|Year : 2021 | Volume
| Issue : 2 | Page : 71-80
Long-term androgen-induced nonalcoholic fatty liver disease in a polycystic ovary syndrome mouse model is related to mitochondrial dysfunction
Peng Cui1, Jie-Mei Shi2, Tong Ma2, Lin Rao3, Xiao-Yu Tong2, Wei Hu2, Xiao-Qing Xu2, Fei-Fei Zhang4, Xin Li4, Håkan Billig5, Linus R Shao5, Yi Feng2
1 Department of Integrative Medicine and Neurobiology, State Key Lab of Medical Neurobiology, Institute of Acupuncture Research (WHO Collaborating Center for Traditional Medicine), Institute of Integrative Medicine, Institute of Brain Science, School of Basic Medical Sciences, Fudan University, Shanghai 200032; Department of Obstetrics and Gynecology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2 Department of Integrative Medicine and Neurobiology, State Key Lab of Medical Neurobiology, Institute of Acupuncture Research (WHO Collaborating Center for Traditional Medicine), Institute of Integrative Medicine, Institute of Brain Science, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
3 Nursing Department, the International Peace Maternal and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, China
4 Department of Gynecology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011, China
5 Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, the Sahlgrenska Academy, University of Gothenburg, Gothenburg 40530, Sweden
|Date of Submission||18-Dec-2020|
|Date of Decision||11-Jan-2021|
|Date of Acceptance||23-May-2021|
|Date of Web Publication||08-Jul-2021|
Department of Integrative Medicine and Neurobiology, State Key Lab of Medical Neurobiology, Institute of Acupuncture Research (WHO Collaborating Center for Traditional Medicine), Institute of Integrative Medicine, Institute of Brain Science, School of Basic Medical Sciences, Fudan University, Shanghai 200032
Source of Support: None, Conflict of Interest: None
Objective: Metabolic disorders are markedly common in women with polycystic ovary syndrome (PCOS), and nonalcoholic fatty liver disease (NAFLD) is observed in 30%–55% of all PCOS patients. Many studies have reported that autophagy and apoptosis, which are closely related to mitochondrial function, play important roles in the development of NAFLD. Therefore, in this study, we aimed to explore the role of mitochondrial dysfunction caused by liver apoptosis and autophagy imbalance in the development of NAFLD in a PCOS mouse model.
Methods: We used a dihydrotestosterone (DHT)-induced PCOS model to mimic the pathological process of hyperandrogenism. Hematoxylin and eosin and Oil Red O staining assays were used to observe the pathological changes in the liver. Western blotting and quantitative real-time polymerase chain reaction were used to perform mitochondrion-related assays.
Results: Hepatic steatosis and different degrees of inflammation were observed in the DHT-treated mice. The expression of molecules involved in the respiratory chain and aerobic respiration process was altered. The levels of the key molecules associated with apoptosis and autophagy were abnormal.
Conclusions: Androgens may play a role in the process of hepatic steatosis development by affecting mitochondrial function and subsequently inducing apoptosis and autophagy. Such phenomena might be involved in the pathogenesis of NAFLD in women with PCOS.
Keywords: Apoptosis; Autophagy; Mitochondrial Function; Nonalcoholic Fatty Liver Disease; Polycystic Ovary Syndrome
|How to cite this article:|
Cui P, Shi JM, Ma T, Rao L, Tong XY, Hu W, Xu XQ, Zhang FF, Li X, Billig H, Shao LR, Feng Y. Long-term androgen-induced nonalcoholic fatty liver disease in a polycystic ovary syndrome mouse model is related to mitochondrial dysfunction. Reprod Dev Med 2021;5:71-80
|How to cite this URL:|
Cui P, Shi JM, Ma T, Rao L, Tong XY, Hu W, Xu XQ, Zhang FF, Li X, Billig H, Shao LR, Feng Y. Long-term androgen-induced nonalcoholic fatty liver disease in a polycystic ovary syndrome mouse model is related to mitochondrial dysfunction. Reprod Dev Med [serial online] 2021 [cited 2021 Jul 31];5:71-80. Available from: https://www.repdevmed.org/text.asp?2021/5/2/71/320884
| Introduction|| |
Polycystic ovary syndrome (PCOS) is an endocrine disorder, which affects approximately 5%–20% of women.,, Hyperandrogenism and insulin resistance are two major pathological features of PCOS and are closely related to the occurrence of this disease and associated complications.,, Recent clinical studies have reported that approximately 30%–55% of PCOS patients also experience nonalcoholic fatty liver disease (NAFLD),, with evidence showing that PCOS patients have a 2.54-fold increased prevalence of NAFLD compared to healthy women. NAFLD is clinically characterized by excessive lipid deposition in hepatocytes, and the primary PCOS-related type of NAFLD is accompanied by varying degrees of inflammation. Mitochondria are the center of lipid metabolism and maintain the balance of energy and metabolism in cells by continuously regulating key transcription factors. Mitochondrial dysfunction can lead to abnormal lipid metabolism, resulting in fat deposition. Many studies have shown that the imbalance between lipid synthesis and decomposition in hepatocytes plays an important role in the occurrence of fatty liver., Increased synthesis of triglycerides and cholesterol and reduced β-oxidation of fatty acids lead to the occurrence of fatty liver., These changes are considered to be associated with mitochondrial dysfunction, which is in turn related to the pathogenesis of NAFLD. Recent studies have suggested that hyperandrogenism can cause NAFLD;, however, the precise mechanisms involved remain unclear.
