Download Comparative analysis of mono(2-ethylhexyl)phthalate (MEHP) and

Comparative microarray analyses of mono(2-ethylhexyl)phthalate impacts on fat
cell bioenergetics and adipokine network
Subtitle: MEHP enhances energy metabolism-related gene expression in adipocytes
Huai-chih Chiang,a Chih-Hong Wang,b Szu-Ching Yeh,a Yi-Hua Lin,a Ya-Ting Kuo,a
Chih-Wei Liao,a Feng-Yuan Tsai,a Wei-Yu Lin,b Wen-Han Chuang,b and Tsui-Chun
Tsou,a,*
a
National Institute of Environmental Health Sciences, National Health Research
Institutes, Zhunan, Miaoli 350, Taiwan
b
Department of Biological Science and Technology, National Chiao Tung University,
Hsinchu 300, Taiwan
* Corresponding author: Tsui-Chun Tsou
National Institute of Environmental Health Sciences, National Health Research
Institutes, 35 Keyan Road, Zhunan, Miaoli 350, Taiwan; Tel.: +886-37-246-166 ext.
36511; fax: +886-37-587-406; E-mail: tctsou@nhri.org.tw
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Abstract
Cellular accumulation of mono(2-ethylhexyl)phthalate (MEHP) has been
recently demonstrated to disturb fat cell energy metabolism; however, the underlying
mechanism remained unclear. The study aimed to determine how MEHP influenced
fat cell transcriptome and how the changes might contribute to bioenergetics. Because
of the pivotal role of PPARγ in energy metabolism of fat cells, comparative
microarray analysis of gene expression in 3T3-L1 adipocytes treated with both MEHP
and rosiglitazone was performed. Pathway enrichment analysis and gene ontology
(GO) enrichment analysis revealed that both treatments caused up-regulation of genes
involved in PPAR signaling/energy metabolism-related pathways and down-regulation
of genes related to adipokine/inflammation signals. MEHP/rosiglitazone-treated
adipocytes exhibited increased levels of lipolysis, glucose uptake, and glycolysis; the
gene expression profiles provided molecular basis for the functional changes.
Moreover, MEHP was shown to induce nuclear translocation and activation of PPARγ.
The similarity in gene expression and functional changes in response to MEHP and
rosiglitazone suggested that MEHP influenced bioenergetics and adipokine network
mainly via PPARγ. Importantly, adipokine levels in C57BL/6J mice with
di(2-ethylhexyl)phthalate (DEHP) treatments provided in vivo evidence for
microarray results. On the basis of correlation between gene expression and functional
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assays, possible involvements of genes in bioenergetics of MEHP-treated adipocytes
were proposed.
Keywords: phthalates; endocrine disruptor; energy metabolism; PPARγ; adipocytes
Abbreviations: βAR, β-adrenergic receptor; DAVID, Database for Annotation,
Visualization and Integrated Discovery; DEHP, di(2-ethylhexyl)phthalate; FABPs,
fatty acid-binding proteins; GO, gene ontology; HFD, high-fat diet; KEGG, Kyoto
Encyclopedia of Genes and Genomes; MEHP, mono(2-ethylhexyl)phthalate; Mlycd,
malonyl-CoA decarboxylase; NCD, normal chow diet; NEFA, non-esterified fatty
acids; OCR, oxygen consumption rate; PANTHER, Protein Analysis through
Evolutionary Relationships; PCA, principal component analysis; PDC, pyruvate
dehydrogenase complex; PDH, pyruvate dehydrogenase; PPRE, PPAR response
element; REVIGO, Reduce + Visual Gene Ontology; T2DM, type 2 diabetes mellitus;
WAT, white adipose tissue
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Introduction
Phthalates, commonly used as softeners in plastics, are the most ubiquitous
environmental pollutants. Phthalates are easily released from plastics due to a lack of
covalent bonds between plastics and phthalates. Accumulating epidemiological
studies have revealed the association between phthalate exposure and prevalence of
metabolic diseases including obesity and its complications, i.e., type 2 diabetes
mellitus (T2DM) and insulin resistance (James-Todd et al. 2012; Kim et al. 2013;
Wang et al. 2013); the association is evident especially for di(2-ethylhexyl)phthalate
(DEHP). In animal studies, DEHP accelerates atherosclerosis in apolipoprotein
E-deficient mice (Zhao et al. 2014) and maternal exposure to DEHP deregulates blood
pressure, adiposity, cholesterol metabolism, and social interaction in mouse offspring
(Lee et al. 2015). These experimental and epidemiological studies strongly suggests
that phthalate exposure is a critical health issue.
Adipose tissue is a metabolically dynamic organ, acting as a regulator in
maintenance of fatty acid homeostasis and adipokine network. Upon ingestion of
phthalates in human body, the majority of phthalates (≈90%) is excreted in the first 24
h and monoesters are the major metabolites (Koch et al. 2012). However, adipose
tissue is found to be major storage sites for lipophilic pollutants, including phthalates.
Phthalate accumulation in adipose tissue has been demonstrated in both the general
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human population (Zhang et al. 2003) and the phthalate-treated rats (Zeng et al. 2013).
