Exposure to p,p'-DDE enhances differentiation of 3T3-L1 preadipocytes in a model of sub-optimal differentiation

Abstract

The incidence of obesity is increasing worldwide at an alarming rate. Recently, exposure to environmental contaminants, especially organochlorines such as p,p'-dichlorodiphenyldichloroethylene (DDE), has been implicated as a possible causative factor in the increasing obesity epidemic. The objective of this study was to evaluate the ability of DDE to alter adipogenesis in a model of sub-optimal differentiation. 3T3-L1 preadipocytes were induced to differentiate in the presence of DDE (0.01-100μM) using a sub-optimal differentiation cocktail. Eight days after the initiation of differentiation, adipogenesis was assessed through neutral lipid staining, triglyceride accumulation, and expression of markers of terminal differentiation. Exposure to DDE induced a concentration dependent increase in intracellular neutral lipid accumulation as determined by Oil Red O staining and triglyceride assay. Alterations in lipid accumulation were accompanied by upregulation of genetic markers of differentiation. DDE (10μM) enhanced expression of fatty acid binding protein 4 and Sterol regulatory element-binding protein-1c at the 2.5 and 20μM concentrations. DDE (2.5, 10, and 20μM) induced upregulation of leptin and fatty acid synthase, as compared to sub-optimal vehicle control (0.05% ethanol). Our results indicate that DDE is capable of enhancing adipogenesis and intracellular lipid accumulation in 3T3-L1 cells through upregulation of molecular targets responsible for lipid storage.

Keywords: 3T3-L1; Adipogenesis; Organochlorine insecticides; p,p′-DDE.

Fig. 1.

3T3-L1 exposure protocol. Sub-optimal differentiation protocol for 3T3-L1 cells.

Fig. 2.. DDE enhances neutral lipid accumulation.

Representative photomicrograph of adipocytes differentiated using sub-optimal cocktail in the presence of vehicle (A), 20 μM DDE (B) compared with fully differentiated adipocytes (C). Cells were stained with Oil Red O and dye was extracted and measured spectrophotometrically at 520 nm (D). Data (n = 4–7) are expressed as mean fold change compared to vehicle +/− standard error (SEM). Statistical significance was determined by one-way ANOVA with Duncan’s Studentized range test for multiple comparisons. Means with different letters indicate significant difference at P < 0.05.

Fig. 3.. DDE enhances triglyceride accumulation.

Data (n = 5) are expressed as mean triglyceride (mg TG/mg protein) +/− standard error (SEM). Triglyceride content was extracted and measured using commercial reagents. Statistical significance was determined by one-way ANOVA with Duncan’s Studentized range test for multiple comparisons. Means with different letters indicate significant difference at P< 0.05

Fig. 4.. DDE upregulates mediators of differentiation.

Data (n = 3) are expressed as mean fold change compared to undifferentiated cells (CS) +/− standard error (SEM). Differentiating 3T3 cells were exposed to increasing concentrations of DDE (2.5,10, and 20 μM), standard differentiation cocktail (FULL), or Vehicle (V; 0.05% ethanol). Markers and mediators of differentiation were measured by RT-qPCR. Statistical significance was determined by one-way ANOVA. Means with different symbols indicate significant difference at P< 0.05.