The endocrine disruptor DEHP and the ECS: analysis of a possible crosstalk

Abstract

Studies of the last decade associated the environmental contamination by di-(2-ethylhexyl)-phthalate (DEHP) with obesity and endocrine malfunction. DEHP was found to interact with several receptors - among them are receptors of the endocannabinoid system (ECS) with high expression levels in adipose tissue. Furthermore, the correlation for BMI and body fat to the serum endocannabinoid level raises the question if the obesogenic and endocrine-disrupting DEHP effects are mediated via the ECS. We therefore characterized the ECS in a human cell model of adipogenesis using the SGBS preadipocytes to subsequently investigate if DEHP exposure affects the intrinsic ECS. The receptors of the ECS and the endocannabinoid-metabolizing enzymes were upregulated during normal adipogenesis, accompanied by an increasing secretion of the adipokines adiponectin and leptin. DEHP affected the secretion of both adipokines but not the ECS, suggesting DEHP to alter the endocrine function of adipocytes without the involvement of the intrinsic ECS.

Keywords: DEHP; SGBS; adipocytes; endocannabinoid system; endocrine disruptor; leptin.

Figure 1

Adipokines, their receptors and the glucose transporters in the SGBS cell model during normal and DEHP-exposed adipogenesis. The secretion of adiponectin and leptin was measured (A). Additionally, the gene expression of their receptors, adiponectin receptor 2 (ADIPOR2) and leptin receptor (LepR) (B), and the glucose transporters GLUT1 and GLUT4 (C) was evaluated in SGBS cells during adipogenic differentiation with and without DEHP. Absolute mRNA expression is presented copy number per 1000 molecules TBP. n = 6 for secretion; n = 8 for mRNA expression; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 for comparing normal adipogenesis to day 0; ^#P ≤ 0.05 for comparing the unexposed and DEHP-exposed group.

Figure 2

The endocannabinoid system (ECS) in the SGBS cell model during normal and DEHP-exposed adipogenesis. Western blot analyses of CB₁, FAAH, MAGL, NAPE-PLD, DAGLalpha and DAGLbeta comparing unexposed and mature (day 8) DEHP-exposed SGBS cells were normalized to the endogenous reference beta-ACTIN and GAPDH, respectively (A). Gene and protein expression of the receptors transient receptor potential vanilloid 1 (TRPV1) (B) and the cannabinoid receptor 1 (CNR1, CB₁) (C), the enzymes fatty acid amide hydrolase (FAAH) (D), monoacylglycerol lipase (MAGL) (E), N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) (F) and diacylglycerol lipase alpha (DAGL) (G) were determined in SGBS cells with and without DEHP exposure. For DAGL, the protein expression of both isoforms was evaluated with DAGLalpha as blank columns and DAGLbeta as patterned columns within one figure (G). Absolute mRNA expression is presented copy number per 1000 molecules TBP. n ≥ 8 for gene expression; n ≥ 4 for protein expression; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 for comparing normal adipogenesis to day 0; ^#P ≤ 0.05 for comparing the unexposed and DEHP-exposed group.

Figure 3

The localization of the ECS in the SGBS cell model with and without DEHP treatment. Immunohistochemical staining of CB₁ (A), FAAH (B), MAGL (C), NAPE-PLD (D), DAGLalpha (E) and DAGLbeta (F) in SGBS cells at day 0, 4 and 8 after treatment with DMSO or DEHP (scale bar = 100 µm).