Adipogenic Effects and Gene Expression Profiling of Firemaster® 550 Components in Human Primary Preadipocytes

Abstract

Background: Exposure to flame retardants has been associated with negative health outcomes including metabolic effects. As polybrominated diphenyl ether flame retardants were pulled from commerce, human exposure to new flame retardants such as Firemaster® 550 (FM550) has increased. Although previous studies in murine systems have shown that FM550 and its main components increase adipogenesis, the effects of FM550 in human models have not been elucidated.

Objectives: The objectives of this study were to determine if FM550 and its components are active in human preadipocytes, and to further investigate their mode of action.

Methods: Human primary preadipocytes were differentiated in the presence of FM550 and its components. Differentiation was assessed by lipid accumulation and expression of peroxisome proliferator-activated receptor γ (PPARG), fatty acid binding protein (FABP) 4 and lipoprotein lipase (LPL). mRNA was collected for Poly (A) RNA sequencing and was used to identify differentially expressed genes (DEGs). Functional analysis of DEGs was undertaken in Ingenuity Pathway Analysis.

Results: FM550 triphenyl phosphate (TPP) and isopropylated triphenyl phosphates (IPTP), increased adipogenesis in human primary preadipocytes as assessed by lipid accumulation and mRNA expression of regulators of adipogenesis such as PPARγ, CCAAT enhancer binding protein (C/EBP) α and sterol regulatory element binding protein (SREBP) 1 as well as the adipogenic markers FABP4 LPL and perilipin. Poly (A) RNA sequencing analysis revealed potential modes of action including liver X receptor/retinoid X receptor (LXR/RXR) activation, thyroid receptor (TR)/RXR, protein kinase A, and nuclear receptor subfamily 1 group H members activation.

Conclusions: We found that FM550, and two of its components, induced adipogenesis in human primary preadipocytes. Further, using global gene expression analysis we showed that both TPP and IPTP likely exert their effects through PPARG to induce adipogenesis. In addition, IPTP perturbed signaling pathways that were not affected by TPP. https://doi.org/10.1289/EHP1318.

Figure 1.

Effects of Firemaster 550®, and its components TPP and IPTP, on lipid accumulation in differentiating human preadipocytes. Human primary preadipocytes were induced to differentiate for 14 d in the presence of MI and $1\mathrm{\mu }\mathrm{M}$ dexamethasone (MID) or MI and $5\mathrm{\mu }\mathrm{M}$ troglitazone (MIT) supplemented with either FM550 ($0–200\mathrm{\mu }\mathrm{M}$) or its components (TPP, IPTP, TBB, and TBPH; $0–20\mathrm{\mu }\mathrm{M}$). At day 14 of differentiation, lipid accumulation was quantified by Nile red staining normalized to DAPI. Data from all treatments, normalized to their respective control condition (MID or MIT), are graphically presented as $\text{mean}\pm \text{SEM}$ of four separate donor samples. *$p<0.05$ compared with respective controls, as assessed by one-way ANOVA with Dunnett’s post hoc tests.

Figure 2.

Effects of Firemaster 550®, and its components TPP and IPTP, on FABP4 protein expression in the presence of dexamethasone or troglitazone. Human primary preadipocytes were induced to differentiate for 14 d in the presence of MI and $1\mathrm{\mu }\mathrm{M}$ dexamethasone (MID) or MI and $5\mathrm{\mu }\mathrm{M}$ troglitazone (MIT) supplemented with either FM550 ($0–200\mathrm{\mu }\mathrm{M}$) (A), or its components (TPP and IPTP; $0–20\mathrm{\mu }\mathrm{M}$) (B, C). Equal amounts of solubilized cellular proteins were separated by SDS-PAGE and immunoblotted with antibodies against FABP4 and ACTB as a loading control. Densitometric data from four separate donor samples, normalized to loading control, are graphically presented as $\text{means}\pm \text{SEM}$. *$p<0.05$ compared with respective controls, as assessed by one-way ANOVA with Dunnett’s post hoc tests.

Figure 3.

Effects of TPP and IPTP on the mRNA expression levels of transcriptional regulators of adipogenesis and adipogenic markers in differentiating human preadipocytes. Human primary preadipocytes were induced to differentiate for 4, 6, 9, and 12 d in the presence of $1\mathrm{\mu }\mathrm{M}$ dexamethasone (MID) supplemented with either TPP ($20\mathrm{\mu }\mathrm{M}$) or IPTP ($20\mathrm{\mu }\mathrm{M}$). RNA was isolated and the mRNA levels of adipogenic markers, PPARG (A), CEBPA (B), FABP4 (C), LPL (D), PLIN1 (E), and SREBF1 (F) were quantified by real-time qPCR. Levels were normalized to endogenous ACTB mRNA, and expressed as a fold over the control condition (MID) for each time point. Results from five separate donor samples are graphically presented as $\text{means}\pm \text{SEM}$. #$p<0.05$ for TPP-treated cells, and *$p<0.05$ for IPTP-treated cells compared with MID controls, as assessed by one-way ANOVA with Dunnett’s post hoc tests.

Figure 4.

Differentially expressed genes (DEGs) affected by TPP, IPTP, and troglitazone. Human primary preadipocytes were differentiated in the presence of MID supplemented with $20\mathrm{\mu }\mathrm{M}$ TPP, $20\mathrm{\mu }\mathrm{M}$ IPTP, or $5\mathrm{\mu }\mathrm{M}$ troglitazone. At day 6 of differentiation, RNA was collected from five donors and used for RNA-seq analysis. The overlap of significant ($-1.5\le \text{fold}\text{change}\ge 1.5$, FDR $p<0.05$) DEGs between all treatments are depicted in a Venn diagram (A). Venn diagrams were produced using the on-line tool Venny (http://bioinfogp.cnb.csic.es/tools/venny/). (B) Hierarchical cluster analysis of the fold change for all DEGs from IPTP, troglitazone, TPP having an FDR of $p<0.05$ and fold change $>1.5$. The distance is metric $1-$ the Spearman correlation, values greater than 5 were truncated strictly for the color scale.

Figure 5.

Validation of selected differentially expressed genes (DEGs) by RT-qPCR. Human primary preadipocytes were differentiated in the presence of MID supplemented with $20\mathrm{\mu }\mathrm{M}$ TPP or $20\mathrm{\mu }\mathrm{M}$ IPTP. At day 6 of differentiation, RNA was collected for RNA-seq analysis. The mRNA levels of select DEGs from RNA-seq analysis were quantified by RT-qPCR. Levels were normalized to endogenous ACTB mRNA, and expressed as a fold over the control condition (MID) for each treatment. Results from five separate donor samples are graphically presented as $\text{means}\pm \text{SEM}$. *$p<0.05$, **$p<0.01$, and ***$p<0.001$ for TPP- and IPTP- treated samples compared with control; #$p<0.05$, ##$p<0.01$, and ###$p<0.001$ for TPP-treated compared with IPTP-treated samples, as assessed by one-way ANOVA with Tukey’s post hoc tests.