Mono(2-ethylhexyl)phthalate (MEHP), the primary metabolite of DEHP, promotes
differentiation of preadipocytes and to induce obesity in mice, possibly via PPARγ
activation (Feige et al. 2007; Campioli et al. 2011; Hao et al. 2012). Disturbed lipid
metabolism has been observed in both human primary adipocytes with MEHP
treatments (Ellero-Simatos et al. 2011) and neonatal rat cardiomyocytes with DEHP
treatments (Posnack et al. 2012). Our recent study found that cellular MEHP
accumulation disturbed energy metabolism, including lipolysis, glucose uptake, and
glycolysis, in 3T3-L1 adipocyte (Chiang et al. 2016). These studies provide clear
evidence reveling that MEHP not only enhances adipogenic differentiation of
preadipocytes via PPARγ but also markedly interferes with energy metabolism in
mature adipocytes.
Fat cells proliferate in childhood, but not in adulthood; fat cell numbers are set
and generally stay constant through adult life (Spalding et al. 2008). Therefore, for
both fat cell biology and obesity development, the influences of phthalates on
preadipocytes and mature adipocytes could be fundamentally different. Using a
special phthalate exposure procedure during adipogenesis, we demonstrated effects of
MEHP on energy metabolism in mature adipocytes (Chiang et al. 2016). However, the
possible involvement of PPARγ in the process remains unclear, mainly due to the
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difficulty in conducting DNA/RNA transfection of mature adipocytes with the
standard lipid-based techniques. Also, inhibition of PPARγ with specific inhibitors
usually results in poor adipogenesis, which makes the following functional assays of
mature adipocytes impossible. Through observations of differential expression for
thousands of genes across multiple conditions, microarray analysis can be a promising
technique to address the roles of PPARγ in the MEHP-induced energy disturbance in
mature adipocytes.
The study combines both in vitro cell models (microarray analysis and functional
assays) and in vivo animal models to determine phthalate effects on adipocytes,
mainly focusing on metabolism of glucose and fatty acids for energy production and
adipokine network. To the best of our knowledge, this is the first study demonstrating
comprehensive expression profiles of genes involved in energy metabolism pathways,
which clearly highlights several scientific interests for future phthalate studies.
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Materials and methods
Chemicals and cells
The information on chemicals and cells was described in detail in Supplemental
Material.
Induction of adipogenesis and MEHP/rosiglitazone treatments
Adipogenesis of mouse 3T3-L1 cells was induced following the standard
procedure as described (Hsu et al. 2010). Figure 1a shows the procedures of
adipogenesis and MEHP/rosiglitazone treatments and the details were described in
Supplemental Material.
Microarray analysis
Following the standard DIM induction from D0 to D5, 3T3-L1 adipocytes were
treated with DMSO (0.1%) (the vehicle control), MEHP (100 μM), or rosiglitazone (2
µM) from D5 to D11 (Figure 1a). RNA samples were collected at D5 (Control_D5),
D8 (DMSO_D8, MEHP_D8, and Rosig._D8), and D10 (DMSO_D10, MEHP_D10,
and Rosig._D10) from three independent experiments. RNA samples were prepared
with RNAspin Mini Kit (25-0500-87) (GE Healthcare Life Sciences). RNA samples
(3 µg) were sent to the Microarray Core Laboratory at NHRI for cDNA
synthesis/labeling and then were hybridized onto the GeneChip® Mouse Gene 2.0 ST
Array (Affymetrix, Inc.); the array contains a total of 35,240 RefSeq transcripts and
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26,515 RefSeq (Entrez) genes. Data analyses were conducted by software of Partek
Genomics Suite (http://www.partek.com/); principal component analysis (PCA) was
used to simplify the analysis and visualization of multidimensional microarray data
sets and relevance network was used to determine the linkage between two treatments.
Venn diagrams were performed by GeneVenn (http://genevenn.sourceforge.net/), a
web application for comparison and visualization of microarray data.
Pathway enrichment analysis was performed by using the Database for
Annotation, Visualization and Integrated Discovery (DAVID) online analysis tool
(http://david.abcc.ncifcrf.gov/) with BioCarta, Kyoto Encyclopedia of Genes and
Genomes (KEGG), and Protein Analysis through Evolutionary Relationships
(PANTHER) pathway databases. The pathways with fold enrichment ≥1.5 (vs.
DMSO_D10) were selected. Gene ontology (GO) enrichment analysis was performed
by using the DAVID online analysis tool based on the category of biological processes.
The pathways with fold enrichment ≥1.5 (vs. DMSO_D10) were selected as the
candidate GO: biological process terms. Following removal of redundant GO terms
by using Reduce + Visual Gene Ontology (REVIGO) (http://revigo.irb.hr/), the top
ten GO: biological process terms were shown. Heatmaps were visualized by
MultiExperiment Viewer downloaded from the TM4 microarray software suite
(http://www.tm4.org/). The color-coded scale for the normalized expression value,
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log2 (fold change), is shown at the bottom of each figure (the red indicates
up-regulated genes and the green indicates the down-regulated genes). qPCR was
used to validate microarray data as described in detail in Supplemental Material.
Determination of glycerol, triglycerides, non-esterified fatty acids (NEFA), and
lactate
Cellular glycerol, triglycerides, and NEFA were extracted as previously described
(Samuel et al. 2013). Briefly, to completely dissolve the intracellular lipids, cells were
collected in 5% NP-40 for lysis at 90°C for 5 min and then cool to RT for at least
three times. Following centrifugation at 15,000 rpm for 5 min to remove cell debris,
supernatants were collected for analysis by using glycerol assay (GY105) (Randox),
triglycerides assay (TR213) (Randox), NEFA-C assay (279-75401) (Wako Pure
Chemical).