The dihydrotestosterone (DHT)-induced PCOS mouse model has been widely used in the study of PCOS. These mice display many phenotypes associated with PCOS, including increased body weight, androgen levels, and visceral and genital fat coefficients, as well as changes in fat cell morphology., However, in contrast to the disorder in women, most studies have confirmed the absence of insulin resistance in PCOS-like mice., Therefore, this allows us to explore whether hyperandrogenemia alone can cause mitochondrial dysfunction in liver cells, which is of considerable significance for understanding the occurrence of NAFLD in patients with PCOS.
Autophagy is a process by which cells are able to self-renew and maintain a healthy state through the phagocytosis of abnormal or damaged structures. In contrast, apoptosis is the process of programmed self-destruction of a cell under the control of apoptotic factors. Many studies have shown that autophagy and apoptosis are associated with mitochondrial dysfunction in NAFLD.,, Impaired autophagy prevents the clearance of excessive lipid droplets, leading to the occurrence of NAFLD., Hepatocyte apoptosis is accompanied by an increase in triglyceride and cholesterol synthesis and a decrease in fatty acid β-oxidation.,, Therefore, mitochondrial dysfunction, as well as changes in apoptosis and autophagy, may be involved in the development of NAFLD.
The aim of this study was to investigate whether NAFLD developed in a PCOS-like mouse model and observe the changes in mitochondrial function, including the expression of protein molecules related to mitochondrial respiratory chain function and peroxide production. Furthermore, changes in the levels of key molecules related to autophagy and apoptosis in hepatocytes were recorded.
| Materials and Methods|| |
Female, specific pathogen-free, C57BL/6 mice (21 days old), weighing 14 ± 1 g, were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). All animals were housed under a 12-h light/12-h dark cycle at 22°C ± 2°C and 45%–55% humidity, with free access to food and water. All animal procedures were approved by the Animal Ethics Committee of Fudan University and met the requirements of the relevant animal ethics association (No. 20160901).
To prepare the DHT slow-release bio-tubes, 10 mg of DHT powder was added to the prepared sterile silicone tubes using negative pressure suction, following which the tubes were sealed with a sterile plug. The DHT dose used in this study was based on a previous study. The tube walls contained micropores that ensured continuous slow release of the drug.
Mice of comparable body weight were randomly divided into control and DHT-treated groups (n = 7/group). DHT tubes were implanted under the skin of the neck, and each tube contained 10 mg of DHT, which was released over a 12-week period to induce PCOS in the mice. Body weight and estrous cycle status of the mice were recorded throughout the period of 12 weeks.
Oral glucose tolerance test
The oral glucose tolerance test (OGTT) was performed the day before sampling. Before the test, the animals were fasted for 12 h. After sterilization and analgesia of the tail, the tail tip was gently punctured, and blood sugar was tested using the second drop of blood (ACCU-CHEK Performa, Roche). Animals were then fed 50% dextro-glucose (2 g/kg body weight), and blood glucose levels were measured at 30, 60, 90, and 120 min.
Serum levels of endocrine markers, steroid hormones, lipid profiles, and liver functional markers were measured. Progesterone, estradiol, total testosterone, follicle-stimulating hormone, luteinizing hormone, sex hormone-binding globulin, and C-reactive protein levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Sino-UK Institute of Biological Technology, Beijing, China) with a STAT FAX 2100 Microplate Reader (Awareness Technology Inc., USA). The levels of triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, insulin, aspartate transaminase, and alanine aminotransferase (ALT) were measured using colorimetric kits (BioSino Bio-Technology & Science Inc., China) with a BS-420 Chemistry Analyzer (Mindray, China).
Quantitative real-time polymerase chain reaction
Total RNA was extracted from liver tissues using TRIzol reagent (Life Technologies, CA, USA) according to the manufacturer's protocol, and single-stranded cDNA was synthesized from each sample (1 μg) using PrimeScript RT Master Mix (Cat#RR036A, Takara Bio, Inc., Shiga, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using an ABI PRISM 7900 Sequencing System (Applied Biosystems, Foster City, CA, USA). PCR parameters were set according to the manufacturer's protocols, and amplification was performed using the SYBR Premix Ex Taq kit (Cat# RR420A, Takara Bio, Inc.). For each sample, duplicate reactions were carried out in 384-well plates, and all primers were checked to ensure the purity of the target gene. The expression of all genes was normalized to that of GAPDH and U87, and the similarity in expression of the two housekeeping genes was used to ensure the quality of the measurements. Total DNA was extracted from liver tissues using PrimeScript RT Master Mix (Takara Bio, Inc.) according to the manufacturer's protocol for mitochondrial DNA copy number analysis. A total of 1 μg of DNA from each sample was used in the subsequent qRT-PCR assay, and all other parameters and methods in the amplification process were the same as those used for RNA quantification. The two normalized housekeeping genes were Ywhaz and 36B4, and the relative gene expression was determined using the 2−△△CT method. Detailed information for the primers is shown in [Supplementary Table 1].
Mouse liver tissues were lysed using radio-immunoprecipitation assay buffer supplemented with cOmplete Mini protease and PhosSTOP phosphatase inhibitor cocktail tablets. Equal amounts (20 μg) of protein for each treatment group were resolved on 4%–15% Tris-Glycine eXtended stain-free gels (Bio-Rad Laboratories GmbH, Munich, Germany) and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were probed with primary antibodies [Supplementary Table 2] in 0.01 mol/L Tris-buffered saline supplemented with Triton X-100 (TBST) containing 5% (w/v) nonfat dry milk, followed by HRP-conjugated secondary antibody. When necessary, the PVDF membranes were stripped using Restore PLUS western blot stripping buffer (Thermo Scientific, Rockford, IL, USA) for 15 min at 25°C, washed twice in TBST, and then re-probed. Ultraviolet activation of the stain-free gel on a ChemiDoc MP Imaging System (Bio-Rad) was used to control the loading. PVDF membranes were scanned and quantified using Image Laboratory (Version 5.0, Bio-Rad, Sweden), and all specific protein band densities were normalized to those of the total protein loading control.
