Differential effects of estrogen/androgen on the prevention of nonalcoholic fatty liver disease in male rat

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1 Differential effects of estrogen/androgen on the prevention of nonalcoholic fatty liver disease in male rat Hua Zhang 1, Yuanwu Liu 1, Li Wang 1,2, Zhen Li 3, Hongwen Zhang 4, Jihua Wu 4, Nafis Rahman 5,6, Yangdong Guo 3, Defa Li 7, Ning Li 1, Ilpo Huhtaniemi 8, Suk Ying Tsang 9, George F Gao 10, Xiangdong Li 1* 1 State Key Laboratory of the Agro-Biotechnology, College of Biological Sciences, China Agricultural University, China; 2 College of Agriculture and Biotechnology, China Agricultural University, China; 3 State Key Laboratory of Plant Physiology and Biochemistry, China Agricultural University, China; 4 Department of General Surgery, The 306 th Hospital of PLA, Beijing, China; 5 Department of Physiology, Institute of Biomedicine, University of Turku, Finland; 6 Departments of Cell Biology, Human Molecular Genetics, Ob/Gyn, Florida International University School of medicine, Miami, FL, USA; 7 State key laboratory of Animal Nutrition, China Agricultural University, China; 8 Department of Surgery and Cancer, Imperial College London, London, United Kingdom; 9 Department of Biochemistry, Chinese University of Hong Kong, China; 10 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, China. Running title: Estrogen/androgen and fatty liver disease *Corresponding authors & reprint requests: Dr. Xiangdong Li, Ph.D, Professor State Key Laboratory of the Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing , China Tel/Fax: ; xiangdongli@cau.edu.cn 1

2 ABBREVIATIONS: E2, estradiol; DHT, 5 -dihydrotestosterone; P-ACC, acetyl coenzyme A carboxylase; ERα, estrogen receptor α; CPT1, carnitine palmitotyltransferase1; P-HMGCR, 3-hydroxy-3-methyl-glutaryl-CoA reductase; AR, androgen receptor,; tam, tamoxifen;arko, knockout mouse for aromatase; αerko, knockout mouse for ERα; αβerko, double ER α & β knockout mouse; TG, triglyceride; ArKO, aromatase knockout mouse; ARKO, androgen receptor knockout mouse; ORX, orchidectomized; HFD, high fat diet; SD, Sprague-Dawley; IR, insulin resistance, MMP, mitochondrial membrane potential; ALT, alanine aminotransferase; HDL-c, high density lipoprotein cholersterol; LDL-c, low density lipoprotein cholersterol; ORO, Oil Red O; SREBP-1c, sterol regulatory element-binding protein-1c; FAS, fatty acid synthase; ACC, Acetyl-CoA carboxylase; SCD1, stearoyl-coenzyme A desaturase 1; PPAR, peroxisome proliferator-activated receptor ; VLCAD, very long-chain acyl-coa dehydrogenase; MCAD, medium-chain acyl-coa dehydrogenase; ACO1, acyl-coa oxidase 1; UCP2,uncoupling protein 2; PO, palmitic acid +oleic acid 2

3 ABSTRACT It is important to clarify the distinct contributions of estrogen/estrogen receptors (ERs) and/or androgen/androgen receptor (AR) signaling, as well as their reciprocal effects on the regulation of hepatic lipid homeostasis. We studied the molecular mechanisms underlying the preventive effects of estradiol (E2); dihydrotestosterone (DHT); or E2+DHT on high-fat diet induced non-alcoholic fatty liver disease (NAFLD) in an orchidectomized (ORX) Sprage-Dawly (SD) rat model. E2 is shown to be associated with decreased fatty acid synthesis in hepatic zone 3-specific manner by increasing the phosphorylation of acetyl coenzyme-a carboxylase (P-ACC) via an ERα-mediated pathway. DHT is shown to be associated with decreased lipid accumulation and cholesterol synthesis in hepatic zone 1-specific manner by increasing expression of carnitine palmitotyltransferase1 (CPT1) and phosphorylation of 3-hydroxy-3-methyl-glutaryl-CoA reductase (P-HMGCR) via an AR-mediated pathway. E2+DHT showed an additive positive effect and normalized all the 3 impaired zones of the liver. Gene expression changes in human severe liver steatosis were similar to those of experimental rat NAFLD. Steroids reversed the histopathological NAFLD changes, likely by decreasing fatty acid and cholesterol synthesis and increasing β-oxidation. The diverse steroid effects (ER/AR) on NAFLD prevention in males indicate the potential applicability of ER/AR modulators for NAFLD treatment. Supplementary key words: Non-alcoholic steatohepatitis; Lipid metabolism; estrogen; androgen 3

4 INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) is strongly linked to central obesity, insulin resistance (IR), and metabolic syndrome (1), and its increasing prevalence is estimated at 20-30% among the adult population in industrialized countries (2). Although estrogen and androgen are important regulators of lipid homeostasis (3, 4), data on their possible influence on the prevention or treatment of NAFLD is still scarce. Clinically, tamoxifen (Tam) is widely used for the treatment of estrogen-responsive breast cancer, while its frequent side effect is the development of NAFLD. Forty-three percent of Tam-treated breast cancer patients develop steatosis within the first 2 years of treatment (5). Human estrogen receptor (ER) and aromatase deficiency are very rare clinical conditions (6, 7). While men with ERα mutation developed severe steatosis (7), the three adult men reported with aromatase deficiency had impaired lipid metabolism, and only one presenting with hepatic steatosis (8). Aromatase-knockout mouse (ArKO) (3, 4), ERα-knockout (αerko) mouse (9), and the double ER knockout (αβerko) mouse (10), all display elevated triglyceride (TG) levels (11). These clinical and experimental studies have shown the significant role of the estrogen signaling pathway in lipid homeostasis (3, 4). Regardless of the importance of estrogen in NAFLD, testosterone treatment studies have showed visceral-fat reduction in men (12, 13) and improvement of NAFLD (14). Androgen receptor knockout (ARKO) male mice develop late-onset obesity (15, 16). The liver-specific ARKO male mice have increased IR and steatosis, with decreased β-oxidation, upon high-fat diet (HFD) (17). Clinically, increased IR and impaired glucose tolerance have been observed in men with testosterone deficiency (18). However, the specific role of the androgen/ar signaling for the regulation of lipid metabolism in men is still largely unknown. ArKO male mice are characterized by a marked hepatic steatosis and consistently high serum testosterone levels (reviewed in (19)). Accordingly, αerko mice also have higher serum testosterone levels (reviewed in (20)). In contrast, serum E2 levels of ARKO mice are either elevated (21) or normal (15) implying an enhanced or sufficient supply of systemic estrogen action. It is thus anticipated that the estrogen/er or androgen/ar pathways play key roles in lipid homeostasis. Clinical studies on effects of androgens on lipid metabolism (12, 13) have used aromatizable androgens, resulting in a mixture of ER and AR signal actions (22). It is important to clarify the distinct contributions of estrogen/ers and/or androgen/ar signaling in the regulation of hepatic lipid homeostasis. 4

