Study On The Mechanism Of Abnormal Autophagy And Lipid Metabolism Mediated By MTOR In The Development Of Non-alcoholic Fatty Liver Disease Induced By Bisphenol A And Fructose Exposure In Early Life

Objective: Non-alcoholic fatty liver disease(NAFLD)is rapidly becoming one of the most important causes of liver disease worldwide.The increasing incidence of NAFLD is related to the prevalence of obesity and obesity-related metabolic disorders.Over the past 30 years,with the increasing rate of childhood obesity,the incidence of childhood NAFLD is also increasing rapidly.A recent Meta-analysis reported that the prevalence of NAFLD was 7.6% in general children and 34.2% in obese children.The prevalence of NAFLD in children in the United States was 3%-11%,6.3% and 3.4% in Asia and China,respectively.The prevalence of obesity was 19.5%,and 10% of them had fatty liver.The prevalence of NAFLD in overweight and obese children was 44.5%.Therefore,NAFLD exists not only in obese people,but also in people with normal body weight.The etiology and mechanism of NAFLD morbidity are complex and unclear,which is characterized by liver fat accumulation.Therefore,too much dietary energy intake and too little activity are often considered to be related to obesity,abnormal lipid metabolism and NAFLD.Recent studies have shown that bisphenol A(BPA)and fructose exposure can increase the risk of NAFLD,but the specific mechanism is not clear.BPA is one of the endocrine disrupting chemicals(EDCs)with estrogenic activity and the highest yield.The intake of BPA is mainly produced by food and drink,but it can also be exposed by continuous contact with the skin through heat-sensitive paper receipts.As a result of these multiple sources and daily exposure,humans are exposed to low doses of BPA on a wide and sustained basis.A growing body of evidence suggests that BPA exposure increases the risk of obesity,insulin resistance(IR),and dyslipidemia.Fructose is a common monosaccharide,isomer of glucose,which is abundant in fruits and honey in free form.With the rapid development of food processing industry,high fructose corn syrup(HFCS),which is produced by fermentation of corn,has been widely used as a sweetener in almost all soft drinks,cakes and packaged foods since the 1970 s because of its low production cost and long shelf life.HFCS is composed of 55% fructose,41% glucose and 4% complex polysaccharides.Even in the absence of obesity,high fructose intake can lead to serious health problems,such as liver fat accumulation,dyslipidemia and NAFLD,which may be related to increased liver fat de novo lipogenesis(DNL),endoplasmic reticulum stress and increased expression of fat synthesis genes.Recent studies have shown that elevated levels of BPA in urine are associated with NAFLD in adults and adolescents.Excessive intake of high fructose diet is considered to be one of the main causes of NAFLD in adults and children.Considering that BPA and fructose are often exposed to food and beverage at the same time in daily life,and whether combined exposure to BPA and fructose during pregnancy and lactation will aggravate the development of NAFLD in offspring is still unclear,so the impact of BPA and fructose on the development of NAFLD and its mechanism need further study.There is growing evidence that the activation of mammalian target protein of rapamycin(mTOR)signaling pathway leads to obesity,T2 DM and malignant tumors.mTOR is the downstream target of phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)pathway,which can regulate cell growth and autophagy,and lipid synthesis through peroxisome proliferator-activated receptor ?(PPAR?)and sterol regulatory element-binding protein 1(SREBP1).AMP-activated protein kinase(AMPK)is known as the switch of energy regulation in vivo.It controls a variety of cellular processes such as lipid metabolism by inhibiting hepatic lipogenesis and stimulating fatty acid oxidation.The activation of AMPK can not only lead to the inhibition of mTOR activity to activate autophagy,but also directly activate autophagy through ULK1.Impaired autophagy is associated with a variety of diseases,such as T2 DM,obesity,neurodegeneration,cancer and aging.Enhanced inflammatory response has also been proved to be a key inducement of metabolic diseases such as NAFLD.The inflammatory response is characterized by activation of inflammatory signaling pathways,such as nuclear factor ?B(NF-?B)signaling pathways,and abnormal production of pro-inflammatory cytokines,including IL-1?,IL-6,and TNF-?.NF-?B signaling pathway plays an important role in metabolic diseases,including NAFLD,especially in liver inflammation.In conclusion,this study was designed to investigate the role of mTOR-mediated autophagy and abnormal lipid metabolism in the development of NAFLD induced by early life exposure to BPA and fructose.In order to explore the high incidence of NAFLD in children in recent years and provide experimental basis for taking targeted preventive measures,and also provide a theoretical basis for further formulating the exposure threshold of BPA and the appropriate intake of fructose.Methods: Thirty pregnant SD rats were randomly divided into control group,1 ?g/mL BPA(LBPA)group,10 ?g/m L BPA(HBPA)group,10% fructose(Fructose)group and 1 ?g/m L BPA + 10% fructose(LBPA + Fructose)group.From gestational day 6(GD6),pregnant rats in each group drank water containing BPA and/or fructose until the end of lactation.The offspring were weaned at PND21 and separated into male and female cages.Within 5 weeks after weaning,the offspring drank water without BPA and/or fructose.During the whole experiment,the mother and offspring were exposed to 24 ± 1°C temperature,45 ± 5% humidity and 12 hours of day and night cycle.The food intake and water intake of the mother and offspring were recorded every day,and the body weight of them were recorded every week.At the end of the experiment,two female mice and two male mice were randomly selected