Accepted Manuscript
Autophagy as a compensation mechanism participates in ethanol- induced fetal adrenal dysfunction in female rats
The Premier International Journal in Toxicology
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Toxicology and Applied Pharmacology
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Hegui Huang, Lian Liu, Jing Li, Chunyan Zhu, Xiaoyu Xie, Ying Ao, Hui Wang
| PII: | S0041-008X(18)30085-1 |
| DOI: | doi:10.1016/j.taap.2018.03.007 |
| Reference: | YTAAP 14190 |
| To appear in: | Toxicology and Applied Pharmacology |
| Received date: | 26 December 2017 |
| Revised date: | 1 March 2018 |
| Accepted date: | 6 March 2018 |
Please cite this article as: Hegui Huang, Lian Liu, Jing Li, Chunyan Zhu, Xiaoyu Xie, Ying Ao, Hui Wang , Autophagy as a compensation mechanism participates in ethanol- induced fetal adrenal dysfunction in female rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ytaap(2018), doi:10.1016/j.taap.2018.03.007
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Autophagy as a compensation mechanism participates in ethanol-induced fetal adrenal dysfunction in female rats
Hegui Huang 1,3, Lian Liu 1, Jing Li 1, Chunyan Zhu 1, Xiaoyu Xie 1, Ying Ao 1,2*, Hui Wang 1,2* Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China;
2 Hubei Provincial Key Laboratory of Developmentally Originated Disorder, Wuhan 430071, China;
3 Department of Pharmacy, Wuhan No.1 Hospital, Wuhan 430022, China.
*Corresponding authors: Hui Wang, Ph.D., Telephone: +86-13627232557, E-mail: wanghui19@whu.edu.cn; Ying Ao, Ph.D., Telephone: +86-13995592855, E-mail: yingao@whu.edu.cn.
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Abbreviations:
AKT1, serine/threonine kinase; Atg, autophagy-related genes; CR, corticoid receptors; GD, gestational day; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GR, glucocorticoid receptor; HPA, hypothalamic-pituitary-adrenal; HE, hematoxylin and eosin; IUGR, intrauterine growth retardation; IGF1, insulin-like growth factor 1; IGF1R, insulin-like growth factor 1 receptor; LC3B, microtubule-associated protein light chain 3 beta; MR, mineralocorticoid receptor; NAFLD, non-alcoholic fatty liver diseases; P450scc, cytochrome P450 cholesterol side chain cleavage; PEE, prenatal ethanol exposure; PI3K, phosphatidylinositol 3 kinase ; SF1, steroidogenic factor 1; StAR, steroidogenic acute regulatory protein; P62, protein 62; TEM, transmission electron microscopy; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; 11ß-HSD2, 11ß-hydroxysteroid dehydrogenase type 2; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase.
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ABSTRACT
Autophagy plays a vital role in embryonic development and cell differentiation. Our previous study demonstrated that prenatal ethanol exposure (PEE) resulted in intrauterine growth retardation (IUGR) and adrenal developmental toxicities in rat offspring. The present study focused on PEE-induced autophagy as an underlying mechanism and its biological significance in female fetal rats. Female fetuses in the PEE group exhibited lower body weights and suffered adrenal structural abnormalities compared to the controls. Cell proliferation was inhibited, the insulin-like growth factor 1 (IGF1) pathway was reduced, and autophagy was activated in the glands of female fetal rats. Ethanol increased the ratio of microtubule-associated protein light chain 3 beta-II (LC3B-II) to LC3B-I in vitro, and it reduced cortisol levels in time- and concentration-dependent manners in human adrenocortical carcinoma cells (NCI-H295A). Bafilomycin Al inhibited autophagy, steroidogenic factor 1 (SF1) protein and steroidogenesis in the present study. Rapamycin with ethanol up-regulated autophagy and SF1 expression and activated steroidogenesis when compared with ethanol alone. In addition, ethanol inhibited IGF1 receptor (IGF1R) and phospho-mTOR (Ser2448) expression in a concentration-dependent manner. These results demonstrate that PEE activated autophagy in fetal adrenal glands, and the underlying mechanism may be associated with inhibition of the IGF1R/phospho-mTOR (Ser2448) pathway. Autophagy may be a compensatory mechanism for the PEE-induced inhibition of fetal adrenal steroidogenesis to maintain fetal adrenal development.
Keywords: adrenal steroido genesis; fetal adrenal development; hypo thalamic-pituitary-adrenal axis; intrauterine growth retardation
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1 Introduction
Intrauterine growth retardation (IUGR) refers to embryonic or fetal growth and development limitations due to adverse prenatal environments and manifests as the dysfunction of multiple organs, growth retardation, and low birth weight. IUGR causes fetal distress, perinatal death and neonatal asphyxia, and it induces adverse effects in the physical and mental development of offspring after birth and increases their susceptibility to metabolic syndromes (e.g., fatty liver disease and diabetes) in adulthood (Eleftheriades et al., 2006). An adverse intrauterine environment is one of the major causes of IUGR. Ethanol is one of the most widely consumed pharmacological agents of abuse (Harris et al., 2008). Approximately half (50%) of women of reproductive age are binge drinkers, and nearly 7% of pregnant women in some communities in North America consume alcoholic beverages (Muhajarine et al., 1997; Williams and Gloster, 1999). Prenatal ethanol exposure (PEE) may produce diverse adverse fetal outcomes and long-term effects on an individual’s endocrine and metabolic functions (Lundsberg et al., 1997; Mullally et al., 2011; Murphy et al., 2013). Our previous study demonstrated that PEE produced IUGR and inhibited the development of fetal hypothalamic-pituitary-adrenal (HPA) axis function in offspring (Liang et al., 2011). PEE-induced dysfunction of the HPA axis in rats may persist post-birth to adulthood (Huang et al., 2015), which results in a high susceptibility to non-alcoholic fatty liver disease (NAFLD) and osteoarthritis in adulthood when fed a high-fat diet (Shen et al., 2014; Ni et al., 2015). The underlying mechanism we proposed involves an “HPA axis-related neuroendocrine metabolic programming alteration” (Xia et al., 2014).
Adrenal glands are terminal effector organs of the HPA axis that are responsible for the synthesis and secretion of glucocorticoids (cortisol in humans and primates and corticosterone in rodents), which is necessary for embryonic development (Vieau et al., 2007). Adverse intrauterine environments impair the development of the fetal adrenal glands (Meaney et al., 2007). Many studies indicated that the developmental retardation of adrenal gland in the individual with low-birth weight increased the risk of developing metabolic syndrome in later life (Ong, 2005; Marciniak et al., 2011). Our previous studies (Shen et al., 2014; Huang et al., 2015; Niet al., 2015; Xia et al., 2014) also demonstrated that PEE induced IUGR, developmental abnormalities of fetal
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multiple tissues (e.g., adrenal gland, liver, hippocampus and articular cartilage) and susceptibility to adult metabolic syndrome. The underlying mechanism may be associated with the intrauterine programming alteration of multiple tissue function caused by over-exposure to maternal glucocorticoids (GC) (Liang et al., 2011), which showed a GC-dependent changes in the function of multiple tissue after birth. These results suggested that the adrenal developmental abnormality induced by PEE plays an important role in the occurrence and development of fetal-originated adult diseases.
A complex multi-enzyme process mediates steroidogenesis in which cholesterol is converted to biologically active steroid hormones. Steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side chain cleavage (P450SCC) are the limiting enzymes in this process. Our previous study found that the expression of Star and P450scc ribonucleic acid (RNA) was inhibited, and the level of adrenal corticosterone was reduced in PEE-treated male fetal rats, which indicated reduced fetal adrenal steroidogenesis following PEE (Huang et al., 2015). The suppression effect of PEE on fetal adrenal steroidogenesis was maintained to adulthood, and it was characterized by intrauterine programming alterations of adrenal steroidogenesis in rat male offspring (Huang et al., 2015).