Hematoxylin and eosin and Oil Red O staining
Liver tissues were fixed in 4% paraformaldehyde (PFA) at 4°C overnight. The fixed tissues were washed with phosphate-buffered saline (PBS) and transferred to alcohol for dehydration before embedding in paraffin and slicing. The paraffin sections were dewaxed in xylene twice for 5 min each time. The slices were placed in 100%, 95%, 85%, 75%, and 50% (v/v) alcohol for 5 min each time and then washed three times with PBS or 5 min each time. The sections were stained with hematoxylin and eosin (H and E) separately.
Tissues were fixed overnight in 4% PFA at 4°C, washed three times with PBS, and then incubated at room temperature with 20%, 40%, 80%, and 100% 1,2-propylene glycol (Sinopharm Group Co., Ltd., China) for 15 min each time. The tissues were next incubated in Oil Red O solution (Wuhan Guge Biological Co., Ltd., China) overnight at 4°C. Samples were then rinsed in absolute propylene glycol for 60 min and washed with decreasing concentrations of 1,2-propylene glycol solution for approximately 30 min. Finally, the tissues were washed with PBS and photographed under a bright-field dissecting microscope (Leica DM LB2 microscope, Germany).
Determination of malondialdehyde and xanthine oxidase
The levels of reactive oxygen species (ROS) were measured using ELISA kits for malondialdehyde (A003-1) and xanthine oxidase (A002), both from Nanjing Jiancheng Bioengineering Institute, China. Mouse liver tissues were lysed in 100% ethanol, in a 1:9 tissue to lysis buffer ratio, according to the manufacturer's instructions.
Data are presented as the mean ± standard error of the mean for all experiments. Statistical analyses were performed using SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). Datasets were first assessed for normal distribution using the Shapiro–Wilk test. Differences between control and DHT-treated groups were determined using the unpaired Student's t-test for normally distributed data and Kruskal–Wallis test followed by the Mann–Whitney U-test. Results were considered statistically significant at P < 0.05.
| Results|| |
Dihydrotestosterone-treated mice develop nonalcoholic fatty liver disease
Our findings indicate that the mice developed metabolic abnormalities following 12 weeks of DHT administration. As shown in [Table 1], DHT-treated mice increased body weight compared with the controls, but there was no significant change in liver weight. Despite the change in body weight, there was no difference in the levels of various hormones between the two groups. H and E staining and Oil Red O staining of liver tissues showed an increase in the number of lipid components deposited around the nucleus, and there was an increase in red lipid droplets in the cytoplasm, suggesting the occurrence of lipid deposition in hepatocytes [Figure 1]a. While the levels of circulating total triglycerides decreased in DHT-treated mice [Table 1], triglyceride and cholesterol levels in the liver increased [Figure 1]b and [Figure 1]c. There was no difference of free fatty acids (FFAs) between two groups [Figure 1]d.
|Table 1: Metabolic parameters, endocrine and steroid hormone levels in dihydrotestosterone -treated polycystic ovary syndrome -like mice|
Click here to view
|Figure 1: Changes in liver morphology, lipid content, and gene expression and changes in inflammation-associated protein and gene expression in DHT-treated mice. (a) Representative H and E staining (upper panels) and Oil Red O staining (lower panels) for the control and DHT-treated groups. (b-d) Quantification of liver triglyceride, total cholesterol, and free fatty acid levels. (e and f) mRNA levels of genes associated with lipid metabolism were determined by qRT-PCR. (g) Western blotting analysis of NF-κing analysis of NF-f gin mice. Total proteins were used as loading controls. (h) mRNA expression of key inflammatory factors in mice. Data are shown as the mean ± SEM, n = 7/group, differences in the control and DHT-treated groups were determined by the unpaired Student's t-test, *P < 0.05, - P < 0.01, scale bar = 100 μm. Srebp1: Sterol regulatory element-binding protein 1; Acc1: Acetyl-CoA carboxylase 1; Pparg: Peroxisome proliferator activated receptor gamma; Fasn: Fatty acid synthase; Pparα: Peroxisome proliferator activated receptor alpha; Cpt1α: Carnitine palmitoyltransferase 1 alpha; Acox1: Acyl-CoA oxidase 1; Hmgcs: Hydroxymethylglutaryl-CoA synthase; DHT: Dihydrotestosterone; qRT-PCR: Quantitative real-time polymerase chain reaction; H and E: Hematoxylin and eosin; SEM: Standard error of the mean.|
Click here to view
We further detected the expression of transcriptional regulators and genes encoding key enzymes related to lipid synthesis. The expression levels of the lipid synthesis genes Srebp1, Acc1, and Pparg were increased in the DHT-treated mice [Figure 1]e, and the mRNA levels of genes encoding lipid decomposition-related factors, Pparα and Acox1, decreased in DHT-treated mice compared with the controls [Figure 1]f. Taken together, these results suggest that long-term DHT exposure causes hepatic lipid synthesis and deposition in mice.
Lipid deposition usually leads to a wide range of inflammatory reactions. Circulating ALT levels were increased in DHT-treated mice, indicating impaired liver function [Table 1]. Furthermore, Western blotting results showed that the expression of key factors of the NF-κB signaling pathway at the protein level remained unchanged in DHT-treated mice [Figure 1]g; however, Il-6 and Il-1β mRNA levels increased significantly [Figure 1]h. These results suggest extensive inflammation in hepatocytes and liver damage in DHT-treated mice.
