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. 2014 Jul 23;9(7):e102991.
doi: 10.1371/journal.pone.0102991. eCollection 2014.

Inhibitory action of benzo[α]pyrene on hepatic lipoprotein receptors in vitro and on liver lipid homeostasis in mice

Hamed Layeghkhavidaki  1 , Marie-Claire Lanhers  1 , Samina Akbar  1 , Lynn Gregory-Pauron  1 , Thierry Oster  1 , Nathalie Grova  2 , Brice Appenzeller  2 , Jordane Jasniewski  3 , Cyril Feidt  1 , Catherine Corbier  1 , Frances T Yen  4
Affiliations

Inhibitory action of benzo[α]pyrene on hepatic lipoprotein receptors in vitro and on liver lipid homeostasis in mice

Hamed Layeghkhavidaki et al. PLoS One. .

Abstract

Background: Dyslipidemia associated with obesity often manifests as increased plasma LDL and triglyceride-rich lipoprotein levels suggesting changes in hepatic lipoprotein receptor status. Persistent organic pollutants have been recently postulated to contribute to the obesity etiology by increasing adipogenesis, but little information is available on their potential effect on hepatic lipoprotein metabolism.

Objective: The objective of this study was to investigate the effect of the common environmental pollutant, benzo[α]pyrene (B[α]P) on two lipoprotein receptors, the LDL-receptor and the lipolysis-stimulated lipoprotein receptor (LSR) as well as the ATP-binding cassette transporter A1 (ABCA1) using cell and animal models.

Results: LSR, LDL-receptor as well as ABCA1 protein levels were significantly decreased by 26-48% in Hepa1-6 cells incubated (<2 h) in the presence of B[α]P (≤1 µM). Real-time PCR analysis and lactacystin studies revealed that this effect was due primarily to increased proteasome, and not lysosomal-mediated degradation rather than decreased transcription. Furthermore, ligand blots revealed that lipoproteins exposed to 1 or 5 µM B[α]P displayed markedly decreased (42-86%) binding to LSR or LDL-receptor. B[α]P-treated (0.5 mg/kg/48 h, i.p. 15 days) C57BL/6J mice displayed higher weight gain, associated with significant increases in plasma cholesterol, triglycerides, and liver cholesterol content, and decreased hepatic LDL-receptor and ABCA1 levels. Furthermore, correlational analysis revealed that B[α]P abolished the positive association observed in control mice between the LSR and LDL-receptor. Interestingly, levels of other proteins involved in liver cholesterol metabolism, ATP-binding cassette transporter G1 and scavenger receptor-BI, were decreased, while those of acyl-CoA:cholesterol acyltransferase 1 and 2 were increased in B[α]P-treated mice.

