- Email: [email protected]
High-fat diet aggravates 2,2′,4,4′-tetrabromodiphenyl ether-inhibited testosterone production via DAX-1 in Leydig cells in rats
Accepted Manuscript High-fat diet aggravates 2,2′,4,4′-tetrabromodiphenyl etherinhibited testosterone production via DAX-1 in Leydig cells in rats
Zhan Zhang, Yongquan Yu, Hengsen Xu, Chao Wang, Minghui Ji, Jun Gu, Lu Yang, Jiansheng Zhu, Huibin Dong, Shou-Lin Wang PII: DOI: Reference:
S0041-008X(17)30114-X doi: 10.1016/j.taap.2017.03.010 YTAAP 13891
To appear in:
Toxicology and Applied Pharmacology
Received date: Revised date: Accepted date:
15 October 2016 2 March 2017 10 March 2017
Please cite this article as: Zhan Zhang, Yongquan Yu, Hengsen Xu, Chao Wang, Minghui Ji, Jun Gu, Lu Yang, Jiansheng Zhu, Huibin Dong, Shou-Lin Wang , High-fat diet aggravates 2,2′,4,4′-tetrabromodiphenyl ether-inhibited testosterone production via DAX-1 in Leydig cells in rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ytaap(2017), doi: 10.1016/ j.taap.2017.03.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT High-fat diet aggravates 2,2’,4,4’-tetrabromodiphenyl ether-inhibited testosterone production via DAX-1 in Leydig cells in rats
Zhan Zhang1,2,4, Yongquan Yu1,2,4, Hengsen Xu2, Chao Wang1,2, Minghui Ji2, Jun Gu2,
State Key Lab of Reproductive Medicine, Institute of Toxicology, Nanjing Medical
SC
1
RI
PT
Lu Yang1,2, Jiansheng Zhu1,2, Huibin Dong3, Shou-Lin Wang1,2,*
University, 101 Longmian Avenue, Nanjing 211166, P. R. China Key Lab of Modern Toxicology of Ministry of Education, School of Public Health,
NU
2
3
MA
Nanjing Medical University, 101 Longmian Avenue, Nanjing 211166, P. R. China Changzhou Center for Disease Control and Prevention, 203 Taishan Road,
D
Changzhou, 2013022, P. R. China
These authors contributed equally to this study
*
To whom correspondence should be addressed: Key Lab of Modern Toxicology of
CE
PT E
4
AC
Ministry of Education, School of Public Health, Nanjing Medical University, 101 Longmian Avenue, Nanjing 211166, P. R. China Tel: +86-25-8686-8417 Fax: +86-25-8686-8499 E-mail: [email protected]
1
ACCEPTED MANUSCRIPT Abbreviations: BDE47, 2,2’, 4,4’-tetrabromodiphenyl ether; PBDEs, polybrominated diphenyl ethers; ND, normal diet; HFD, high-fat diet; BAT, brown adipose tissue; WAT, white adipose tissue; CHOL, total cholesterol; TG, triglycerides; ELISA, enzyme-linked
PT
immunosorbent assay; 3β-HSD, 3β-hydroxysteroid dehydrogenase; StAR,
RI
steroidogenic acute regulatory protein; DAX-1, dosage-sensitive sex reversal-adrenal
AC
CE
PT E
D
MA
NU
SC
hypoplasia congenita critical region of the X chromosome gene 1.
2
ACCEPTED MANUSCRIPT Abstract Growing evidence has revealed that a high-fat diet (HFD) could lead to disorders of glycolipid metabolism and insulin-resistant states, and HFDs have been associated with the inhibition of testicular steroidogenesis. Our previous study demonstrated that
PT
2,2′,4,4′-tetrabromodiphenyl ether (BDE47) could increase the risk of diabetes in
RI
humans and reduce testosterone production in rats. However, whether the HFD affects
SC
BDE47-inhibited testosterone production by elevating insulin levels and inducing related pathways remains unknown. In male rats treated with BDE47 by gavage for 12
NU
weeks, the HFD significantly increased the BDE47 content of the liver and testis and
MA
increased the weight of the adipose tissue; increased macrovesicular steatosis in the liver and the levels of triglycerides, fasting glucose and insulin; further aggravated the
D
disruption of the seminiferous epithelium; and lowered the level of testosterone,
PT E
resulting in fewer sperm in the epididymis. Of note, the HFD enhanced BDE47-induced DAX-1 expression and decreased the expression levels of StAR and
CE
3β-HSD in the testicular interstitial compartments in rats. In isolated primary Leydig
AC
cells from rats, BDE47 or insulin increased DAX-1 expression, decreased the expression of StAR and 3β-HSD, and reduced testosterone production, which was nearly reversed by knocking down DAX-1. These results indicated that the HFD aggravates BDE47-inhibited testosterone production through hyperinsulinemia, and the accumulation of testicular BDE47 that induces the up-regulation of DAX-1 and the subsequent down-regulation of steroidogenic proteins, i.e., StAR and 3β-HSD, in Leydig cells. 3
ACCEPTED MANUSCRIPT Keywords: High-fat diet; 2,2′,4,4′-tetrabromodiphenyl ether; DAX-1; insulin;
AC
CE
PT E
D
MA
NU
SC
RI
PT
testosterone; primary Leydig cells
4
ACCEPTED MANUSCRIPT Introduction Male reproductive problems, such as poor semen quality, low testosterone levels, cryptorchidism, and testicular cancer, are widespread and have recently been the focus of several basic and clinical research projects (Skakkebaek et al., 2016). Many
PT
environmental and lifestyle factors may negatively affect spermatogenesis and male
RI
fertility (Barazani et al., 2014). With rapid economic growth and its associated
SC
industrialization, urbanization, and lifestyle changes, i.e. high-fat diets (HFDs), and low physical activity, diabetes has reached epidemic proportions in the Chinese
NU
population (Xu et al., 2013). HFD could induce an increase in body weight, impair
MA
glucose tolerance and cause insulin resistance (Skovso, 2014; Jung et al., 2015); the HFD could also impair male fertility by reducing the quality of the semen and sperm
D
and by reducing testosterone levels (Fan et al., 2015).
PT E
Polybrominated diphenyl ethers (PBDEs) have been used as flame-retardant chemicals in various products worldwide (Linares et al., 2015). Among the PBDEs,
CE
2,2′,4,4′-tetrabromodiphenyl ether (BDE47) is the dominant congener in animal and
AC
human tissue (Hites, 2004; Mhadhbi et al., 2012). Our recent study has shown that environmental exposure to BDE47 increased the risk of diabetes by activating the diabetes pathway and upregulating genes involved in glucose transport (Zhang et al., 2016). In addition, BDE47 disrupts spermatogenesis, impairs mitochondrial function and induces the apoptosis of early leptotene spermatocytes in rats (Huang et al., 2015a). Our previous study also revealed that BDE47 could cause a striking disruption of the normal cellular organization of the seminiferous epithelium and 5
ACCEPTED MANUSCRIPT decrease the serum testosterone level (Zhang et al., 2013b). However, the mechanism by which BDE47 inhibits testosterone production is still unclear. Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1) is required for human spermatogenesis because it is a
PT
crucial regulator of steroidogenesis in mammals (Lardone et al., 2011; Stickels et al.,
RI
2015). For instance, DAX-1 is a repressor of several steroidogenic enzymes, blocking
SC
steroid production at multiple levels and decreasing the formation of steroids (Maris et al., 2015). High insulin levels are associated with low testosterone levels in type 2
NU
diabetes (Haffner et al., 1994; Grossmann et al., 2010). In healthy men, lower total
MA
testosterone is associated with insulin resistance (Yeap et al., 2009; Biswas et al., 2010). Insulin might directly repress steroidogenesis by inducing the orphan nuclear
D
receptor DAX-1 in testicular Leydig cells (Ahn et al., 2013). Thus, BDE47 might
PT E
induce high insulin levels and subsequently inhibit testosterone production via DAX-1.
CE
In the present study, adult male rats were treated with BDE47 by gavage for 3
AC
months while being fed the normal diet (ND) or HFD. The effects of BDE47 on lipometabolism and glycometabolism in rats fed the HFD were investigated. Next, testosterone levels, histopathologic changes in the testis and epididymis, and the expression of steroidogenic proteins in rat testis were evaluated. Furthermore, the primary Leydig cells of rats were isolated and used to explore the roles of DAX-1 in the effects of HFD on BDE47-inhibited testosterone production. This study attempts to illustrate the molecular mechanism of the effects of HFD and/or BDE47 on 6
ACCEPTED MANUSCRIPT testosterone production, to enhance the knowledge of metabolic syndrome in relation to reduced male reproduction and to provide evidence-based information for assessing the risks of environmental pollutants, particularly in the presence of the HFD.
PT
Materials and methods
RI
Chemicals and reagents
SC
BDE47 (purity ≥ 98.7%) was purchased from Chemservice (West Chester, PA, USA). Corn oil and collagenase I were purchased from Sigma-Aldrich (St. Louis, MO, USA).
NU
The enzyme-linked immunosorbent assay (ELISA) kit for rat insulin was obtained
MA
from Cusabio Biotech Co., Ltd (Wuhan, China). Dulbecco's Modified Eagle’s Medium/ Nutrient Mixture F-12 (DMEM/F-12), fetal bovine serum (FBS) and
D
collagen-coated culture plates were purchased from GIBCO-BRL (Grand Island, NY,
PT E
USA). Antibodies specific for StAR and DAX-1 and an enhanced chemiluminescence (ECL) immunoblotting assay kit were purchased from Cell Signaling Technology
CE
(Danvers, MA, USA). Antibodies specific for GAPDH and 3β-HSD were obtained
AC
from Santa Cruz Biotechnology (Santa Cruz, CA). A cell counting kit-8 (CCK-8), penicillin, streptomycin, gentamicin, a bicinchoninic acid (BCA) protein assay kit, and bovine serum albumin (BSA) were obtained from Beyotime Institute of Biotechnology (Shanghai, China). The ND (15% calories from fat) and HFD (45% calories from fat) were obtained from Jiangsu Medicience Ltd. (Yangzhou, China). Animal treatment and sample collection Male Sprague-Dawley rats (weighing 180-200 g) were obtained from Shanghai SLAC 7
ACCEPTED MANUSCRIPT Laboratory Animal Co., Ltd (Shanghai, China). The animals were maintained in 12-h/12-h light/dark cycles at ambient temperature (22C) and 55% relative humidity. Two rats were placed in a plastic Macrolon cage with stainless steel covers and wood shavings, and sterilized food and tap water were provided ad libitum. The rats were
PT
allowed to acclimate for 1 week before the experiments began. Thirty-six rats were
RI
randomly divided into six groups and were gavaged for 12 weeks: group 1 (ND
SC
control group) was fed a normal diet; groups 2 and 3 were treated with BDE47 (dissolved in corn oil) at 0.03 and 20 mg/kg. body weight (BW)/day, respectively;
NU
group 4 (HFD control group) was fed a high-fat diet; and groups 5 and 6 were fed
MA
HFD plus BDE47 at 0.03 or 20 mg/kg.bw/day, respectively. The ND and HFD control groups were given the same volume of corn oil, and the BDE47 doses were designed
D
based on our previous study (Zhang et al., 2013b). BW was quantified weekly, and
PT E
the rats were euthanized by CO2 asphyxiation at the study endpoint for serum collection and harvesting of tissues, including the liver, pancreas, testis, epididymis,
CE
brown adipose tissue (BAT), and the retroperitoneal and gonadal white adipose tissue
AC
(WAT). Except the serum that was used for fasting glucose measurements, all other samples were frozen and stored at -80C until analysis. During the experimental period, the care and use of the animals followed the guidelines of the Animal Care and Welfare Committee of Nanjing Medical University. Detection of BDE47 in the liver and testes of rats The sample extraction, clean-up and quality control for analysis were performed as described in our previous publication (Zhang et al., 2013b). The samples were 8
ACCEPTED MANUSCRIPT analyzed with a gas chromatograph-mass spectrometer (GC/MS) (Thermo Finnigan DSQ, USA) using BDE77 as an internal standard. Briefly, 1 μL of sample was injected using the splitless injection mode with a splitless time of 1 min. The mass spectrometer was operated using negative chemical ionization. Selected ion
PT
monitoring (SIM) was employed to determine individual peaks. The following pair of
RI
ions was monitored for each target compound, with the first ion being used to quantify
SC
an ion and the second ion for peak identification: 486, 326. Representative GC/MS chromatograms for each PBDE were from calibration mixtures (Accustandard, New
NU
Haven, CT, USA).