Macroautophagy (herein referred to as autophagy) is a dynamic process with several sequential stages (i.e., initiation, elongation, maturation and degradation) that are controlled by a group of autophagy-related genes (Atg) and their respective ATG proteins. Autophagy plays an important role in cell differentiation and embryonic development (Kuma et al., 2007), and autophagy activation is detected in various neonatal tissues (Kuma et al., 2004); however, whether autophagy is involved in changes of fetal adrenal steroidogenesis in IUGR offspring rats following PEE and its biological significance are currently not known. The insulin-like growth factor 1 (IGF1) pathway modulates diverse physiological activities in various organs (e.g., adrenal glands), including the stimulation of cell proliferation, differentiation and metabolism (Baquedano et al., 2005; Roberts et al., 2008; Netchine et al., 2009). The IGF1 signaling pathway stimulates adrenal growth (Baquedano et al., 2005) and up-regulates steroidogenic factor 1 (SF1) and a variety of steroid synthesis enzymes (Raha et al., 2007; Sirianni et al., 2007). IGF1 up-regulated phosphatidylinositol 3 kinase (PI3K)
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and its downstream molecules, including serine/threonine kinase (AKT1) and mammalian targets of rapamycin (mTOR), which inactivated autophagy (Mammucari et al., 2008). However, it is still unknown whether the IGF1-autophagy signaling pathway is involved in the regulation of fetal adrenal steroidogenesis.
Notably, we found that female offspring with IUGR were more susceptible to the suppression effect of PEE on birth weight and fetal adrenal steroidogenesis. The present study used an established PEE-induced IUGR model in rat PEE (Shen et al., 2014; Ni et al., 2015) to confirm the existence and mechanism of adrenal autophagy. We also examined the biological significance of autophagy on adrenal steroidogenesis in female offspring. This study elucidates the underlying mechanisms and the biological significance of adrenal autophagy during prenatal adverse environments and explains the adrenal developmental origin of adult diseases.
2 Material and methods
2.1 Drugs and reagents
Diethyl ether (catalogue number: 10009318) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ethanol (catalogue number: 1000918) was obtained from Zhen Xin Co., Ltd. (Shanghai, China). Corticosterone enzyme-linked immunosorbent assay (ELISA) kits (catalogue number: EC3001-1) were purchased from Assaypro, Inc. (St. Charles, MO, USA). The anti-Ki67 antibody (product code: ab15580) was obtained from Abcam (Cambridge, UK). The anti-SF1 (product code: sc-28740), anti-IGF1 (product code: sc9013) and anti-GAPDH antibodies (product code: sc-25778) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Bafilomycin A1 (product code: B1793), rapamycin (product code: R0395), anti-protein 62 (P62) antibody (product code: P0067), anti-beclin 1 antibody (product code: B6186), anti-phospho-mTOR antibody (Ser2448) (product code: SAB4504476), and the anti- microtubule-associated protein light chain 3 beta (LC3฿) antibody (product code: L7543) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay kit (catalogue number: G5421) was obtained from Cayman Chemical Co. (Ann Arbor,
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Michigan, USA). Quantitative real-time polymerase chain reaction (qRT-PCR) kits (catalogue number: RR037Q) and reverse transcription supplies (catalogue number: 639505) were obtained from Takara Biotechnology Co., Ltd. (Dalian, China). SYBR Green dye (catalogue number: 208052) was provided by Applied Biosystems and Thermo Fisher Scientific (ABI) (Foster City, CA, USA). All of the primers used in this study were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals and agents were of analytical grade.
2.2 Animals and treatment
Specific pathogen-free Wistar rats [No. 2012-2014, license number: SCXK (Hubei), certification number: 42000600002258] weighing 258 ± 17 g (male) and 209 ± 12 g (female) were purchased from the Experimental Center of the Hubei Medical Scientific Academy (Wuhan, China). Animal experiments were performed at the Center for Animal Experiments of Wuhan University (Wuhan, China), which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The Committee on the Ethics of Animal Experiments of the Wuhan University School of Medicine approved the protocol (permit number: 14016). All animal experimental procedures were performed in accordance with the Committee on Animal Research and Ethics Guidelines of the Chinese Animal Welfare Committee. Animals were housed in cages equipped with wire-mesh floors in an air-conditioned environment under standard conditions (light cycle: 12 h light-dark cycle; 10-15 air changes per hour; room temperature: 18-22℃; relative humidity: 40%-60%) and allowed free access to standard tap water and rat chow. All rats were fed ad libitum for one week prior to experimentation, and two female rats were placed together in a cage with a single male rat overnight for mating. Mating was confirmed by the appearance of sperm in vaginal smears, and the day of mating was regarded as gestational day (GD) 0.
Pregnant female rats were placed in individual cages. Pregnant female rats were divided randomly into two groups, the control and PEE groups. Rats in the PEE group were intragastrically administered ethanol (4 g/kg·d) to establish the IUGR model from GD9 to GD20 (Shen et al., 2014). Rats in the control group were treated with the same volume (10 ml/kg) of distilled water. Animals were placed in a separate quiet room on GD20 and anesthetized using isoflurane. Pregnant rats were
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euthanized rapidly after the disappearance of the righting reflex via cutting of the left carotid artery with a sharp pair of scissors. The maternal blood was collected. Pregnant rats with 10-14 live fetuses were selected from each group (n = 12 for each group). The uterus was quickly dissected, and fetuses were quickly separated from the uterus. Fetuses were dried on filter paper and weighed quickly. The IUGR rate was calculated according to previously reported criteria for IUGR (Shen et al., 2014). Serum samples of female fetuses from 3 littermates were collected, pooled together and frozen immediately at -80℃ for analyses. Female fetal adrenal glands were dissected using fine forceps. Three pairs of fetal adrenal glands were selected from different litters and fixed routinely via transcardial perfusion fixation (Yoshida and Ikuta, 1984) for histological, ultra-structural, and immunocytochemical examination. Six pairs of fetal adrenal glands from two litters were pooled into one sample for homogenization for gene expression analyses, and all pooled samples were immediately frozen and stored at -80℃ until examination.
2.3 Cell culture
A subline of human adrenocortical carcinoma cells (NCI-H295A) were cultured in RPMI-1640 medium (catalogue number: 11875085, Gibco, NY, USA) supplemented with 2% fetal bovine serum (catalogue number: 10438034, Gibco, NY, USA), 0.1% insulin-transferrin-selenium (catalogue number: 11884, Sigma, MO, USA) and penicillin/streptomycin (catalogue number: 15140122, Gibco, NY, USA). Our previous study proposed that the mean serum ethanol level of the fetuses was 58±5 mM at 4 g/kg.d PEE (Shen et al., 2014). Therefore, NCI-H295 A cells were washed twice with phosphate buffered solution (PBS) (pH 7.4) when the cells reached 40% to 50% confluence, starved with non-serum medium overnight, and treated with ethanol.
2.4 MTS assay
The NCI-H295A cells were seeded at a density of 1 × 104 cells per well in 96-well plates. Cells were treated with ethanol (0-120 mM) when 40% to 50% confluent for 8-72 h. The medium was removed from each well, and the cells were washed with PBS twice. The cultures were incubated with medium (100 ul) and MTS (20 ul) at 37°℃ for another 2 h. The optical density was measured at 570 nm using a microplate reader (Shimadzu, Kyoto, Japan), and the relative cell viability values were normalized as a percentage relative to the untreated, control cells.
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2.5 Hormonal level measurements
Serum IGF1 and corticosterone levels were measured using ELISA kits following the manufacturer’s protocol. The inter- and intra-assay coefficients of variation for IGF1 were 9.1% and 5.6%, respectively. The cross-reactivity of the IGF1 ELISA was less than the 0.5% observed with available related molecules. The inter- and intra-assay coefficients of variation for corticosterone were 7.2% and 5.0%, respectively. The cross-reactivity for the corticosterone ELISA was 2% for aldosterone and 2% for progesterone.
2.6 Histological and ultra-microstructural measurements
The adrenal glands were fixed in a solution of 4% paraformaldehyde overnight at 4℃ and subsequently embedded in paraffin. Histological sections (5-um thick) were prepared and stained routinely using hematoxylin and eosin (HE). Each 5th section of the series was saved, observed and photographed under an Olympus AH-2 light microscope (Olympus, Tokyo, Japan). The cross-sectional areas of the fetal adrenal cortex were determined planimetrically, and the cross-sectional areas of the adrenocortical zones were obtained using the Photo Imaging System (Nikon H550S, Japan). Transcardial perfusion fixation was performed, and transmission electron microscopy (TEM) was used to investigate the microstructure of the fetal adrenal glands (Yoshida and Ikuta, 1984). Fetal adrenal glands were fixed with 2.5% glutaraldehyde buffered to pH 7.4 with 0.1-M phosphate-buffered saline for 2 h at 4℃ and post-fixed with 1% osmium tetroxide for 1 h. Samples were dehydrated through a graded series of ethanol and embedded in Epon 812. The treated NCI-H295A cells were harvested and processed as described previously (Hansson et al., 2004). Approximately 50-nm thickness ultra-thin sections were cut using a LKB-V ultra-microtome (Bromma, Sweden) and stained dually with lead citrate and uranyl acetate. Stained sections were examined using a Hitachi H600 transmission electron microscope (Hitachi, Tokyo, Japan). Digital images were acquired using a computer (Dell, Texas, USA).