5 It was impossible to breed homozygous double knockout ER/AR mice for this study, due to the infertility of ARKO male mice (23), subfertility of ARKO females (23), and sexual immaturity in both genders of homozygous αerko mice. To analyze the actions of ER and AR signaling pathways on NAFLD, we depleted endogenous sex steroids by orchidectomy (ORX) male Sprague-Dawley (SD) adult rats on HFD and assessed the specific impacts of testosterone and/or estradiol deficiency and the effect of E2, 5α-dihydrotestosterone (DHT; a non-aromatizable androgen), or E2+DHT supplementation in this inducible NAFLD model. In order to demonstrate this NAFLD model to human relevance, we studied the same molecular changes observed in this NAFLD rat model in samples of severe liver steatosis in men. 5

6 MATERIALS AND METHODS Experimental design Two-month-old male SD rats (200±20g) (Vital-River, Beijing, China) were orchidectomized (ORX) and fed with either normal diet (ND, n=10) or high-fat diet (HFD, n=30/group). HFD ORX rats received either vehicle (corn oil) or E2, DHT or E2+DHT treatment once daily subcutaneously for 75 days, starting 7-days post-orx (n=30/group). We also had wild type gonad intact (WT) SD rats on HFD that received vehicle (corn oil) or E2, DHT or E2+DHT (n=6/group) as an internal control group for the ORX rats. The China Agricultural University Ethics Committee approved the animal experiments. The treatment design is shown in Table 1. The rats were orchidectomized (ORX) under general anesthesia with 4.0% isoflurane. The testes were removed through a small incision in the scrotum and incision place sutured. The ORX had a 7-day time period to recover from the surgery before the treatments started. Food and water were provided ad libitum and animals were housed in a room with controlled light (12h light 12h darkness). The high fat diet (HFD) was prepared according to our previous study (24). The 3.0 mg/kg/d dose of DHT was chosen as it was shown that this dose can restore the weight of the ventral prostate (VP) in ORX rats (control: 0.062±0.13 g/vp; ORX: 0.02±0.01 g/vp; DHT: 0.070±0.32 g/vp). The 1.0 mg/kg/d dose of E2 was chosen as this dose was shown to prevent ORX-induced trabecular bone loss, as reported by Moverate-Skrtic et al. (25). Subcutaneous (s.c.) injections were given to all animals always at the same time at 16:00 hrs. Chemicals E2, ICI 182,780 (ICI, an ER antagonist), palmitic acid (PA), oleic acid (OA), DHT, flutamide (Flu, an AR antagonist) and DMSO were purchased from Sigma Aldrich, CA. 4,4,4 -(4-Propyl-[1H]-pyrazole-1,3,5-triyl) triphenol (PPT; an ERα selective agonist) and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN; an ERβ selective agonist) were purchased from Tocris Biosciences, Ellisville, MO. Sample collection and histological analysis After overnight fasting for 12 h, rats were anesthetized with intraperitoneal pentobarbital 6

7 (50 mg/kg), and sacrificed by cardiac puncture. All the animals were sacrificed in the morning hours (8:00-9:00). The serum samples were stored at -20 C. In order to minimize the variability of the treatment response for rat liver lobes, we followed the optimal sampling of rat liver tissue methodology, validated by Foley et al (26). Briefly, the left lateral lobe of rat liver was divided into 3 parts, 1/3 was used for histopathological and immunohistochemical analyses, 1/3 was homogenized for RNA and protein analyses, and 1/3 for biochemical /steroid etc. measurement analyses. Liver tissues were fixed with 4% paraformaldehyde (PFA), with portions snap-frozen in liquid nitrogen and stored at -80 C. For histological examinations, PFA-fixed liver tissues were embedded in paraffin and stained with hematoxylin and eosin (H&E), or the frozen sections of liver were stained with Oil-Red-O (ORO) as described previously (n=30/ all the HF diet groups, n=10/ ND group) (24). The histology of liver zonation was determined according to S.P.S. Monga (ed.), Molecular Pathology of Liver Diseases, Molecular Pathology Library 5 (27). Briefly, hepatic acinus representing a liver lobule that is divided into 3 regions based on their proximity to the distributing veins: Zone 1, cells closest to the vessels (portal triad); Zone 2, cells in between portal triad and central vein; Zone 3, cells near the central vein. Human liver specimens Samples from liver cancer male patients (ages years) were collected with informed consents in the 306 th Hospital of PLA, China. The Ethics Committee of the 306 th Hospital of PLA approved the study. Samples consisted of 5 individual patient cases of milder/subnormal liver steatosis and 5 individual patient cases of severe liver steatosis. Due to lacking of normal human liver samples, the milder/subnormal liver steatosis specimens were used as controls. Serum concentrations of E2, DHT, aminotransferase, and serum static lipid profile Serum E2 and DHT were measured as described in previous studies (28, 29). Serum lipid parameters were measured with a lipid measuring kit (Wako Pure Chemical Industries Ltd, Osaka, Japan) according to the protocol provided with kit. Serum levels of alanine aminotransferase (ALT) were measured as a biochemical marker of hepatic function using a multiparametric analyzer (AU 5400, Olympus, Japan). The cholesterol ester for serum was measured with Cholesterol/Cholesteryl Ester Quantitation Kit (cat. KA0829, Abnova, Walnut, CA., USA) according to the protocol provided by the manufacturer. 7

8 Liver gene expression analysis by RT-PCR Total RNA was extracted from the frozen liver tissues using the acid guanidinium method (30). RT-PCR was performed as described previously (31); primer pairs are shown in Supporting Information Table 1. Briefly, all the samples were run individually in RT-reactions in triplicates and normalized to β-actin. Transcripts were measured using RT-PCR, and then the data were presented with densitometric analysis as a mean of the three times in triplicates. The relative values were quantified using NIH Image J 1.34 program ( Western blot analysis Protein was extracted from the frozen liver tissue samples using the RIPA method and quantified by a Bio-Rad protein assay kit (cat ) (BioRad, San Diego, CA). Protein samples were separated by SDS-PAGE and then transferred to a PVDF membrane as described previously (24). Antibodies and dilutions are presented in Supporting Information Table 2. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 20 min. Antibody binding was visualized by using a Supersignal West Pico detection kit (Pierce, Rockford, IL). Peroxidase-conjugated secondary antibodies included goat anti-rabbit IgG diluted at 1:2000 (Zymed, San Francisco, CA), goat anti-mouse IgG diluted at 1:4000 (Pierce, Rockford, IL), or rabbit anti-sheep IgG diluted 1:2000 (Sigma, Beijing, China). Taking each sample in triplicates in three independent experiments western-blotting assays. The films were scanned and quantified using NIH Image 1.34 program ( Fatty acid treatment and preparation of conditioned medium- In vitro treatment on BRL3A rat hepatocyte cell line All the in vitro experiments in this study were carried on in rat hepatocyte BRL3A cell line (32). The rat hepatocyte BRL3A cell line was purchased from the Chinese Academy of Sciences Cell Bank (under the licence of ATCC, cat. CRL-1442) and cultured using DMEM/Ham s F-12 (Sigma, Beijing, China) with 10% fetal bovine serum (Sigma, Beijing, China). The treatment groups were shown in Table 2. Cells were treated for 24 h. The individual experiments were repeated three times. Cells were exposed to medium containing 1% free fatty acid-free bovine serum albumin (Sigma-Aldrich, CA), and fat loaded with 1 mm PA 8