MA
Measurement of serum biochemical parameters and testosterone in rats Serum parameters, such as the levels of total cholesterol (CHOL), triglycerides (TG)
D
and fasting glucose, were measured routinely as a single measurement on an
PT E
autoanalyzer (Hitachi 7100, Japan) using standard kits from the Jiancheng Bio-engineering Research Institute (Nanjing, China) (Zhang et al., 2013a). The serum
CE
concentration of testosterone (T) was determined using [125I]-T-specific
AC
radioimmunoassay kits according to the manufacturer’s instructions (Beijing North Institute of Biological Technology, Beijing, China) (Zhang et al., 2013b). Histological observation of rat tissues After euthanization, the testes, epididymis, liver, pancreas and adipose tissue of the rats were fixed in 4% paraformaldehyde and embedded in paraffin, and 5-μm cross-sections were prepared and stained with hematoxylin and eosin (H&E). In the testis, giant cells were identified by their characteristic features, with multiple round 9
ACCEPTED MANUSCRIPT spermatid nuclei (≥2) in a syncytial body present in the lumen. Images were captured using an optical microscope (Axioskop 2 Plus, Carl Zeiss, Hamburg, Germany). Isolation, purification and culture of primary Leydig cells in rats Leydig cells were isolated and cultured as previously described (Klinefelter et al.,
PT
1987; Klinefelter and Ewing, 1988) with slight modifications. Briefly, the testes of
RI
rats (8 weeks old) were decapsulated, placed in 100 mL of PBS containing
SC
collagenase I (0.5 mg/mL) at 34C and shaken for 15 min. The digested testes were passed through a 100-μm nylon mesh, and the Leydig cells were sequentially purified
NU
by 21%, 23%, 32%, and 60% Percoll gradient separation. The Leydig cells were
MA
located in the second layer from the bottom. The final purity of the Leydig cells was determined to be >90% following staining with 3β-HSD, a typical marker for Leydig
D
cells. The isolated Leydig cells were resuspended in DMEM/F12 containing 5% FBS,
further experiments.
PT E
5 μg/mL gentamicin, 50 U/mL penicillin, and 50 μg/mL streptomycin and used for
CE
Treatment of primary Leydig cells from rats with BDE47
AC
The cells were plated at a density of 105/cm2 in six-well plates at 34°C in 5% CO2 overnight and then were treated with 0-150 μM BDE47 to screen for the non-observed cytotoxic concentration using the CCK-8 assay as previously described (Zhang et al., 2013b). Then, the medium and cell lysates were used to determine the testosterone levels and the expression of DAX-1, StAR and 3β-HSD after treatment with BDE47 or insulin for 24 h. Knockdown of DAX-1 in primary Leydig cells from rats 10
ACCEPTED MANUSCRIPT Small interfering RNAs (siRNAs) were used to confirm the role of DAX-1 in the synthesis of testosterone in primary Leydig cells treated with BDE47 or insulin. DAX-1 siRNA (5’-GCAGCAUCUUAUACAGCUU-3’, 3’-CGUCGUAGAAUAUGUCGAA-5’) and one negative scrambled control were
PT
synthesized by RiboBio Co., Ltd (Guangzhou, China). The cells (3×105 per dish) were
RI
plated in 35-mm dishes and cultured overnight, and then DAX-1 siRNA (si-DAX)
SC
was transfected into the cells for 24 h using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Subsequently, the cells
NU
were treated with BDE47 or insulin for an additional 24 h. The medium was used to
of DAX-1, StAR and 3β-HSD.
MA
determine testosterone levels, and the cell lysates were used to measure the expression
D
Determination of DAX-1 expression by immunocytochemistry and
PT E
immunohistochemistry assays
The expression levels of DAX-1 in the primary Leydig cells and the rat testis were
CE
determined by immunocytochemistry and immunohistochemistry based on our
AC
previous studies (Yang et al., 2013; Zhang et al., 2014). For immunocytochemistry, isolated Leydig cells were attached to a glass slide using cytospin centrifugation and then fixed with 4% paraformaldehyde, washed with phosphate-buffered saline (PBS), and incubated with 0.2% Triton X-100. Then, the cells were incubated overnight with the DAX-1 primary antibody in 1% BSA at 4°C followed by the appropriate fluorescence-conjugated secondary antibody at RT. Finally, the cells were mounted and observed under a confocal microscope (LSM710; Carl Zeiss, Hamburg, 11
ACCEPTED MANUSCRIPT Germany). For immunohistochemistry, deparaffinized and hydrated testis sections (5 μm) were treated with 3% hydrogen peroxide (H2O2) for 5 min, rinsed with PBS for 15 min, immunostained using a StreptAvidin Biotin Complex kit (Boster BioEngineering, Wuhan, China) and examined using an Axioskop 2 Plus optical
PT
microscope (Carl Zeiss, Hamburg, Germany).
RI
Determination of the expression levels of DAX-1, StAR and 3β-HSD by
SC
immunoblotting assays
Testis tissue and primary Leydig cell lysates (50-100 μg) were separated by sodium
NU
dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a
MA
polyvinylidene fluoride membrane (Millipore, Bedford, MA). Using specific antibodies for DAX-1, StAR and 3β-HSD, the protein-immune complexes were
D
detected using an ECL immunoblotting assay kit and were exposed to Kodak X-Omat
Statistical analysis
PT E
film. GAPDH was used as an internal control.
CE
Significant differences among different doses in same group were evaluated with the
AC
Student-Newman-Keuls multiple comparison test using SPSS software version 17.0 (Chicago, IL, USA), while the differences in different groups at the same dose were analyzed with one-way analysis of variance (ANOVA). The data for the BDE47 content in the liver and testis are shown as box and whisker plots (minimum to maximum), and the other data were expressed as the means± standard deviation (SD). Differences were considered statistically significant at p≤ 0.05.
12
ACCEPTED MANUSCRIPT Results HFD aggravates the accumulation of adipose tissue in rats treated with BDE47 After a 12-week treatment with BDE47 (0.03 or 20 mg/kg/d) by gavage, BDE47 was found in the rat liver and testis; the HFD could aggravate the accumulation of BDE47
PT
in the liver, and particularly in the testis (Fig. 1A). During the treatment, body weight
RI
was not altered by BDE47, but the HFD caused an increase, as expected (Fig. S1).
SC
Additionally, neither BDE47 nor the HFD changed the relative weight of the candidate organs, including the liver, pancreas, testis and epididymis (Fig. S2).
NU
However, treatment with both doses of BDE47 increased the relative weight of the
MA
retroperitoneal WAT, the gonadal WAT and BAT in rats fed with the HFD, while there were no changes in lipid accumulation in rats treated with BDE47 but given ND
D
feed (Fig. 1B, C, D). Additionally, the number of neonatal dikaryotic adipocytes
PT E
increased after treatment with 20 mg/kg BDE47, most remarkably in the HFD rats (Fig. S3).
CE
HFD aggravates the disruption of glycolipid metabolism in rats treated with BDE47
AC
Serum TG in BDE47-treated rats increased and was enhanced by the HFD (Fig. 2A), whereas no changes in CHOL were observed in rats treated with BDE47 or the HFD (Fig. 2B). Similar to its effect on TG, BDE47 (20 mg/kg/d) significantly increased fasting blood glucose and serum insulin levels in rats, which were also enhanced by the HFD (Fig. 2C, D). No histopathological changes were observed in the pancreas of rats treated with BDE47 or a HFD alone, but exocrine glands transfers were found in the HFD rats treated with 20 mg/kg BDE47 (Fig. S4). Notably, histopathological 13
ACCEPTED MANUSCRIPT analysis showed obvious microvesicular and macrovesicular steatosis in the livers of rats treated with BDE47; in particular, the 20 mg/kg/d BDE47 treatment plus the HFD exacerbated this phenotype (Fig. 2E). HFD aggravates the inhibition of testosterone production and damages the testis
PT
and epididymis in rats treated with BDE47
RI
Similar to our previous study, both doses of BDE47 reduced testosterone
SC
concentrations in ND rats, and this reduction was significantly exacerbated by the HFD (Fig. 3A). Histological examination of the testes of rats exposed to BDE47
NU
revealed a striking disruption of the normal cellular organization of the seminiferous
MA
epithelium. In the lumen, 20 mg/kg BDE47 significantly increased the number of multinucleated giant cells that arose from spermatocytes and aborted meiosis, and
D
they appeared as a syncytium. Abundant vacuolar spaces in the seminiferous
PT E
epithelium were found after BDE47 treatment, particularly with the HFD (Fig. 3B). Similarly, the HFD further reduced the number of epididymal sperm in rats treated
CE
with BDE47 (Fig. 3C).
AC
HFD aggravates the changes in the expression of DAX-1 and steroidogenic proteins in the testis of rats treated with BDE47 As shown in Fig. 4A, BDE47 treatment resulted in the down-regulation of StAR and 3β-HSD, and this response was enhanced by the HFD. Conversely, DAX-1 expression was up-regulated after BDE47 exposure and even more so in the HFD rats (Fig. 4A). These observations were further confirmed through immunochemistry on testicular sections, which revealed that DAX-1 was clearly expressed in the testicular interstitial 14
ACCEPTED MANUSCRIPT compartments after 20 mg/kg/d BDE47 treatment; this expression was also enhanced by the HFD (Fig. 4B). DAX-1 mediates the inhibition of testosterone in primary Leydig cells treated with BDE47 or insulin
PT
The results of 3β-HSD staining showed that primary Leydig cells were successfully
RI
isolated from rat testes (Fig. S5), with 7.1×106 cells per testis, an average of 93.7%
SC
viability, and an approximate purity of 90%. As shown in Fig. 5A, no cytotoxic effects were found using 20 μM BDE47, the concentration used in further
NU
experiments. Similar to the results in rats, BDE47 resulted in the down-regulation of
MA
StAR and 3β-HSD (Fig. 5B). However, BDE47 induced the up-regulation of DAX-1 in Leydig cells, as observed using Western blotting (Fig. 5B, C) and
D
immunofluorescence (Fig. 5D).
PT E
As shown in Figures 6A and 6B, BDE47 or insulin inhibited testosterone production in Leydig cells in a dose-dependent manner. In addition, both BDE47 and
CE
insulin induced the expression of DAX-1, but these effects were nearly eliminated by
AC
si-DAX-1 (Fig. 6C). Interestingly, both BDE47 and insulin significantly reduced testosterone production, and this effect was almost completely reversed by siDAX-1 (Fig. 6D).
Discussion The present study demonstrated that the HFD aggravated the high glucose and insulin levels, the disruption of the seminiferous epithelium and the low testosterone levels in 15
ACCEPTED MANUSCRIPT rats treated with BDE47. The HFD not only enhanced the BDE47-induced abnormalities in glycolipid metabolism and subsequent hyperinsulinemia but also increased the accumulation of BDE47 in the testis. High levels of both insulin and BDE47 directly up-regulated DAX-1, subsequently down-regulated the steroidogenic
PT
proteins, i.e., 3β-HSD and StAR, and ultimately inhibited testosterone production in
RI
the Leydig cells of rats. Therefore, the HFD aggravated BDE47-inhibited testosterone
SC
production by upregulating DAX-1.