Dihydrotestosterone-treated mice show no insulin resistance
Fasting insulin level was increased in DHT-treated mice, whereas the OGTT showed no difference between basal blood glucose levels at 30, 60, 90, and 120 min. Similarly, there was no difference in the area under curve or homeostasis model assessment insulin resistance score in DHT-treated mice compared with the controls, suggesting that DHT exposure did not induce insulin resistance [Table 1]. In addition, there was no differences in insulin pathway key factors in the liver except Pan-Akt [Figure 2]a.
|Figure 2: Metabolic and androgen changes in DHT-treated mice. (a) Representative Western bloting analysis of key insulin signaling molecules in mice. Total protein served as loading control. (b) Representative Western blotting analysis of AR, p21, and p27 in hepatic cells of mice. Total protein served as loading control. (c) Representative mRNA analysis of AR, p21, and p27 in the ovaries and uteri of mice. Data are presented as the mean ± SEM, n = 7/group, and differences in the control and DHT-treated groups were determined using the unpaired Student's t-test, *P < 0.05, - P < 0.01. DHT: Dihydrotestosterone; SEM: Standard error of the mean; AR: Androgen receptor.|
Click here to view
To determine the effect of androgen on DHT-treated mice, the levels of androgen receptor (AR), p21, and p27 in the liver, uterus, and ovary were measured. As shown in [Figure 2]b and [Figure 2]c, the expression of AR was increased in the uteri of DHT-treated mice. However, there were no significant differences in AR, p21, and p27 levels in the liver tissue between the control and DHT-treated mice. The AR expression difference in the liver tissue and classic reproductive tissues might be due to variances of sensitivity and reactivity to excess dose and exposure time of DHT.
Mitochondrial dysfunction in the hepatocytes of dihydrotestosterone-treated mice
There is a close relationship between mitochondrial function and the respiratory chain in the development of hepatic inflammation, and as shown in [Figure 3]a, the expression of two mitochondrial DNA copy number markers, ND4 and D-loop, was decreased in DHT-treated mice. In addition, the expression of the mitochondrial fusion-related gene, Mfn1, and the mitochondrial fission-related gene, Drp1, was increased in DHT-treated mice [Figure 3]b. The mRNA level of Pgc1α, which is a key transcription factor that regulates the biogenesis of mitochondria, was decreased in DHT-treated mice compared to the controls [Figure 3]c, and the expression levels of UCP1 and the antioxidant, SOD1, were decreased in DHT-treated mice [Figure 3]d. These results suggest that the mitochondrial DNA copy number and dynamics are significantly affected by DHT.
|Figure 3: Mitochondrial dysfunction in DHT-treated mice. (a) Expression of mitochondrial DNA copy number genes. (b) Expression of genes associated with mitochondrial fission and fusion. (c) Expression of the key transcriptional factors regulating the biogenesis of mitochondria. (d) Representative Western blotting analysis of mitochondrial functional markers in mice. (e) Representative Western blotting analysis of OXPHOS complexes in the mitochondrial respiratory chain in mice. Total proteins were used as loading controls. Data are shown as the mean ± SEM, n = 7/group; differences in the control and DHT-treated groups were determined using the unpaired Student's t-test, *P < 0.05, - P < 0.01. HSP60: Heat shock protein 60; SDHA: Succinate dehydrogenase complex subunit A; VDAC: Voltage-dependent anion channel; PHB1: Prohibitin 1; PDH: Pyruvate dehydrogenase; SOD1: Superoxide dismutase 1; UCP1: Uncoupling protein 1; DHT: Dihydrotestosterone; SEM: Standard error of the mean.|
Click here to view
Western blotting was used to detect the expression of respiratory chain complexes, which are crucial regulatory enzymes involved in aerobic respiration in the mitochondria. Our results showed that the expression levels of mitochondrial complex I and complex III in DHT-treated mice were lower than those in the control mice [Figure 3]e.
The above-mentioned findings suggest that mitochondrial dysfunction occurred, and the levels of markers of enzymes and enzymatic products associated with oxidative stress were detected by ELISA. Moreover, the levels of primary molecules related to energy status were measured by Western blotting [Supplementary Figure 1]. Taken together, these results indicate that mitochondrial dysfunction and ROS production occur in DHT-treated mice.
Imbalance between apoptosis and autophagy in the hepatocytes of dihydrotestosterone -treated mice
The presence of factors involved in apoptosis and autophagy was determined by qRT-PCR and Western blotting. The expression of the proapoptotic gene, Bax, was increased, while the levels of the antiapoptotic factors did not change in DHT-treated mice [Figure 4]a. The expression of cytochrome c and caspase-3 in the hepatocytes of DHT-treated mice was not different from that in the controls [Figure 4]b, while the levels of autophagy-related proteins, Atg5 and Atg12, were increased in the hepatocytes of DHT-treated mice [Figure 4]c, suggesting an imbalance between hepatic apoptosis and autophagy in DHT-treated mice.
|Figure 4: mRNA expression of pro-apoptotic and anti-apoptotic genes and protein expression of apoptosis and autophagy markers in DHT-treated mice. (a) The expression levels of the anti-apoptotic genes, Bcl-2 and Bcl-xl, and the pro-apoptotic genes, Bax and Bak, were quantified by qRT-PCR. (b) Representative Western blotting analysis of the apoptosis markers, Cyto c and Casp-3, in the control and DHT-treated groups. (c) Representative Western blotting analysis of autophagy-related proteins in control and DHT-treated mice. Atg: Autophagy-related proteins. Total proteins were used as loading controls. Data are shown as the mean ± SEM, n = 7/group; differences in the control and DHT-treated groups were determined by the unpaired Student's t-test, *P < 0.05 , - P < 0.01. Cyto c: Cytochrome c; DHT: Dihydrotestosterone; SEM: Standard error of the mean.|
Click here to view
In the present study, 12 weeks of DHT exposure in mice led to an increase in body weight and an increase in lipid synthesis and deposition in the liver, while the majority of steroid and glycolipid indexes remained unchanged. Extensive inflammatory responses were observed in the liver and Il-6 and Il-1β mRNA levels were increased, thus providing the evidence of impaired liver function. Mitochondrial function was disrupted following DHT administration and the number of hepatic mitochondria decreased. Finally, an imbalance between mitochondrial apoptosis and autophagy was found, which might be the main cause of hepatic steatosis in PCOS-like mice.