Lipolytic activity in adipocytes was determined by glycerol release assay. For
analysis of the basal lipolytic activity, cells were replenished with fresh DMEM-HG
with 10% FBS and cultured for another 24 h, the basal lipolytic activity was
determined by levels of glycerol released in culture medium. For analysis of the
β-adrenergic receptor (βAR)-induced lipolytic activity, following starvation in
DMEM-LG with 0.1% BSA for 2 h, cell were treated with the βAR agonist
isoproterenol (1 µM) for 4 h and culture medium was collected for analysis by using
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glycerol assay kit (GY105) (Randox). Moreover, culture medium was collected at
D11 for determination of lactate production. Following centrifugation at 15,000 rpm
for 5 min to remove cell debris, supernatants were collected for analysis by using
lactate assay (LC2389) (Randox).
Cellular uptake of exogenous glucose and palmitate
To determine cellular uptake of exogenous glucose and NEFA, adipocytes were
subjected to glucose/pyruvate-free DMEM supplemented with glucose (5 mM) or
palmitate
(BSA-conjugated)
(200
μM),
a
model
NEFA.
Preparation
of
BSA-conjugated palmitate was described in Supplemental Material. At the indicated
time points, 100 μl of culture medium samples were collected. Following
centrifugation at 10,000 rpm for 5 min to remove cell debris, supernatants were kept
on ice for the following determination of glucose and palmitate by using glucose
assay (GL2623) (Randox) and NEFA-C assay (279-75401) (Wako Pure Chemical),
respectively.
Animal treatments
Male C57BL/6J mice, aged 4 weeks purchased from the National Laboratory
Animal Center (Taipei, Taiwan), were housed in the Laboratory Animal Center in
National Health Research Institutes. The mice were kept in individually ventilated
cages, at controlled temperatures of 25 ± 1 °C, with a 12 : 12 light-dark cycle (lights
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on at 6:00 AM), and on ad libitum food and water intake. Following acclimation for 2
weeks, the mice were fed with normal chow diet (NCD) (10 kcal% fat) (D12450B) or
high-fat diet (HFD) (60 kcal% fat) (D12492) (Research Diets Inc., New Brunswick,
NJ, USA) for 10 weeks. Then, the mice with both diets were treated with DEHP (1
mg/kg body weight) or corn oil (the vehicle controls) daily by gavage for 25 weeks.
After the treatments, mice were euthanized with continuous CO2 inhalation to cardiac
arrest following the policy of the American Veterinary Medical Association (AVMA).
Blood samples were collected by cardiac puncture for enzyme-linked immunosorbent
assay (ELISA) (see Supplemental Material in detail). The animal protocol was
approved by the Institutional Animal Care and Use Committee (IACUC) at NHRI
(NHRI-IACUC-099092). All animal studies were performed following the National
Institutes of Health Guide for the Care and Use of Laboratory Animals.
Statistical analyses
All qualitative data were from at least three independent experiments. Relative
quantitative data were expressed as fold changes and presented as mean ± SD.
Statistical significance was determined by using student’s t-test and a value of p <
0.05 was considered statistically significant. Trend test for evaluating the
dose-dependent effect of chemicals was conducted by the nonparametric
Jonckheere–Terpstra test in SPSS Version 18.
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Results
Comparative microarray analyses
Comparative microarray analysis of gene expression in 3T3-L1 adipocytes with
both MEHP and rosiglitazone treatments was performed as described in Figure 1a.
First, PCA analysis was used to simplify analysis and visualization of
multidimensional microarray data. PCA analysis of seven samples revealed the clear
separation of DMSO- and MEHP/rosiglitazone-treated data sets (Figure 1b, left panel),
indicating that the gene expression patterns in MEHP/rosiglitazone-treated cells were
different from that in the vehicle control. Moreover, the distinct separation of
MEHP_D8 and MEHP_D10 data sets suggested a marked gene expression change in
MEHP-treated cells during adipogenesis from D8 to D10. Next, relevance networks
were used to compute comprehensive pair-wise measures of similarity for all genes in
microarray data sets to find genetic regulatory networks (Figure 1b, right panel),
where relevant links of transcriptomic patterns of MEHP_D10 were relatively lower
than the others, suggesting the unique influence of MEHP on gene expression at D10.
A total of 711 genes with up- or down-regulation at D10 (by ≥ 2-fold change vs.
DMSO_D10) were summarized in the Venn diagrams (Figure 1c), including 346
up-regulated genes (202 genes in MEHP_D10, 185 genes in Rosig._D10, and 41
overlapped genes) and 365 down-regulated genes (175 genes in MEHP_D10, 256
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genes in Rosig._D10, and 66 overlapped genes).
Functional ontology analyses
To assign biological
significance of the 711
genes in
response to
MEHP/rosiglitazone, both pathway enrichment analysis and GO enrichment analysis
were performed. Results of pathway enrichment analysis were summarized in Figure
2a. In the up-regulated genes (Figure 2a, top panel), the pathways involved in PPAR
signaling, unsaturated fatty acid biosynthesis, fatty acid metabolism, and
glycolysis/gluconeogenesis were markedly enriched in both MEHP_D10 and
Rosig._D10. All these biosynthesis/metabolism pathways have been previously
demonstrated as PPARγ-mediated biological functions in adipocytes (Sharma and
Staels 2007). In the down-regulated genes (Figure 2a, bottom panel), the pathways
involved in sulfur metabolism, complement signaling, β3 adrenergic receptor signaling,
starch and sucrose metabolism, systemic lupus erythematosus, glutathione metabolism,
and heterotrimeric G-protein signaling/Giα and Gsα mediated signaling were enriched
in both adipocytes.