PBDEs are ubiquitous in various environmental matrices and organisms. As is
NU
well known, BDE47 is a typical lipophilic chemical that accumulates in adipose-rich
MA
tissues. The concentrations of BDE47 were the highest in the adipose tissue of rats or mice treated with at least one dose of BDE47 (Sanders et al., 2006). In general, a
D
HFD could significantly increase weight gain and fat accumulation (Liu et al., 2016),
PT E
which indicated that a HFD might increase the accumulation of BDE47 in target tissues. In the present study, a HFD aggravated the accumulation of BDE47 in the
CE
liver and in the testis. In addition,BDE47 could disturb energy metabolism, and more
AC
than 1 μg/L BDE47 could induce higher levels of glucose and ATP in the earthworm Eisenia fetida (Ji et al., 2013). As reported in our previous study, environmental exposure to BDE47 could increase the risk of diabetes in the Chinese population (Zhang et al., 2016). In the present study, BDE47 significantly increased the weight of the adipose tissue, increased macrovesicular steatosis in the liver and the levels of triglycerides, fasting glucose and insulin, which was aggravated by the HDF. These findings were also supported by a previous study that the HFD aggravated the glucose 16
ACCEPTED MANUSCRIPT homeostasis disorder caused by chronic exposure to bisphenol A (BPA) (Ding et al., 2014). BDE47 has been identified as a developmental, reproductive, and neurological toxicant and a disrupter of multiple endocrine systems in rats (Emond et al., 2010); it
PT
also decreased the frequency of the induction of spawning and fertilization in male
RI
zebrafish (Huang et al., 2015b). Consistent with our previous study (Zhang et al.,
SC
2013b), the present study showed that BDE47 could reduce serum testosterone levels, cause vacuolar spaces in the seminiferous epithelium and decrease the number of
NU
sperm in the epididymis. Our previous study had shown that BPA could induce
MA
spermatogenesis disorders mainly through decreasing androgen receptor expression (Qiu et al., 2013), and the HFD could aggravate BPA-induced impaired
D
spermatogenesis (Nanjappa et al., 2014; Tarapore et al., 2016). In the present study,
PT E
the HFD aggravated BDE47-induced the reduction of testosterone, the disruption of the seminiferous epithelium, resulting in fewer sperm in the epididymis. These results
CE
indicated that metabolic disorders induced by BDE47 could contribute to male
AC
reproductive damage.
Testosterone, which is produced by Leydig cells in response to LH, plays a pivotal role in the initiation and maintenance of spermatogenesis by affecting androgen receptors in the Sertoli cells. Thus, an abnormal reduction in testosterone levels can cause testicular atrophy, accompanied by reduced numbers of germ cells and, ultimately, azoospermia (Awoniyi et al., 1989; Smith and Walker, 2014). Testicular steroidogenesis is mediated by LH through multiple signaling pathways, 17
ACCEPTED MANUSCRIPT including the steroidogenic acute regulatory protein (StAR), a cholesterol side chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) (Miller, 1988). Polychlorinated biphenyls (PCBs) could act directly on Leydig cells to diminish testosterone production by inhibiting gene expression of steroidogenic
PT
enzymes including StAR, P450scc and 3β-HSD (Murugesan et al., 2007). A previous
RI
study revealed that benzo[a]pyrene could decrease epididymal sperm quality, possibly
SC
by disturbing testosterone production, indicating that StAR should be a major target among steroidogenic proteins (Chung et al., 2011). These studies support our present
NU
results, which showed that BDE47 inhibits testosterone by decreasing the expression
MA
of StAR and 3β-HSD in the primary Leydig cells and after an in vivo exposure. DAX-1 plays a key role in the development and maintenance of reproductive
D
function. Indeed, the expression pattern of DAX-1 is restricted to tissues directly
PT E
involved in steroid hormone production (Lalli and Sassone-Corsi, 2003). DAX-1 blocks the rate-limiting step in steroid biosynthesis by repressing StAR as well as by
CE
inhibiting both P450scc and 3β-HSD (Maris et al., 2015). In the present study, BDE47
AC
upregulated the expression of DAX-1 in vitro and in vivo, and knocking down DAX-1 significantly reversed BDE47-inhibited testosterone production in the primary Leydig cells, suggesting that BDE47 could directly upregulate DAX-1, leading to the inhibition of testosterone production; this phenomenon was also enhanced by a HFD by increasing BDE47 accumulation in the testis. Insulin can directly bind to insulin receptors in the Leydig cell membrane, leading to the up-regulation of DAX-1 and resulting in liver receptor homolog-1 (LRH-1)-mediated testicular steroidogenesis 18
ACCEPTED MANUSCRIPT (Ahn et al., 2013). In the present study, the knockdown of DAX-1 could significantly reverse the insulin-mediated inhibition of testosterone production in primary Leydig cells, indicating that BDE47 induced a high insulin level and subsequently inhibited testosterone production by upregulating DAX-1; this response was aggravated by the
PT
HFD.
RI
In conclusion, our study demonstrated that the HFD aggravated BDE47-inhibited
SC
testosterone production through hyperinsulinemia and that the accumulation of testicular BDE47 targeted the steroidogenic pathway via DAX-1. The inhibition of
NU
steroidogenesis through the insulin-mediated induction of DAX-1 provides new
MA
insights into the relationship between metabolic disorders and male reproductive damage caused by environmental exposure to lipophilic xenobiotics, particularly with
PT E
D
an unhealthy lifestyle, including a high-fat diet.
Funding
CE
This work was supported by the National Natural Science Foundation of China
AC
(81372956, 81573194), Natural Science Foundation of Jiangsu province (BK20151555), Technology & Development Fund of Nanjing Medical University (2014NJMUZD002), Six talents peak project of Jiangsu province (DG216D5047), 333 Advance Talents Projects of Jiangsu Province [BRA2014285], Natural Science Foundation of Jiangsu Universities (14KJA330002), National 973 Program (2009CB941701), Science and Technique Foundation for Youths by Health and Family Planning Commission of Changzhou (QN201603), and a project funded by the 19
ACCEPTED MANUSCRIPT Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China.
Author Disclosure Statement
AC
CE
PT E
D
MA
NU
SC
RI
PT
No competing financial interests exist.
20
ACCEPTED MANUSCRIPT References Ahn, S.W., Gang, G.T., Kim, Y.D., Ahn, R.S., Harris, R.A., Lee, C.H., Choi, H.S., 2013. Insulin directly regulates steroidogenesis via induction of the orphan nuclear receptor DAX-1 in testicular Leydig cells. J Biol Chem 288,
PT
15937-15946.
RI
Awoniyi, C.A., Santulli, R., Sprando, R.L., Ewing, L.L., Zirkin, B.R., 1989.
SC
Restoration of advanced spermatogenic cells in the experimentally regressed rat testis: quantitative relationship to testosterone concentration within the
NU
testis. Endocrinology 124, 1217-1223.
MA
Barazani, Y., Katz, B.F., Nagler, H.M., Stember, D.S., 2014. Lifestyle, environment, and male reproductive health. Urol Clin North Am 41, 55-66.
D
Biswas, M., Hampton, D., Turkes, A., Newcombe, R.G., Aled Rees, D., 2010.
PT E
Reduced total testosterone concentrations in young healthy South Asian men are partly explained by increased insulin resistance but not by altered adiposity.
CE
Clin Endocrinol (Oxf) 73, 457-462. Chung, J.-Y., Kim, Y.-J., Kim, J.Y., Lee, S.G., Park, J.-E., Kim, W.R., Yoon, Y.-D.,
AC
Yoo, K.S., Yoo, Y.H., Kim, J.-M., 2011. Benzo[a]pyrene Reduces Testosterone Production in Rat Leydig Cells via a Direct Disturbance of Testicular Steroidogenic Machinery. Environmental Health Perspectives 119, 1569-1574. Ding, S., Fan, Y., Zhao, N., Yang, H., Ye, X., He, D., Jin, X., Liu, J., Tian, C., Li, H., Xu, S., Ying, C., 2014. High-fat diet aggravates glucose homeostasis disorder caused by chronic exposure to bisphenol A. J Endocrinol 221, 167-179.
21
ACCEPTED MANUSCRIPT Emond, C., Raymer, J.H., Studabaker, W.B., Garner, C.E., Birnbaum, L.S., 2010. A physiologically based pharmacokinetic model for developmental exposure to BDE-47 in rats. Toxicol Appl Pharmacol 242, 290-298. Fan, Y., Liu, Y., Xue, K., Gu, G., Fan, W., Xu, Y., Ding, Z., 2015. Diet-induced obesity
PT
in male C57BL/6 mice decreases fertility as a consequence of disrupted
RI
blood-testis barrier. PLoS One 10, e0120775.
SC
Grossmann, M., Gianatti, E.J., Zajac, J.D., 2010. Testosterone and type 2 diabetes. Curr Opin Endocrinol Diabetes Obes 17, 247-256.
NU
Haffner, S.M., Karhapaa, P., Mykkanen, L., Laakso, M., 1994. Insulin resistance,
MA
body fat distribution, and sex hormones in men. Diabetes 43, 212-219. Hites, R.A., 2004. Polybrominated diphenyl ethers in the environment and in people:
D
a meta-analysis of concentrations. Environ Sci Technol 38, 945-956.
PT E
Huang, S., Cui, Y., Guo, X., Wang, L., Li, S., Lu, Y., Bi, Y., Huang, X., Lin, M., Xia, Y., Wang, S., Wang, X., Zhou, Z., Sha, J., 2015a. 2,2',4,4'-Tetrabromodiphenyl
CE
ether disrupts spermatogenesis, impairs mitochondrial function and induces
AC
apoptosis of early leptotene spermatocytes in rats. Reprod Toxicol 51, 114-124.
Huang, Y., Zhu, G., Peng, L., Ni, W., Wang, X., Zhang, J., Wu, K., 2015b. Effect of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on sexual behaviors and reproductive function in male zebrafish (Danio rerio). Ecotoxicol Environ Saf 111, 102-108. Ji, C., Wu, H., Wei, L., Zhao, J., Lu, H., Yu, J., 2013. Proteomic and metabolomic 22
ACCEPTED MANUSCRIPT analysis of earthworm Eisenia fetida exposed to different concentrations of 2,2',4,4'-tetrabromodiphenyl ether. J Proteomics 91, 405-416. Jung, T.W., Hwang, H.J., Hong, H.C., Yoo, H.J., Baik, S.H., Choi, K.M., 2015. BAIBA attenuates insulin resistance and inflammation induced by palmitate or
PT
a high fat diet via an AMPK-PPARdelta-dependent pathway in mice.
RI
Diabetologia 58, 2096-2105.
SC
Klinefelter, G.R., Ewing, L.L., 1988. Optimizing testosterone production by purified adult rat Leydig cells in vitro. In Vitro Cell Dev Biol 24, 545-549.
NU
Klinefelter, G.R., Hall, P.F., Ewing, L.L., 1987. Effect of luteinizing hormone
MA
deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 36, 769-783.
D
Lalli, E., Sassone-Corsi, P., 2003. DAX-1, an unusual orphan receptor at the
PT E
crossroads of steroidogenic function and sexual differentiation. Mol Endocrinol 17, 1445-1453.