2.7 Immunohistochemistry measurements
Immunohistochemical procedures were performed using streptavidin-peroxidase (SP)-conjugation as described in the manufacturer’s protocol. Paraffin-embedded tissues were cut into serial sections at a thickness of 10 um and incubated with primary antibodies diluted for
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optimization (Ki67 1:50; LC3B 1:100; and Beclin 1 1:100). The primary antibody was replaced with non-immune rabbit IgG for a negative control. We calculated the mean optical density in five random fields for each protein in the fetal adrenals. All images were captured using an Olympus AH-2 Light Microscope (Olympus, Tokyo, Japan). The analysis of the digital images was performed using Olympus software (Olympus, Tokyo, Japan).
2.8 qRT-PCR measurement
Total RNA was isolated from pooled fetal adrenal glands using the TRIzol reagent according to the manufacturer’s specifications. Isolated RNA concentrations and purity were determined using a spectrophotometer (Nano Drop 2000C, Thermo), and the RNA concentration was adjusted to 1 ug/ul. Isolated RNA was immediately stored in diethyl pyrocarbonate-H2O (DEPC-H2O) at -80°℃ until used.
Single-strand cDNA for qRT-PCR data analysis was prepared from 2 ug of total RNA according to the protocol of the Exscript RT reagent kit. Primers for Sf1, Star and Igfl were designed using Primer Premier 5.0 software (PREMIER Biosoft International, CA, USA), and the sequence of each primer was queried using the National Center for Biotechnology Information (2009) BLAST database for homology comparison. The designs of other primers used are described in our previous studies (Xu et al., 2012a; Xu et al., 2012b; Ping et al., 2014). Table 1 lists the oligonucleotide sequences of the primers. Relative standard curves were constructed for target genes (Table 1) using the corresponding qRT-PCR products isolated using a DNA extraction kit with different concentrations ranging from 10 pg to 10,000 pg per reaction. Analyses of all qRT-PCR reactions were performed in 96-well optical reaction plates using an ABI Step One qRT-PCR Thermal Cycler (ABI Stepone, NY, USA) in 20 ul of reaction mixture. The expression of the housekeeping gene glyceraldehyde phosphate dehydrogenase (Gaphd) was also determined to quantify the gene transcripts more precisely, and each sample was normalized to the Gapdh mRNA level. The following polymerase chain reaction cycling conditions were used: 95℃ (pre-denaturation) for 30 s, 95°C (denaturation) for 5 s, the annealing conditions for each gene listed in Table 1, and 72°℃ (elongation) for 30 s.
2.9 Western blot analysis
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Supernatants were harvested and stored at -80℃ until used. Treated cells were rinsed quickly with ice-cold PBS (pH 7.4) and lysed for 30 min at 4℃ in radio immunoprecipitation assay lysis buffer (catalogue number: 89900, Thermo Scientific, MA, USA) containing protease inhibitors. The cell lysates were centrifuged at 14,000 g for 10 min at 4℃. Cell protein extracts were prepared using a protein extract kit, and protein concentrations were determined using a bicinchoninic acid protein assay kit (catalogue number: BCA1, Sigma, MO, USA). Aliquots of the cell lysates were mixed with 5x loading buffer with 2-mercaptoethanol, and boiled at 100℃ for 5 min for direct immunoblotting. Samples were loaded onto 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Separated proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) using a BioRad gel system. Membranes were blocked for 2 h at room temperature with 5% bovine serum albumin in tris buffered saline with tween 20 (TBST) (25 mM Tris-HC1, 50 mM NaCl, 0.05% Tween-20), washed and incubated with primary antibodies (anti-LC3B, 1:1,000; anti-P62, 1:5,000; anti-SF1, 1:800; anti-IGF1R, 1:800; anti-phospho-mTOR (Ser2448), 1:2000; anti-GAPDH, 1:6,000) overnight at 4℃. The membranes were washed extensively, incubated with a fluorescent-conjugated secondary antibody (1:6000 dilution) for 2 h and washed with TBST. Specific bands were detected using the enhanced chemiluminescence assay. Digital images were acquired using a chemiluminescence immunoassay system (FUSION, Vilber, France). Quantification of band intensity was performed using Olympus software (Olympus, Tokyo, Japan).
2.10 Statistical analysis
Statistical analyses were performed using SPSS 17 (SPSS Science Inc., Chicago, Illinois) and Graph Pad Prism 5 software (Graph Pad Software Inc., La Jolla, CA, USA). All data presented are expressed as the means ± S.E.M. and were evaluated8 using independent sample t-tests for comparisons between two groups. One-way analysis of variance (ANOVA) followed by a post hoc Dunnett t-test was used to compare multiple groups. The proportion of affected animals per litter was initially calculated, and the IUGR rate was defined as the arcsine square root transformed prior to one-way ANOVA (Tan et al., 2012). The results are presented as group mean values of the litter proportions ± S.E.M. A P-value less than 0.05 was considered statistically significant.
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3 Results
3.1 In vivo study
3.1.1 Body weights, adrenal histological examination and steroidogenesis
A significant decrease in female fetal body weight (P<0.01, Fig. 1A) was observed compared to control, and the corresponding IUGR rate was 85.4% (P<0.01, Fig. 1B) with ethanol administration (4 g/kg.d). HE staining revealed cytoplasmic swelling of the fetal adrenal cortex in the PEE group (Fig. 1C). The maximum entire cross-sectional area of the fetal adrenal glands was decreased in the PEE group (P<0.05, Fig. 1D). The number of cortical cells that were immunohistochemically positive for Ki67 was reduced in the fetal rat adrenals in the PEE group (P<0.01, Fig. 1E,1F). PEE treatment significantly decreased the mRNA expression of fetal adrenal Sf1, Star, P450scc, P450c21 and P450cl1 (P<0.05, P<0.01, Fig. 1G-1L). Fetal adrenal corticosterone level was lower in the PEE group (50.01% decrease) on ELISA compared to the control group (P<0.05, Fig. 1M). These results indicated that the female offspring in the PEE group showed developmental toxicity and inhibited steroidogenesis.
3.1.2 Changes in the IGF1 pathway and autophagy in fetal adrenal glands
Serum levels of IGF1 and Igflr and Aktl expression were decreased markedly in the PEE group compared to controls (P<0.05, P<0.01, Fig. 2A-2C). Immunohistochemical staining showed that the expression of the Beclin 1 and LC36 proteins were increased significantly in the PEE group (P<0.05, P<0.01, Fig. 2D-2G). Ultra-structural observation revealed that autophagic vacuoles in adrenal cortex cells was increased in the PEE group (P<0.01, Fig. 2H,2I). These vacuoles are special vesicles with a double-membrane structure. These results indicated that PEE inhibited the IGF1 signaling pathway and induced autophagy in fetal adrenal glands.
3.2 In vitro study
3.2.1 Effects of ethanol on cell viability and steroidogenesis in adrenocortical cells
We evaluated the direct effect of ethanol on cytotoxicity and steroidogenesis on cells in vitro.
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The cell number in the ethanol group was comparable with controls after a 24 h exposure to ethanol (Fig. 3A), while its decreased after the treatment of ethanol at 30, 60 and 120 mM for 72 h (P<0.05, P<0.01, Fig. 3B). The mRNA expression of SF1, StAR, P450SCC, P450C21 and P450C11 was decreased after an 8 h exposure to ethanol at 60 and 120 mM (P<0.05, P<0.01, Fig. 3C-3H). Cortisol concentration was also decreased following an 8 h exposure to ethanol at 60 and 120 mM (P<0.01, Fig. 3I). Cortisol concentration was decreased in the medium following ethanol (60 mM) exposure for 8, 12 and 24 h (P<0.01, Fig. 3J). These results indicate that ethanol inhibited steroidogenesis in NCI-H295A cells.