Classically, androgens can act directly through the AR or indirectly, following aromatization via the estrogen receptor (ER). DHT is a nonaromatic androgen; it does not induce an increase in downstream estrogen levels. Therefore, direct AR-driven mechanisms are key to driving the development of PCOS metabolic traits. Zhang et al. used high-fat diet and DHT-induced male rat model and found that DHT significantly increased AR expression in the liver after 75 days treatment. Dai et al.found that AR signaling plays an important role in the development of liver diseases and that DHT can initiate cell cycle arrest and apoptosis in androgen-sensitive liver cells. In our present study, the expression of AR in the uterus was increased, while the expression of AR and downstream p21 and p27 in the liver remained unchanged. This might be due to the different time points and sex difference of DHT exposure.
Hyperandrogenism and insulin resistance are two major pathological features of PCOS. Hyperandrogenism alone can lead to NAFLD, and women with PCOS with insulin resistance or obesity are at higher risk of NAFLD. Moreover, approximately 10%–20% of nonobese women with PCOS still develop NAFLD, and it is associated with visceral fat, dietary composition, intestinal microbiota, and genetic factors., Many studies have shown metabolic disorders in DHT-treated mice, including weight gain, increased adipocyte volume in the liver, and fat accumulation., In our study, fasting insulin levels was increased in DHT-treated mice, but the fasting blood glucose levels, OGTT results, and the expression of insulin signaling pathway components were not affected by prolonged DHT exposure. This suggests that PCOS-like mice only have a pathological state of hyperandrogenism. The lack of insulin resistance in mice may be associated with sensitivity differences in the insulin pathway in different organs. In a previous study, insulin resistance was not detected through the analysis of insulin-sensitive tissues, such as the liver, muscle, and fat. Another study only found that blood glucose levels were impaired, a phenomenon which was associated with decreased glucose transporters (GLUTs) expression. Our study also showed that DHT-treated mice exhibited increased body weight, but NAFLD could not be explained only by obesity. A clinical study showed that women with hyperandrogenic PCOS had an increased risk of hepatic steatosis, even after statistical correction for differences in fat volume.
The imbalance between lipid synthesis and fatty acid decomposition in hepatocytes plays an important role in the development of NAFLD., Studies have shown that approximately 59% of the lipid sources come from FFAs, which are produced by the decomposition of adipose tissue, and only 25% of lipids are produced by hepatocytes themselves., Disorders of hepatocyte-origin lipid synthesis and fatty acid decomposition play important roles in the development of NAFLD., Our results showed that long-term high DHT levels increased lipid accumulation in the hepatocytes and elevated the level of hepatic triglycerides and cholesterol. However, we did not observe significant differences in liver FFA levels between the control and DHT-treated mice [Figure 1]. This suggests that hepatic steatosis mainly developed due to increased triglyceride and cholesterol levels in the liver. However, the total circulating triglyceride levels decreased in DHT-treated mice in our study. The source and the synthesis of hepatocyte triglycerides are different, and thus, the changes in triglyceride levels in the blood and liver are not necessarily synchronous. Fatty acid oxidation and export of triglycerides are two possible mechanisms to reduce hepatic lipid content. Changes in triglyceride level in the liver can be due to the ability or inability to export compromised triglycerides. Serum levels of triglycerides may, in part, be related to the beneficial effects of androgens on lipid metabolism. In addition, we found that the expression of key enzymes and regulatory factors controlling lipid synthesis was increased, while the levels of key enzymes regulating fatty acid β-oxidation appeared to be increased or decreased. This demonstrated that abnormal lipid synthesis and decomposition in DHT-treated mouse hepatocytes were related to the occurrence of NAFLD, and excessive lipid deposition in hepatocytes could lead to cytotoxicity and inflammation.
The function of mitochondria is mainly reflected in the process of aerobic respiration, which is closely related to lipid synthesis and decomposition. Electron transfer through the mitochondrial respiratory chain is the main manifestation of mitochondrial aerobic respiratory function, and respiratory chain dysfunction leads to changes in the redox state of the cell and abnormal energy production. It has been suggested that lipid deposition and inflammation in hepatocytes may cause abnormal mitochondrial function during the development of NAFLD, which is manifested through the production of peroxides and subsequent cell damage. However, the specific mechanism remains unclear.,,, Our study also showed abnormal expression of mitochondrial respiratory chain complexes in DHT-treated mice. Changes in the expression of molecules related to mitochondrial aerobic respiration and mitochondrial oxidative phosphorylation suggest abnormal electron transport and aerobic respiration processes in PCOS-like mice. However, there was no obvious correlation between the oxidative and antioxidant processes in DHT-treated mice. Our results showed that there was no significant increase in oxidative stress, although the electron transfer process was abnormal in DHT-treated mice. These results suggest that hyperandrogenism is involved in PCOS-related mitochondrial aerobic respiratory dysfunction in NAFLD and that abnormal lipid metabolism in hepatocytes is closely related to mitochondrial aerobic respiratory dysfunction.