CE
Lardone, M.C., Parada-Bustamante, A., Ebensperger, M., Valdevenito, R., Kakarieka,
AC
E., Martinez, D., Pommer, R., Piottante, A., Castro, A., 2011. DAX-1 and DAX-1A expression in human testicular tissues with primary spermatogenic failure. Mol Hum Reprod 17, 739-746. Linares, V., Belles, M., Domingo, J.L., 2015. Human exposure to PBDE and critical evaluation of health hazards. Arch Toxicol 89, 335-356. Liu, D., Archer, N., Duesing, K., Hannan, G., Keast, R., 2016. Mechanism of fat taste perception: Association with diet and obesity. Prog Lipid Res 63, 41-49. 23
ACCEPTED MANUSCRIPT Maris, P., Campana, A., Barone, I., Giordano, C., Morelli, C., Malivindi, R., Sisci, D., Aquila, S., Rago, V., Bonofiglio, D., Catalano, S., Lanzino, M., Ando, S., 2015. Androgens inhibit aromatase expression through DAX-1: insights into the molecular link between hormone balance and Leydig cancer development.
PT
Endocrinology 156, 1251-1262.
RI
Mhadhbi, L., Fumega, J., Boumaiza, M., Beiras, R., 2012. Acute toxicity of
SC
polybrominated diphenyl ethers (PBDEs) for turbot (Psetta maxima) early life stages (ELS). Environ Sci Pollut Res Int 19, 708-717.
NU
Miller, W.L., 1988. Molecular biology of steroid hormone synthesis. Endocr Rev 9,
MA
295-318.
Murugesan, P., Balaganesh, M., Balasubramanian, K., Arunakaran, J., 2007. Effects of
D
polychlorinated biphenyl (Aroclor 1254) on steroidogenesis and antioxidant
PT E
system in cultured adult rat Leydig cells. J Endocrinol 192, 325-338. Nanjappa, M.K., Ahuja, M., Dhanasekaran, M., Coleman, E.S., Braden, T.D., Bartol,
CE
F.F., Bird, R.C., Wanders, D., Judd, R.L., Akingbemi, B.T., 2014. Bisphenol A
AC
regulation of testicular endocrine function in male rats is affected by diet. Toxicol Lett 225, 479-487. Qiu, L.L., Wang, X., Zhang, X.H., Zhang, Z., Gu, J., Liu, L., Wang, Y., Wang, X., Wang, S.L., 2013. Decreased androgen receptor expression may contribute to spermatogenesis failure in rats exposed to low concentration of bisphenol A. Toxicol Lett 219, 116-124. Sanders, J.M., Chen, L.J., Lebetkin, E.H., Burka, L.T., 2006. Metabolism and 24
ACCEPTED MANUSCRIPT disposition of 2,2',4,4'- tetrabromodiphenyl ether following administration of single or multiple doses to rats and mice. Xenobiotica 36, 103-117. Skakkebaek, N.E., Rajpert-De Meyts, E., Buck Louis, G.M., Toppari, J., Andersson, A.M., Eisenberg, M.L., Jensen, T.K., Jorgensen, N., Swan, S.H., Sapra, K.J.,
PT
Ziebe, S., Priskorn, L., Juul, A., 2016. Male Reproductive Disorders and
RI
Fertility Trends: Influences of Environment and Genetic Susceptibility.
SC
Physiol Rev 96, 55-97.
Skovso, S., 2014. Modeling type 2 diabetes in rats using high fat diet and
NU
streptozotocin. J Diabetes Investig 5, 349-358.
MA
Smith, L.B., Walker, W.H., 2014. The regulation of spermatogenesis by androgens. Semin Cell Dev Biol 30, 2-13.
D
Stickels, R., Clark, K., Heider, T.N., Mattiske, D.M., Renfree, M.B., Pask, A.J., 2015.
PT E
DAX1/NR0B1 was expressed during mammalian gonadal development and gametogenesis before it was recruited to the eutherian X chromosome. Biol
CE
Reprod 92, 22.
AC
Tarapore, P., Hennessy, M., Song, D., Ying, J., Ouyang, B., Govindarajah, V., Leung, Y.K., Ho, S.M., 2016. High butter-fat diet and bisphenol A additively impair male rat spermatogenesis. Reprod Toxicol. doi: 10.1016/j.reprotox.2016.09.008. Xu, Y., Wang, L., He, J., Bi, Y., Li, M., Wang, T., Wang, L., Jiang, Y., Dai, M., Lu, J., Xu, M., Li, Y., Hu, N., Li, J., Mi, S., Chen, C.S., Li, G., Mu, Y., Zhao, J., Kong, L., Chen, J., Lai, S., Wang, W., Zhao, W., Ning, G., China Noncommunicable 25
ACCEPTED MANUSCRIPT Disease Surveillance, G., 2013. Prevalence and control of diabetes in Chinese adults. JAMA 310, 948-959. Yang, X., Zhang, Z., Wang, X., Wang, Y., Zhang, X., Lu, H., Wang, S.L., 2013. Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial
PT
cells to aflatoxin B1-induced DNA damage. Toxicol Appl Pharmacol 270,
RI
114-121.
SC
Yeap, B.B., Chubb, S.A., Hyde, Z., Jamrozik, K., Hankey, G.J., Flicker, L., Norman, P.E., 2009. Lower serum testosterone is independently associated with insulin
NU
resistance in non-diabetic older men: the Health In Men Study. Eur J
MA
Endocrinol 161, 591-598.
Zhang, Z., Li, S., Liu, L., Wang, L., Xiao, X., Sun, Z., Wang, X., Wang, C., Wang, M.,
D
Li, L., Xu, Q., Gao, W., Wang, S.L., 2016. Environmental exposure to BDE47
PT E
is associated with increased diabetes prevalence: Evidence from community-based case-control studies and an animal experiment. Sci Rep 6,
CE
27854.
AC
Zhang, Z., Lu, H., Huan, F., Meghan, C., Yang, X., Wang, Y., Wang, X., Wang, X., Wang, S.L., 2014. Cytochrome P450 2A13 mediates the neoplastic transformation of human bronchial epithelial cells at a low concentration of aflatoxin B1. Int J Cancer 134, 1539-1548. Zhang, Z., Sun, Z.Z., Xiao, X., Zhou, S., Wang, X.C., Gu, J., Qiu, L.L., Zhang, X.H., Xu, Q., Zhen, B., Wang, X., Wang, S.L., 2013a. Mechanism of BDE209-induced impaired glucose homeostasis based on gene microarray 26
ACCEPTED MANUSCRIPT analysis of adult rat liver. Arch Toxicol 87, 1557-1567. Zhang, Z., Zhang, X., Sun, Z., Dong, H., Qiu, L., Gu, J., Zhou, J., Wang, X., Wang, S.L., 2013b. Cytochrome P450 3A1 mediates 2,2',4,4'-tetrabromodiphenyl
AC
CE
PT E
D
MA
NU
SC
RI
PT
ether-induced reduction of spermatogenesis in adult rats. PLoS One 8, e66301.
27
ACCEPTED MANUSCRIPT Figure legends Figure 1. Effects of the HFD on the accumulation of BDE47 and relative weight of adipose tissues in rats treated with BDE47. (A) BDE47 content in the liver and testis. The data are shown as box and whisker plots (minimum to maximum). (B)
PT
Ratio of the retroperitoneal WAT weight and body weight (BW). (C) Ratio of the
RI
gonadal WAT weight and BW. (D) Ratio of the BAT weight and BW. The data were
SC
expressed as the means± SD of 6 rats per group. #p < 0.05 compared with the corresponding vehicle control; *p< 0.05, **p < 0.01 compared with the corresponding
MA
NU
BDE47 treatment in the ND group.
Figure 2. Effects of the HFD on glycolipid metabolism in rats treated with
D
BDE47. (A) Serum TG. (B) Serum CHOL. (C) Fasting glucose. (D) Serum insulin. (E)
PT E
Morphological observations of liver tissue. The tissue was stained with hematoxylin and eosin (H&E) and was observed using optical microscopy. Blue arrow, lipid
p < 0.05 compared with the corresponding vehicle control; *p < 0.05, **p < 0.01
AC
#
CE
droplets; bar, 100 μm. The data were expressed as the means± SD of 6 rats per group.
compared with the corresponding BDE47 treatment in the ND group.
Figure 3. Effects of the HFD on the serum testosterone and the morphological changes in testes and epididymides of rats treated with BDE47. (A) Serum testosterone levels. (B) Morphological observations of the testis and seminiferous tubule architecture. (C) Morphological observations of epididymis. The tissue was 28
ACCEPTED MANUSCRIPT stained with H&E and observed using optical microscopy. Blue arrow, giant cells in the testis; red arrow, vacuolar spaces in the seminiferous epithelium; black arrow, reduced sperm count; bar, 100 μm. The data were expressed as the means± SD of 6 rats per group. #p < 0.05, ##p < 0.01 compared with the corresponding vehicle control;
RI
PT
*p < 0.05 compared with the corresponding BDE47 treatment in the ND group.
SC
Figure 4. Effects of the HFD on the expression of DAX-1, StAR, and 3β-HSD in the testis of rats treated with BDE47. (A) The expression levels of DAX-1, StAR,
NU
and 3β-HSD using immunoblotting assays. Lysates from the testes (80 μg) were
MA
prepared, and GAPDH was used as an internal reference. The densitometric analysis was conducted for the protein bands using ImageJ software. The data are expressed as
p < 0.001 compared with the corresponding vehicle control; *p < 0.05, **p < 0.01,
PT E
###
D
the mean ± SD of three independent experiments with triplicates. #p < 0.05, ##p < 0.01,
***p < 0.001 compared with the corresponding BDE47 treatment in the ND group. (B)
CE
The expression of DAX-1 using an immunohistochemical assay. The localization of
AC
DAX-1 was observed in the testicular interstitial compartments.
Figure 5. Effects of BDE47 on cell viability and DAX-1 expression in the primary Leydig cells of rats. (A) Cells viability using a CCK-8 assay. The cells were treated with BDE47 for 48 h, and the data were expressed as the means± SE of three independent experiments with six replicates; (B) The expression levels of DAX-1, StAR, and 3β-HSD using immunoblotting assays. Cell lysates (50 μg) were prepared, 29
ACCEPTED MANUSCRIPT and GAPDH was used as an internal reference. (C) The densitometric analysis was conducted for the protein bands using ImageJ software. The data are expressed as the mean ± SD of three independent experiments with triplicates. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle control. (D) The expression of DAX-1 using
RI
PT
an immunofluorescence assay.
SC
Figure 6. Effects of DAX-1 knockdown on DAX-1 expression and testosterone secretion in primary Leydig cells from rats treated with BDE47 or insulin. (A)
NU
The effects of BDE47 on the testosterone concentration in the cell culture medium. (B)
MA
The effects of insulin on the testosterone in the cell culture medium. The cells were treated with BDE47 or insulin for 24 h, and the testosterone levels in the supernatant
D
were determined using ELISA. (C) The effects of si-DAX-1 on DAX-1 expression in
PT E
cells treated with BDE47 or insulin, the densitometric analysis was conducted for the protein bands using ImageJ software. The data are expressed as the mean ± SD of
CE
three independent experiments with triplicates. (D) The effects of si-DAX-1 on the
AC
testosterone secretion of cells treated with BDE47 or insulin. The cells were pretreated with si-DAX-1 for 48 h and were then treated with 20 μM BDE47 or 80 nM insulin (1 μU/ml= 6.00 pmol/L) for an additional 24 h. The data were expressed as the means± SE of three independent experiments with six replicates. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the vehicle control; *p < 0.05, **p < 0.01 compared with the BDE47 or insulin treatment alone.
