Combined transcriptomic-(1)H NMR metabonomic study reveals that monoethylhexyl phthalate stimulates adipogenesis and glyceroneogenesis in human adipocytes

Abstract

Adipose tissue is a major storage site for lipophilic environmental contaminants. The environmental metabolic disruptor hypothesis postulates that some pollutants can promote obesity or metabolic disorders by activating nuclear receptors involved in the control of energetic homeostasis. In this context, monoethylhexyl phthalate (MEHP) is of particular concern since it was shown to activate the peroxisome proliferator-activated receptor γ (PPARγ) in 3T3-L1 murine preadipocytes. In the present work, we used an untargeted, combined transcriptomic-(1)H NMR-based metabonomic approach to describe the overall effect of MEHP on primary cultures of human subcutaneous adipocytes differentiated in vitro. MEHP stimulated rapidly and selectively the expression of genes involved in glyceroneogenesis, enhanced the expression of the cytosolic phosphoenolpyruvate carboxykinase, and reduced fatty acid release. These results demonstrate that MEHP increased glyceroneogenesis and fatty acid reesterification in human adipocytes. A longer treatment with MEHP induced the expression of genes involved in triglycerides uptake, synthesis, and storage; decreased intracellular lactate, glutamine, and other amino acids; increased aspartate and NAD, and resulted in a global increase in triglycerides. Altogether, these results indicate that MEHP promoted the differentiation of human preadipocytes to adipocytes. These mechanisms might contribute to the suspected obesogenic effect of MEHP.

Figure 1

Transcriptomic effects of MEHP on human adipocytes. Human preadipocytes (n = 5) were differentiated in vitro into adipocytes for 11 days and then treated with MEHP (100 μM) or DMSO (0.1%, vehicle) for 24 h. (A) PCA analysis was conducted using expression data from the entire microarray (32 322 probesets). Black: DMSO, red: MEHP. (B, C) Gene ontology analyses were conducted on the 142 significantly upregulated and on the 85 downregulated transcripts, using DAVID (B) and KEGG (C) databases. P < 0.05 was used as cutoff for selecting significantly enriched biological pathways.

Figure 2

¹H NMR-based metabonomic investigation MEHP effects on human adipocytes. (A) Representative ¹H NMR spectra (600 MHz) derived from an aqueous extract of human adipocytes. (B) Plot of O-PLS-DA coefficients related to the discrimination between ¹H NMR spectra from aqueous extracts of human adipocytes treated with MEHP (n = 5, top) vs. DMSO (n = 5, bottom) for 48 h. O-PLS-DA parameters: R²X = 0.65, R²Y = 0.94, Q²Y = 0.73, p = 9 × 10^–3, one predictive and one orthogonal component. (C) Representative ¹H NMR spectra (600 MHz) derived from an organic extract of human adipocytes. uk: unknown compound. (D) Plot of O-PLS-DA coefficients related to the discrimination between ¹H NMR spectra from organic extracts of human adipocytes treated with MEHP (n = 5, top) vs. DMSO (n = 5, bottom) for 48 h. O-PLS-DA parameters: R²X = 0.93, R²Y = 0.98, Q²Y = 0.68, p = 0.03, one predictive and two orthogonal components. Leu: leucine; Ile: isoleucine; Val: valine; Glu: glutamate; Gln: glutamine; Asp: aspartate; TMA: trimethylamine; Cre: creatine; PC: phosphocholine; uk: unknown compound; PEA: phosphoethanolamine; NMN: N-methylnicotinamide; NAD: nicotinamide adenine dinucleotide; Tyr: tyrosine; Phe: phenylalanine.

Figure 3

Intracellular triglycerides in human adipocytes. Human preadipocytes (n = 5) were differentiated in vitro into adipocytes for 11 days and then treated with MEHP (100 μM) or DMSO (0.1%, vehicle) for 24 or 48 h. Black: DMSO, red: MEHP. *: P < 0.05 compared to DMSO-treated cells, Wilcoxon signed-rank test.

Figure 4

MEHP induced early transcriptomic alterations in human adipocytes. mRNA expression of genes involved in adipocyte differentiation (A), glycerol metabolism (B), and other lipid-related metabolic pathways (C) in MEHP-treated (100 μM) or DMSO-treated (0.1%) human adipocytes. Data represent fold changes compared to DMSO-treated human adipocytes (mean ± SEM, n = 5). *: P < 0.05, **: p < 0.01 compared to DMSO-treated cells, Wilcoxon signed-rank test.

Figure 5

MEHP induced early metabolic alterations in human adipocytes. (A) General description of the glyceroneogenesis and NEFA reesterification pathways in adipocytes (adapted from ref (25)). (B) PEPCK-C protein expression in human adipocytes treated with MEHP (100 μM) or DMSO (0.1%) for 18 h. Representative Western blot. (C) NEFA release in the culture medium of human adipocytes treated with MEHP (100 μM) or DMSO (0.1%) for 18 h and fasted for 3 h. Results represent a mean ± SEM for three replicates per individual (n = 5). *: P < 0.05 compared to DMSO-treated cells, Wilcoxon signed-rank test.