3.2.2 Ethanol induced autophagy activation in adrenocortical cells
The protein expression ratio of LC3B-II/LC3B-I was increased following treatment with 60 mM ethanol for 8, 12 and 24 h (P<0.01, Fig. 4A,4B), and 60 and 120 mM ethanol for 8 h (P<0.05, Fig. 4C,4D). Ultra-structural analysis of adrenocortical cells revealed that autophagic vacuoles (Avi, black arrow) in adrenal cortex cells was increased after 60 mM ethanol exposure for 24 h (P<0.01, Fig. 4E,4F). These results suggest that ethanol induced autophagy in adrenocortical cells.
3.2.3 The IGF1R/phospho-mTOR pathway mediated ethanol-induced autophagy in adrenocortical cells
Rapamycin (200 nM) increased the protein expression ratio of LC3-II/LC3B-I (P<0.05, Fig. 5A,5D) and reduced P62 levels (P<0.01, Fig. 5A,5C). Treatment with 60 mM ethanol and rapamycin for 8 h also increased the ratio of LC3B-II/LC3B-I (P<0.05, Fig. 5A,5D) and reduced P62 levels (P<0.01, Fig. 5A,5C); however, bafilomycin A1 (10 nM) and ethanol with bafilomycin Al increased the LC3B-II/LC3B-I ratio and P62 (P<0.01, 5A,5C,5D). Ethanol with rapamycin (200 nM) increased the expression of SF1 protein and the cortisol level in the media compared with the ethanol group (P<0.05, P<0.01, Fig. 5A,5B,5E). Treatment with ethanol and bafilomycin A1 alone or in combination for 8 h did not affect cell number, but SF1 protein and the cortisol level were remarkably reduced in all ethanol- and/or bafilomycin A1-treated groups (P<0.01, Fig. 5A,5B,5E). These results suggest that autophagy could compensate for the ethanol-induced inhibition of steroido genesis in adrenocortical cells. The mTOR pathway is an important signaling pathway that regulates autophagic activity. Inhibition of the IGF1 signaling pathway generally results in
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autophagic activation. Therefore, we further investigated the effect of ethanol on the IGF1R/mTOR pathway. We treated cells with ethanol (15, 30 and 60 mM) for 8 h and found that ethanol inhibited the expression of IGF1R protein and reduced the phosphorylation level of mTOR (Ser2448) and the expression of the downstream protein P62 (P<0.05, P<0.01, Fig. 5F-5I). Taken together, these results suggest that the inhibition of the IGF1R/ phospho-mTOR (Ser2448) pathway is involved in ethanol-induced autophagy activation, and autophagy is a compensatory mechanism of the ethanol-induced inhibition of steroidogenesis.
4 Discussion
4.1 Ethanol dose selection and PEE-induced adrenal structural and functional abnormalities
Maternal alcohol intake during pregnancy is a well-known risk factor for IUGR (May et al., 2005). The average blood alcohol level is approximately 33 mM in humans after consumption of 3 to 5 drinks of liquor containing 6.7% (v/v) ethanol (Gohlke et al., 2005). This level is observed in moderate to severe alcoholics and ranges from 20 to 170 mM (Pantazis et al., 1992). Our previous work demonstrated that serum ethanol levels in fetal rats following the administration of 4 g/kg.d ethanol to pregnant rats were approximately 58 mM (Shen et al., 2014). Therefore, the dose of ethanol in our research (4 g/kg.d) in vivo is attainable in the daily lives of humans. NCI-H295A cells originated from originated from the adrenal cortex of an African American female patient with adrenocortical carcinoma, and the cell line can produce steroids and regulate the enzymes involved in human adrenal steroidogenesis in a manner similar to human fetal adrenal cells (Dardis and Miller, 2003). NCI-H295A cells provide an ideal environment to investigate the function of fetal human adrenal glands (Samandari et al., 2007). The range of ethanol concentrations in the present study (15~120 mM) was based on the mean ethanol level (58±5 mM) in fetal rat blood in our previous study (Shen et al., 2014). Therefore, the results of this study are helpful to address the molecular mechanisms of female fetal adrenal developmental toxicity in PEE-induced IUGR.
Fetal rodent adrenal development originates from the adreno-gonadal primordium (Ikeda et al., 1994), and it differentiates into distinct zones: the zona glomerulosa, the zona fasciculata, the zona reticularis and the undifferentiated cell zone (Mitani, 2014). Steroid biosynthetic enzymes (e.g.,
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StAR, P450scc and 3B-HSD) participate in the biosynthesis of steroid hormones in the adrenal cortex. SF1 is a key transcription factor that is essential for the regulation of normal adrenal development and function (Bassett et al., 2005). SF1 directly binds to the response element site in the promoter regions of genes encoding StAR, P450SCC and 3B-HSD (Hu et al., 2001; Ben-Zimra et al., 2002). The Ki67 protein is a cell proliferation-associated nuclear antigen, and it is strictly associated with cell proliferation in fetal organs (Mickiewicz et al., 2012; Unek et al., 2014). Our previous studies found that the expression of Star and P450scc was decreased in the fetal adrenals of the PEE group (Liang et al., 2011). The present study found a decrease in the maximum entire cross-sectional area of female fetal adrenals with reduced cell proliferation (i.e., the number of Ki67-positive nuclei) in the PEE group. The expression of SF1 and steroid synthetic enzymes and adrenal corticosterone production were all decreased. These results indicate that PEE induced adrenal structural and functional abnormalities in female fetal rats.
4.2 Autophagy is a compensatory mechanism to the ethanol-induced inhibition of steroidogenesis
The activation of autophagy and its biological function are associated with the regulation of ATG and its signaling pathway. The conversion of LC3B from the cytosolic form (LC3B-I) to the autophagosome-associated form (LC3B-II) suggests an increase in the formation of autophagosomes. P62 is a cargo protein that is involved in the degradation of misfolded proteins, and it is degraded in lysosomes after fusion of autophagosomes and lysosomes (Jiang and Mizushima, 2015). Rapamycin is a well-known inhibitor of mTOR (Mammucari et al., 2008), and it inhibits the phosphorylation of mTOR, which consequently activates autophagy. Bafilomycin Al is a potent and selective inhibitor of the late phase of autophagy, and it inhibits the fusion of autophagosomes and lysosomes, which may increase P62 levels. In the present study, PEE could induce autophagy in the adrenals in female fetuses with the increasing expression of Beclin 1 and LC3B proteins and the increasing number of autophagic vacuoles in the PEE group. Interestingly, we found that male fetuses also showed similar change with the increasing expression of Beclin 1 and LC3ß proteins and the increasing number of autophagic vacuoles (Fig. S1A-F), which suggested the activation of autophagy by PEE both in female and male offspring. In vitro studies, ethanol increased the ratio of LC3-II/LC3B-I in vitro and decreased cortisol levels in a time- and
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concentration-dependent manner. Ethanol with rapamycin enhanced autophagy, and ethanol with bafilomycin A1 inhibited the degradation of autophagy. These results suggest that PEE could induce adrenal autophagy in fetal rats.
Numerous reports suggest that autophagy plays a crucial role in differentiation and embryonic development (Kuma et al., 2007). Autophagy is activated in various tissues after birth (Kuma et al., 2004). Atg (e.g., Beclin 1, Atg3 and Atg5)-deficient mice die in the uterus or within 1 d of delivery, and tissue-specific Atg knockout restricts the development of the nervous system, inhibits adipose differentiation and induces beta-cell dysfunction and islet damage (Jung et al., 2008; Singh et al., 2009). Potential autophagy enhancers protect against fipronil-induced apoptosis in SH-SY5Y cells, and the inhibition of autophagy promotes reactive oxygen species generation and deteriorates ethanol-induced cell death (Cunningham and Bailey, 2001). Autophagy activation is a neuroprotective response to alleviate ethanol toxicity (Chen et al., 2012).
The relationship between autophagy and fetal adrenal development is still unclear. It was known that autophagy compensates for impaired mitochondrial impairments and energy metabolism (Knuppertz and Osiewacz, 2017). It was also reported that the synthesis of testosterone in Leydig cells was impaired in the testes following autophagy inhibition (Weckman et al., 2014). In the present study, female offspring in the PEE group showed fetal developmental toxicity and steroidogenesis inhibition, accompanied by autophagy enhancement in the PEE group. In the experiment in vitro, ethanol increased the autophagy and decreased cortisol levels in the time- and concentration-dependent manner. Furthermore, the inhibition of autophagy by bafilomycin Al aggravated the reduction of SF1 expression and steroidogenesis caused by ethanol. In contrast, the activation of autophagy by rapamycin relieved the inhibition effects of ethanol on SF1 expression and steroidogenesis. These results suggest that autophagy as a compensation mechanism participates in ethanol-induced fetal adrenal dysfunction.