30
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 1
31
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 2
32
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 3
33
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 4
34
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 5
35
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 6
36
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Graphical abstract
37
ACCEPTED MANUSCRIPT Highlights ► High-fat diet (HFD) aggravates the accumulation of BDE47 in liver and testis in rats ► HFD aggravates BDE47-inhibited testosterone production via DAX-1 in Leydig cells ► HFD enhances BDE47-induced the disorder of glycolipid metabolism and hyperinsulinemia
AC
CE
PT E
D
MA
NU
SC
RI
PT
► Both hyperinsulinemia and accumulation of BDE47 inhibited testosterone via DAX-1
38
Zhan Zhang, Yongquan Yu, Hengsen Xu, Chao Wang, Minghui Ji, Jun Gu, Lu Yang, Jiansheng Zhu, Huibin Dong, Shou-Lin Wang PII: DOI: Reference:
S0041-008X(17)30114-X doi: 10.1016/j.taap.2017.03.010 YTAAP 13891
To appear in:
Toxicology and Applied Pharmacology
Received date: Revised date: Accepted date:
15 October 2016 2 March 2017 10 March 2017
Please cite this article as: Zhan Zhang, Yongquan Yu, Hengsen Xu, Chao Wang, Minghui Ji, Jun Gu, Lu Yang, Jiansheng Zhu, Huibin Dong, Shou-Lin Wang , High-fat diet aggravates 2,2′,4,4′-tetrabromodiphenyl ether-inhibited testosterone production via DAX-1 in Leydig cells in rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ytaap(2017), doi: 10.1016/ j.taap.2017.03.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT High-fat diet aggravates 2,2’,4,4’-tetrabromodiphenyl ether-inhibited testosterone production via DAX-1 in Leydig cells in rats
Zhan Zhang1,2,4, Yongquan Yu1,2,4, Hengsen Xu2, Chao Wang1,2, Minghui Ji2, Jun Gu2,
State Key Lab of Reproductive Medicine, Institute of Toxicology, Nanjing Medical
SC
1
RI
PT
Lu Yang1,2, Jiansheng Zhu1,2, Huibin Dong3, Shou-Lin Wang1,2,*
University, 101 Longmian Avenue, Nanjing 211166, P. R. China Key Lab of Modern Toxicology of Ministry of Education, School of Public Health,
NU
2
3
MA
Nanjing Medical University, 101 Longmian Avenue, Nanjing 211166, P. R. China Changzhou Center for Disease Control and Prevention, 203 Taishan Road,
D
Changzhou, 2013022, P. R. China
These authors contributed equally to this study
*
To whom correspondence should be addressed: Key Lab of Modern Toxicology of
CE
PT E
4
AC
Ministry of Education, School of Public Health, Nanjing Medical University, 101 Longmian Avenue, Nanjing 211166, P. R. China Tel: +86-25-8686-8417 Fax: +86-25-8686-8499 E-mail: [email protected]
1
ACCEPTED MANUSCRIPT Abbreviations: BDE47, 2,2’, 4,4’-tetrabromodiphenyl ether; PBDEs, polybrominated diphenyl ethers; ND, normal diet; HFD, high-fat diet; BAT, brown adipose tissue; WAT, white adipose tissue; CHOL, total cholesterol; TG, triglycerides; ELISA, enzyme-linked
PT
immunosorbent assay; 3β-HSD, 3β-hydroxysteroid dehydrogenase; StAR,
RI
steroidogenic acute regulatory protein; DAX-1, dosage-sensitive sex reversal-adrenal
AC
CE
PT E
D
MA
NU
SC
hypoplasia congenita critical region of the X chromosome gene 1.
2
ACCEPTED MANUSCRIPT Abstract Growing evidence has revealed that a high-fat diet (HFD) could lead to disorders of glycolipid metabolism and insulin-resistant states, and HFDs have been associated with the inhibition of testicular steroidogenesis. Our previous study demonstrated that
PT
2,2′,4,4′-tetrabromodiphenyl ether (BDE47) could increase the risk of diabetes in
RI
humans and reduce testosterone production in rats. However, whether the HFD affects
SC
BDE47-inhibited testosterone production by elevating insulin levels and inducing related pathways remains unknown. In male rats treated with BDE47 by gavage for 12
NU
weeks, the HFD significantly increased the BDE47 content of the liver and testis and
MA
increased the weight of the adipose tissue; increased macrovesicular steatosis in the liver and the levels of triglycerides, fasting glucose and insulin; further aggravated the
D
disruption of the seminiferous epithelium; and lowered the level of testosterone,
PT E
resulting in fewer sperm in the epididymis. Of note, the HFD enhanced BDE47-induced DAX-1 expression and decreased the expression levels of StAR and
CE
3β-HSD in the testicular interstitial compartments in rats. In isolated primary Leydig
AC
cells from rats, BDE47 or insulin increased DAX-1 expression, decreased the expression of StAR and 3β-HSD, and reduced testosterone production, which was nearly reversed by knocking down DAX-1. These results indicated that the HFD aggravates BDE47-inhibited testosterone production through hyperinsulinemia, and the accumulation of testicular BDE47 that induces the up-regulation of DAX-1 and the subsequent down-regulation of steroidogenic proteins, i.e., StAR and 3β-HSD, in Leydig cells. 3
ACCEPTED MANUSCRIPT Keywords: High-fat diet; 2,2′,4,4′-tetrabromodiphenyl ether; DAX-1; insulin;
AC
CE
PT E
D
MA
NU
SC
RI
PT
testosterone; primary Leydig cells
4
ACCEPTED MANUSCRIPT Introduction Male reproductive problems, such as poor semen quality, low testosterone levels, cryptorchidism, and testicular cancer, are widespread and have recently been the focus of several basic and clinical research projects (Skakkebaek et al., 2016). Many
PT
environmental and lifestyle factors may negatively affect spermatogenesis and male
RI
fertility (Barazani et al., 2014). With rapid economic growth and its associated
SC
industrialization, urbanization, and lifestyle changes, i.e. high-fat diets (HFDs), and low physical activity, diabetes has reached epidemic proportions in the Chinese
NU
population (Xu et al., 2013). HFD could induce an increase in body weight, impair
MA
glucose tolerance and cause insulin resistance (Skovso, 2014; Jung et al., 2015); the HFD could also impair male fertility by reducing the quality of the semen and sperm
D
and by reducing testosterone levels (Fan et al., 2015).
PT E
Polybrominated diphenyl ethers (PBDEs) have been used as flame-retardant chemicals in various products worldwide (Linares et al., 2015). Among the PBDEs,
CE
2,2′,4,4′-tetrabromodiphenyl ether (BDE47) is the dominant congener in animal and
AC
human tissue (Hites, 2004; Mhadhbi et al., 2012). Our recent study has shown that environmental exposure to BDE47 increased the risk of diabetes by activating the diabetes pathway and upregulating genes involved in glucose transport (Zhang et al., 2016). In addition, BDE47 disrupts spermatogenesis, impairs mitochondrial function and induces the apoptosis of early leptotene spermatocytes in rats (Huang et al., 2015a). Our previous study also revealed that BDE47 could cause a striking disruption of the normal cellular organization of the seminiferous epithelium and 5
ACCEPTED MANUSCRIPT decrease the serum testosterone level (Zhang et al., 2013b). However, the mechanism by which BDE47 inhibits testosterone production is still unclear. Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1) is required for human spermatogenesis because it is a
PT
crucial regulator of steroidogenesis in mammals (Lardone et al., 2011; Stickels et al.,
RI
2015). For instance, DAX-1 is a repressor of several steroidogenic enzymes, blocking
SC
steroid production at multiple levels and decreasing the formation of steroids (Maris et al., 2015). High insulin levels are associated with low testosterone levels in type 2
NU
diabetes (Haffner et al., 1994; Grossmann et al., 2010). In healthy men, lower total
MA
testosterone is associated with insulin resistance (Yeap et al., 2009; Biswas et al., 2010). Insulin might directly repress steroidogenesis by inducing the orphan nuclear
D
receptor DAX-1 in testicular Leydig cells (Ahn et al., 2013). Thus, BDE47 might
PT E
induce high insulin levels and subsequently inhibit testosterone production via DAX-1.
CE
In the present study, adult male rats were treated with BDE47 by gavage for 3
AC
months while being fed the normal diet (ND) or HFD. The effects of BDE47 on lipometabolism and glycometabolism in rats fed the HFD were investigated. Next, testosterone levels, histopathologic changes in the testis and epididymis, and the expression of steroidogenic proteins in rat testis were evaluated. Furthermore, the primary Leydig cells of rats were isolated and used to explore the roles of DAX-1 in the effects of HFD on BDE47-inhibited testosterone production. This study attempts to illustrate the molecular mechanism of the effects of HFD and/or BDE47 on 6
ACCEPTED MANUSCRIPT testosterone production, to enhance the knowledge of metabolic syndrome in relation to reduced male reproduction and to provide evidence-based information for assessing the risks of environmental pollutants, particularly in the presence of the HFD.
PT
Materials and methods
RI
Chemicals and reagents
SC
BDE47 (purity ≥ 98.7%) was purchased from Chemservice (West Chester, PA, USA). Corn oil and collagenase I were purchased from Sigma-Aldrich (St. Louis, MO, USA).
NU
The enzyme-linked immunosorbent assay (ELISA) kit for rat insulin was obtained
MA
from Cusabio Biotech Co., Ltd (Wuhan, China). Dulbecco's Modified Eagle’s Medium/ Nutrient Mixture F-12 (DMEM/F-12), fetal bovine serum (FBS) and
D
collagen-coated culture plates were purchased from GIBCO-BRL (Grand Island, NY,
PT E
USA). Antibodies specific for StAR and DAX-1 and an enhanced chemiluminescence (ECL) immunoblotting assay kit were purchased from Cell Signaling Technology
CE
(Danvers, MA, USA). Antibodies specific for GAPDH and 3β-HSD were obtained
AC
from Santa Cruz Biotechnology (Santa Cruz, CA). A cell counting kit-8 (CCK-8), penicillin, streptomycin, gentamicin, a bicinchoninic acid (BCA) protein assay kit, and bovine serum albumin (BSA) were obtained from Beyotime Institute of Biotechnology (Shanghai, China). The ND (15% calories from fat) and HFD (45% calories from fat) were obtained from Jiangsu Medicience Ltd. (Yangzhou, China). Animal treatment and sample collection Male Sprague-Dawley rats (weighing 180-200 g) were obtained from Shanghai SLAC 7
ACCEPTED MANUSCRIPT Laboratory Animal Co., Ltd (Shanghai, China). The animals were maintained in 12-h/12-h light/dark cycles at ambient temperature (22C) and 55% relative humidity. Two rats were placed in a plastic Macrolon cage with stainless steel covers and wood shavings, and sterilized food and tap water were provided ad libitum. The rats were
PT
allowed to acclimate for 1 week before the experiments began. Thirty-six rats were
RI
randomly divided into six groups and were gavaged for 12 weeks: group 1 (ND
SC
control group) was fed a normal diet; groups 2 and 3 were treated with BDE47 (dissolved in corn oil) at 0.03 and 20 mg/kg. body weight (BW)/day, respectively;
NU
group 4 (HFD control group) was fed a high-fat diet; and groups 5 and 6 were fed
MA
HFD plus BDE47 at 0.03 or 20 mg/kg.bw/day, respectively. The ND and HFD control groups were given the same volume of corn oil, and the BDE47 doses were designed
D
based on our previous study (Zhang et al., 2013b). BW was quantified weekly, and
PT E
the rats were euthanized by CO2 asphyxiation at the study endpoint for serum collection and harvesting of tissues, including the liver, pancreas, testis, epididymis,
CE
brown adipose tissue (BAT), and the retroperitoneal and gonadal white adipose tissue
AC
(WAT). Except the serum that was used for fasting glucose measurements, all other samples were frozen and stored at -80C until analysis. During the experimental period, the care and use of the animals followed the guidelines of the Animal Care and Welfare Committee of Nanjing Medical University. Detection of BDE47 in the liver and testes of rats The sample extraction, clean-up and quality control for analysis were performed as described in our previous publication (Zhang et al., 2013b). The samples were 8
ACCEPTED MANUSCRIPT analyzed with a gas chromatograph-mass spectrometer (GC/MS) (Thermo Finnigan DSQ, USA) using BDE77 as an internal standard. Briefly, 1 μL of sample was injected using the splitless injection mode with a splitless time of 1 min. The mass spectrometer was operated using negative chemical ionization. Selected ion
PT
monitoring (SIM) was employed to determine individual peaks. The following pair of
RI
ions was monitored for each target compound, with the first ion being used to quantify
SC
an ion and the second ion for peak identification: 486, 326. Representative GC/MS chromatograms for each PBDE were from calibration mixtures (Accustandard, New
NU
Haven, CT, USA).