Results of GO enrichment analysis were summarized in Figure 2b. In the
up-regulated genes (Figure 2b, top panel), both adipocytes exhibited higher scores in
metabolism-related biological processes, i.e., glycerol, pyruvate, and lipid metabolism.
In the down-regulated genes (Figure 2b, bottom panel), the pathways involved in
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dietary excess response, immune/inflammation-related responses (i.e., cytokine
stimulus response, acute inflammatory response, and complement activation), and fat
cell differentiation were significantly enriched in both adipocytes. It was noted that
expression
patterns
of
those
genes
particularly
involved
in
biosynthesis/metabolism-related processes were highly similar between MEHP_D10
and Rosig._D10 (Figure 2). The finding might provide a mechanistic explanation for
our previous studies that MEHP disturbed energy metabolism in fat cells (Chiang et al.
2016).
Up-regulation of PPAR signaling-related genes
The genes involved in PPAR signaling pathway were significantly up-regulated in
both MEHP_D10 and Rosig._D10 (Figure 2a). Analysis of microarray data revealed
that a total of 16 PPAR signaling-related genes were up-regulated in
MEHP/rosiglitazone-treated adipocytes (vs. DMSO) (Figure 3a). The genes were
functionally categorized into six groups, i.e., nuclear receptor, transcriptional
coactivator, fatty acid transport, fatty acid β-oxidation, adipocyte differentiation, and
gluconeogenesis/glyceroneogenesis. The details of microarray data were summarized
in Table S1 in Supplemental Material. Moreover, qPCR analysis of 10 genes (Ppara,
Ppargc1a, Fabp3, Fabp5, Olr1, Acaa2, Acox1, Plin2, Gyk, and Pck1) was used to
validate the microarray data, confirming the higher PPAR signaling-related gene
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expression in MEHP/rosiglitazone-treated adipocytes (Figure 3b).
Up-regulation of energy metabolism-related genes
Microarray analysis also revealed that, in MEHP/rosiglitazone treated adipocytes,
the genes involved in both lipid/glucose metabolism (Figure 2) and PPAR pathways
(Figures 2 and 3) were markedly up-regulated. Whether the gene expression caused
the disturbed energy metabolism in MEHP-treated adipocytes (Chiang et al. 2016)
was the major concern here. Therefore, energy metabolism-related genes were
categorized based on GO annotation (GO: biological process); a total of 37 energy
metabolism-related genes shown in the heatmap were functionally categorized into six
groups, i.e., fatty acid β-oxidation, glucose metabolism, energy production, fatty acid
synthesis, triglyceride synthesis, and lipid droplet-associated proteins (Figure 4a).
MEHP/rosiglitazone-treated adipocytes exhibited higher expression levels of the most
energy metabolism-related genes in both time-dependent (Figure 4a, left panel) and
treatment-dependent manners (Figure 4a, right panel) (see the details of microarray
data in Table S2 in Supplemental Material). It was critical to determine whether the
gene expression caused any energy metabolism-related functional change in
adipocytes.
First, following supplementation of culture medium with glucose or a model
NEFA, palmitate, the cellular consumption rates of exogenous fuel substrates were
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determined. Results in Figure 4b revealed that, after the supplementation for ≥ 6 h and
≥ 0.5 h, levels of glucose and palmitate left in culture medium of MEHP-treated
adipocytes were significantly lower than that of the vehicle controls (DMSO),
respectively. In lipolysis, triglycerides are hydrolyzed into glycerol and NEFA for
energy. Second, lipolytic activity in adipocytes was evaluated with cellular changes of
the three lipid molecules. As shown in Figure 4c, glycerol levels in adipocyte with
MEHP and rosiglitazone treatments were significantly increased by 1.87 folds and
2.42 folds (vs. DMSO), respectively, meanwhile triglycerides and NEFA levels did
not change significantly. Lipolytic activity was also determined with glycerol release
assay. Levels of glycerol released from adipocytes with MEHP and rosiglitazone
treatments were significantly increased by 2.56 folds and 2.21 folds (vs. DMSO),
respectively (Figure 4d, left panel). In the presence of the βAR agonist isoproterenol,
the glycerol changes were not significant (Figure 4d, right panel). Finally, glucose
metabolism in adipocytes was evaluated with glucose uptake and glycolysis. In
MEHP/rosiglitazone-treated adipocytes, both basal and insulin-induced glucose
uptake levels were significantly enhanced by 2–3 folds (vs. DMSO/basal) (Figure 4e,
left panel). Glycolytic activity, as determined by cellular release of lactate, in
adipocytes with MEHP and rosiglitazone treatments were significantly increased by
3.02 folds and 3.58 folds (vs. DMSO), respectively (Figure 4e, right panel).
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Taken
together,
MEHP-treated
adipocytes
consume
more
exogenous
glucose/fatty acids and exhibit higher activities in both lipolysis (in a
βAR-independent manner) and glucose metabolism, suggesting a higher energy
metabolism activity in the adipocytes. Evidently, both MEHP and rosiglitazone cause
similar changes in energy metabolism-related gene expression/biological functions in
adipocytes.