4.3 The inhibition of the IGF1R/mTOR pathway induced adrenal autophagy
Low blood IGF1 is the major determinant for the induction of IUGR (Klammt et al., 2008; Agrogiannis et al., 2014). The IGF1 signaling pathway plays a vital role in a variety of basic physiological activities in the adrenal glands, including the stimulation of pre- and post-natal
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adrenal growth (Baquedano et al., 2005). This pathway also up-regulates SF1 and a diverse set of steroid biosynthetic enzymes (Raha et al., 2007; Sirianni et al., 2007), whereas IGF1R defects leads to growth retardation and the dysfunction of the adrenals in mice (Pitetti et al., 2013). The mTOR protein is a point of convergence for many upstream stimuli and pathways in autophagy regulation (Yang et al., 2010). An abundance of nutrients activates mTOR-dependent signaling and leads to the suppression of autophagy. Stress conditions, such as starvation, inhibit mTOR and consequently activate autophagy (Yang et al., 2010). IGF1 activates the PI3K/AKT1/mTOR pathway, which results in the inhibition of autophagy (Zhao et al., 2007; Mammucari et al., 2008). PEE induced autophagy activation in the present study and caused a developmental retardation of fetal adrenal glands, which impaired gland function. Serum IGF1 levels and the expression of IGF1 pathway proteins (e.g., IGF1R and AKT1) were reduced in fetal adrenal glands. Ethanol affected the expression of IGF1R and phospho-mTOR (Ser2448) in concentration-dependent manners in vitro. These results suggest that PEE inhibits the IGF1R/phospho-mTOR (Ser2448) signaling pathway and consequently activates autophagy, which compensates for the ethanol-induced inhibition of steroidogenesis in fetal adrenal glands.
5 Conclusion
In conclusion, we demonstrated for the first time that PEE enhanced adrenal autophagy in female fetal rats. The activated autophagy compensates for the ethanol-induced inhibition of steroidogenesis, and its underlying mechanism is associated with inhibition of the IGF1R/phospho-mTOR (Ser2448) signaling pathway (Fig. 6). This study provides novel insight on the mechanism and biological significance of fetal adrenal autophagy to help explain the adrenal developmental origin of adult diseases during adverse environments (e.g., alcohol).
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Conflict of interest
The authors declare no conflicts of interest.
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Acknowledgements
Human adrenal NCI-H295A cells were kindly provided by Dr. W.L. Miller (Department of Pediatrics and the Metabolic Research Unit, University of California, San Francisco, California).
This work was funded by grants from the National Key Research and Development Program of China [grant numbers 2017YFC1001300], the National Natural Science Foundation of China [grant numbers 81430089, 81501269, 81673524, 81703241], and the Hubei Province Health and Family Planning Scientific Research Project [grant numbers WJ2017C0003].
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References
Agrogiannis, G.D., Sifakis, S., Patsouris, E.S., Konstantinidou, A.E., 2014. Insulin-like growth factors in embryonic and fetal growth and skeletal development (Review). Molecular medicine reports 10, 579-584.
Baquedano, M.S., Berensztein, E., Saraco, N., Dorn, G.V., de Davila, M.T., Rivarola, M.A., Belgorosky, A., 2005. Expression of the IGF system in human adrenal tissues from early infancy to late puberty: implications for the development of adrenarche. Pediatric research 58, 451-458.
Bassett, M.H., Mayhew, B., Rehman, K., White, P.C., Mantero, F., Arnaldi, G., Stewart, P.M., Bujalska, I., Rainey, W.E., 2005. Expression profiles for steroidogenic enzymes in adrenocortical disease. The Journal of clinical endocrinology and metabolism 90, 5446-5455.
Ben-Zimra, M., Koler, M., Orly, J., 2002. Transcription of cholesterol side-chain cleavage cytochrome P450 in the placenta: activating protein-2 assumes the role of steroidogenic factor-1 by binding to an overlapping promoter element. Molecular endocrinology 16, 1864-1880.
Chen, G., Ke, Z.J., Xu, M., Liao, M.J., Wang, X., Qi, Y.L., Zhang, T., Frank, J.A., Bower, K.A., Shi, X.L., Luo, J., 2012. Autophagy is a protective response to ethanol neurotoxicity. Autophagy 8, 1577-1589.
Cunningham, C.C., Bailey, S.M., 2001. Ethanol consumption and liver mitochondria function. Biological signals and receptors 10, 271-282.
Dardis, A., Miller, W.L., 2003. Dexamethasone does not exert direct intracellular feedback on steroidogenesis in human adrenal NCI-H295A cells. The Journal of endocrinology 179, 131-142.
Eleftheriades, M., Creatsas, G., Nicolaides, K., 2006. Fetal growth restriction and postnatal development. Annals of the New York Academy of Sciences 1092, 319-330.
Gohlke, J.M., Griffith, W.C., Faustman, E.M., 2005. A systems-based computational model for dose-response comparisons of two mode of action hypotheses for ethanol-induced neurodevelopmental toxicity. Toxicological sciences : an official journal of the Society of Toxicology 86, 470-484.
Hansson, A., Hance, N., Dufour, E., Rantanen, A., Hultenby, K., Clayton, D.A., Wibom, R., Larsson, N.G., 2004. A switch in metabolism precedes increased mitochondrial biogenesis in respiratory chain-deficient mouse hearts. Proceedings of the National Academy of Sciences of the United States of America 101, 3136-3141.
Harris, R.A., Trudell, J.R., Mihic, S.J., 2008. Ethanol’s molecular targets. Science signaling 1, re7. Hu, M.C., Hsu, N.C., Pai, C.I., Wang, C.K., Chung, B., 2001. Functions of the upstream and proximal steroidogenic factor 1 (SF-1)-binding sites in the CYP11A1 promoter in basal transcription and hormonal response. Molecular endocrinology 15, 812-818.
Huang, H., He, Z., Zhu, C., Liu, L., Kou, H., Shen, L., Wang, H., 2015. Prenatal ethanol exposure-induced adrenal developmental abnormality of male offspring rats and its possible intrauterine programming mechanisms. Toxicology and applied pharmacology 288, 84-94.
Ikeda, Y., Shen, W.H., Ingraham, H.A., Parker, K.L., 1994. Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Molecular endocrinology 8, 654-662.
Jiang, P., Mizushima, N., 2015. LC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells. Methods 75, 13-18.
Jung, H.S., Chung, K.W., Kim, J.W., Kim, J., Komatsu, M., Tanaka, K., Nguyen, Y.H., Kang, T.M., Yoon, K.H., Kim, J.W., Jeong, Y.T., Han, M.S., Lee, M.K., Kim, K.W., Shin, J., Lee, M.S., 2008. Loss of Autophagy Diminishes Pancreatic beta Cell Mass and Function with Resultant
THE
ACCEPTED MANUSCRIPT
Hyperglycemia. Cell metabolism 8, 318-324.
Klammt, J., Pfaffle, R., Werner, H., Kiess, W., 2008. IGF signaling defects as causes of growth failure and IUGR. Trends in endocrinology and metabolism: TEM 19, 197-205.
Knuppertz, L., Osiewacz, H.D., 2017. Autophagy compensates impaired energy metabolism in CLPXP-deficient Podospora anserina strains and extends healthspan. Aging cell 16, 704-715.
Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., Ohsumi, Y., Tokuhisa, T., Mizushima, N., 2004. The role of autophagy during the early neonatal starvation period. Nature 432, 1032-1036.
Kuma, A., Matsui, M., Mizushima, N., 2007. LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization. Autophagy 3, 323-328.
Liang, G., Chen, M., Pan, X.L., Zheng, J., Wang, H., 2011. Ethanol-induced inhibition of fetal hypothalamic-pituitary-adrenal axis due to prenatal overexposure to maternal glucocorticoid in mice. Experimental and toxicologic pathology : official journal of the Gesellschaft fur Toxikologische Pathologie 63, 607-611.
Lundsberg, L.S., Bracken, M.B., Saftlas, A.F., 1997. Low-to-moderate gestational alcohol use and intrauterine growth retardation, low birthweight, and preterm delivery. Annals of epidemio logy 7, 498-508.