MA
Measurement of serum biochemical parameters and testosterone in rats Serum parameters, such as the levels of total cholesterol (CHOL), triglycerides (TG)
D
and fasting glucose, were measured routinely as a single measurement on an
PT E
autoanalyzer (Hitachi 7100, Japan) using standard kits from the Jiancheng Bio-engineering Research Institute (Nanjing, China) (Zhang et al., 2013a). The serum
CE
concentration of testosterone (T) was determined using [125I]-T-specific
AC
radioimmunoassay kits according to the manufacturer’s instructions (Beijing North Institute of Biological Technology, Beijing, China) (Zhang et al., 2013b). Histological observation of rat tissues After euthanization, the testes, epididymis, liver, pancreas and adipose tissue of the rats were fixed in 4% paraformaldehyde and embedded in paraffin, and 5-μm cross-sections were prepared and stained with hematoxylin and eosin (H&E). In the testis, giant cells were identified by their characteristic features, with multiple round 9
ACCEPTED MANUSCRIPT spermatid nuclei (≥2) in a syncytial body present in the lumen. Images were captured using an optical microscope (Axioskop 2 Plus, Carl Zeiss, Hamburg, Germany). Isolation, purification and culture of primary Leydig cells in rats Leydig cells were isolated and cultured as previously described (Klinefelter et al.,
PT
1987; Klinefelter and Ewing, 1988) with slight modifications. Briefly, the testes of
RI
rats (8 weeks old) were decapsulated, placed in 100 mL of PBS containing
SC
collagenase I (0.5 mg/mL) at 34C and shaken for 15 min. The digested testes were passed through a 100-μm nylon mesh, and the Leydig cells were sequentially purified
NU
by 21%, 23%, 32%, and 60% Percoll gradient separation. The Leydig cells were
MA
located in the second layer from the bottom. The final purity of the Leydig cells was determined to be >90% following staining with 3β-HSD, a typical marker for Leydig
D
cells. The isolated Leydig cells were resuspended in DMEM/F12 containing 5% FBS,
further experiments.
PT E
5 μg/mL gentamicin, 50 U/mL penicillin, and 50 μg/mL streptomycin and used for
CE
Treatment of primary Leydig cells from rats with BDE47
AC
The cells were plated at a density of 105/cm2 in six-well plates at 34°C in 5% CO2 overnight and then were treated with 0-150 μM BDE47 to screen for the non-observed cytotoxic concentration using the CCK-8 assay as previously described (Zhang et al., 2013b). Then, the medium and cell lysates were used to determine the testosterone levels and the expression of DAX-1, StAR and 3β-HSD after treatment with BDE47 or insulin for 24 h. Knockdown of DAX-1 in primary Leydig cells from rats 10
ACCEPTED MANUSCRIPT Small interfering RNAs (siRNAs) were used to confirm the role of DAX-1 in the synthesis of testosterone in primary Leydig cells treated with BDE47 or insulin. DAX-1 siRNA (5’-GCAGCAUCUUAUACAGCUU-3’, 3’-CGUCGUAGAAUAUGUCGAA-5’) and one negative scrambled control were
PT
synthesized by RiboBio Co., Ltd (Guangzhou, China). The cells (3×105 per dish) were
RI
plated in 35-mm dishes and cultured overnight, and then DAX-1 siRNA (si-DAX)
SC
was transfected into the cells for 24 h using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Subsequently, the cells
NU
were treated with BDE47 or insulin for an additional 24 h. The medium was used to
of DAX-1, StAR and 3β-HSD.
MA
determine testosterone levels, and the cell lysates were used to measure the expression
D
Determination of DAX-1 expression by immunocytochemistry and
PT E
immunohistochemistry assays
The expression levels of DAX-1 in the primary Leydig cells and the rat testis were
CE
determined by immunocytochemistry and immunohistochemistry based on our
AC
previous studies (Yang et al., 2013; Zhang et al., 2014). For immunocytochemistry, isolated Leydig cells were attached to a glass slide using cytospin centrifugation and then fixed with 4% paraformaldehyde, washed with phosphate-buffered saline (PBS), and incubated with 0.2% Triton X-100. Then, the cells were incubated overnight with the DAX-1 primary antibody in 1% BSA at 4°C followed by the appropriate fluorescence-conjugated secondary antibody at RT. Finally, the cells were mounted and observed under a confocal microscope (LSM710; Carl Zeiss, Hamburg, 11
ACCEPTED MANUSCRIPT Germany). For immunohistochemistry, deparaffinized and hydrated testis sections (5 μm) were treated with 3% hydrogen peroxide (H2O2) for 5 min, rinsed with PBS for 15 min, immunostained using a StreptAvidin Biotin Complex kit (Boster BioEngineering, Wuhan, China) and examined using an Axioskop 2 Plus optical
PT
microscope (Carl Zeiss, Hamburg, Germany).
RI
Determination of the expression levels of DAX-1, StAR and 3β-HSD by
SC
immunoblotting assays
Testis tissue and primary Leydig cell lysates (50-100 μg) were separated by sodium
NU
dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a
MA
polyvinylidene fluoride membrane (Millipore, Bedford, MA). Using specific antibodies for DAX-1, StAR and 3β-HSD, the protein-immune complexes were
D
detected using an ECL immunoblotting assay kit and were exposed to Kodak X-Omat
Statistical analysis
PT E
film. GAPDH was used as an internal control.
CE
Significant differences among different doses in same group were evaluated with the
AC
Student-Newman-Keuls multiple comparison test using SPSS software version 17.0 (Chicago, IL, USA), while the differences in different groups at the same dose were analyzed with one-way analysis of variance (ANOVA). The data for the BDE47 content in the liver and testis are shown as box and whisker plots (minimum to maximum), and the other data were expressed as the means± standard deviation (SD). Differences were considered statistically significant at p≤ 0.05.
12
ACCEPTED MANUSCRIPT Results HFD aggravates the accumulation of adipose tissue in rats treated with BDE47 After a 12-week treatment with BDE47 (0.03 or 20 mg/kg/d) by gavage, BDE47 was found in the rat liver and testis; the HFD could aggravate the accumulation of BDE47
PT
in the liver, and particularly in the testis (Fig. 1A). During the treatment, body weight
RI
was not altered by BDE47, but the HFD caused an increase, as expected (Fig. S1).
SC
Additionally, neither BDE47 nor the HFD changed the relative weight of the candidate organs, including the liver, pancreas, testis and epididymis (Fig. S2).
NU
However, treatment with both doses of BDE47 increased the relative weight of the
MA
retroperitoneal WAT, the gonadal WAT and BAT in rats fed with the HFD, while there were no changes in lipid accumulation in rats treated with BDE47 but given ND
D
feed (Fig. 1B, C, D). Additionally, the number of neonatal dikaryotic adipocytes
PT E
increased after treatment with 20 mg/kg BDE47, most remarkably in the HFD rats (Fig. S3).
CE
HFD aggravates the disruption of glycolipid metabolism in rats treated with BDE47
AC
Serum TG in BDE47-treated rats increased and was enhanced by the HFD (Fig. 2A), whereas no changes in CHOL were observed in rats treated with BDE47 or the HFD (Fig. 2B). Similar to its effect on TG, BDE47 (20 mg/kg/d) significantly increased fasting blood glucose and serum insulin levels in rats, which were also enhanced by the HFD (Fig. 2C, D). No histopathological changes were observed in the pancreas of rats treated with BDE47 or a HFD alone, but exocrine glands transfers were found in the HFD rats treated with 20 mg/kg BDE47 (Fig. S4). Notably, histopathological 13
ACCEPTED MANUSCRIPT analysis showed obvious microvesicular and macrovesicular steatosis in the livers of rats treated with BDE47; in particular, the 20 mg/kg/d BDE47 treatment plus the HFD exacerbated this phenotype (Fig. 2E). HFD aggravates the inhibition of testosterone production and damages the testis
PT
and epididymis in rats treated with BDE47
RI
Similar to our previous study, both doses of BDE47 reduced testosterone
SC
concentrations in ND rats, and this reduction was significantly exacerbated by the HFD (Fig. 3A). Histological examination of the testes of rats exposed to BDE47
NU
revealed a striking disruption of the normal cellular organization of the seminiferous
MA
epithelium. In the lumen, 20 mg/kg BDE47 significantly increased the number of multinucleated giant cells that arose from spermatocytes and aborted meiosis, and
D
they appeared as a syncytium. Abundant vacuolar spaces in the seminiferous
PT E
epithelium were found after BDE47 treatment, particularly with the HFD (Fig. 3B). Similarly, the HFD further reduced the number of epididymal sperm in rats treated
CE
with BDE47 (Fig. 3C).
AC
HFD aggravates the changes in the expression of DAX-1 and steroidogenic proteins in the testis of rats treated with BDE47 As shown in Fig. 4A, BDE47 treatment resulted in the down-regulation of StAR and 3β-HSD, and this response was enhanced by the HFD. Conversely, DAX-1 expression was up-regulated after BDE47 exposure and even more so in the HFD rats (Fig. 4A). These observations were further confirmed through immunochemistry on testicular sections, which revealed that DAX-1 was clearly expressed in the testicular interstitial 14
ACCEPTED MANUSCRIPT compartments after 20 mg/kg/d BDE47 treatment; this expression was also enhanced by the HFD (Fig. 4B). DAX-1 mediates the inhibition of testosterone in primary Leydig cells treated with BDE47 or insulin
PT
The results of 3β-HSD staining showed that primary Leydig cells were successfully
RI
isolated from rat testes (Fig. S5), with 7.1×106 cells per testis, an average of 93.7%
SC
viability, and an approximate purity of 90%. As shown in Fig. 5A, no cytotoxic effects were found using 20 μM BDE47, the concentration used in further
NU
experiments. Similar to the results in rats, BDE47 resulted in the down-regulation of
MA
StAR and 3β-HSD (Fig. 5B). However, BDE47 induced the up-regulation of DAX-1 in Leydig cells, as observed using Western blotting (Fig. 5B, C) and
D
immunofluorescence (Fig. 5D).
PT E
As shown in Figures 6A and 6B, BDE47 or insulin inhibited testosterone production in Leydig cells in a dose-dependent manner. In addition, both BDE47 and
CE
insulin induced the expression of DAX-1, but these effects were nearly eliminated by
AC
si-DAX-1 (Fig. 6C). Interestingly, both BDE47 and insulin significantly reduced testosterone production, and this effect was almost completely reversed by siDAX-1 (Fig. 6D).
Discussion The present study demonstrated that the HFD aggravated the high glucose and insulin levels, the disruption of the seminiferous epithelium and the low testosterone levels in 15
ACCEPTED MANUSCRIPT rats treated with BDE47. The HFD not only enhanced the BDE47-induced abnormalities in glycolipid metabolism and subsequent hyperinsulinemia but also increased the accumulation of BDE47 in the testis. High levels of both insulin and BDE47 directly up-regulated DAX-1, subsequently down-regulated the steroidogenic
PT
proteins, i.e., 3β-HSD and StAR, and ultimately inhibited testosterone production in
RI
the Leydig cells of rats. Therefore, the HFD aggravated BDE47-inhibited testosterone
SC
production by upregulating DAX-1.