Down-regulation of adipokine-related genes
White adipose tissue (WAT) is emerging as an active regulator in control of
various physiologic processes, where adipokines provide an elaborate network for
systemic communication. The genes involved in cytokine/inflammation/complement
activation-related responses (Figure 2b, bottom panel) were here collectively
classified as adipokine-related genes. The heatmap of 55 adipokine-related genes was
summarized in Figure 5a (see the details in Table S3 in Supplemental Material).
Clearly, the majority of adipokine-related genes, e.g., Lep, Adipsin, Vegfc, Ccl2, Ccl6,
Fgf7, and Csf1, in MEHP/rosiglitazone-treated adipocytes were down-regulated (vs.
DMSO). It was noted that several important adipokine genes, such as Adipor2, Fgf21,
Angptl4, and Ghrh, were up-regulated in MEHP/rosiglitazone-treated adipocytes (vs.
DMSO). Microarray results were further validated with qPCR analysis of 5 genes
(Adipsin, Lep, Adipor2, Fgf21, and Angptl4) (Figure 5b). Expression patterns of the
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adipokine-related genes in response to both MEHP and rosiglitazone are highly
similar, supporting the hypothesis that MEHP disturbs adipokine network via PPARγ.
Following ingestion, DEHP is quickly metabolized into MEHP in vivo. Mouse
models with both normal chow diet (NCD) and high-fat diet (HFD) were subjected to
DEHP treatments (1 mg/kg body weight/day) (Figure 5c) to address MEHP effects on
adipokine network in vivo. After the treatments, body weight of HFD-mice was
significantly increased by 36% than that of NCD-mice; the DEHP treatment caused
on significant change on body weight of both mouse models (data not shown). ELISA
analysis revealed that plasma levels of Fgf21 in HFD-mice were lower than that in
NCD-mice and DEHP treatments significantly enhanced Fgf21 levels in both mouse
models (Figure 5d, left panel). Plasma Angptl4 levels in HFD-mice were significantly
higher than that in NCD-mice; in the presence of DEHP, Angptl4 levels in HFD-mice
were further increased (Figure 5d, right panel). It is clear that DEHP treatments result
in the higher Fgf21 and Angptl4 levels in NCD-mice and/or HFD-mice, which is
consistent with the higher mRNA levels of both genes in MEHP-treated adipocytes
(Figures 5a and 5b). The results provide in vivo evidence for MEHP impacts on
systemic regulation via adipokine network.
Activation of PPARγ by MEHP
PPARγ is highly expressed in fat tissue and its activation orchestrates both
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triglyceride/fatty acid metabolism and adipocyte differentiation (Sharma and Staels
2007). The present microarray results suggested the potential involvement of PPARγ
in energy metabolism in MEHP-treated adipocytes. It was necessary to determine
whether MEHP directly induced PPARγ expression and its activation. qPCR analysis
revealed that PPARγ mRNA levels in vehicle controls (DMSO) and MEHP-treated
adipocytes at D11 were increased by 3.39 folds and 2.89 folds (vs. Control_D5),
respectively (Figure 6a); no significant differences were detected between the two
adipocytes. Immunoblot analysis with total protein samples indicated that MEHP up
to 100 µM caused no significant change in both PPARγ1 and PPARγ2 isoforms
(Figure 6b, top panel), whereas analysis with nuclear fraction samples revealed
that MEHP significantly enhanced nuclear translocation of PPARγ2 in a
dose-dependent manner (Figure 6b, bottom panel). Genetic analysis has
demonstrated that PPARγ2 is more potent than PPARγ1 in transcriptional
activation of the genes involved in adipogenesis (Mueller et al. 2002). By using a
recombinant
PPAR
response
element
(PPRE)-driven
luciferase
cell
line,
Huh7-PPRE-Luc (Tsai et al. 2014), PPAR transcriptional activity in response to
MEHP (100 µM) and rosiglitazone (2 µM) was induced by 1.92 folds and 2.64 folds,
respectively (Figure 6c). Clearly, MEHP can act as a PPARγ agonist for PPARγ
nuclear translocation/activation.
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The microarray analysis revealed the highly similar expression patterns of genes
involved in PPAR signaling, energy metabolism, and adipokine network between
MEHP- and rosiglitazone-treated adipocytes. Significant correlation between gene
expression profiles and energy-metabolism-related functional changes suggests the
pivotal roles of PPARγ in regulation of energy metabolism in MEHP-treated
adipocytes. Importantly, expression of adipokine-related genes, i.e., Fgf21 and
Angptl4, in MEHP-treated adipocytes was confirmed with plasma samples from
DEHP-treated mice, providing in vivo evidence for an adipokine-mediated systemic
regulation in response to DEHP/MEHP exposure.
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Discussion
By using microarray analysis of gene expression in MEHP/rosiglitazone-treated
adipocytes, it was noted that expression patterns of significant amounts of genes were
highly similar between the two cells (Figure 1). Following pathway enrichment
analysis, we found that PPAR signaling pathway stood on the top of up-regulated
genes in MEHP/rosiglitazone-treated adipocytes (Figure 2a). PPARγ, a key player in
adipogenesis, acts as a metabolic regulator primarily involved in lipid and
carbohydrate metabolism (Savage 2005). Both MEHP and rosiglitazone treatments
resulted in similar expression patterns of the genes involved in PPAR signaling
(Figure 3), energy metabolism (Figure 4), and adipokine network (Figure 5). The
results clearly suggested the central role of PPARγ in regulation of energy metabolism
in MEHP-treated adipocytes.