9 and OA or DMSO (vehicle control). Mitochondrial membrane potential measurement A mitochondrial staining kit, allowing detection of mitochondrial potential changes was purchased from Sigma (Catalog # CS0390), and the mitochondrial membrane potential (MMP) was measured. JC-1 dye was used as an internal control, and the experiments were performed in triplicate. The cell were suspended ( cells per ml) in complete medium (the medium used for cell growth containing the required supplements) with 1 ml of Staining Solution, and incubated for 20 min at 37 C CO 2 incubator. The cells were collected and washed with 5 ml of the ice-cold 1 JC-1 Staining Buffer. Fluorescence intensity was determined by fluorescence spectrophotometer. The red fluorescence of the aggregated JC-1 represents intact mitochondria, and the green fluorescence of the monomeric JC-1 represents disrupted mitochondria. The ratio of Red and green fluorescence intensity can reflect the level of mitochondrial membrane potential. The individual experiments were repeated in three times. Static ATP level measurements Static ATP levels in total liver homogenate were determined using a commercially available bioluminescence assay (Sigma, Beijing, China). Homogenate (20 µg protein) was added to a reaction mix containing 1.25 µg/ml luciferase and 0.1 mm luciferin. Luminescence was determined using the SpectraMax 5 luminometer (Molecular Devices, Sunnyvale, CA). The amount of ATP in the experimental samples was calculated from a standard ATP. Duplicates were performed for each sample. The results are expressed as nmol of ATP per mg of protein. Measurement of static levels of fatty acids from liver using Gas Chromatography (GS) for lipogenesis analysis Due to difficulties with experimental equipment for measuring the dynamic levels of free fatty acid of rat liver samples, we could only measure the static levels of fatty acids by GC. Briefly, about g of frozen liver samples were placed in glass test tubes. 4 ml of acetyl chloride and methanol mixture (1:10) were added to the test tube with 1 ml hexane and 1 ml stearic acid (1 mg/ml, as internal standard). The test tubes were sealed and placed in 80 C 9

10 water bath for 2 h. After that, the samples were cooled to room temperature and 5 ml of potassium carbonate solution (7%) was added to the reaction mixture. The mixture was then centrifuged at 1200 rpm for 5 min and the supernatant was collected for GC analysis. An Agilent 6890 GC (Agilent, Santa Clara, CA, USA) with HP-88 column (100m 0.25mm 0.20µm, Agilent) and FID (flame ionization detector) was used for quantitative analysis of the fatty acids. The GC inlet temperature was set at 260 C and the detector temperature was set at 270 C. The temperature program raised the oven temperature from 100 C to 220 C in an hour. The fatty acids were identified based on retention times of fatty acid standards. Quantification was achieved by comparing the integrated peak area of each fatty acid with that of the internal standard. Measurement of De Novo Lipogenesis The measurement of lipogenesis was performed as described previously (33). Briefly, BRL3A hepatocytes were cultured and treated as described above. For the final 4 h or 6 h treatment, cells were labeled with 1-14 C acetic acid (1 μci, PerkinElmer Inc., US) and incubated in a shaker. Cells were washed 4 times with PBS before lysis in 0.5% Triton X-100. The lipid fraction was extracted by the addition of 10 times the volume of chloroform, methanol and water (8:4:3 v/v). Samples were centrifuged (3000 rpm, 20 min), and the lower chloroform phase was collected. The chloroform phase was evaporated to dryness. The residue was re-dissolved in scintillation cocktail. 14 C incorporation was measured by a Beckman LS6500 scintillation counter. Immnunohistochemical and Immunofluorescence visualization assay For immunohistochemical detection of P-ACC, streptavidin-biotin-peroxidase complex method was used. 5 μm thick sections from paraffin embedded liver tissues were deparaffinized and rehydrated in xylene and ethanol, then they were placed in 10 mmol/l citrate buffer (ph 6.0), and boiled in a microwave oven for antigen retrieval, four periods of 4 min each were used. The sections were treated with 3% H 2 O 2 in PBS (ph 7.2~7.4) for 10 min and blocked with normal goat serum (1:10 dilution) for 30 min. The sections were then incubated in 37 C for 2 h with P-ACC antibody (rabbit polyclonal IgG; Cell Signaling Technology, Inc, #3661, at 1:400 dilution). The primary antibody bound was detected by using biotinylated goat anti-rabbit IgG, (1:200 dilution) followed by incubation with 10

11 horseradish peroxidase streptavidin. After staining with diaminobenzidine, the slides were counter stained with hematoxylin, dehydrated and mounted. The frozen liver sections were fixed in 4% paraformaldehyde in PBS for 10 min, rinsed in PBS, and then blocked for 60 min at room temperature in 10% normal rabbit serum (TNFα) or goat serum (IL-6) in PBS. The sections were then incubated for overnight at 4 C with TNFα antibody (goat polyclonal IgG; Santa Cruz Biotechnology, Inc, sc-1348, at 1:100 dilution) or IL-6 antibody (Rabbit polyclonal IgG; Abcam, Inc, ab6672, at 1:500 dilution). The primary antibody bound was detected by using FITC conjugated rabbit anti-goat IgG (1:50 dilution) or FITC conjugated goat anti-rabbit IgG (1:50 dilution). The sections were slightly counterstained with DAPI (1:1000 in PBS) and mounted with cover glass. Confocal laser microscopy Confocal laser microscope setup (NIKON, Japan) was used to detect emission light emitted from FITC or DAPI. The sections were exposed to a 488 nm (FITC) or 408 nm (DAPI) excitation wavelength. Four images with 2 m intervals in the A-axis were collected with a confocal scanner equipped with ECLIPSE C1Si laser system (Nikon) coupled with a Nikon EZ-C1 program. Semi-quantification of Oil-Red-O staining and immunofluorescence by Image ProPlus6.0 software The semi-quantification of staining of the Oil-Red-O (ORO) for liver tissue and hepatic cell lines, and immunofluorescence of TNFα and IL-6 for liver tissues were analyzed by using Image Pro plus 6.0 (IPP 6.0, Media Cybernetics). Integrated optical density (IOD) of positive staining area and total areas of sections were calculated. The ratio of IOD and total area (IOD / total area) is as a protein (TNFα or IL-6) expression semi-quantification or ORO-staining quantification. To eliminate variations, the sections were captured under the same conditions for all the tissues or cells. Statistical analysis One-way-ANOVA and Dunnett s post hoc tests were performed for statistical analyses using the SPSS Package (SPSS Inc., Chicago, IL). P-values<0.05 were regarded as statistically significant. Values are presented as mean±sem. 11

