DEHP deregulates adipokine levels and impairs fatty acid storage in human SGBS-adipocytes

Abstract

DEHP is a plasticizer which has been used in plastic products of everyday use for decades. Studies in mice and murine cell culture models identified DEHP as an endocrine disruptor that may also act as an obesogen. As this is of high concern in respect of the worldwide obesity epidemic, our aim is the translation of these findings into a human model system. On the basis of DOHaD, we investigated the influence of an environmentally relevant dose of DEHP [50 µg/ml] on adipogenesis in the human cell culture model SGBS. Pre-adipocytes were exposed to DEHP and differentiated into mature adipocytes. At different stages of differentiation, markers of adipogenesis like GLUT4, FABP4, LPL and PPARs, and of signaling pathways like AMPK/ACC2, JAK/STAT and MAPK were analyzed. Functional markers like adipokine secretion and triglyceride content as well as ROS production were measured in mature adipocytes. We found significantly lower expression levels of adipogenic markers, a reduction in lipid accumulation, higher leptin- and reduced adiponectin levels in the supernatant of treated adipocytes. Moreover, ROS production was significantly elevated after DEHP-exposure. In conclusion, DEHP led to lower grade of adipogenic differentiation in human SGBS-adipocytes under the chosen conditions.

Figure 1

Determination of MEHP in cell supernatants (A) and cell lysates (B) by HPLC: The SGBS cells were exposed to DEHP [50 µg/ml] for the whole period of adipogenic differentiation (d0–d14). Samples were taken at d4 and d14 for analysis by HPLC. Statistics: a = DEHP versus corresponding DMSO control; b = DEHP d4 versus DEHP d14; c = DMSO d4 versus DMSO d14; ANOVA (Student-Newman-Keuls Method), significance: p > 0.05; N = 4, n = 1 (4 pooled wells each).

Figure 2

Analysis of ROS-production after DEHP-exposure: SGBS cells were exposed to DEHP from d0–d4 and subsequently differentiated into adipocytes. For the ROS-measurement (A), the medium was changed to DMEM/F12 with 10 µM H2DCFDA at d4 and incubated for 30 min. As the positive assay control H₂O₂ (25 µM) was used, as well as an untreated control. Statistics: a = DEHP/H₂O₂ versus control; b = DEHP/H₂O₂ versus DMSO control; ANOVA (Student-Newman-Keuls Method); N = 2, n = 9.

Figure 3

Triglyceride content of adipocytes after DEHP-exposure: SGBS cells were exposed to DEHP from d0–d4 and subsequently differentiated into adipocytes. For the triglyceride measurement (A) the cells were lysed at d8 in a special lysis buffer. Statistics: Student’s t-test (Wilcoxon rank-sum test); N = 2, n = 8. Light microscopy with a 4-fold magnification of Oil Red O stained adipocytes at d8 (B); scale bar = 100 µm.

Figure 4

Adiponectin and leptin and their receptors after DEHP-exposure: SGBS cells were exposed to DEHP from d0-d4 and subsequently differentiated into adipocytes. For the adiponectin (A) and leptin ELISA (B) the cells were analyzed at d8. Statistics: Student’s t-test (Wilcoxon rank-sum test); N = 6, n = 1 (4 pooled wells each). For the analysis of mRNA expression of ADIPOR2 (C) and LEPR (D) samples were taken at d0, d4 and d8 of differentiation. The housekeeping gene for standardsation was the TATA-box binding protein (TBP). The caption “mRNA expression/mol. TBP” means expression of target gene per molecule(s) TBP. Statistics: Student’s t-test (Wilcoxon rank-sum test); N = 8, n = 1 (4 pooled wells each).

Figure 5

Impact of DEHP on AMPK/ACC2 pathway: SGBS cells were exposed to DEHP from d0-d4 and subsequently differentiated into adipocytes. For western blot analysis of pAMPK/AMPK (A), pACC2/ACC2 (B), pACC2 (C), ACC2 (D) and CPT1 (F) samples were taken at d0, d4 and d8 of differentiation. Statistics: Student’s t-test (Wilcoxon rank-sum test); N ≥ 4, n = 1 (4 pooled wells each). For the analysis of mRNA expression of ACC2 (E) samples were taken at d0, d4 and d8 of differentiation. Statistics: Student’s t-test (Wilcoxon rank-sum test); N = 8, n = 1 (4 pooled wells each).

Figure 6

Impact of DEHP on JAK/STAT and ERK1/2 pathway: SGBS cells were exposed to DEHP from d0-d4 and subsequently differentiated into adipocytes. For western blot analysis of pSTAT3α/STAT3α (A), SOCS3 (B), pERK1/ERK1 (C), pERK2/ERK2 (D) samples were taken at d0, d4 and d8 of differentiation. Statistics: Student’s t-test (Wilcoxon rank-sum test); N ≥ 4, n = 1 (4 pooled wells each).

Figure 7

Protein amount of PPARα and PPARγ after DEHP-exposure: SGBS cells were exposed to DEHP from d0-d4 and subsequently differentiated into adipocytes. For western blot analysis of PPARα (A) and PPARγ (B) samples were taken at d0, d4 and d8 of differentiation. Statistics: Student’s t-test (Wilcoxon rank-sum test); N ≥ 4, n = 1 (4 pooled wells each).

Figure 8

Expression of adipocyte markers after DEHP exposure: SGBS cells were exposed to DEHP from d0–d4 and subsequently differentiated into adipocytes. For the analysis of mRNA expression of FABP4 (A), LPL (B), GLUT4 (C), CD36 (D), LIPE (E) and ATGL (F) samples were taken at d0, d4 and d8 of differentiation; The housekeeping gene for standardization was the TATA-box binding protein (TBP) and the abbreviation “mol. TBP” means molecules TBP. Statistics: Student’s t-test N ≥ 4, n = 1 (4 pooled wells).

Figure 9

Measurement of free glycerol in SGBS derived adipocytes: The cells have been differentiated as described before and have been treated with DEHP and DMSO as control as followed: treatment a: exposure from d0–d4 and sampling at d4; treatment b: exposure at d4 for 3h and sampling at d4; treatment c: exposure from d0–d4 and sampling at d8. Statistics: Student’s t-test, N = 4, n = 3.

Figure 10

Immunohistochemistry for GLUT4 in SGBS-adipocytes: SGBS cells were exposed to DEHP from d0–d4 and subsequently differentiated into adipocytes. For the analysis of GLUT4 localization within the adipocytes, the cells have been fixed with PFA at d8, stained for GLUT4 (HRP-conjugated antibody – brown staining) and counterstained with hematoxylin (blue staining). The figure shows representative micrographs at × 200 magnification of DEHP treated adipocytes and DMSO controls, as well as the negative control without GLUT4 but secondary antibody. The scale bar indicates 50 µm.