The present gene expression profiles provide molecular clues on how MEHP
influences energy metabolism in adipocytes. Following uptake, MEHP activates
PPARγ and its downstream genes, primarily transcription factor Ppara (PPARα) and
transcriptional coactivators Ppargc1a (PGC-1α) and Ppargc1b (PGC-1β) (Figure 3).
PPARγ activates different panels of gene expression via coordination with PPARα
(Park et al. 2012), PGC-1α (Powell et al. 2007), and PGC-1β (Deng et al. 2011).
Particularly, the PGC-1 coactivator family plays a central role in control of
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mitochondrial biogenesis and fatty acid β-oxidation (Vega et al. 2000; Kleiner et al.
2012; Enguix et al. 2013). Results in Figure 3 suggest the involvement of PPARγ in
fatty acid transport, fatty acid β-oxidation, adipocyte differentiation, and
gluconeogenesis/glyceroneogenesis in MEHP-treated adipocytes.
MEHP-treated adipocytes consumed more fatty acids (Figure 4b, right panel).
Up-regulation of the genes involved in fatty acid transport and fatty acid β-oxidation
in MEHP/rosiglitazone-treated adipocytes (Figures 3 and 4a) provides a possible
scenario for the higher cellular fatty acid uptake. The fatty acid transport-related genes
include Olr1, Fabp3, Fabp5, and the solute carrier family 25 (Slc25a20, Slc25a22,
Slc25a34, and Slc25a54). Olr1 encodes a cell-surface receptor for endocytosis of
oxidized low density lipoprotein (Mehta and Li 1998). Fatty acid-binding proteins
(FABPs), e.g., Fabp3 and Fabp5, facilitate fatty acid transport to mitochondria for
β-oxidation (Furuhashi and Hotamisligil 2008). The solute carrier family 25, e.g.,
Slc25a20, has been reported as a mitochondrial fatty acid transporter (Palmieri 2004).
Fatty acids are transported into mitochondria for energy production via fatty acid
β-oxidation; the genes involved in β-oxidation include Acox1, Acaa1b, Acaa2, Hadha,
Hsdl2, Echdc1, and Eci1 (Figure 4a). Clearly, in MEHP-treated adipocytes, the higher
fatty acid consumption combined with the higher fatty acid transport/β-oxidation may
lead to the higher mitochondrial respiration for energy. Moreover, the higher fatty acid
22
demand in MEHP-treated adipocytes is supported by the up-regulation of genes
involved in fatty acid synthesis, including Acacb, Elovl3, Ephx2, Mlycd, and Cyp4f17
(Figure 4a).
MEHP-treated adipocytes also consumed more glucose (Figure 4b, left panel)
and exhibited a higher glycolytic activity (Figure 4e). The genes involved in glucose
metabolism, including glucose transport (Slc2a1), glycolysis (Fbp2, Gapdh, and
Pgam1), and gluconeogenesis/glyceroneogenesis (Pdk2, Pdk4, Pck1, and Gyk), were
up-regulated in MEHP-treated adipocytes (Figure 4a), suggesting that the gene
expression may contribute to the higher glucose uptake/glycolysis in the cells.
Moreover, it was noted that, in MEHP-treated adipocytes, the higher glucose uptake
was accompanied with a proportional lactate production (Figure 4e), indicating that
the majority of glucose uptake is metabolized into lactate, with limited energy
production via mitochondrial respiration.
Therefore, fatty acids could be the major fuel for the higher oxygen consumption
rate (OCR) in MEHP-treated adipocytes that we previously reported (Chiang et al.
2016). It is suggested that two enzyme activities, Pdk2/Pdk4 and Mlycd, (Figure 4a)
are responsible for the preferential fatty acid utilization for mitochondrial energy
production. First, Pdk4 has been reported to inhibit pyruvate dehydrogenase (PDH),
the first component enzyme of pyruvate dehydrogenase complex (PDC) (Zhang et al.
23
2014). PDH/PDC bridges the glycolysis metabolic pathway to the TCA cycle;
inactivation of PDH/PDC prevents conversion of pyruvate to acetyl CoA and thus
switches glucose catabolism to fatty acid oxidation (Zhang et al. 2014). Second,
Mlycd encodes an enzyme of malonyl-CoA decarboxylase (Mlycd). Mlycd catalyzes
the conversion of malonyl-CoA to acetyl-CoA and prevents accumulation of
malonyl-CoA, a potent inhibitor of mitochondrial fatty acid uptake (Foster 2012).
Therefore, up-regulation of Mlycd in MEHP-treated adipocytes suggests facilitation of
mitochondrial fatty acid uptake and β-oxidation. Our findings support the hypothesis
that MEHP-treated adipocytes prefer to utilize fatty acids rather than glucose in
mitochondrial respiration for energy.
Lipolysis involves hydrolysis of triglycerides into glycerol and NEFA and plays
critical roles in systemic supply of fatty acids for energy. Up-regulation of the lipid
metabolism-related genes (Figures 3 and 4a) supported the higher lipolytic activity
detected in MEHP/rosiglitazone-treated adipocytes (Figures 4c and 4d). It was noted
that the lipolytic activity was regulated in a βAR-independent manner (Figure 4d).