12 RESULTS Body and liver weights of ORX SD rats on HFD after different treatments ORX SD male rats on HFD received either vehicle, E2, DHT or E2+DHT (n=30/group). An additional control group consisted of ORX-SD male rats on ND (n=10). A 2.5-month HFD increased the body and liver weights compared to normal diet ORX controls (p<0.05) (Table 3A). E2 and E2+DHT significantly decreased the body and liver weights (p<0.05), whereas DHT induced only a mild decrease in these weights, as compared to HFD-treated controls (Table 3A). Relative peri-kidney fat weight did not differ between the studied groups (Table 3A). We also checked wild type (WT) gonad intact SD rats on either ND, or HFD which received identical treatments ORX (Table 1). E2, DHT and E2+DHT did not increase significantly the total body (350.3±18.12, 405.4±8.2, 380.2±4.1 respectively) and liver weights (11.2±0.11, 13.4±1.09, 10.8±0.22 respectively) compared with ND intact control (375.5±21.48 and 10.8±0.69). Treatment effects in intact rats were milder than in ORX HFD control and treated groups (Table 3A). Therefore, ORX HFD treatment groups were further compared to only control ORX HFD group, where the changes were more severe. Administration of estrogen/androgen could prevent NAFLD progression in rat liver Severe steatosis characterized by macrovacuolar focal necrosis and inflammation was observed in zones 1, 2 and 3 in most (26/30 rats) of the HFD ORX controls (Fig. 1B1-3) (n=30/group). ORX rats treated with E2 exhibited reversible phenotype, but macrovacuolar changes still were observed in periportal zone (zone-1) compared to controls, while less pronounced microvesicular steatosis occurred in perivenous zone (zone-3) (Fig. 1C1-3). In contrast to E2-treatment, DHT (Fig. 1D1-3) decreased the level of steatosis in periportal zone (zone-1), but still some macrovacuolar steatosis were observed in perivenous zone (zone-3) compared to the HFD ORX controls. E2+DHT treatment exhibited the best preventive histopathologic response with apparently normal liver cells in zones 2 and 3, with only some of microvesicular steatosis in zone-1 (Fig. 1E1-3). Portal inflammation was absent in the E2, DHT, and E2+DHT treated liver samples (Fig. 1C1-3, 1D1-3, 1E1-3). The accumulation of lipids in hepatocytes was further confirmed by ORO-staining and morphometric quantification analysis in S-Fig. 1. The histopathological changes in gonad intact HFD rats were very similar to the HFD ORX treated rats after the different treatments, however the changes were much milder (S-Fig. 2). 12

13 Serum E2 and DHT levels E2, DHT and E2+DHT treatments increased serum E2 and DHT levels compared to control ORX HFD control groups (Table 3A). There was no statistically significant difference between the levels of ND control vs. E2 ( vs ) or ND control vs. DHT treated (p<0.08), most likely due to the high individual variance. Serum parameters and lipid profiles and intra-hepatic static levels of palmitic, palmitoleic and oleic acids The HFD significantly increased the serum alanine aminotransferase (ALT) levels compared to the ORX ND control (p<0.05) (Table 3A). There was a significant decrease (p<0.01) in serum ALT levels in SD rats treated with E2 or E2+DHT compared to the HFD controls (Table 3A). Transporting the endogenous cholesterol into liver is the biological function of HDL, and transporting cholesterol into extrahepatic tissues is the biological function of LDL. Thus, hepatic HDL-c and LDL-c are important biological parameters of lipid balance. The HFD itself significantly increased (p<0.01) total hepatic and serum cholesterol, triglyceride (TG) and LDL-c levels, and decreased the HDL-c levels compared to the ND in ORX male rats (Table 3A). E2 and E2+DHT treatments reduced serum TG and increased HDL-c levels (p<0.05 compared to the HFD control levels) (Table 3A). DHT and E2+DHT significantly reduced serum LDL-c levels (Table 3A). Treatment with E2, DHT, and E2+DHT decreased serum total cholesterol and cholesterol ester, respectively (Table 3A). ApoB100 was significantly decreased after all treatments (Table 3A), highlighting the additive positive treatment effect. Furthermore, E2-, DHT-, and E2+DHT-treatment significantly decreased the production of palmitic acid and palmitoleic acid, as well as oleic acid by hepatic metabolic analysis for lipogenesis (Table 3B). GC measurement of free fatty acids in rat liver could only reflect the static level of changes. Genetic profiling of lipogenesis is shown in Fig. 2. Local ER/AR pathways in the rat liver E2 increased ERα expression significantly compared to non-treated rats on HFD controls (Figs. 2A and 3A). ERβ signal was undetectable in livers of all groups. DHT significantly increased AR expression (p<0.01), compared to control ORX HFD mice (Figs. 2B and 3A). Interestingly, upon E2/DHT treatment, a reciprocal suppression of ERα was found with DHT, 13

14 and of AR with E2 (Figs. 2A-B and 3A). Estrogen and androgen may regulate the expression of genes involved in de novo synthesis of fatty acids and β-oxidation Among the key hepatic lipogenic genes, the expression of sterol regulatory element-binding protein-1c (SREBP-1c), stearoyl-coa desaturase 1 (SCD1) and fatty acid synthase (FAS) were similar in all treatment groups, except for a significant increase of FAS in the HFD control group compared to ND (Fig. 2C). E2, DHT or E2+DHT downregulated the FAS compared to HFD. Acetyl-CoA carboxylase (ACC) was significantly increased in the HFD groups compared to ND (Fig. 2C). We also analyzed mrna expression of the key genes associated with β-oxidation. The expression levels of peroxisome proliferator-activated receptor α (PPARα), very long-chain acyl-coa dehydrogenase (VLCAD), medium-chain acyl-coa dehydrogenase (MCAD), and acyl-coa oxidase 1 (ACO1) did not change in any of the treated groups. However, the expression of uncoupling protein2 (UCP2) was significantly downregulated (p<0.05) in E2, DHT and E2+DHT-treated groups compared to HFD controls (Figs. 2D, 3A). Carnitine palmitotyltransferase 1 (CPT1) was increased by DHT and E2+DHT treatment compared to HFD controls (Figs. 2D, 3A). No significant changes were observed in genes involved in TG synthesis (S-Fig. 3). Estrogen and androgen may improve MMP and static ATP levels An increase of MMP (Fig. 2E) and static ATP levels (Fig. 2F) in E2, DHT and E2+DHT groups compared to the ORX HFD control was found. Both MMP and static ATP significantly decreased with HFD control groups compared with ND control group (Fig. 2E-F). Steroid treatments significantly decreased UCP2 and increased MMP and static ATP levels in the ORX rat liver on HFD (Fig. 2E-F). Downregulation of lipogenesis by ACC phosphorylation was probably due to estrogen action in rat liver E2, DHT and E2+DHT treatments significantly increased the phosphorylation of the rate-limiting enzyme in de novo fatty acid synthesis, ACC (P-ACC), compared to HFD controls (Fig. 3A and S-Fig. 4), although it seemed ACC enzyme was not regulated at the 14

