Influence And Mechanism Of Androgen On Hepatic Fat Accumulation In Polycystic Ovary Syndrome

Objective:Polycystic ovary syndrome（PCOS）is the most common reproductive,endocrine,and metabolic disorder among women of childbearing age.The most common clinical manifestations of PCOS include chronic anovulation,hirsutism or acne,menstrual cycle disorder,and infertility,which are closely related to obesity,insulin resistance,and abnormal lipid metabolism.Up to 80%of PCOS patients are overweight or obese.Recent studies have shown that the prevalence of nonalcoholic fatty liver disease（NAFLD）in PCOS patients is significantly higher than that in the normal population,and this phenomenon is more common in obese PCOS patients.However,the risk factors for NAFLD in obese PCOS and the mechanisms influencing it are not fully understood.Hyperandrogenemia（HA）is a major endocrine characteristic of PCOS.Androgens exert their effects by binding to androgen receptors（AR）.HA is present in approximately80%of patients with PCOS,and HA is an independent risk factor for hepatic steatosis in patients with PCOS.However,the exact mechanism by which HA exacerbates hepatic steatosis in patients with PCOS has not yet been fully elucidated.Hepatic triglyceride（TG）metabolism is closely associated with the development of hepatic steatosis and NAFLD.TG metabolism in the liver includes fatty acid intake,TG synthesis,fatty acid oxidation,lipolysis,and TG secretion.Abnormalities in any process of hepatic TG metabolism may increase the risk of NAFLD.Previous studies have shown that androgens can increase hepatic TG synthesis and exacerbate hepatic steatosis in PCOS by increasing the expression of Fas,SCD,ACC1,ACC2,and SREBP1c in hepatocytes,and enhancing SCAP-SREBP1 interactions.However,the effects of androgens on hepatic fatty acid oxidation,lipolysis,as well as TG secretion have not been investigated.Therefore,we designed（1）a clinical study to observe the changes in the controlled attenuation parameter（CAP）of liver and its influencing factors in obese PCOS patients,and to investigate the prevalence and risk factors of NAFLD in obese PCOS patients.（2）A PCOS rat model was constructed by animal experiments and the androgen receptor blocker flutamide（Flu）was given to intervene and observe the effects of androgens on liver fat accumulation and TG metabolism in PCOS rats;（3）A rat model of PCOS was constructed by animal experiments,and a dihydrotestosterone（DHT）-induced human Hep G2 cell model was constructed by cellular experiments to explore the mechanism of androgen down-regulation of microsomal triglyceride transfer protein（MTTP）expression in the liver of PCOS.The above studies aim to further explore the effects and possible mechanisms of androgens on hepatic fat accumulation in PCOS,so as to provide new directions and ideas for the management and treatment of PCOS combined with NAFLD in clinical work.Methods:Part 1:Forty-one obese PCOS patients attending the endocrine clinic of the Shengjing Hospital of China Medical University from October 2020 to January 2022were enrolled in the study.Twenty non-PCOS women matched for age and body mass index（BMI）were selected as control subjects during the same period.General data,body composition,biochemical parameters,sex hormones,and liver CAP in the two groups were collected and compared.Factors associated with elevated CAP in obese PCOS patients are analysed and prevalence and risk factors for NAFLD in obese PCOS patients are explored.Liver CAP was measured using Fibro Touch.Part 2:A PCOS rat model was constructed by gavage with letrozole in combination with a high-fat diet,and androgen receptor blocker Flu was administered to observe the effects of androgens on hepatic fat accumulation and triglyceride metabolising enzymes in PCOS rats.24 rats were divided into control group,high-fat diet（HFD）group,PCOS group and PCOS+Flu group.Vaginal smears were taken to observe the estrous cycle of the rats,and