Among the genes involved in triglyceride synthesis in adipocytes (Figure 4a), Ces1d
encodes the major non-hormone-sensitive/βAR-independent lipase, carboxylesterase
1D (Soni et al. 2004). Expression levels of Ces1d in MEHP_D10 and Rosig._D10
were increased by 1.31 folds and 1.69 folds (vs. DMSO_D10), respectively; the
24
higher Ces1d expression may lead to the higher βAR-independent lipolytic activity.
Moreover, Aqp7 deficiency leads to triglyceride accumulation in adipocytes,
indicating the role of Aqp7 in glycerol release (Hibuse et al. 2005). Thus, the higher
Aqp7 expression in MEHP/rosiglitazone-treated adipocytes (Figure 3) may also
contribute to the higher glycerol release (Figure 4d).
Obesity is a major etiological factor for metabolic complication, such as T2DM.
Adipose tissue functions as a key endocrine organ mainly via adipokine network;
adipokines play important roles in both inflammatory responses and systemic
regulation of metabolism (Ouchi et al. 2011). The majority of adipokine-related genes
in MEHP/rosiglitazone-treated adipocytes were down-regulated (Figure 5a), which is
generally consistent with a previous rosiglitazone study (Wang et al. 2007).
Expression levels of Lep in adipocytes treated with MEHP (30 µM and 100 µM) and
rosiglitazone (2 µM) were markedly decreased by 80%, 92%, and 96%, respectively
(Figure 5b). Leptin, predominantly synthesized in WAT, plays critical roles in control
of appetite and energy balance. Lep-deficient (ob/ob) mouse, an animal model of
T2DM, exhibits uncontrolled food intake and thus rapidly develops obesity (Muzzin
et al. 1996). Therefore, down-regulation of Lep in MEHP/rosiglitazone-treated
adipocytes provides a potential linkage between MEHP exposure/TZD therapy and
obesity. Importantly, up-regulation of two specific adipokine-related genes (Fgf21 and
25
Angptl4) was presented here with both in vitro (microarray and qPCR) and in vivo
(animal) studies (Figure 5). FGF21 plays pivotal roles in control of glucose
homeostasis and body weight (Kharitonenkov et al. 2005; Dutchak et al. 2012).
Angtl4 acts as a regulator of glucose and lipid metabolism (Xu et al. 2005).
Microarray data listed in Figure 5a provide comprehensive information about how
phthalate exposures may influence systemic regulation via adipokine network.
26
Conclusion
Taken together, on the basis of causal correlation between the microarray-based
gene expression profiles and the energy metabolism-related functional assays, a
proposed model of MEHP effects on bioenergetics and adipokine network in
adipocytes is schematically summarized in Figure 7. Upon uptake in adipocytes,
MEHP functions as a PPARγ agonist for PPARγ activation. The activated PPARγ is
translocated into nucleus and promotes the PPARγ-mediated gene expression. PPARγ
also cooperate with different coactivators, such as PPARα, PGC1α, and PGC1β, to
regulate expression of different panels of the genes involved in control of energy
metabolism (i.e., glycolysis, gluconeogenesis/glyceroneogenesis, lipolysis, fatty acid
β-oxidation, and TCA cycle) and adipokine network. The MEHP-treated adipocytes
exhibit significant increases in fatty acid consumption, glucose uptake, and
lipolytic activity. The higher fatty acid consumption combined with the higher
fatty acid transport/β-oxidation may lead to the higher mitochondrial respiration
for energy. The higher glucose uptake accompanied with a proportional lactate
production indicates that the majority of glucose uptake is metabolized into
lactate, with limited energy production via mitochondrial respiration. It is noted
that the higher expression of Pdk2/Pdk4 and Mlycd genes in MEHP-treated
adipocytes may result in preferential utilization of fatty acids rather than glucose
27
in mitochondrial respiration. Moreover, changes in adipokine profile and
lactate/glycerol levels in circulation can also disturb systemic metabolic
homeostasis.
28
Acknowledgements
This work was supported by grants from the Ministry of Science and Technology
(101-2314-B-400-003-MY3, 102-2811-B-400-015, and 103-2811-B-400-022) and the
National Health Research Institutes (EO-103-PP-03 and EO-104-PP-03) in Taiwan.
29
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Figure legends
Fig.
1.
Comparative
microarray
analysis
of
gene
expression
in
MEHP/rosiglitazone-treated adipocytes. (a) Procedures of cell treatments in this study.
A total of seven samples were collected for microarray analysis. CF, confluence; Dex,
dexamethasone;
DIM,
differentiation-inducing
medium;
IBMX,
3-isobutyl-1-methylxanthine; Ins, insulin; Rosig, rosiglitazone. (b) PCA analysis (left
panel) was used to simplify analysis and visualization of multidimensional microarray
data. Relevance network (right panel) was used to determine the linkage between two
treatments. (c) Venn diagrams of the 711 genes with significant up- or
down-regulation in MEHP_D10 or Rosig._D10 (vs. DMSO_D10). There were 346
up-regulated genes, i.e., 202 genes in MEHP_D10 and 185 genes in Rosig._D10,
with 41 overlapped genes. There were 365 down-regulated genes, i.e., 175 genes
in MEHP_D10 and 256 genes in Rosig._D10, with 66 overlapped genes.
Fig. 2. Microarray data biological process classification. (a) Pathway enrichment
analysis and (b) GO enrichment analysis were performed with a total of 711 genes
with significant changes in either MEHP_D10 or Rosig._D10 (vs. DMSO_D10) as
described in detail in Fig. 1c. Value in parenthesis indicates the number of genes
in
each
MEHP_D10
or
Rosig._D10
37
group.