Conclusions: B[α]P demonstrates inhibitory action on LSR and LDL-R, as well as ABCA1, which we propose leads to modified lipid status in B[α]P-treated mice, thus providing new insight into mechanisms underlying the involvement of pollutants in the disruption of lipid homeostasis, potentially contributing to dyslipidemia associated with obesity.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of B[α]P on LSR, LDL-R and ABCA1 protein and mRNA levels in Hepa1-6 cells.
A) Hepa1-6 cells were incubated at 37°C for up to 2 h in the absence or presence of the indicated concentrations of B[α]P, after which cell lysates and were prepared as described in Methods. Representative immunoblots (for LSR and ABCA1, results of 1-h incubation are shown; for LDL-R, results for 2-h incubation are shown) and corresponding densitometric analyses using β-tubulin as internal control are shown (one-way ANOVA, *P<0.05, **P<0.01, as compared to 0 µM B[α]P, n = 3 different cell preparations using duplicate or triplicate wells). B) mRNA levels of LSR, LDLR and ABCA1 were determined in cells incubated with the indicated concentrations of B[α]P using real-time PCR, as described in Materials and Methods. Results are shown (n = 4 in triplicate) of LSR, LDLR and ABCA1 mRNA expression relative to HPRT, used as reference housekeeping gene. It should be noted that there was no significant changes in HPRT expression under the different conditions (**P<0.01 as compared to 0 µM B[α]P).
Figure 2
Figure 2. Lactacystin effect on B[α]P-induced decrease of LSR, LDL-R and ABCA1 protein levels in Hepa1-6 cells.
Hepa1-6 cells were preincubated for 1 h at 37°C with 10 µM lactacystin, followed by 1-h incubation with 0.1 µM B[α]P with lactacystin still maintained in the cell medium. Immunoblots were performed on cell lysates, to detect LSR, LDL-R and ABCA1, and are shown with corresponding densitometric analyses (two-way ANOVA, *P<0.05, **P<0.01, vs cells incubated in absence of lactacystin and B[α]P; # P<0.05 vs cells incubated with lactacystin alone, n = 3 different wells per treatment).
Figure 3
Figure 3. Chloroquine effect on B[α]P-induced decrease of LSR, LDL-R protein levels in Hepa1-6 cells.
Hepa1-6 cells were preincubated for 2 h at 37°C with 25 µM chloroquine, followed by 1-h incubation with 0.1 µM B[α]P with chloroquine still maintained in the cell medium. Immunoblots were performed on cell lysates, to detect LSR and LDL-R, and are shown with corresponding densitometric analyses (two-way ANOVA, *P<0.05, **P<0.01, vs cells incubated in absence of chloroquine and B[α]P; ## P<0.01 vs cells incubated with chloroquine alone, n = 3 different wells per treatment).
Figure 4
Figure 4. B[α]P effect on (A) body weight gain, (B) food intake, (C) plasma lipids, (D) lipoprotein profile in mice.
Male 11 week-old C57BL/6J mice were i.p. injected every 48 h with vehicle alone (□, n = 9) or vehicle containing 0.5 mg/kg B[α]P (▪, n = 9) from day 1 to day 15 and monitored for (A) weight gain (two-way ANOVA, *P<0.05 B[α]P group as compared to control group), and (B) food intake. C) Plasma total cholesterol (TC) and triglycerides (TG) were measured on Day 0 and Day 16 in plasma of 4-h fasted animals (two-way ANOVA*P<0.05, **P <0.01 B[α]P Day 16 vs control Day 16, # P<0.05; B[α]P Day 16 vs B[α]P Day 0). D) Lipoprotein profiles were obtained from pooled plasma samples obtained on day 16 using gel filtration chromatography as described in Materials and Methods. Fractions were analyzed for TC (upper panel) and TG (lower panel) content. The elution of TG-rich lipoproteins (TGRL), LDL and HDL are indicated by arrows.
Figure 5
Figure 5. Effect of B[α]P on mouse hepatic LDL-R, LSR, and ABCA1 protein and mRNA levels.
A) Immunoblots to detect LDL-R, LSR, and ABCA1 were performed on protein solubilized from liver total membranes isolated from control and B[α]P-treated mice. Blots are shown with corresponding densitometric analysis (Student's t-test, *P<0.05, **P<0.01 B[α]P as compared to control groups, n = 7 for each group). B) mRNA levels of liver LSR, LDLR and ABCA1 isolated from control and B[α]P-treated mice were determined using real-time PCR, as described in Materials and Methods. Results are shown for LSR, LDLR and ABCA1 mRNA expression relative to HPRT, used as reference housekeeping gene. There was no significant changes in HPRT expression under the different conditions (**P<0.01 as compared to control mice; triplicate determinations of n = 4 per group).
Figure 6
Figure 6. Scatter plot between hepatic LSR and LDL-R in mice.
Distribution of individual values for liver membrane LSR and LDL-R protein levels are shown for mice from control (□) and B[α]P-treated (▪) groups.
Figure 7
Figure 7. Effect of B[α]P on lipoprotein binding to (A) LSR and (B) LDL-R.
Solubilized proteins from rat liver plasma membranes were separated on SDS-PAGE under non-reducing conditions and immobilized onto nitrocellulose membrane. For LSR, nitrocellulose membranes were pre-incubated with 0.8 mM oleate in order to activate the LSR complex. Strips were incubated at 37°C for 1 h with VLDL or LDL pre-incubated with 0, 1, or 5 µM B[α]P Strips were then washed and immunoblots were performed using anti-apoB or anti-apoE antibodies to identify LDL or VLDL, as indicated. Immunoblots using anti-LSR or anti-LDL-R antibodies were performed to verify the presence of the LSR complex or LDL-R (left strip for A and B, respectively). Densitometric analysis and representative blots are shown here of ligand blots performed on two different preparations of lipoproteins.
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Grants and funding

These studies were supported by grants from the French Ministry of Higher Education and Research, and from the University of Lorraine SF (Structure Fédérative) EFABA. HL and SA were supported by thesis fellowships from the Avicenna Research Institute, Iran, and Pakistan Ministry of Research, respectively. The authors hereby state that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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