Apoptosis and autophagy are two physiological processes that occur in response to pathological conditions, and both are closely related to mitochondrial function. It has been reported that increased production of stress-related peroxides in hepatocytes could lead to changes in mitochondrial membrane permeability and transition potential, which might further lead to the release of pro-apoptotic factors or the activation of caspase-3., Apoptosis plays an important role in the progression of NAFLD. Caspase-3 activation and hepatocyte apoptosis are prominent features of NAFLD and have been shown to be correlated with disease severity. A previous study showed that Bax inhibitor-1 was associated with hepatic lipid accumulation, which was related to apoptosis caused by endoplasmic reticulum stress and ROS accumulation. In the present study, the expression of Bax was increased in DHT-treated mouse hepatocytes, but the expression and activation ratio of caspase-3 in DHT-treated mice did not increase. Thus, more details about the apoptotic effect of PCOS-like mice on hepatocytes need to be further investigated in the future. Autophagy is regulated by the energy status of the cell to improve the utilization of intracellular substances when energy supply is deficient. Atg7 is an important autophagy-related protein, and when deficient in mice, it can lead to increased lipid deposition. In addition, an in vitro study by others showed that 3-methyladenine inhibited autophagy in hepatocytes and led to increased lipid deposition, while agonists of autophagy increased the degradation of intracellular lipids. Therefore, autophagy may play a protective role against abnormal lipid deposition in hepatocytes. In the current study, there was no significant increase in the levels of important autophagy-related molecules (e.g., microtubule-associated protein 1 light chain 3), suggesting that the protective effect of autophagy was weakened in PCOS-like mice.
In conclusion [Figure 5], hyperandrogenism appears to be involved in the development of NAFLD in PCOS-like mice, and its mechanism may be related to aerobic respiration disorders, such as mitochondrial respiratory chain dysfunction, and an imbalance between apoptosis and autophagy. Exploring the direct relationship between hyperandrogenism and NAFLD remains a significant challenge. Although the number of animals in the experimental and control groups was small, our study confirmed that hyperandrogenism can lead to NAFLD, and this result may provide ideas for future clinical treatments. Women with PCOS, particularly those with hyperandrogenism, should pay attention to whether they have developed NAFLD and should undergo regular physical examinations from an early age. In clinical treatment, physicians should pay attention to patients with hyperandrogenemia and take particular care in controlling their hormone levels to prevent the occurrence of NAFLD.
|Figure 5: Summary of possible mechanisms behind the development of NAFLD in PCOS patients. NAFLD: Nonalcoholic fatty liver disease; PCOS: Polycystic ovary syndrome.|
Click here to view
Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.
We thank the National Natural Science Foundation of China, the Chinese Special Fund for Postdocs, the Development Project of Shanghai Peak Disciplines-Integrated Chinese and Western Medicine, the Swedish Medical Research Council, and the Hjalmar Svensson Foundation for supporting this study.
Financial support and sponsorship
This work was supported by the National Natural Science Foundation of China (No. 81673766 and 81973945 to YF, No. 81572555 to XL), the Chinese Special Fund for Postdocs (No. 2014T70392), and the Swedish Medical Research Council (No. 10380), the Swedish Federal Government under the LUA/ALF agreement (No. ALFGBG-147791 to HB and LS).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab 2004;89:2745-9. doi: 10.1210/jc.2003-032046.
Goodarzi MO, Azziz R. Diagnosis, epidemiology, and genetics of the polycystic ovary syndrome. Best Pract Res Clin Endocrinol Metab 2006;20:193-205. doi: 10.1016/j.beem.2006.02.005.
Escobar-Morreale HF. Polycystic ovary syndrome: Definition, aetiology, diagnosis and treatment. Nat Rev Endocrinol 2018;14:270-84. doi: 10.1038/nrendo.2018.24.
Diamanti-Kandarakis E, Dunaif A. Insulin resistance and the polycystic ovary syndrome revisited: An update on mechanisms and implications. Endocr Rev 2012;33:981-1030. doi: 10.1210/er.2011-1034.
Catteau-Jonard S, Dewailly D. Pathophysiology of polycystic ovary syndrome: The role of hyperandrogenism. Front Horm Res 2013;40:22-7. doi: 10.1159/000341679.
Rosenfield RL, Ehrmann DA. The pathogenesis of polycystic ovary syndrome (PCOS): The hypothesis of PCOS as functional ovarian hyperandrogenism revisited. Endocr Rev 2016;37:467-520. doi: 10.1210/er. 2015-1104.
Vassilatou E. Nonalcoholic fatty liver disease and polycystic ovary syndrome. World J Gastroenterol 2014;20:8351-63. doi: 10.3748/wjg.v20.i26.8351.
Makri E, Tziomalos K. Prevalence, etiology and management of non-alcoholic fatty liver disease in patients with polycystic ovary syndrome. Minerva Endocrinol 2017;42:122-31. doi: 10.23736/S0391-1977.16.02564-5.
Rocha AL, Faria LC, Guimarães TC, Moreira GV, Cândido AL, Couto CA, et al.
Non-alcoholic fatty liver disease in women with polycystic ovary syndrome: Systematic review and meta-analysis. J Endocrinol Invest 2017;40:1279-88. doi: 10.1007/s40618-017-0708-9.
Scarpulla RC, Vega RB, Kelly DP. Transcriptional integration of mitochondrial biogenesis. Trends Endocrinol Metab 2012;23:459-66. doi: 10.1016/j.tem.2012.06.006.
Cree-Green M, Bergman BC, Coe GV, Newnes L, Baumgartner AD, Bacon S, et al.
Hepatic steatosis is common in adolescents with obesity and PCOS and relates to de novo
lipogenesis but not insulin resistance. Obesity (Silver Spring) 2016;24:2399-406. doi: 10.1002/oby.21651.