The
common
biological
functions/pathways found in both treatments are underlined. p-values by Fisher
extract test were indicated.
Fig. 3. Up-regulation of PPAR signaling-related genes in MEHP/rosiglitazone-treated
adipocytes. (a) Heatmap of mRNA levels of 16 PPAR signaling-related genes in
response to DMSO (the vehicle control), MEHP, and rosiglitazone. (b) qPCR
validation of microarray gene expression in adipocytes treated with DMSO, MEHP
(30 and 100 μM), or rosiglitazone (2 μM). Data are expressed as relative mRNA
levels (vs. Control_D5) and presented as mean ± SD (n ≥ 4). *p < 0.05, **p < 0.01,
***p < 0.001 vs. DMSO. P-values for trend were calculated by nonparametric
Jonckheere-Terpstra test.
Fig. 4. Higher energy metabolism activity in MEHP/rosiglitazone-treated adipocytes.
(a) Heatmap of mRNA levels of 37 energy metabolism-related genes in response to
DMSO (the vehicle control), MEHP, and rosiglitazone in time-dependent (vs.
Control_D5) or treatment-dependent (vs. DMSO) manners. Energy metabolism
activity in adipocytes treated with DMSO, MEHP (100 μM), or rosiglitazone (2 μM)
was analyzed with (b) cellular uptake of exogenous glucose/palmitate, (c and d)
lipolysis, and (e) glucose metabolism. Lipolytic activity was determined with (c)
38
cellular levels of triglyceride, NEFA, and glycerol as well as (d) glycerol release assay.
Glucose metabolism activity was evaluated with glucose uptake (see Supplemental
Material in detail) and lactate production. Data are presented as mean ± SD (n ≥ 4). *p
< 0.05, **p < 0.01, ***p < 0.001 vs. DMSO of each time point (for b) or vs. DMSO
(for c, d, and e).
Fig. 5. Down-regulation of adipokine-related genes in MEHP/rosiglitazone-treated
adipocytes. (a) Heatmap of mRNA levels of 55 adipokine-related genes in response to
DMSO (the vehicle control), MEHP, and rosiglitazone. (b) Microarray gene
expression was validated by qPCR as described in Figure 3b. Data are expressed as
relative mRNA levels (vs. Control_D5) and presented as mean ± SD (n ≥ 4). *p < 0.05,
**p < 0.01, ***p < 0.001 vs. DMSO. P-values for trend were calculated by
nonparametric Jonckheere-Terpstra test. (c) Male C57BL/6J mice were fed with
normal chow diet (NCD) or high-fat diet (HFD) for 10 weeks. Then, both mice were
treated with DEHP (1 mg/kg body weight) or corn oil (the vehicle control) daily by
gavage for 25 weeks. (d) After treatments, serum samples were collected for analysis
of Fgf21 and Angptl4 by ELISA. Data are presented as mean ± SD (NCD, n = 2;
NCD-DEHP, n = 5; HFD, n = 5; HFD-DEHP, n = 3). *p < 0.05 vs. NCD; †p < 0.05,
HFD-DEHP vs. HFD.
39
Fig. 6. Activation of PPARγ by MEHP. (a) PPARγ mRNA levels in adipocytes treated
with DMSO (the vehicle control) or MEHP (100 μM) were determined by qPCR.
Data are expressed as relative mRNA levels (vs. Control_D5) and presented as mean
± SD (n = 4). (b) PPARγ levels in both total (top panel) and nuclear protein samples
(bottom panel) from adipocytes treated with DMSO (the vehicle control) or MEHP
(30 and 100 µM) were determined by immunoblot analysis with α-tubulin and lamin
B as loading controls, respectively (see Supplemental Material in detail).
Representative immunoblots are shown. Data are expressed as relative PPARγ protein
levels (vs. loading controls) and presented as mean ± SD (n = 3). *p < 0.05 vs. DMSO.
(c) Huh7-PPRE-Leu cells, carrying a PPRE-driven luciferase gene, were left untreated
or treated with DMSO (the vehicle control), MEHP (100 µM), or rosiglitazone (2 µM)
for 24 h; luciferase activity in the cells was determined (see Supplemental Material in
detail). Data are expressed as relative luciferase activity (vs. untreated control) and
presented as mean ± SD (n = 4). ***p < 0.001 vs. DMSO.
Fig. 7. A proposed schematic diagram for MEHP impacts on fat cell bioenergetics and
adipokine network. Functional correlation between the microarray-based gene
expression and the energy metabolism-related functional assays suggested the pivotal
40
role of PPARγ in control of energy metabolism and adipokine network in
MEHP-treated
adipocytes.
Transcription
factors/coactivators,
transporters,
adipokines, and potential enzymes/proteins involved in energy metabolism pathways
(i.e., glycolysis, lipolysis, gluconeogenesis/glyceroneogenesis, esterification, fatty
acid β-oxidation, and TCA cycle) are depicted. Fuel molecules and their metabolites
are underlined. Arrow (→) shows the flow direction of metabolites and pathways.
Arrow to bar (→|) indicates inhibition of enzyme activity or protein function. Dashed
lines represent a potential PPARγ-mediated regulation. Red color codes for
up-regulated genes, increased metabolites, or activated pathways; green color codes
for the opposite. Black color codes for no significant changes. Genes in frame indicate
absent in the array.
41