B [a] P exposure caused a significant weight gain and increased fat mass without detectable changes... and insulin receptors The mechanisms of adrenergic receptor signalling proceed via stimulatory and inhibitory G-proteins that control adenylate cyclase activity and thus cAMP formation Insulin