15 level of gene expression by DHT (Fig. 2C and 3A). Abundant P-ACC protein expression was further confirmed by Immunohistochemistry and morphometric quantification showing highest abundance in zone-3 localization (Fig. 4A-F). E2, DHT and E2+DHT treatments enhanced the protein phosphorylation, i.e. P-ACC/total ACC levels (S-Fig. 4). These results suggested that E2 is involved in the suppression of lipogenesis through P-ACC. Downregulation of cholesterol biosynthesis by increasing HMGCR phosphorylation was associated most likely with androgen action Treatment with DHT resulted in a marked decrease in the concentrations of total cholesterol, LDL-c and ApoB. Marked increase of HMGCR phosphorylation in DHT and E2+DHT groups (Fig. 3A) and enhanced phosphorylation related to protein levels, i.e. P-HMGCR /total HMGCR levels (S-Fig. 4) was observed. Estrogen and androgens are associated with the prevention of NASH progression by amelioration of TNFα and IL-6 Tumor necrosis factor-α (TNFα) (Fig. 3B) and Interleukin-6 (IL-6) (Fig. 3C) were significantly downregulated in all HFD ORX rats treated with E2, DHT, E2+DHT compared to the HFD control. Conversely, TNFα and IL-6 (Fig. 3B-3C) were upregulated in ORX HFD group compared to ND (p<0.05). These alterations (Fig. 3D) were confirmed by immunofluorescence visualization. E2, DHT, E2+DHT significantly decreased the signals of TNFα and IL-6 (shows only FITC/DAPI signal) (Fig. 3E-3F). Molecular actions of estrogen and androgen in a rat BRL3A hepatocyte cell line in vitro With 330 µmol/l PA and 670 µmol/l OA, the rat BRL3A hepatocyte controls formed abundant reddish ORO-positive cells, while the E2 and DHT treatments resulted in scant ORO staining (Fig. 5A). Addition of 1 μm ICI or Flu to the BRL3A cells reflected the red staining comparable to controls (Fig. 5A). The combination of E2+DHT totally blocked the ORO-positive formation (Fig. 5A) and morphometric quantification is shown in Fig. 5B. Furthermore, treatment with E2, DHT, and E2+DHT significantly decreased the production of total cholesterol and cholesterol ester in BRL3A hepatocytes (S-Table 3). Due to insufficient sensitivity of the assays, no data could be obtained on TG, HDL-c and LDL-c. 15

16 Treatment of the rat BRL3A hepatocytes in vitro for 24 h with E2, E2+DHT and PPT, resulted in upregulation of ERα (Fig. 5C). This ERα upregulation was blocked by ICI (p<0.05, E2+ICI vs.e2 or PPT), but DPN did not induce any changes (Fig. 5C). Additionally, AR expression was significantly upregulated by DHT and E2+DHT and downregulated by Flu (Fig. 5D). To verify the E2/ER and/or DHT/AR mediated blockage of TG, we analyzed genes associated with de novo fatty acid synthesis and β-oxidation in vitro in BRL3A cells. The expression of SREBP-1c, SCD1 and FAS were similar among the treated groups (S-Fig. 5A). E2 significantly upregulated the P-ACC levels, which were significantly suppressed by ICI. DHT alone did not alter the level of P-ACC in hepatocytes (Fig. 5E). The expression of PPARα, VLCAD, MCAD, and ACO1 were similar among groups (S-Fig. 5B). DHT significantly increased CPT1 and this DHT-mediated increase could be disrupted by Flu (Fig. 5F). UCP2 expression was significantly downregulated (p<0.05) by E2 and DHT (Fig. 5G), and this suppression was reversed by ICI and Flu (Fig. 5G). Interestingly, UCP2 protein was nearly absent after the treatment with E2+DHT (Fig. 5G). DHT and E2+DHT significantly increased the P-HMGCR (Fig. 5H), and this increase could be significantly suppressed by Flu. There was an increase in mitochondrial membrane potential after E2, DHT and E2+DHT compared to control (Fig. 5I). Moreover, E2, DHT and E2+DHT increased intracellular static ATP, which was significantly suppressed by Flu (Fig. 5J). Furthermore, we measured the incorporation rate for de novo lipogenesis by using of 14 C-labeled acetic acid method in hepatocytes. Treatment with E2, DHT, and E2+DHT significantly decreased the incorporation of 14 C from the lipid-containing phase (Fig. 6A). In this de novo lipogenesis by 1-14 C labeling acetic acid procedure, we traced the total radiation that can reflect the lipogenesis. Acetate is the precursor of lipid synthesis, and fatty acid is the substrate of sterols and lipid synthesis. The 14 C-acetate labeling procedure done in BRL3A cells reflected lipogenesis, although the neutral solvent extraction method used hereby could have also collected labeled sterols and other lipids, but fatty acids would likely predominate among radio labeled lipids. Gene expression changes in liver steatosis in men are similar to NAFLD rat model In order to demonstrate the clinical relevance of this NAFLD rat model for human patients, we analyzed human liver samples for pathological changes (Fig. 6B-C) and protein 16

17 expressions of ERα, AR, P-ACC, ACC, UCP2, HMGCR and P-HMGCR in severe (Fig. 6D; n=5) and milder/subnormal (Fig. 6D; n=5) male NAFLD. The expression of ERα and AR were markedly decreased in severe steatotic livers compared to the subnormal control ones (Fig. 6D-E). Decreased P-ACC (Fig. 6D-E), increased UCP2 and decreased P-HMGCR expression could be observed in severe steatotic livers compared to mild/subnormal controls (Fig. 6D-E). There was no significant difference in between the subnormal/milder control and severe groups in serum lipid parameters, T and E2, as all the serum parameters from the controls were of abnormal range (S-Fig. 6)... 17

18 DISCUSSION Numerous clinical and experimental studies have demonstrated that estrogen (19, 36) or androgen (25, 37) elicits favorable effects on reducing body fat mass and improving the liver steatosis. Recently, it has been shown that E2 deficiency accelerated NASH progression in ovariectomized mice fed high fat high cholesterol diet and that this effect was improved by estrogen therapy (38). We have demonstrated recently in a pilot study that strong suppression of the actions of endogenous estrogens and/or androgens by Tam, Flu, or Tam/Flu induced NASH in adult intact male SD rats receiving HFD (but never with ND) (24). In this current study, we depleted endogenous steroids by ORX and supplemented with E2, DHT or E2+DHT, to study the precise differential action of estrogen and androgens on HFD treated NAFLD. We hereby demonstrated that the downregulation of lipogenesis with E2 treatment in abundancy in zone-3 might be associated with ERα-mediated increase in ACC phosphorylation. Accordingly, E2 treatment significantly decreased serum TG levels in ORX SD rats and upregulated ERα. We have shown previously that Tam treatment significantly downregulated ERα and increased TG levels in intact SD rat liver. As we could not detect any ERβ expression (24), this estrogen effect must be mediated by the ERα pathway. This ERα-mediated inhibition of lipogenesis was due to increased P-ACC that we confirmed by treating hepatocytes with PPT and E2. Accordingly, a recent study has shown that Tam promotes hepatic steatosis by increasing lipogenesis through the reduction of P-ACC (39). DHT treatment significantly increased CPT-1 expression with the highest abundancy in zone-1 through significant increase of β-oxidation and decrease of cholesterol biosynthesis by HMGCR phosphorylation via the AR pathway. Interestingly, our study also revealed enhanced protective effects of E2 and DHT on liver lipid metabolism. These additive effects of E2 and DHT treatments on lipid homeostasis were demonstrated by normalization of liver histology in zones-1, 2 and 3, as measured by decreased lipogenesis via P-ACC. This was further confirmed by decreased de novo lipogenesis, which in turn decreased serum TG levels, and normalized β-oxidation with increased MMP and static ATP both in vivo and in vitro. Although our results show mitochondrial activity is involved in ER/AR of lipid homeostatis, putative role of oxidative stress triggering inflammation associated to NASH cannot be ruled out and needs to be further checked. We found cholesterol biosynthesis was decreased as indicated by P-HMGCR (which was further confirmed by the significant decrease in total hepatic and serum cholesterol, LDL-c, and serum ApoB concentrations in E2/DHT groups). A 18