Simões I, Fontes A, Pinton P, Zischka H, Wieckowski MR. Mitochondria in non-alcoholic fatty liver disease. Int J Biochem Cell Biol 2018;95:93-9. doi: 10.1016/j.biocel.2017.12.019.
Shimano H, Sato R. SREBP-regulated lipid metabolism: Convergent physiology-divergent pathophysiology. Nat Rev Endocrinol 2017;13:710-30. doi: 10.1038/nrendo.2017.91.
Ju J, Huang Q, Sun J, Jin Y, Ma W, Song X, et al.
Correlation between PPAR-α methylation level in peripheral blood and inflammatory factors of NAFLD patients with DM. Exp Ther Med 2018;15:1474-8. doi: 10.3892/etm.2017.5530.
Ju J, Huang Q, Sun J, Zhao X, Guo X, Jin Y, et al.
Correlation between PPAR-α methylation level in peripheral blood and atherosclerosis of NAFLD patients with DM. Exp Ther Med 2018;15:2727-30. doi: 10.3892/etm.2018.5730.
Kim JJ, Kim D, Yim JY, Kang JH, Han KH, Kim SM, et al.
Polycystic ovary syndrome with hyperandrogenism as a risk factor for non-obese non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2017;45:1403-12. doi: 10.1111/apt.14058.
Kumarendran B, O'Reilly MW, Manolopoulos KN, Toulis KA, Gokhale KM, Sitch AJ, et al.
Polycystic ovary syndrome, androgen excess, and the risk of nonalcoholic fatty liver disease in women: A longitudinal study based on a United Kingdom primary care database. PLoS Med 2018;15:e1002542. doi: 10.1371/journal.pmed.1002542.
van Houten EL, Kramer P, McLuskey A, Karels B, Themmen AP, Visser JA. Reproductive and metabolic phenotype of a mouse model of PCOS. Endocrinology 2012;153:2861-9. doi: 10.1210/en.2011-1754.
Caldwell AS, Middleton LJ, Jimenez M, Desai R, McMahon AC, Allan CM, et al.
Characterization of reproductive, metabolic, and endocrine features of polycystic ovary syndrome in female hyperandrogenic mouse models. Endocrinology 2014;155:3146-59. doi: 10.1210/en.2014-1196.
van Houten EL, Visser JA. Mouse models to study polycystic ovary syndrome: A possible link between metabolism and ovarian function? Reprod Biol 2014;14:32-43. doi: 10.1016/j.repbio.2013.09.007.
Czaja MJ, Ding WX, Donohue TM Jr., Friedman SL, Kim JS, Komatsu M, et al.
Functions of autophagy in normal and diseased liver. Autophagy 2013;9:1131-58. doi: 10.4161/auto.25063.
Cichoż-Lach H, Michalak A. Oxidative stress as a crucial factor in liver diseases. World J Gastroenterol 2014;20:8082-91. doi: 10.3748/wjg.v20.i25.8082.
Khambu B, Yan S, Huda N, Liu G, Yin XM. Autophagy in non-alcoholic fatty liver disease and alcoholic liver disease. Liver Res 2018;2:112-9. doi: 10.1016/j.livres. 2018.09.004.
Dong S, Jia C, Zhang S, Fan G, Li Y, Shan P, et al.
The REGγ proteasome regulates hepatic lipid metabolism through inhibition of autophagy. Cell Metab 2013;18:380-91. doi: 10.1016/j.cmet.2013.08.012.
Garcimartin A, Lopez-Oliva ME, Santos-Lopez JA, Garcia-Fernandez RA, Macho-Gonzalez A, Bastida S, et al.
Silicon alleviates nonalcoholic steatohepatitis by reducing apoptosis in aged wistar rats fed a high-saturated fat, high-cholesterol diet. J Nutr 2017;147:1104-12. doi: 10.3945/jn.116.243204.
Lee S, Kim S, Hwang S, Cherrington NJ, Ryu DY. Dysregulated expression of proteins associated with ER stress, autophagy and apoptosis in tissues from nonalcoholic fatty liver disease. Oncotarget 2017;8:63370-81. doi: 10.18632/oncotarget.18812.
Kanda T, Matsuoka S, Yamazaki M, Shibata T, Nirei K, Takahashi H, et al.
Apoptosis and non-alcoholic fatty liver diseases. World J Gastroenterol 2018;24:2661-72. doi: 10.3748/wjg.v24.i25.2661.
Aflatounian A, Edwards MC, Rodriguez Paris V, Bertoldo MJ, Desai R, Gilchrist RB, et al.
Androgen signaling pathways driving reproductive and metabolic phenotypes in a PCOS mouse model. J Endocrinol 2020;245:381-95. doi: 10.1530/JOE-19-0530.
Zhang H, Liu Y, Wang L, Li Z, Zhang H, Wu J, et al.
Differential effects of estrogen/androgen on the prevention of nonalcoholic fatty liver disease in the male rat. J Lipid Res 2013;54:345-57. doi: 10.1194/jlr.M028969.
Dai R, Yan D, Li J, Chen S, Liu Y, Chen R, et al.
Activation of PKR/eIF2α signaling cascade is associated with dihydrotestosterone-induced cell cycle arrest and apoptosis in human liver cells. J Cell Biochem 2012;113:1800-8. doi: 10.1002/jcb.24051.
Petta S, Ciresi A, Bianco J, Geraci V, Boemi R, Galvano L, et al.
Insulin resistance and hyperandrogenism drive steatosis and fibrosis risk in young females with PCOS. PLoS One 2017;12:e0186136. doi: 10.1371/journal.pone.0186136.