enzyme linked immunosorbent assay（ELISA）was used to detect the changes of serological indexes and biochemical indexes of liver tissues in the rats.Hematoxylin-eosin（H&E）staining was used to observe the morphology of the ovaries and liver of rats,and oil red O staining was used to observe the fat accumulation in the liver of rats.Western blot（WB）was used to detect the changes of key enzymes of TG secretion,fatty acid oxidation and lipolysis,including MTTP,carnitine palmitoyl transferase 1A（CPT1A）,adipose triglyceride lipase（ASTL）,and fat lipase（ASTL）,in the rat liver.triglyceride lipase（ATGL）,hormone sensitive triglyceride lipase（HSL）.Part 3:Experimental studies were conducted using an PCOS rat model and a DHT-induced human Hep G2 cell model.Twenty-four female Sprague–Dawley rats were randomly divided into four groups:control,HFD,PCOS,and PCOS+Flu.The expression of AR,small heterodimer partner（SHP）,liver receptor homolog-1（LRH-1）and MTTP in the liver tissues of each group was detected by WB.Hep G2 cells were divided into four groups:control,DHT,DHT+negative control（NC）,and DHT+si-SHP groups.The interaction between AR and SHP in DHT-induced Hep G2 cells was examined using co-inmunoprecipitation（Co-IP）.The m RNA and protein expression of AR,SHP,LRH-1 and MTTP in each group of Hep G2 cells were detected using real-time polymerase chain reaction（RT-PCR）and WB.The lipid content in each group of Hep G2cells was detected by BODIPY ^493/503fluorescent probe,and the concentration of TG in each group of Hep G2 cells was detected by spectrophotometry.The accumulation of fat in each group of Hep G2 cells was observed using oil red O staining.Results:Part 1:1.NAFLD was more common in the Obese PCOS group than in the control group（75.61%vs.45.00%,P=0.018）.2.Compared to the control group,the obese PCOS group showed apparent increases in alanine transaminase（ALT）,aspartate transaminase（AST）,CAP,total cholesterol（TC）,triglyceride（TG）,low-density lipoprotein cholesterol（LDL-C）,totle testosterone（TT）,free androgen index（FAI）,fasting insulin（FIns）,and homeostasis model assessment-insulin resistance（HOMA-IR）（P<0.05）,along with decreased high-density lipoprotein cholesterol（HDL-C）and sex hormone binding globulin（SHBG）levels（P<0.05）.3.Liver CAP in Obese PCOS patients had a positive correlation with ALT,AST,TG,TT,FAI,FIns,and HOMA-IR,and a negative correlation with SHBG（P<0.05）.4.TG,TT,FIns,and HOMA-IR were risk factors for NAFLD,while TT was an independent risk factor for NAFLD in obese PCOS patients.Part 2:1.Compared to the control and HFD groups,the PCOS rats had significantly higher body weight（PCOS vs.Control vs.HFD,295.08±8.67 vs.242.67±5.79 vs.265.17±9.68 g,P<0.05）and higher serum TT（PCOS vs.Control vs.HFD,279.03±15.34 vs.242.75±16.19 vs.254.17±18.34 pg/m L,P<0.05）at 4 weeks of modelling.Vaginal smears showed that PCOS rats lost their normal estrous cycle and remained in interestrous phase,and that their ovaries had a polycystic pattern.2.After 8 weeks of modelling,serum TT levels were further elevated in rats of PCOS group（PCOS vs.Control vs.HFD,313.23±21.57 vs.248.56±15.34 vs.264.49±14.37 pg/m L,P<0.05）.Compared with the control and HFD groups,hepatocyte steatosis and TG accumulation were more pronounced in the PCOS group,with significantly higher fat area（PCOS vs.Control vs.HFD,40.48±0.90 vs.2.24±1.11 vs.24.37±3.39 a.u.,P<0.05）and liver weight（PCOS vs.Control vs.HFD,11.58±0.62 vs.8.29±0.49 vs.9.68±0.50 g,P<0.05）,significantly higher liver weight as a percentage of body weight（PCOS vs.Control vs.HFD,3.01±0.07%vs.2.64±0.11%vs.2.87±0.08%,P<0.05）,significantly higher serum ALT levels（PCOS vs.Control vs.HFD,105.85±5.77 vs.84.28±4.87 vs.92.81±4.60 U/L,P<0.05）and AST levels（PCOS vs.Control vs.HFD,33.80±3.56 vs.24.34±1.74 vs.28.80±3.48 U/L,P<0.05）.TG levels were significantly increased in liver tissue of PCOS rats（PCOS vs.Control vs.HFD,5.02±0.66 vs.3.17±0.46 vs.3.94±0.69 nmol/L,P<0.05）,while very low density