19 clinical study on transdermal DHT in older men with partial androgen deficiency demonstrated decreased total and LDL-c and unchanged HDL-c and TG (40), which supports our findings that DHT decreases cholesterol biosynthesis to some extent. Interestingly, DHT treatment did not decrease serum E2 levels in that study (40), which may also implicate that the balance of estrogen and androgen levels is crucial for the maintenance of lipid homeostasis in males. In line with this induced NAFLD rat model, we detected decreased levels of ERα and AR and P-ACC in steatosis patients compared to subnormal/milder controls. Using subnormal/mild liver as control could have caused an underestimation of the alterations in gene expression. Despite these subnormal controls, significantly increased UCP2 and decreased P-HMGCR were observed in severe human liver steatosis. These preliminary findings highlight the need for a large-scale clinical investigation on estrogen and androgen actions on human NAFLD. Taken together, we showed here a possible molecular mechanism explaining the additive positive effects of estrogen and androgen in the prevention of NAFLD (as illustrated in Fig. 7). The estrogen/erα pathway mainly suppresses hepatic steatosis by decreasing lipogenesis mostly in zone-3, through the induction of P-ACC, and in turn, improving mitochondrial function by inhibiting proton leakage (Fig. 7 black-arrows). The androgen/ar pathway mainly suppresses hepatic steatosis, both by increasing CPT-1-mediated β-oxidation mainly in zone-1 and decreasing cholesterol biosynthesis (Fig. 7 white-arrows). Altogether, applications of estrogen and androgen modulate hepatic lipid metabolism by decreasing lipogenesis and cholesterol biosynthesis, and by increasing β-oxidation; both pathways subsequently normalize the MMP and ATP production and prevent NASH by downregulating TNFα and IL-6 as well. Our findings suggest that the combined administration of selective ER and AR modulators (SERMs and SARMs) could be a potential therapeutic approach for NAFLD. 19

20 ACKNOWLEDGEMENT We thank Dr Stanford Chan from Imperial College London, for revising the English language of our revised manuscript. AUTHOR CONTRIBUTION STATEMENT Li X designed and made the study concept; Zhang H., Liu.Y, Li Z, Wang L performed experiments; Zhang Ho and Wu J performed histopathological analysis; Zhang H, Rahman N, Guo Y, Li D, Li N, Huhtaniemi I, Tsang S, Gao G and Li X analyzed and interpreted the results; Zhang H., Rahman N, Huhtaniemi I and Li X wrote the manuscript. FINANCIAL SUPPORT This work was supported by grants from Ministry of Science and Technology No.SQ2011AAJY and No.2011CB944103, Natural Science Foundation of Beijing No to X.Li; and National Natural Science Foundation of China No to GF.Gao. 20

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22 expenditure and lipolytic activity but show normal insulin sensitivity with high adiponectin secretion. Diabetes;54: Sato, T., Matsumoto, T., Yamada, T., Watanabe, T., Kawano, H., Kato, S Late onset of obesity in male androgen receptor-deficient (AR KO) mice. Biochem Biophys Res Commun;300: Lin, H. Y., Yu, I. C., Wang, R. S., Chen, Y. T., Liu, N. C., Altuwaijri, S., Hsu, C. L., Ma, W. L., Jokinen, J., Sparks, J. D., et al Increased hepatic steatosis and insulin resistance in mice lacking hepatic androgen receptor. Hepatology;47: Alexandersen, P., Christiansen, C The aging male: testosterone deficiency and testosterone replacement. An up-date. Atherosclerosis;173: Simpson, E. R., Jones, M. E., Clyne, C Lessons from the ArKO mouse. Aromatase Inhibitors. Edited by B.J.A. Furr Birkhäuser Verlag/Switzerland: Couse, J. F., Korach, K. S Estrogen receptor null mice: what have we learned and where will they lead us? Endocr Rev;20: Walters, K. A., McTavish, K. J., Seneviratne, M. G., Jimenez, M., McMahon, A. C., Allan, C. M., Salamonsen, L. A., Handelsman, D. J Subfertile female androgen receptor knockout mice exhibit defects in neuroendocrine signaling, intraovarian function, and uterine development but not uterine function. Endocrinology;150: Dieudonne, M. N., Pecquery, R., Boumediene, A., Leneveu, M. C., Giudicelli, Y Androgen receptors in human preadipocytes and adipocytes: regional specificities and regulation by sex steroids. Am J Physiol;274:C Walters, K. A., Simanainen, U., Handelsman, D. J Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. Hum Reprod Update;16: Mu, Y., She, R., Zhang, H., Dong, B., Huang, C., Lin, W., Li, D., Li, X Effects of estrogen and androgen deprivation on the progression of non-alcoholic steatohepatitis (NASH) in male Sprague-Dawley rats. Hepatol Res;39: Moverare-Skrtic, S., Venken, K., Andersson, N., Lindberg, M. K., Svensson, J., Swanson, C., Vanderschueren, D., Oscarsson, J., Gustafsson, J. A., Ohlsson, C Dihydrotestosterone treatment results in obesity and altered lipid metabolism in orchidectomized mice. Obesity (Silver Spring);14: Foley, J. F., Collins, J. B., Umbach, D. M., Grissom, S., Boorman, G. A., Heinloth, A. N Optimal sampling of rat liver tissue for toxicogenomic studies. Toxicol Pathol;34: Colnot, S., Perret, C Liver zonation. Molecular Pathology of Liver Diseases, Molecular Pathology Library 5, Springer New York, Dordrecht Heidelberg London: Li, X., Nokkala, E., Yan, W., Streng, T., Saarinen, N., Warri, A., Huhtaniemi, I., Santti, R., Makela, S., Poutanen, M Altered structure and function of reproductive organs in transgenic male mice overexpressing human aromatase. Endocrinology;142: Shen, Z. J., Lu, Y. L., Chen, Z. D., Chen, F., Chen, Z Effects of androgen and ageing on gene expression of vasoactive intestinal polypeptide in rat corpus cavernosum. BJU Int;86: Sambrook, J., Russel, D Molecular Cloning: A Laboratory Manual, 3rd ed.:

23 31. Li, X., Strauss, L., Kaatrasalo, A., Mayerhofer, A., Huhtaniemi, I., Santti, R., Makela, S., Poutanen, M Transgenic mice expressing p450 aromatase as a model for male infertility associated with chronic inflammation in the testis. Endocrinology;147: Coon, H. G., Weiss, M. C A quantitative comparison of formation of spontaneous and virus-produced viable hybrids. Proc Natl Acad Sci U S A;62: Yecies, J. L., Zhang, H. H., Menon, S., Liu, S., Yecies, D., Lipovsky, A. I., Gorgun, C., Kwiatkowski, D. J., Hotamisligil, G. S., Lee, C. H., et al Akt stimulates hepatic SREBP1c and lipogenesis through parallel mtorc1-dependent and independent pathways. Cell Metab;14: Ferre, P., Satabin, P., Decaux, J. F., Escriva, F., Girard, J Development and regulation of ketogenesis in hepatocytes isolated from newborn rats. Biochem J;214: Kim, J. Y., Hickner, R. C., Cortright, R. L., Dohm, G. L., Houmard, J. A Lipid oxidation is reduced in obese human skeletal muscle. Am J Physiol Endocrinol Metab;279:E Ohlsson, C., Hellberg, N., Parini, P., Vidal, O., Bohlooly, M., Rudling, M., Lindberg, M., Warner, M., Angelin, B., Gustafsson, J Obesity and disturbed lipoprotein profile in estrogen receptor-β-deficient male mice. Biochem Biophys Res Commun;278: Vandenput, L., Mellstrom, D., Lorentzon, M., Swanson, C., Karlsson, M. K., Brandberg, J., Lonn, L., Orwoll, E., Smith, U., Labrie, F., et al Androgens and glucuronidated androgen metabolites are associated with metabolic risk factors in men. J Clin Endocrinol Metab;92: Kamada, Y., Kiso, S., Yoshida, Y., Chatani, N., Kizu, T., Hamano, M., Tsubakio, M., Takemura, T., Ezaki, H., Hayashi, N., et al Estrogen deficiency worsens steatohepatitis in mice fed high-fat and high-cholesterol diet. Am J Physiol Gastrointest Liver Physiol;301:G Cole, L. K., Jacobs, R. L., Vance, D. E Tamoxifen induces triacylglycerol accumulation in the mouse liver by activation of fatty acid synthesis. Hepatology;52: Ly, L. P., Jimenez, M., Zhuang, T. N., Celermajer, D. S., Conway, A. J., Handelsman, D. J A double-blind, placebo-controlled, randomized clinical trial of transdermal dihydrotestosterone gel on muscular strength, mobility, and quality of life in older men with partial androgen deficiency. J Clin Endocrinol Metab;86:

24 FIGURE LEGENDS Fig. 1 Histopathology of the liver tissues by hematoxylin and eosin (H/E) staining from ORX SD male rats (n=30/for each HF diet group; n=10/normal diet group) (A1-3) A representative liver specimen from the ORX normal diet control group. Dotted arrow depicts the liver zonation; zone 1, cells closest to the portal triad; zone 2, cells in between portal triad and central vein; zone 3, cells near central vein (A2). (B1-3) Liver cells with marked fat accumulation in the ORX HFD control in a representative liver specimen. Arrows in B3 depicts the inflammation, and the empty arrows depict fatty liver cells; 1, and 3 represent zones 1 and 3. (C1-3) A representative liver specimen from the E2-treated group (E2 1.0mg/kg); zones 2 and 3 of liver lobules contain normal appearing liver cells rimmed with marked fatty changes in zone 3. (D1-3) A representative liver specimen from the DHT-treated group (DHT 3.0mg/kg); zones 1 and 2 of liver lobules contain normal appearing liver cells rimmed with marked fatty changes in zone 1. Arrows in D3 depicts the normalized hepatocyte and the empty arrows depict the fatty liver cell; (E1-3) Liver from the E2+DHT-treated group (1.0 mg/kg E2+3.0 mg/kg DHT); zones 2 and 3 of liver lobules contain normal appearing liver cells rimmed with lesser fatty changes in zone 1. Middle and right panels represent higher magnifications of dotted box areas of left panel; Bars, 50 m. Fig. 2 Gene expressions of ERα and AR and genes related to de novo synthesis of fatty acids, β-oxidation and mitochondrial parameters. (n=15/for each HF diet group; n=10/normal diet group) Gene expression of (A) ERα, (B) AR and different components of lipogenesis (C) and -oxidation (D) at mrna levels by RT-PCR. (E) The mitochondrial membrane potential from the treatments, and (F) production of ATP from liver cells. Data from treatment groups are compared to HFD control group (or HFD compared to ND). * P<0.05, **P<0.01, and ***P< NC, normal diet; C-HFD, control high fat diet. 24

25 Fig. 3 Alterations of liver-proteins, IL-6 and TNFα levels after different treatments. (n=15/for each HF diet group; n=10/normal diet group) (A) Protein levels in ORX rat livers after different treatments analyzed by Western blot ( -actin as internal control). mrna expression of (B) TNF and (C) IL-6 by RT-PCR and TNF protein (D, left panel) and IL-6 protein (D, right panel) in liver by FITC/DAPI immunofluorescence after different treatments. Green fluorescence is mainly shown in HFD groups (TNF, IL-6). Cells were counterstained with DAPI (4,6-diamidino-2-phenylindole). (E and F) Semi-quantification of the positive signals for TNF (D, left panel) and IL6 (D, right panel). Data (B,C,E,F) from E2, DHT, and E2+DHT groups are compared to HFD control (or HFD compared to ND). ***P< Dose for E2 is 1.0 mg/kg and for DHT is 3.0 mg/kg. NC, normal diet; C-HFD, control of high fat diet. Fig. 4 (A-F) Zone-specific immunohistochemical assay for P-ACC in SD rat liver tissues (n=30/for each HF diet group; n=10/normal diet group) (A1-2) A representative specimen liver from the ORX normal diet control group. Dotted arrow depicts the liver zonation; zone 1, cells closest to the portal triad; zone 2, cells in between portal triad and central vein; zone 3, cells near central vein (A2). (B1-2) Liver cells with marked fat accumulation in the ORX HFD control. (C1-2) A representative specimen liver from the E2-treated group; brownish stained cells were observed in cytosol in zones 2 and 3 of liver lobules, represent the P-ACC positive cells. (D1-2) A representative specimen liver from the DHT-treated group; only few brownish cytosol stained cells were observed in zone 3 of liver lobules in the liver samples. (E1-2) A representative specimen liver from the E2+DHT-treated group; zones 2 and 3 of liver lobules contain strong brownish cytosol stained cells, while no positive signal was observed among hepatocytes near zone 1. Right panels represent higher magnifications of dotted box areas of left panel; 1, and 3 represent zones 1 and 3. (F) Morphometric quantifiation analysis of the P-ACC stained areas of the liver shown as Mean±SEM, *** P<0.001 versus C-HFD, control of high fat diet. 25