Mammucari, C., Schiaffino, S., Sandri, M., 2008. Downstream of Akt: Fox03 and mTOR in the regulation of autophagy in skeletal muscle. Autophagy 4, 524-526.
Marciniak, B., Patro-Malysza, J., Poniedziałek-Czajkowska, E., Kimber-Trojnar, Z., Leszczynska-Gorzelak, B., Oleszczuk, J., 2011. Glucocorticoids in pregnancy. Current pharmaceutical biotechnology 12, 750-757.
May, P.A., Gossage, J.P., Brooke, L.E., Snell, C.L., Marais, A.S., Hendricks, L.S., Croxford, J.A., Viljoen, D.L., 2005. Maternal risk factors for fetal alcohol syndrome in the Western cape province of South Africa: a population-based study. American journal of public health 95, 1190-1199.
Meaney, M.J., Szyf, M., Seckl, J.R., 2007. Epigenetic mechanisms of perinatal programming of hypothalamic-pituitary-adrenal function and health. Trends in molecular medicine 13, 269-277.
Mickiewicz, M., Zabielski, R., Grenier, B., Le Normand, L., Savary, G., Holst, J.J., Oswald, I.P., Metges, C.C., Guilloteau, P., 2012. Structural and functional development of small intestine in intrauterine growth retarded porcine offspring born to gilts fed diets with differing protein ratios throughout pregnancy. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society 63, 225-239.
Mitani, F., 2014. Functional zonation of the rat adrenal cortex: the development and maintenance. Proceedings of the Japan Academy. Series B, Physical and biological sciences 90, 163-183.
Muhajarine, N., D’Arcy, C., Edouard, L., 1997. Prevalence and predictors of health risk behaviours during early pregnancy: Saskatoon Pregnancy and Health Study. Canadian journal of public health = Revue canadienne de sante publique 88, 375-379.
Mullally, A., Cleary, B.J., Barry, J., Fahey, T.P., Murphy, D.J., 2011. Prevalence, predictors and perinatal outcomes of peri-conceptional alcohol exposure — retrospective cohort study in an urban obstetric population in Ireland. BMC pregnancy and childbirth 11, 27.
Murphy, D.J., Mullally, A., Cleary, B.J., Fahey, T., Barry, J., 2013. Behavioural change in relation to alcohol exposure in early pregnancy and impact on perinatal outcomes — a prospective cohort study. BMC pregnancy and childbirth 13, 8.
Netchine, I., Azzi, S., Houang, M., Seurin, D., Perin, L., Ricort, J.M., Daubas, C., Legay, C., Mester, J., Herich, R., Godeau, F., Le Bouc, Y., 2009. Partial primary deficiency of insulin-like
ACCEPTED MANUSCRIPT
growth factor (IGF)-I activity associated with IGF1 mutation demonstrates its critical role in growth and brain development. The Journal of clinical endocrinology and metabolism 94, 3913-3921.
Ni, Q., Wang, L., Wu, Y., Shen, L., Qin, J., Liu, Y., Magdalou, J., Chen, L., Wang, H., 2015. Prenatal ethanol exposure induces the osteoarthritis-like phenotype in female adult offspring rats with a post-weaning high-fat diet and its intrauterine programming mechanisms of cholesterol metabolism. Toxicology letters 238, 117-125.
Ong, K., 2005. Adrenal function of low-birthweight children. Endocrine development 8, 34-53.
Pantazis, N.J., Dohrman, D.P., Luo, J., Goodlett, C.R., West, J.R., 1992. Alcohol reduces the number of pheochromocytoma (PC12) cells in culture. Alcohol 9, 171-180.
Ping, J., Wang, J.F., Liu, L., Yan, Y.E., Liu, F., Lei, Y.Y., Wang, H., 2014. Prenatal caffeine ingestion induces aberrant DNA methylation and histone acetylation of steroidogenic factor 1 and inhibits fetal adrenal steroidogenesis. Toxicology 321, 53-61.
Pitetti, J.L., Calvel, P., Romero, Y., Conne, B., Truong, V., Papaioannou, M.D., Schaad, O., Docquier, M., Herrera, P.L., Wilhelm, D., Nef, S., 2013. Insulin and IGF1 receptors are essential for XX and XY gonadal differentiation and adrenal development in mice. PLOS genetics 9, e1003160.
Raha, D., Nehar, S., Paswan, B., Rebuffat, P., Neri, G., Naskar, R., Kumari, K., Raza, B., Rao, N.V., Macchi, C., Sen, N.S., Nussdorfer, G.G., Ahmad, M.F., 2007. IGF-I enhances cortisol secretion from guinea-pig adrenal gland: in vivo and in vitro study. International journal of molecular medicine 20, 91-95.
Roberts, C.T., Owens, J.A., Sferruzzi-Perri, A.N., 2008. Distinct actions of insulin-like growth factors (IGFs) on placental development and fetal growth: lessons from mice and guinea pigs. Placenta 29 Suppl A, S42-47.
Samandari, E., Kempna, P., Nuoffer, J.M., Hofer, G., Mullis, P.E., Fluck, C.E., 2007. Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3beta-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities. The Journal of endocrinology 195, 459-472.
Shen, L., Liu, Z., Gong, J., Zhang, L., Wang, L., Magdalou, J., Chen, L., Wang, H., 2014. Prenatal ethanol exposure programs an increased susceptibility of non-alcoholic fatty liver disease in female adult offspring rats. Toxicology and applied pharmacology 274, 263-273.
Singh, R., Xiang, Y., Wang, Y., Baikati, K., Cuervo, A.M., Luu, Y.K., Tang, Y., Pessin, J.E., Schwartz, G.J., Czaja, M.J., 2009. Autophagy regulates adipose mass and differentiation in mice. The Journal of clinical investigation 119, 3329-3339.
Sirianni, R., Chimento, A., Malivindi, R., Mazzitelli, I., Ando, S., Pezzi, V., 2007. Insulin-like growth factor-I, regulating aromatase expression through steroidogenic factor 1, supports estrogen-dependent tumor Leydig cell proliferation. Cancer research 67, 8368-8377.
Tan, Y., Liu, J., Deng, Y., Cao, H., Xu, D., Cu, F., Lei, Y., Magdalou, J., Wu, M., Chen, L., Wang, H., 2012. Caffeine-induced fetal rat over-exposure to maternal glucocorticoid and histone methylation of liver IGF-1 might cause skeletal growth retardation. Toxicology letters 214, 279-287.
Unek, G., Ozmen, A., Ozekinci, M., Sakinci, M., Korgun, E.T., 2014. Immunolocalization of cell cycle proteins (p57, p27, cyclin D3, PCNA and Ki67) in intrauterine growth retardation (IUGR) and normal human term placentas. Acta histochemica 116, 493-502.
Vieau, D., Sebaai, N., Leonhardt, M., Dutriez-Casteloot, I., Molendi-Coste, O., Laborie, C., Breton, C., Deloof, S., Lesage, J., 2007. HPA axis programming by maternal undernutrition in the male rat offspring. Psychoneuroendocrinology 32 Suppl 1, S16-20.
Weckman, A., Di Ieva, A., Rotondo, F., Syro, L.V., Ortiz, L.D., Kovacs, K., Cusimano, M.D., 2014. Autophagy in the endocrine glands. Journal of molecular endocrinology 52, R151-163.
ACCEPTED MANUSCRIPT
Williams, R.J., Gloster, S.P., 1999. Knowledge of fetal alcohol syndrome (FAS) among natives in Northern Manitoba. Journal of studies on alcohol 60, 833-836.
Xia, L.P., Shen, L., Kou, H., Zhang, B.J., Zhang, L., Wu, Y., Li, X.J., Xiong, J., Yu, Y., Wang, H., 2014. Prenatal ethanol exposure enhances the susceptibility to metabolic syndrome in offspring rats by HPA axis-associated neuroendocrine metabolic programming. Toxicology letters 226, 98-105.
Xu, D., Wu, Y., Liu, F., Liu, Y.S., Shen, L., Lei, Y.Y., Liu, J., Ping, J., Qin, J., Zhang, C., Chen, L.B., Magdalou, J., Wang, H., 2012a. A hypothalamic-pituitary-adrenal axis-associated neuroendocrine metabolic programmed alteration in offspring rats of IUGR induced by prenatal caffeine ingestion. Toxicology and applied pharmacology 264, 395-403.