lipoprotein（VLDL）levels were significantly decreased（PCOS vs.Control vs.HFD,12.32±1.16 vs.14.96±1.03 vs.16.51±1.05 mmol/L,P<0.05）.3.After administration of Flu to block the effects of androgens,PCOS rats showed a significant decrease in serum TT levels（PCOS+Flu vs.PCOS,269.54±17.88 vs.313.23±21.57 pg/ml,P<0.05）.Hepatic steatosis and TG accumulation were reduced in the PCOS group,with a significant decrease in fat area（PCOS+Flu vs.PCOS,27.49±0.74 vs.40.48±0.90 a.u.,P<0.05）.Liver weight（PCOS+Flu vs.PCOS,9.74±0.52 vs.11.58±0.62 g,P<0.05）and liver weight as a percentage of body weight（PCOS+Flu vs.PCOS,2.81±0.04%vs.3.01±0.07%,P<0.05）were significantly reduced in the PCOS group,serum ALT（PCOS+Flu vs.PCOS,93.42±5.83 vs.105.85±5.77 U/L,P<0.05）and AST（PCOS+Flu vs.PCOS,29.61±3.86 vs.33.80±3.56 U/L,P<0.05）levels were decreased,liver TG levels were reduced（PCOS+Flu vs.PCOS,4.11±0.62 vs.5.02±0.66 nmol/L,P<0.05）,and liver VLDL levels were increased（PCOS+Flu vs.PCOS,13.97±1.73 vs.12.32±1.16 mmol/L,P<0.05）.4.WB analysis showed that there was no significant difference in the expression of CPT1A,HSL and ATGL in the liver tissues of rats in each group（P>0.05）.Compared with the control and HFD groups,the expression of AR in liver tissues of PCOS group was significantly elevated,while the expression of MTTP was significantly decreased（P<0.05）.After the administration of Flu,the expression of AR was significantly decreased and the expression of MTTP was significantly increased（P<0.05）.Part 3:1.Compared with the control and the HFD groups,the expression of AR and SHP in the liver tissue of the PCOS group increased（P<0.05）,and the expression of LRH-1 and MTTP decreased（P<0.05）.After administration of Flu to block the effect of androgen,hepatic AR expression decreased（P<0.05）,SHP expression decreased（P<0.05）,and LRH-1 and MTTP expression increased（P<0.05）.2.Compared to the control group,Co-IP revealed protein interactions between AR and SHP in DHT-induced Hep G2 cells.3.RT-PCR and WB results showed that the m RNA and protein expression of AR and SHP were significantly increased in the DHT group（P<0.05）,as well as the m RNA and protein expression of LRH-1 and MTTP were decreased（P<0.05）.After transfection with si-SHP,there was no significant difference in AR expression in the DHT+si-SHP group compared with the DHT group（P>0.05）,SHP expression was significantly reduced（P<0.05）,while the expression of LRH-1 and MTTP was significantly increased（P<0.05）.4.Compared to the control group,oil red O staining showed significantly increased accumulation of triglycerides in DHT-induced Hep G2cells;BODIPY ^493/503fluorescent probe assay suggested that the mean fluorescence intensity（MFI）was significantly higher in the DHT group than in the control group（DHT vs.Control,47.70±1.62 vs.35.32±4.07 a.u.,P<0.05）,and the TG concentration was significantly higher than that of the control group（DHT vs.Control,0.14±0.04 vs.0.05±0.01 mmol/g prot,P<0.05）.When transfected with si-SHP thereby down-regulating SHP expression,compared to the DHT group,TG accumulation was significantly reduced in the DHT+si-SHP group,and MFI（DHT+si-SHP vs.DHT,38.45±2.95 vs.47.70±1.62 a.u.,P<0.05）and TG levels（DHT+si-SHP vs.DHT,0.06±0.01 vs.0.14±0.04 mmol/g prot,P<0.05）decreased significantly compared to before.Conclusions:1.Compared wite age-and BMI-matched non-PCOS women,obese PCOS patients had a significantly higher prevalence of NAFLD.2.Insulin resistance,hypertriglyceridemia,and HA aggravate liver steatosis in obese PCOS patients,with total testosterone being an independent risk factor for NAFLD in obese PCOS patients.3.HA exacerbates hepatic fat accumulation in PCOS rats,which may be related to androgens down-regulating the expression of MTTP,a key enzyme for hepatic triglyceride secretion.4.Androgens may reduce hepatic triglyceride secretion and exacerbate hepatic fat accumulation in PCOS by upregulating hepatocyte SHP expression and thereby inhibiting LRH-1/MTTP expression.