from each litter,that is,female/male rats were n = 12/group.Blood glucose and systolic blood pressure were measured in maternal and filial mice,serum HbA1 c,insulin,serum lipids,liver function index,leptin and inflammatory factors were measured in filial mice by ELISA,and liver TG and T-CHO levels in filial mice were measured.Oil red O staining and HE staining were used to observe the pathological changes of liver and adipose tissue,immunohistochemistry was used to detect the expression of LC3 B and p62 protein,RT-PCR was used to detect the expression of lipogenic enzymes related mRNA levels and Western blot was used to detect the expression of lipogenic enzymes,insulin signaling pathway,PI3K/Akt/mTOR signaling pathway,autophagy and NF-?B signaling pathway related protein levels.Results: 1.During the whole pregnancy and lactation,there was no significant difference in maternal body weight among the groups,but the Lee's index,blood glucose and systolic blood pressure increased significantly,and the visceral fat coefficient of LBPA + Fructose group was significantly higher than that of control group and single exposure group.Compared with the control group,the maternal food intake and protein intake in Fructose group and LBPA + Fructose group decreased significantly,while the water intake increased significantly;the total energy intake of W3,W5 and W6 female mice had no significant difference,but the total energy intake of W2 female mice increased significantly and that of W4 female mice decreased significantly.Compared with LBPA group,food intake and protein intake of LBPA + Fructose group decreased significantly,water intake increased significantly,total energy intake of W2 group increased significantly,but total energy intake of W3-W6 group had no significant difference.In female offspring,the body weight of W7 LBPA group and HBPA group were higher than that of control group,and the body weight of W8 LBPA group,HBPA group,Fructose group and LBPA + Fructose group were higher than that of control group;In offspring male mice,compared with the control group,the body weight of each experimental group had no statistical difference.Lee's index,systolic blood pressure,visceral fat coefficient,serum levels of TG,T-CHO,LDL-C,AST,ALT,IL-6 and TNF-? were significantly increased and HDL-C was significantly decreased in the offspring of each experimental group.HOMA-IR in female offspring of Fructose group,LBPA group and LBPA + Fructose group increased significantly.2.Compared with the control group,the liver TG and T-CHO levels in LBPA group,HBPA group,Fructose group and LBPA + Fructose group were significantly increased;compared with LBPA or Fructose group,the liver TG and T-CHO levels of LBPA + Fructose group were significantly higher,and the liver T-CHO levels of LBPA + Fructose group were significantly higher.Pathological examination of liver and adipose tissue showed that the lipid accumulation of hepatocytes increased significantly,the size and volume of adipocytes increased significantly,and the combined exposure group was more serious than the single exposure group.The levels of ER?,Ob-R,ZAG and HSL protein in the liver of female and male offspring decreased significantly,while the levels of PPAR?,SREBP1 and FAS protein increased significantly.The effect of low-dose BPA and fructose combined exposure on the increase of PPAR? and SREBP1 protein levels and the decrease of ER?,ZAG and HSL protein levels in offspring liver was greater than that of single exposure.3.Compared with control group,the protein levels of p-PI3 K,p-Akt(S473),p-Akt(T308),p-mTOR,mTOR,p70S6 K,p-AMPK,p-ULK1,ULK1,Beclin1,LC3 B,p62,TLR4 and NF-?B increased significantly,while the protein levels of InsR,p-IRS-1,IRS-1,TSC1,TSC2 and I?B? decreased significantly.There was no significant difference in the protein levels of PI3 K,Akt and AMPK.Compared with the LBPA group or the Fructose group,the liver InsR,p-IRS-1,IRS-1,TSC1 and TSC2 protein levels of female offspring of the LBPA + Fructose group were significantly reduced,and p-PI3 K,p-Akt(S473),p-Akt(T308),p-mTOR,mTOR,p-AMPK,p-ULK1 and ULK1 protein levels were significantly increased;male offspring p-PI3 K,p-Akt(T308),mTOR,p70S6 K,p-AMPK,P-ULK1,ULK1,Beclin1,p62 and NF-?B protein levels were significantly increased,and p-IRS-1,TSC1 and TSC2 protein levels were significantly decreased.Conclusion: 1.Exposure to BPA and/or fructose in early life can lead to a significant increase in visceral fat content,increased systolic blood pressure,IR,dyslipidemia,liver fat accumulation and NAFLD in both male and female rats,and combined exposure to BPA and fructose can aggravate liver steatosis.The up-regulation of PPAR? and SREBP1 mediated by activation of PI3K/Akt/mTOR signaling pathway promotes fat synthesis and down-regulation of ZAG leads to the decrease of lipolysis.2.Exposure to BPA and/or fructose in early life resulted in activation of liver autophagy associated proteins,but impaired autophagy degradation(increased expression of p62 protein),activation of TLR4/NF-?B inflammatory signaling pathway,downregulation of hepatic InsR,p-IRS-1 and IRS-1 proteins,and IR.Therefore,exposure to BPA and/or fructose in early life resulting in hepatic IR is associated with abnormal autophagy and inflammatory activation.3.Exposure to BPA and/or fructose in early life can down-regulate the expression of ER? and Ob-R protein and up-regulate the expression of AMPK protein in liver,leading to leptin resistance and abnormal energy metabolism,and further aggravate IR in liver.Therefore,exposure to BPA and/or fructose in early life can cause IR through multiple pathways and increase the risk of NAFLD.4.Exposure to BPA and/or fructose in early life can cause visceral fat mass increase,which is related to the increase of adipocyte volume and fat content.Therefore,NAFLD induced by BPA and/or fructose exposure in early life may be related to liver IR and systemic IR,and its mechanism needs further study.