Xu, D., Zhang, B., Liang, G., Ping, J., Kou, H., Li, X., Xiong, J., Hu, D., Chen, L., Magdalou, J., Wang, H., 2012b. Caffeine-induced activated glucocorticoid metabolism in the hippocampus causes hypothalamic-pituitary-adrenal axis inhibition in fetal rats. PloS one 7, e44497.
Yang, L., Li, P., Fu, S., Calay, E.S., Hotamisligil, G.S., 2010. Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance. Cell metabolism 11, 467-478.
Yoshida, Y., Ikuta, F., 1984. Three-dimensional architecture of cerebral microvessels with a scanning electron microscope: a cerebrovascular casting method for fetal and adult rats. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 4, 290-296.
Zhao, J., Brault, J.J., Schild, A., Cao, P., Sandri, M., Schiaffino, S., Lecker, S.H., Goldberg, A.L., 2007. Fox03 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell metabolism 6, 472-483.
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Figure legends
Fig. 1. Effects of prenatal ethanol exposure (PEE) on bodyweight, adrenal histological morphology and steroidogenesis in female fetal rats at gestational day (GD) 20. Pregnant rats were given ethanol (4 g/kg.d) during GD9-20. A: Fetal body weight; B: Intrauterine growth retardation (IUGR) rate (%), n = 12 for weight and IUGR rate (%); C: Fetal adrenal morphology (HE, ×400); D: The maximum cross-sectional area of fetal adrenal glands (mm2); E: Ki67 protein expression (immunohistochemical staining, ×400); F: The number of nuclei-stained cells with Ki67 in the fetal adrenal cortex. n = 6 sections of each group were selected, and five random fields of each section were scored. G-L: The mRNA expression of steroidogenic factor 1 (Sf1), steroidogenic acute regulatory protein (Star), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3B-Hydroxysteroid dehydrogenase (3B-Hsd), steroid 21-hydroxylase (P450c21) and steroid 11ß-hydroxylase (P450cl1), n = 10, six pairs of fetal adrenal glands from two littermates were pooled for homogenization into one sample. M: Fetal adrenal corticosterone content (%), n = 6. Mean ± S.E.M., *P<0.05, ** P<0.01 vs. control. HE: hematoxylin and eosin; Gapdh: glyceraldehyde phosphate dehydrogenase. All experiments were repeated three times.
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Fig. 2. Effects of prenatal ethanol exposure (PEE) on insulin-like growth factor 1 (IGF1) signaling pathway and autophagy in the female fetal adrenal glands at gestational day (GD) 20. Pregnant rats were given doses of ethanol (4 g/kg.d) during GD9-20. The concentration of serum IGF1was detected by ELISA kits. A: Serum IGF1 concentration, n = 3 for serum phenotype (the serum was randomly merged because of the meagerness of fetal blood). B-C: The mRNA expression of Igf1 receptor (Igf1r) and serine/threonine kinase (Akt1), n = 10, six pairs of fetal adrenal glands from two littermates were pooled for homogenization into one sample. D: Beclin 1 protein expression (immunohistochemical staining, ×400); E: Density value of Beclin 1; F: Microtubule-associated protein light chain 3 beta (LC3B) protein expression (immunohistochemical staining, ×400); G: Density value of LC3฿; H: Adrenal cortex cell ultra-structure (TEM, ×40,000); I: Autophagic vacuole (Avi, black arrow) per 100 cells. n = 6, Mean ± S.E.M., *P<0.05, ** P<0.01 vs. control. M: mitochondrion; N: nucleus; TEM: transmission electron microscopy; Gapdh: glyceraldehyde phosphate dehydrogenase. All experiments were repeated three times.
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Fig. 3. Effects of ethanol on adrenocortical cell viability and steroidogenesis in NCI-H295A cells. A: Cell viability after the treatment of ethanol for 24 h; B: Cell viability after the treatment of ethanol for 72 h; C-H: The mRNA expression of steroidogenic factor 1 (SF1), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450SCC), 3B-Hydroxysteroid dehydrogenase (3B-HSD), steroid 21-hydroxylase (P450C21) and steroid 11-hydroxylase (P450C11) after 60 and 120 mM ethanol treatment for 8 h; I: The concentration of cortisol in culture medium after 60 and 120 mM ethanol treatment for 8 h; J: The concentration of cortisol in culture medium after 60 mM ethanol treatment for 8, 12 and 24 h. n = 6, Mean ± S.E.M., *P<0.05, ** P<0.01 vs. control. GAPDH: glyceraldehyde phosphate dehydrogenase; OD: optical density. All experiments were repeated three times.
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Fig. 4. Effect of ethanol on the autophagy of adrenocortical cells in NCI-H295A cells. A: Effect of ethanol (60 mM) on the LC3B-II/LC3B-I ratio (Western blotting) at different time points (8, 12 and 24 h); B: Density value of the LC3B-II/LC3B-I ratio at different time points (8 h, 12 h and 24 h); C: Effect of ethanol on the LC3B-II/LC3B-I ratio (Western blotting) at different concentrations (15, 30, 60 and 120 mM) for 8 h; D: Density value of the LC3B-II/LC3B-I ratio at different concentrations (15, 30, 60 and 120 mM) for 8 h; E: Cell ultra-structure (TEM, ×40,000) after 60 mM ethanol treatment for 24 h; F: Autophagic vacuoles (Avi, black arrow) per 100 cells after 60 mM ethanol treatment for 24 h. n = 4, Mean ± S.E.M., *P<0.05, ** P<0.01 vs. control. LC3B: microtubule-associated protein light chain 3 beta; M: mitochondrion; N: nucleus; TEM: transmission electron microscopy; GAPDH: glyceraldehyde phosphate dehydrogenase. All experiments were repeated three times.
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Fig. 5. Effect of ethanol on insulin-like growth factor 1 receptor (IGF1R) / mammalian target of rapamycin (mTOR) pathway of adrenocortical cells in NCI-H295A cells for 8 h. A: The protein expression levels of steroidogenic factor 1 (SF1), P62, and the LC3B-II/LC3B-I ratio after treatment with ethanol and rapamycin / bafilomycin A1 alone or in combination for 8 h; B-D: Density value of SF1, P62 and the LC3B-II/LC3B-I after ethanol treatment with/without rapamycin or bafilomycin A1; E: The cortisol concentrations in culture medium after treatment with ethanol and rapamycin / bafilomycin Al alone or in combination for 8 h; F: The protein expression of IGF1R, phospho-mTOR (Ser2448), and P62 after ethanol treatment (15, 30 and 60 mM); G-I: Density value ofIGF1R, phospho-mTOR (Ser2448), and P62 after ethanol treatment. n = 4, Mean ± S.E.M., ** P<0.01 vs. control, P<0.01 vs. ethanol group. P62: protein 62; LC3B: microtubule-associated protein light chain 3 beta; GAPDH: glyceraldehyde phosphate dehydrogenase. All experiments were repeated three times.
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Fig. 6. Schematic displaying the effect of prenatal ethanol exposure (PEE) on adrenal autophagy and its biological significance in female fetal rats. PEE inhibits the IGF1R/phospho-mTOR (Ser2448) signaling pathway and consequently activates autophagy, which compensates for the ethanol-induced inhibition of steroidogenesis in fetal adrenal glands. SF1: steroidogenic factor 1; IGF1R: insulin-like growth factors 1 receptor; AKT1: serine/threonine kinase; p-mTOR (Ser2448): mammalian target of rapamycin phosphorylated at serine 2448.