PBDEs are ubiquitous in various environmental matrices and organisms. As is
NU
well known, BDE47 is a typical lipophilic chemical that accumulates in adipose-rich
MA
tissues. The concentrations of BDE47 were the highest in the adipose tissue of rats or mice treated with at least one dose of BDE47 (Sanders et al., 2006). In general, a
D
HFD could significantly increase weight gain and fat accumulation (Liu et al., 2016),
PT E
which indicated that a HFD might increase the accumulation of BDE47 in target tissues. In the present study, a HFD aggravated the accumulation of BDE47 in the
CE
liver and in the testis. In addition,BDE47 could disturb energy metabolism, and more
AC
than 1 μg/L BDE47 could induce higher levels of glucose and ATP in the earthworm Eisenia fetida (Ji et al., 2013). As reported in our previous study, environmental exposure to BDE47 could increase the risk of diabetes in the Chinese population (Zhang et al., 2016). In the present study, BDE47 significantly increased the weight of the adipose tissue, increased macrovesicular steatosis in the liver and the levels of triglycerides, fasting glucose and insulin, which was aggravated by the HDF. These findings were also supported by a previous study that the HFD aggravated the glucose 16
ACCEPTED MANUSCRIPT homeostasis disorder caused by chronic exposure to bisphenol A (BPA) (Ding et al., 2014). BDE47 has been identified as a developmental, reproductive, and neurological toxicant and a disrupter of multiple endocrine systems in rats (Emond et al., 2010); it
PT
also decreased the frequency of the induction of spawning and fertilization in male
RI
zebrafish (Huang et al., 2015b). Consistent with our previous study (Zhang et al.,
SC
2013b), the present study showed that BDE47 could reduce serum testosterone levels, cause vacuolar spaces in the seminiferous epithelium and decrease the number of
NU
sperm in the epididymis. Our previous study had shown that BPA could induce
MA
spermatogenesis disorders mainly through decreasing androgen receptor expression (Qiu et al., 2013), and the HFD could aggravate BPA-induced impaired
D
spermatogenesis (Nanjappa et al., 2014; Tarapore et al., 2016). In the present study,
PT E
the HFD aggravated BDE47-induced the reduction of testosterone, the disruption of the seminiferous epithelium, resulting in fewer sperm in the epididymis. These results
CE
indicated that metabolic disorders induced by BDE47 could contribute to male
AC
reproductive damage.
Testosterone, which is produced by Leydig cells in response to LH, plays a pivotal role in the initiation and maintenance of spermatogenesis by affecting androgen receptors in the Sertoli cells. Thus, an abnormal reduction in testosterone levels can cause testicular atrophy, accompanied by reduced numbers of germ cells and, ultimately, azoospermia (Awoniyi et al., 1989; Smith and Walker, 2014). Testicular steroidogenesis is mediated by LH through multiple signaling pathways, 17
ACCEPTED MANUSCRIPT including the steroidogenic acute regulatory protein (StAR), a cholesterol side chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) (Miller, 1988). Polychlorinated biphenyls (PCBs) could act directly on Leydig cells to diminish testosterone production by inhibiting gene expression of steroidogenic
PT
enzymes including StAR, P450scc and 3β-HSD (Murugesan et al., 2007). A previous
RI
study revealed that benzo[a]pyrene could decrease epididymal sperm quality, possibly
SC
by disturbing testosterone production, indicating that StAR should be a major target among steroidogenic proteins (Chung et al., 2011). These studies support our present
NU
results, which showed that BDE47 inhibits testosterone by decreasing the expression
MA
of StAR and 3β-HSD in the primary Leydig cells and after an in vivo exposure. DAX-1 plays a key role in the development and maintenance of reproductive
D
function. Indeed, the expression pattern of DAX-1 is restricted to tissues directly
PT E
involved in steroid hormone production (Lalli and Sassone-Corsi, 2003). DAX-1 blocks the rate-limiting step in steroid biosynthesis by repressing StAR as well as by
CE
inhibiting both P450scc and 3β-HSD (Maris et al., 2015). In the present study, BDE47
AC
upregulated the expression of DAX-1 in vitro and in vivo, and knocking down DAX-1 significantly reversed BDE47-inhibited testosterone production in the primary Leydig cells, suggesting that BDE47 could directly upregulate DAX-1, leading to the inhibition of testosterone production; this phenomenon was also enhanced by a HFD by increasing BDE47 accumulation in the testis. Insulin can directly bind to insulin receptors in the Leydig cell membrane, leading to the up-regulation of DAX-1 and resulting in liver receptor homolog-1 (LRH-1)-mediated testicular steroidogenesis 18
ACCEPTED MANUSCRIPT (Ahn et al., 2013). In the present study, the knockdown of DAX-1 could significantly reverse the insulin-mediated inhibition of testosterone production in primary Leydig cells, indicating that BDE47 induced a high insulin level and subsequently inhibited testosterone production by upregulating DAX-1; this response was aggravated by the
PT
HFD.
RI
In conclusion, our study demonstrated that the HFD aggravated BDE47-inhibited
SC
testosterone production through hyperinsulinemia and that the accumulation of testicular BDE47 targeted the steroidogenic pathway via DAX-1. The inhibition of
NU
steroidogenesis through the insulin-mediated induction of DAX-1 provides new
MA
insights into the relationship between metabolic disorders and male reproductive damage caused by environmental exposure to lipophilic xenobiotics, particularly with
PT E
D
an unhealthy lifestyle, including a high-fat diet.
Funding
CE
This work was supported by the National Natural Science Foundation of China
AC
(81372956, 81573194), Natural Science Foundation of Jiangsu province (BK20151555), Technology & Development Fund of Nanjing Medical University (2014NJMUZD002), Six talents peak project of Jiangsu province (DG216D5047), 333 Advance Talents Projects of Jiangsu Province [BRA2014285], Natural Science Foundation of Jiangsu Universities (14KJA330002), National 973 Program (2009CB941701), Science and Technique Foundation for Youths by Health and Family Planning Commission of Changzhou (QN201603), and a project funded by the 19
ACCEPTED MANUSCRIPT Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China.
Author Disclosure Statement
AC
CE
PT E
D
MA
NU
SC
RI
PT
No competing financial interests exist.
20
ACCEPTED MANUSCRIPT References Ahn, S.W., Gang, G.T., Kim, Y.D., Ahn, R.S., Harris, R.A., Lee, C.H., Choi, H.S., 2013. Insulin directly regulates steroidogenesis via induction of the orphan nuclear receptor DAX-1 in testicular Leydig cells. J Biol Chem 288,
PT
15937-15946.
RI
Awoniyi, C.A., Santulli, R., Sprando, R.L., Ewing, L.L., Zirkin, B.R., 1989.
SC
Restoration of advanced spermatogenic cells in the experimentally regressed rat testis: quantitative relationship to testosterone concentration within the
NU
testis. Endocrinology 124, 1217-1223.
MA
Barazani, Y., Katz, B.F., Nagler, H.M., Stember, D.S., 2014. Lifestyle, environment, and male reproductive health. Urol Clin North Am 41, 55-66.
D
Biswas, M., Hampton, D., Turkes, A., Newcombe, R.G., Aled Rees, D., 2010.
PT E
Reduced total testosterone concentrations in young healthy South Asian men are partly explained by increased insulin resistance but not by altered adiposity.
CE
Clin Endocrinol (Oxf) 73, 457-462. Chung, J.-Y., Kim, Y.-J., Kim, J.Y., Lee, S.G., Park, J.-E., Kim, W.R., Yoon, Y.-D.,
AC
Yoo, K.S., Yoo, Y.H., Kim, J.-M., 2011. Benzo[a]pyrene Reduces Testosterone Production in Rat Leydig Cells via a Direct Disturbance of Testicular Steroidogenic Machinery. Environmental Health Perspectives 119, 1569-1574. Ding, S., Fan, Y., Zhao, N., Yang, H., Ye, X., He, D., Jin, X., Liu, J., Tian, C., Li, H., Xu, S., Ying, C., 2014. High-fat diet aggravates glucose homeostasis disorder caused by chronic exposure to bisphenol A. J Endocrinol 221, 167-179.
21
ACCEPTED MANUSCRIPT Emond, C., Raymer, J.H., Studabaker, W.B., Garner, C.E., Birnbaum, L.S., 2010. A physiologically based pharmacokinetic model for developmental exposure to BDE-47 in rats. Toxicol Appl Pharmacol 242, 290-298. Fan, Y., Liu, Y., Xue, K., Gu, G., Fan, W., Xu, Y., Ding, Z., 2015. Diet-induced obesity
PT
in male C57BL/6 mice decreases fertility as a consequence of disrupted
RI
blood-testis barrier. PLoS One 10, e0120775.
SC
Grossmann, M., Gianatti, E.J., Zajac, J.D., 2010. Testosterone and type 2 diabetes. Curr Opin Endocrinol Diabetes Obes 17, 247-256.
NU
Haffner, S.M., Karhapaa, P., Mykkanen, L., Laakso, M., 1994. Insulin resistance,
MA
body fat distribution, and sex hormones in men. Diabetes 43, 212-219. Hites, R.A., 2004. Polybrominated diphenyl ethers in the environment and in people:
D
a meta-analysis of concentrations. Environ Sci Technol 38, 945-956.
PT E
Huang, S., Cui, Y., Guo, X., Wang, L., Li, S., Lu, Y., Bi, Y., Huang, X., Lin, M., Xia, Y., Wang, S., Wang, X., Zhou, Z., Sha, J., 2015a. 2,2',4,4'-Tetrabromodiphenyl
CE
ether disrupts spermatogenesis, impairs mitochondrial function and induces
AC
apoptosis of early leptotene spermatocytes in rats. Reprod Toxicol 51, 114-124.
Huang, Y., Zhu, G., Peng, L., Ni, W., Wang, X., Zhang, J., Wu, K., 2015b. Effect of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on sexual behaviors and reproductive function in male zebrafish (Danio rerio). Ecotoxicol Environ Saf 111, 102-108. Ji, C., Wu, H., Wei, L., Zhao, J., Lu, H., Yu, J., 2013. Proteomic and metabolomic 22
ACCEPTED MANUSCRIPT analysis of earthworm Eisenia fetida exposed to different concentrations of 2,2',4,4'-tetrabromodiphenyl ether. J Proteomics 91, 405-416. Jung, T.W., Hwang, H.J., Hong, H.C., Yoo, H.J., Baik, S.H., Choi, K.M., 2015. BAIBA attenuates insulin resistance and inflammation induced by palmitate or
PT
a high fat diet via an AMPK-PPARdelta-dependent pathway in mice.
RI
Diabetologia 58, 2096-2105.
SC
Klinefelter, G.R., Ewing, L.L., 1988. Optimizing testosterone production by purified adult rat Leydig cells in vitro. In Vitro Cell Dev Biol 24, 545-549.
NU
Klinefelter, G.R., Hall, P.F., Ewing, L.L., 1987. Effect of luteinizing hormone
MA
deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 36, 769-783.
D
Lalli, E., Sassone-Corsi, P., 2003. DAX-1, an unusual orphan receptor at the
PT E
crossroads of steroidogenic function and sexual differentiation. Mol Endocrinol 17, 1445-1453.