Leung JC, Loong TC, Wei JL, Wong GL, Chan AW, Choi PC, et al.
Histological severity and clinical outcomes of nonalcoholic fatty liver disease in nonobese patients. Hepatology 2017;65:54-64. doi: 10.1002/hep.28697.
Koo BK, Joo SK, Kim D, Bae JM, Park JH, Kim JH, et al.
Additive effects of PNPLA3 and TM6SF2 on the histological severity of non-alcoholic fatty liver disease. J Gastroenterol Hepatol 2018;33:1277-85. doi: 10.1111/jgh.14056.
Lee G, You HJ, Bajaj JS, Joo SK, Yu J, Park S, et al.
Distinct signatures of gut microbiome and metabolites associated with significant fibrosis in non-obese NAFLD. Nat Commun 2020;11:4982. doi: 10.1038/s41467-020-18754-5.
Andrisse S, Billings K, Xue P, Wu S. Insulin signaling displayed a differential tissue-specific response to low-dose dihydrotestosterone in female mice. Am J Physiol Endocrinol Metab 2018;314:E353-65. doi: 10.1152/ajpendo.00195.2017.
Caldwell AS, Middleton LJ, Jimenez M, Desai R, McMahon AC, Allan CM, et al.
Characterization of reproductive, metabolic, and endocrine features of polycystic ovary syndrome in female hyperandrogenic mouse models. Endocrinology 2014;155:3146-59. doi: 10.1210/en.2014-1196.
Jones H, Sprung VS, Pugh CJ, Daousi C, Irwin A, Aziz N, et al.
Polycystic ovary syndrome with hyperandrogenism is characterized by an increased risk of hepatic steatosis compared to nonhyperandrogenic PCOS phenotypes and healthy controls, independent of obesity and insulin resistance. J Clin Endocrinol Metab 2012;97:3709-16. doi: 10.1210/jc.2012-1382.
Postic C, Girard J. Contribution of de novo
fatty acid synthesis to hepatic steatosis and insulin resistance: Lessons from genetically engineered mice. J Clin Invest 2008;118:829-38. doi: 10.1172/JCI34275.
Ipsen DH, Lykkesfeldt J, Tveden-Nyborg P. Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease. Cell Mol Life Sci 2018;75:3313-27. doi: 10.1007/s00018-018-2860-6.
Duwaerts CC, Maher JJ. Macronutrients and the Adipose-Liver Axis in Obesity and Fatty Liver. Cell Mol Gastroenterol Hepatol 2019;7:749-61. doi: 10.1016/j.jcmgh.2019.02.001.
Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest 2005;115:1343-51. doi: 10.1172/JCI23621.
Tiniakos DG, Vos MB, Brunt EM. Nonalcoholic fatty liver disease: Pathology and pathogenesis. Annu Rev Pathol 2010;5:145-71. doi: 10.1146/annurev-pathol-121808-102132.
Rinella ME. Nonalcoholic fatty liver disease: A systematic review. JAMA 2015;313:2263-73. doi: 10.1001/jama.2015.5370.
Perry RJ, Samuel VT, Petersen KF, Shulman GI. The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes. Nature 2014;510:84-91. doi: 10.1038/nature13478.
Traish AM, Abdou R, Kypreos KE. Androgen deficiency and atherosclerosis: The lipid link. Vascul Pharmacol 2009;51:303-13. doi: 10.1016/j.vph.2009.09.003.
Petrosillo G, Portincasa P, Grattagliano I, Casanova G, Matera M, Ruggiero FM, et al.
Mitochondrial dysfunction in rat with nonalcoholic fatty liver Involvement of complex I, reactive oxygen species and cardiolipin. Biochim Biophys Acta 2007;1767:1260-7. doi: 10.1016/j.bbabio2007.07.011.
Rector RS, Thyfault JP, Uptergrove GM, Morris EM, Naples SP, Borengasser SJ, et al.
Mitochondrial dysfunction precedes insulin resistance and hepatic steatosis and contributes to the natural history of non-alcoholic fatty liver disease in an obese rodent model. J Hepatol 2010;52:727-36. doi: 10.1016/j.jhep.2009.11.030.
Begriche K, Massart J, Robin MA, Bonnet F, Fromenty B. Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease. Hepatology 2013;58:1497-507. doi: 10.1002/hep.26226.
Paradies G, Paradies V, Ruggiero FM, Petrosillo G. Oxidative stress, cardiolipin and mitochondrial dysfunction in nonalcoholic fatty liver disease. World J Gastroenterol 2014;20:14205-18. doi: 10.3748/wjg.v20.i39.14205.
Baker PR 2nd
, Friedman JE. Mitochondrial role in the neonatal predisposition to developing nonalcoholic fatty liver disease. J Clin Invest 2018;128:3692-703. doi: 10.1172/JCI120846.
Feldstein AE, Canbay A, Angulo P, Taniai M, Burgart LJ, Lindor KD, et al.
Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology 2003;125:437-43. doi: 10.1016/s0016-5085(03)00907-7.
Lee HY, Lee GH, Bhattarai KR, Park BH, Koo SH, Kim HR, et al.
Bax Inhibitor-1 regulates hepatic lipid accumulation via ApoB secretion. Sci Rep 2016;6:27799. doi: 10.1038/srep27799.
Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, et al.
Autophagy regulates lipid metabolism. Nature 2009;458:1131-5. doi: 10.1038/nature07976.
Cui P, Hu W, Ma T, Hu M, Tong X, Zhang F, et al.
Long-term androgen excess induces insulin resistance and non-alcoholic fatty liver disease in PCOS-like rats. J Steroid Biochem Mol Biol 2021;208:105829. doi: 10.1016/j.jsbmb.2021.105829.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]