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A
B
☐ Control
☒ PEE
☐ Control
☒ PEE
Fetal body weight (g)
4-
100-
**
**
3
IUGR rate(%)
80-
60-
2-
40-
1-
20
0
T
0
C
Control
PEE
D
Maximum cross-sectional area of fetal adrenal(mm2)
0.8-
☐ Control
PEE
0.6-
0.4-
**
0.2-
0.0
50um
50um
E Control
PEE
F
Number of Ki67-stained nuclei (0.01mm2)
900-
☐ Control
PEE
*
600-
300-
0
O
50um
1
50um
G
H
1
J
Adrenal Sfl expression (of Gapdh )
Adrenal Star expression (of Gapdh)
Adrenal P450scc expression (of Gapdh)
Adrenal 3B -Hsd expression (of Gapdh)
☐ Control
☒ PEE
☐ Control
☒
PEE
☐ Control
PEE
☐ Control
PEE
0.8
4-
1.5
*
1.2
0.6
»ệt
3-
1.0-
0.8
0.4-
2
0.5
0.4
0.2-
1
0.0
0
0.0
0.0
K
L
M
Adrenal P450c21 expression (of Gapdh)
Adrenal P450c11 expression (of Gapdh)
Adrenal corticosterone (%)
☐ Control
☒ PEE
☐
Control
☒
PEE
☐ Control
☒ PEE
6-
15-
150-
*
4
10
100
**
*
2
54
50
0
0
0
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A
B
C
Serum IGF1 concentration (ng/ml)
150-
☐ Control
PEE
PEE
☐ Control
☒ PEE
Adrenal Igflr expression (of Gapdh)
☐ Control
Adrenal Akt1 expression (of Gapdh)
2.0-
0.6-
100-
1.5-
0.4-
*
*
**
1.0-
50-
0.2-
0.5-
0
0.0
0.0
D
E
Control
PEE
0.36-
☐ Control
PEE
Beclin 1 expression (Density value)
**
0.24-
0.12-
0.00
50um
50um
F
Control
PEE
G
0.3-
☐ Control
PEE
LC3B expression (Density value)
*
0.2-
0.1-
50um
50um
0.0
H
Control
PEE
I
Autophagic vacuoles per 100 cells
9-
Control
PEE
**
M
6-
M
AVi
3-
0
0.5um
0.5um
ACCEPTED MANUSCRIPT
A
24 ḥ
B
72 h
0.6-
0.9-
*
*
**
OD490
0.4-
OD490
0.6-
0.2-
0.3-
0.0
0
15
30
60
120
0.0
0
15
30
60
120
Ethanol (mM)
Ethanol (mM)
C
D
E
SF1 mRNA expression (of GAPDH)
StAR mRNA expression (of GAPDH)
P450SCC mRNA expression (of GAPDH)
1.5
1.5
1.5
1.0
*
1.0
**
**
1.0
**
**
0.5
0.5
0.5
**
0.0
Control
Ethanol (60 mM)
Ethanol (120 mM)
0.0
Control
Ethanol (60 mM)
Ethanol (120 mM)
0.0
Control
Ethanol (60 mM)
Etha nol (120 mM)
F
G
H
30 -HSD mRNA expression (of GAPDH)
P450C21 mRNA expression (of GAPDH)
P450C11 mRNA expression (of GAPDH)
1.5
1.5
1.5
1.0
1.0
*
1.0
*
*
0.5
0.5
0.5
0.0
Control
Etanol (60 mM)
Ethanol (120 mM)
0.0
Control
Ethanol (60 mM)
Ethanol (120 mM)
0.0
Control
Ethanol (60 mM)
Etha nol (1 20 mM)
I
J
Supernatant cortisol concentrations (ng/ml)
400
Supernatant cortisol concentrations (ng/ml)
400
300-
300-
**
**
**
**
200-
200-
**
100-
100-
0
T
0
Control
Ethanol (60 mM)
Ethanol (120 mM)
T
Control
Ethanol (8h)
Ethanol (12h)
Ethanol (24h)
ACCEPTED MANUSCRIPT
A
Control (24 h)
Ethanol (60 mM)
B
8 h
12 h
24 h
**
25-
LC3B-IV/LC3B -I ratio (of GAPDH)
20-
**
LC3B-I
15kDa
O
LC3B-II
5-
2.0-
1.5.
1.0-
GAPDH
40kDa
0.5-
0.0
Control
Ethanol (8h)
Ethanol (12h)
Ethanol (24h)
C
Ethanol (mM)
D
0
15
30
60
120
LC3B -11/LC3B -I ratio (of GAPDH)
87
6
*
LC3B-I
LC3B-II
15kDa
A
T
?
GAPDH
40kDa
0
Control
Ethanol (15mM)
Ethanol (30mM)
Ethanol (60mM)
Ethanol (120mM)
E
F
Control
Ethanol
~
control
Etunol
Autophagic vacuoles per 100 cells
40
-
N
NO
M
8
M
10
0
-
0.5um
0.5pm
B
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
A
Ethanol
Rapamycin
Bafilomycin Al
SF1
70kDa
P62
70kDa
LC3B-I
15kDa
LC3B-II
GAPDH
40kDa
B
0.8-
C
2.5-
T
SF1 level (of GAPDH)
0.6-
P62 level (of GAPDH)
2.0-
**
0.4-
1.5-
0.2.
0.4-
**
0.2.
0.0
0.0
Control
Ethanol
Rapa
Ethanol+ Rapa
Baf Al
Ethanol+Baf Al
Central
Ethanol
Rapa
Ethanol+Rapa
Bal Al
Baf Al+Ethanol
D
E
5-
500-
(of GAPDH) LC3B-II/LC3-16 ratio 1
Supernatant cortisol concentrations (ng/ml)
-
400
T
**
**
**
300-
2
200-
100
O
Q
Control
Ethanol
Rapa
Ethanol+Rapa
Baf Al
Baf Al+Ethanol
Control
Ethanol
Rapa
Ethanol+Rapa
Baf Al
Ethanol+Bal Al
F
G
Ethanol (mM)
0
15
30
60
0.8-
p-mTOR(Ser2448)
289kDa
T
IGFIR level (of GAPDH)
0.6
IGFIR
150kDa
-
P62
70kDa
0.2
0.0
GAPDH
40kDa
0 mM
15 mM
30 mM
60 mM
H
I
p-mTOR(ser2448) level (of GAPDH)
1.5-
0.8
P62 level (of GAPDH)
0,6
**
-
0.5.
0.2
-
0.0
0.0
0 mM
15 mM
30 mM
60 ml
8 mM
15mM
30 mM
60 mM
MANUSCRIPT
ACCEPTED
ACCEPTED MANUSCRIPT
IGF1
ethanol
IGF1R
PI3K
AKTI
p-mTOR
P
autophagy
Cargo
Adrenal steroidogenesis
Î
ACCEPTED
ACCEPTED MANUSCRIPT
| Genes | Forward primer | Reverse primer | Product(bp) | Annealing Condition |
|---|---|---|---|---|
| Sf1 | CCAGTACGGCAAGGAAGA | GAGGCTGAAGAGGATGAGGA | 193 | 63℃, 30s |
| Star | GGGAGATGCCTGAGCAAAGC | GCTGGCGAACTCTATCTGGGT | 188 | 65℃, 30s |
| P450scc | GCTGCCTGGGATGTGATTTTC | GATGTTGGCCTGGATGTTCTTG | 156 | 63℃, 30s |
| 3-Hsd | TCTACTGCAGCACAGTTGAC | ATACCCTTATTTTTGAGGGC | 271 | 58℃, 30s |
| P450c21 | AGGAGCTGAAGAGGCACAAG | GAGGTAGCTGCATTCGGTTC | 188 | 63℃, 30s |
| P450c11 | CCCCTTTGIGGATGTGGTAG | CACGCTCTCAGGTTTCAGGT | 188 | 61°℃, 30s |
| Igf1 | GACCAAGGGGCTTTTACTTCAAC | TTTGTAGGCTTCAGCGGAGCAC | 148 | 60℃, 30s |
| Igf1r | GTCCTTCGGGATGGTCTA | TGGCCTTGGGATACTACAC | 195 | 62℃, 30s |
| Akt1 | ATGAGCGACGTGGCTATTGTGAA G | GAGGCCGTCAGCCACAGTCTGGAT G | 156 | 60℃, 30s |
| Gapdf | GCAAGTTCAATGGCACAG | GCCAGTAGACTCCACGACA | 140 | 63℃, 30s |
Sf1: steroidogenic factor 1; Star: steroidogenic acute regulatory protein; P450scc: cytochrome P450 cholesterol side chain cleavage; 3B-Hsd: 30-hydroxysteroid dehydrogenase; P450c21: steroid 21-hydroxylase; P450cl1: steroid 11ß-hydroxylase; Igf1: insulin-like growth factor 1; Igflr. Igfl receptor; Akt1: serine/threonine kinase; Gapdh: glyceraldehyde 3-phosphate dehydrogenase.
ACCEPTED MAN
ACCEPTED MANUSCRIPT
Highligts
· Prenatal ethanol exposure induce adrenal autophag in female fetal rats
· IGF1R/p-mTOR (Ser2448) pathway mediated adrenal autophag induced by ethanol
. Autophagy is a compensatory mechanism to inhibition of fetal adrenal steroidogenesis
ACCEPTED MANUSCRIPT