CE
Lardone, M.C., Parada-Bustamante, A., Ebensperger, M., Valdevenito, R., Kakarieka,
AC
E., Martinez, D., Pommer, R., Piottante, A., Castro, A., 2011. DAX-1 and DAX-1A expression in human testicular tissues with primary spermatogenic failure. Mol Hum Reprod 17, 739-746. Linares, V., Belles, M., Domingo, J.L., 2015. Human exposure to PBDE and critical evaluation of health hazards. Arch Toxicol 89, 335-356. Liu, D., Archer, N., Duesing, K., Hannan, G., Keast, R., 2016. Mechanism of fat taste perception: Association with diet and obesity. Prog Lipid Res 63, 41-49. 23
ACCEPTED MANUSCRIPT Maris, P., Campana, A., Barone, I., Giordano, C., Morelli, C., Malivindi, R., Sisci, D., Aquila, S., Rago, V., Bonofiglio, D., Catalano, S., Lanzino, M., Ando, S., 2015. Androgens inhibit aromatase expression through DAX-1: insights into the molecular link between hormone balance and Leydig cancer development.
PT
Endocrinology 156, 1251-1262.
RI
Mhadhbi, L., Fumega, J., Boumaiza, M., Beiras, R., 2012. Acute toxicity of
SC
polybrominated diphenyl ethers (PBDEs) for turbot (Psetta maxima) early life stages (ELS). Environ Sci Pollut Res Int 19, 708-717.
NU
Miller, W.L., 1988. Molecular biology of steroid hormone synthesis. Endocr Rev 9,
MA
295-318.
Murugesan, P., Balaganesh, M., Balasubramanian, K., Arunakaran, J., 2007. Effects of
D
polychlorinated biphenyl (Aroclor 1254) on steroidogenesis and antioxidant
PT E
system in cultured adult rat Leydig cells. J Endocrinol 192, 325-338. Nanjappa, M.K., Ahuja, M., Dhanasekaran, M., Coleman, E.S., Braden, T.D., Bartol,
CE
F.F., Bird, R.C., Wanders, D., Judd, R.L., Akingbemi, B.T., 2014. Bisphenol A
AC
regulation of testicular endocrine function in male rats is affected by diet. Toxicol Lett 225, 479-487. Qiu, L.L., Wang, X., Zhang, X.H., Zhang, Z., Gu, J., Liu, L., Wang, Y., Wang, X., Wang, S.L., 2013. Decreased androgen receptor expression may contribute to spermatogenesis failure in rats exposed to low concentration of bisphenol A. Toxicol Lett 219, 116-124. Sanders, J.M., Chen, L.J., Lebetkin, E.H., Burka, L.T., 2006. Metabolism and 24
ACCEPTED MANUSCRIPT disposition of 2,2',4,4'- tetrabromodiphenyl ether following administration of single or multiple doses to rats and mice. Xenobiotica 36, 103-117. Skakkebaek, N.E., Rajpert-De Meyts, E., Buck Louis, G.M., Toppari, J., Andersson, A.M., Eisenberg, M.L., Jensen, T.K., Jorgensen, N., Swan, S.H., Sapra, K.J.,
PT
Ziebe, S., Priskorn, L., Juul, A., 2016. Male Reproductive Disorders and
RI
Fertility Trends: Influences of Environment and Genetic Susceptibility.
SC
Physiol Rev 96, 55-97.
Skovso, S., 2014. Modeling type 2 diabetes in rats using high fat diet and
NU
streptozotocin. J Diabetes Investig 5, 349-358.
MA
Smith, L.B., Walker, W.H., 2014. The regulation of spermatogenesis by androgens. Semin Cell Dev Biol 30, 2-13.
D
Stickels, R., Clark, K., Heider, T.N., Mattiske, D.M., Renfree, M.B., Pask, A.J., 2015.
PT E
DAX1/NR0B1 was expressed during mammalian gonadal development and gametogenesis before it was recruited to the eutherian X chromosome. Biol
CE
Reprod 92, 22.
AC
Tarapore, P., Hennessy, M., Song, D., Ying, J., Ouyang, B., Govindarajah, V., Leung, Y.K., Ho, S.M., 2016. High butter-fat diet and bisphenol A additively impair male rat spermatogenesis. Reprod Toxicol. doi: 10.1016/j.reprotox.2016.09.008. Xu, Y., Wang, L., He, J., Bi, Y., Li, M., Wang, T., Wang, L., Jiang, Y., Dai, M., Lu, J., Xu, M., Li, Y., Hu, N., Li, J., Mi, S., Chen, C.S., Li, G., Mu, Y., Zhao, J., Kong, L., Chen, J., Lai, S., Wang, W., Zhao, W., Ning, G., China Noncommunicable 25
ACCEPTED MANUSCRIPT Disease Surveillance, G., 2013. Prevalence and control of diabetes in Chinese adults. JAMA 310, 948-959. Yang, X., Zhang, Z., Wang, X., Wang, Y., Zhang, X., Lu, H., Wang, S.L., 2013. Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial
PT
cells to aflatoxin B1-induced DNA damage. Toxicol Appl Pharmacol 270,
RI
114-121.
SC
Yeap, B.B., Chubb, S.A., Hyde, Z., Jamrozik, K., Hankey, G.J., Flicker, L., Norman, P.E., 2009. Lower serum testosterone is independently associated with insulin
NU
resistance in non-diabetic older men: the Health In Men Study. Eur J
MA
Endocrinol 161, 591-598.
Zhang, Z., Li, S., Liu, L., Wang, L., Xiao, X., Sun, Z., Wang, X., Wang, C., Wang, M.,
D
Li, L., Xu, Q., Gao, W., Wang, S.L., 2016. Environmental exposure to BDE47
PT E
is associated with increased diabetes prevalence: Evidence from community-based case-control studies and an animal experiment. Sci Rep 6,
CE
27854.
AC
Zhang, Z., Lu, H., Huan, F., Meghan, C., Yang, X., Wang, Y., Wang, X., Wang, X., Wang, S.L., 2014. Cytochrome P450 2A13 mediates the neoplastic transformation of human bronchial epithelial cells at a low concentration of aflatoxin B1. Int J Cancer 134, 1539-1548. Zhang, Z., Sun, Z.Z., Xiao, X., Zhou, S., Wang, X.C., Gu, J., Qiu, L.L., Zhang, X.H., Xu, Q., Zhen, B., Wang, X., Wang, S.L., 2013a. Mechanism of BDE209-induced impaired glucose homeostasis based on gene microarray 26
ACCEPTED MANUSCRIPT analysis of adult rat liver. Arch Toxicol 87, 1557-1567. Zhang, Z., Zhang, X., Sun, Z., Dong, H., Qiu, L., Gu, J., Zhou, J., Wang, X., Wang, S.L., 2013b. Cytochrome P450 3A1 mediates 2,2',4,4'-tetrabromodiphenyl
AC
CE
PT E
D
MA
NU
SC
RI
PT
ether-induced reduction of spermatogenesis in adult rats. PLoS One 8, e66301.
27
ACCEPTED MANUSCRIPT Figure legends Figure 1. Effects of the HFD on the accumulation of BDE47 and relative weight of adipose tissues in rats treated with BDE47. (A) BDE47 content in the liver and testis. The data are shown as box and whisker plots (minimum to maximum). (B)
PT
Ratio of the retroperitoneal WAT weight and body weight (BW). (C) Ratio of the
RI
gonadal WAT weight and BW. (D) Ratio of the BAT weight and BW. The data were
SC
expressed as the means± SD of 6 rats per group. #p < 0.05 compared with the corresponding vehicle control; *p< 0.05, **p < 0.01 compared with the corresponding
MA
NU
BDE47 treatment in the ND group.
Figure 2. Effects of the HFD on glycolipid metabolism in rats treated with
D
BDE47. (A) Serum TG. (B) Serum CHOL. (C) Fasting glucose. (D) Serum insulin. (E)
PT E
Morphological observations of liver tissue. The tissue was stained with hematoxylin and eosin (H&E) and was observed using optical microscopy. Blue arrow, lipid
p < 0.05 compared with the corresponding vehicle control; *p < 0.05, **p < 0.01
AC
#
CE
droplets; bar, 100 μm. The data were expressed as the means± SD of 6 rats per group.
compared with the corresponding BDE47 treatment in the ND group.
Figure 3. Effects of the HFD on the serum testosterone and the morphological changes in testes and epididymides of rats treated with BDE47. (A) Serum testosterone levels. (B) Morphological observations of the testis and seminiferous tubule architecture. (C) Morphological observations of epididymis. The tissue was 28
ACCEPTED MANUSCRIPT stained with H&E and observed using optical microscopy. Blue arrow, giant cells in the testis; red arrow, vacuolar spaces in the seminiferous epithelium; black arrow, reduced sperm count; bar, 100 μm. The data were expressed as the means± SD of 6 rats per group. #p < 0.05, ##p < 0.01 compared with the corresponding vehicle control;
RI
PT
*p < 0.05 compared with the corresponding BDE47 treatment in the ND group.
SC
Figure 4. Effects of the HFD on the expression of DAX-1, StAR, and 3β-HSD in the testis of rats treated with BDE47. (A) The expression levels of DAX-1, StAR,
NU
and 3β-HSD using immunoblotting assays. Lysates from the testes (80 μg) were
MA
prepared, and GAPDH was used as an internal reference. The densitometric analysis was conducted for the protein bands using ImageJ software. The data are expressed as
p < 0.001 compared with the corresponding vehicle control; *p < 0.05, **p < 0.01,
PT E
###
D
the mean ± SD of three independent experiments with triplicates. #p < 0.05, ##p < 0.01,
***p < 0.001 compared with the corresponding BDE47 treatment in the ND group. (B)
CE
The expression of DAX-1 using an immunohistochemical assay. The localization of
AC
DAX-1 was observed in the testicular interstitial compartments.
Figure 5. Effects of BDE47 on cell viability and DAX-1 expression in the primary Leydig cells of rats. (A) Cells viability using a CCK-8 assay. The cells were treated with BDE47 for 48 h, and the data were expressed as the means± SE of three independent experiments with six replicates; (B) The expression levels of DAX-1, StAR, and 3β-HSD using immunoblotting assays. Cell lysates (50 μg) were prepared, 29
ACCEPTED MANUSCRIPT and GAPDH was used as an internal reference. (C) The densitometric analysis was conducted for the protein bands using ImageJ software. The data are expressed as the mean ± SD of three independent experiments with triplicates. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle control. (D) The expression of DAX-1 using
RI
PT
an immunofluorescence assay.
SC
Figure 6. Effects of DAX-1 knockdown on DAX-1 expression and testosterone secretion in primary Leydig cells from rats treated with BDE47 or insulin. (A)
NU
The effects of BDE47 on the testosterone concentration in the cell culture medium. (B)
MA
The effects of insulin on the testosterone in the cell culture medium. The cells were treated with BDE47 or insulin for 24 h, and the testosterone levels in the supernatant
D
were determined using ELISA. (C) The effects of si-DAX-1 on DAX-1 expression in
PT E
cells treated with BDE47 or insulin, the densitometric analysis was conducted for the protein bands using ImageJ software. The data are expressed as the mean ± SD of
CE
three independent experiments with triplicates. (D) The effects of si-DAX-1 on the
AC
testosterone secretion of cells treated with BDE47 or insulin. The cells were pretreated with si-DAX-1 for 48 h and were then treated with 20 μM BDE47 or 80 nM insulin (1 μU/ml= 6.00 pmol/L) for an additional 24 h. The data were expressed as the means± SE of three independent experiments with six replicates. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the vehicle control; *p < 0.05, **p < 0.01 compared with the BDE47 or insulin treatment alone.
30
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 1
31
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 2
32
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 3
33
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 4
34
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 5
35
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Fig. 6
36
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Graphical abstract
37
ACCEPTED MANUSCRIPT Highlights ► High-fat diet (HFD) aggravates the accumulation of BDE47 in liver and testis in rats ► HFD aggravates BDE47-inhibited testosterone production via DAX-1 in Leydig cells ► HFD enhances BDE47-induced the disorder of glycolipid metabolism and hyperinsulinemia
AC
CE
PT E
D
MA
NU
SC
RI
PT
► Both hyperinsulinemia and accumulation of BDE47 inhibited testosterone via DAX-1
38