26 Fig. 5 Estrogen and Androgen action on liver steatosis by Oil-Red-O staining, on ER /AR levels and MMP and static ATP level changes in vitro in a rat BRL3A hepatocyte cell line BRL3A rat hepatocytes pre-treated with 330 µmol/l of Palmitic Acid (PA) and 670 µmol/l of Oleic Acid (OA) in ORO staining without (control) and with different treatments (E2, E2+ICI, DHT, DHT+Flu, E2+DHT, PPT, and DPN). (A). E2 and DHT treatments resulted in less dense ORO staining than in control (A). The addition of 100nM ICI or Flu reflected the red staining comparable to the control (A). E2+DHT totally blocked the formation of ORO-positive cells. (B) Semi-quantification of the ORO positive signals. Western blot analysis of proteins (upper panel bands, C-D) and semi-quantification mrna (lower graph panels, C-D, RT-PCR to -actin) and protein levels of ERα (C) and AR (D). Western blot analysis of proteins (upper panel bands, E-H) and semi-quantification mrna (lower graph panels, F, G; RT-PCR to -actin) and protein levels of P-ACC; below P-ACC protein semiquantification levels of ACC (E), CPT1 (F), UCP2 (G), below P-HMGCR protein semiquantification levels of HMGCR (H) to -actin in rat BRL3A hepatocytes. (I) Mitochondrial membrane potential and (J) ATP levels in rat BRL3A hepatocytes. Data from treatment groups were compared to vehicle control. The addition of PPT and DPN is at 10nM. *P<0.05, **P<0.01 and ***P< Fig C acetate incorporation analysis in hepatocytes, and the expression of genes in severe and milder/subnormal human male steatotic liver samples (A) BRL 3A hepatocytes treated with E2 and DHT were labeled with 14 C acetate, and the level of 14 C incorporation into the lipid fraction are shown relative to untreated controls. (B) Milder/subnormal human male steatotic liver sample (n=5). (C) Severe human male steatotic liver sample (n=5). (D) Protein bands of ERα, AR, UCP2, ACC, P-ACC, HMGCR and P-HMGCR and β-actin (as control) by Western blot analysis. Pat 1-4 represents 4 individual patients liver samples. (E) The relative protein levels of ERα, AR, UCP2, P-ACC and P-HMGCR to β-actin. * P<0.05, ** P<0.01, ***P< Pat, Patient. 26

27 Fig. 7 Possible mechanisms of estrogen/androgen action in the prevention of NAFLD/NASH Estrogen/ERα pathway mainly suppresses hepatic steatosis by decreasing lipogenesis in zone 3, through the induction of P-ACC, and improving mitochondrial function by inhibiting proton leakage (black-arrows). Androgen/AR pathway mainly suppresses hepatic steatosis, both by increasing CPT-1-mediated β-oxidation in zone 1 and decreasing cholesterol synthesis (white-arrows). Altogether, estrogen/androgen modulate hepatic lipid metabolism, nd subsequently normalize the mitochondrial function. 27

28 Table 1 Treatment groups for the estrogen and androgen application in castrated (ORX) and intact (non-orx) SD male rats Group Treatment Diet ORX (n) Intact (n) Vehicle Oil Normal diet 10 6 Vehicle Oil High fat diet 30 6 E2 1.0mg/kg High fat diet 30 6 DHT 3.0mg/kg High fat diet 30 6 E2 +DHT 1.0mg/kg + 3.0mg/kg High fat diet 30 6 n, number of animal/group Table 2 In vitro treatment on hepatocytes Group Dose Pre-treatment Control DMSO Non fatty acid Control 1mM (PA:OA=1:2) Fatty acid E2 10nM Fatty acid/ Non fatty acid E2+ICI 10nM+1 M Fatty acid/ Non fatty acid DHT 10nM Fatty acid/ Non fatty acid DHT+Flu 10nM+10nM Fatty acid/ Non fatty acid E2+DHT 10nM+10nM Fatty acid/ Non fatty acid E2+DHT+ICI+Flu 10nM+10nM+1 M+10nM Fatty acid/ Non fatty acid PPT 10nM Fatty acid/ Non fatty acid DPN 10nM Fatty acid/ Non fatty acid 28

29 Table 3 Body and liver weights, serum biochemical markers and lipid and metabolic profile in ORX SD male rats (n=30/in all groups; except in ND group, n=10). The data groups provided with only different letter superscripts (e.g. a vs. b or a vs. b, c etc.) are statistically significantly different (p<0.05), where data from E2/DHT/E2+DHT groups should be mainly compared to HF Control. *, p<0.05, for Control HF diet vs. Normal (N) diet control groups only. A) Body, liver and kidney fat weights, serum biochemical markers, total cholesterol, triglycerides, HDL-c, LDL-c, and E2 and DHT levels in ORX SD rats Normal (N) Diet High fat diet (HF-diet) Control (N) Control (HF) E2 DHT E2+DHT Body weight (g) ± ±10.83 *, a ±17.03 b ±12.60 c ±3.08 b Liver weight (g) 11.74± ±2.26 *, a 14.41±0.34 b 16.92±1.13 b 13.34±0.46 b Relative kidney fat/body weight 0.026± ± a 0.017± a 0.019± a 0.016± a Total cholesterol (mg/dl) 1.84± ±0.27 *, a 3.44±0.27 b 3.05±0.16 b 2.31±0.11 c Cholesterol ester (mg/dl) 1.25± ±0.22 *, a 2.44±0.24 b 1.91±0.19 b 1.79±0.24 b HDL-c (mg/dl) 1.32± ±0.16 *, a 1.31±0.06 b 0.90±0.07 a 1.37±0.08 b LDL-c (mg/dl) 0.57± ±0.07 *, a 1.45±0.56 a 1.25±0.02 b 0.79±0.05 c Triglycerides(mg/dl) 0.49± ±0.08 *, a 1.28±0.05 b 1.49±0.13 a 0.87±0.02 c ApoB100 (mg/dl) 0.082± ±0.01 *, a 0.084±0.02 b 0.075±0.01 b 0.068±0.01 c ALT (U/L) 30.14± ±16.98 *, a 69.48±9.51 b ±13.71 a 53.00±13.67 b Serum E2 (ng/dl) 0.20± ±0.11 a 0.43±0.16 b 0.22±0.13 a 0.41±0.13 b Serum DHT (ng/ml) 0.30± ±0.13 a 0.28±0.18 a 0.44±0.12 b 0.43±0.14 b B) Intra-hepatitic static levels of various fatty acids (palmitic, palmitoleic and oleic acid) from homogenized rat liver tissues with or without different treatments in ORX SD rats (concentration expressed in mg/g tissue) Normal (N) Diet High fat diet (HF-diet) Control (N) Control (HF) E2 DHT E2+DHT Palmitic acid 12.49± ±2.74 *, a 6.82±0.64 b 13.1±2.54 c ±3.2 c Palmitoleic acid 0.81± ±1.14 *, a 5.85±1.06 b 6.05±1.42 b 5.11±0.75 b Oleic acid 6.45± ±6.12 *, a 25.44±4.01 b 62.06±3.7 c 50.67±3.02 c 29

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