The Role Of CD36 In Regulation Of Hepatocyte De Novo Lipogenesis

OBJECTIVE:Non-alcoholic fatty liver disease(NAFLD)is the most common chronic liver disease that occurs in addition to alcohol and other definite liver-damaging factors.The increase of de novo lipogenesis(DNL)in hepatocytes is the main cause of NAFLD.Fatty acid translocase(FAT/CD36),as a fatty acid transporter,is widely expressed in various types of cells and participates in the pathogenesis of NAFLD by promoting the uptake of free fatty acids.However,the relationship of CD36 to DNL has not been reported.Therefore,this topic aims to investigate the effect of CD36 on DNL and its underlying mechanism.METHODS:PART I:A high-fat diet-induced NAFLD model was established in mice and the hepatic CD36 expression was examined.The expression of CD36 mRNA in the NAFLD population in GEO database(GSE151158)was analyzed.A mouse starvation-feeding model and insulin treatment model were established and the expression of hepatic CD36 and hepatic lipogenesis pathway were examined.HepG2 cells were treated with insulin for indicated time(0 h,0.5 h,1 h,3 h,6 h,12 h)and the expression of CD36 and hepatic lipogenesis pathway was detected.PART Ⅱ:The hepatocyte-specific CD36 knockout mice(Cd36lko)and control mice(Cd36fl/fl)were constructed and then were fed with a high-fat diet for 16 weeks to induce NAFLD.Mouse body weight and food consumption were weighed weekly.Glucose tolerance tests(GTTs)and insulin tolerance tests(ITTs)were carried out to access the insulin sensitivity.Serology experiments were carried out to detect the blood glucose level,lipids and liver function.The liver TG and TC contents were analyzed by enzymatic methods.The liver histochemical staining to evaluate liver morphological and pathological changes.We performed RNA sequencing analysis to explore the potential genes for reduced hepatic steatosis in Cd36lko mice and these changes in lipid metabolism pathways were further confirmed by real-time quantitative PCR(Q-PCR)or western blotting.The CD36-overexpressing stable cell line(CD360E)was constructed or primary hepatocytes from Cd36fl/fl and Cd36lko mice were isolated and cultured.The effect of CD36 changes on hepatocyte lipid accumulation and proteins related lipogenesis were analyzed by BODIPY493/503 staining and western blotting.PART Ⅲ:The liver endoplasmic reticulum and Golgi fractions of Cd36fl/fl and Cd36lko mice and the nuclear and cytoplasmic proteins in CD360E and NC cells were isolated,then the expression of SREBP1 were determined by Western blotting,the localization of SREBP1 in the nucleus and Golgi apparatus and the localization of SCAP in the Golgi apparatus in CD36OE cells or primary hepatocytes from Cd36lko mice were observed by immunofluorescence staining.The interaction between CD36 and INSIGs protein in CD36OE cells was detected by co-immunoprecipitation and proximity ligation assay(PLA).HepG2 cells were transfected with HA-full-length SREBP1(FL-SREBP1)alone or co-transfected with Flag-CD36,and the protein expressions of SREBP1 and SREBP1 pathway were detected by Western blotting.HepG2 cells were transfected with small interfering RNA(siRNA)alone or co-transfected to knock down CD36 and/or INSIG2 expression,or CD36OE cells were treated with 25-hydroxycholesterol(25-HC)or betulin,two SREBP/SCAP complex inhibitor,and the changes of SREBP1 pathway and nuclear localization of SREBP1 were detected by western blotting and immunofluorescence staining.RESULTS:PART Ⅰ:The protein and mRNA level of hepatic CD36 was significantly increased in HFD-fed mice.Compared with fasting,feeding,fasted-refeeding,as well as insulin treatment all increased hepatic CD36 expression and activated SREBP1 pathway.The expression of CD36 and SREBP1 pathway were increased by insulin in a time-dependent manner in HepG2 cells.PART Ⅱ:After high-fat diet feeding for 16 w,compared with Cd36fl/fl mice,Cd36lko mice displayed reduced body weight,lower serum TG levels,and decreased area under the insulin tolerance curve,suggesting that improved glucose and lipid metabolism in Cd36lko mice.Furthermore,liver HE staining,Oil Red O staining and quantitative determination of liver TG and TC content showed that hepatic steatosis in was significantly reduced Cd36lko mice.RNA high-throughput sequencing combined with bioinformatics analysis found that the hepatic expression of DNL-related genes such as SREBP1,PPARy,ACLY,ACC and FASN were markly decreased in the Cd36lko mice.Meanwhile,Q-PCR and Western blotting analysis confirmed that hepatic mRNA and protein levels of SREBP1 pathway were reduced in the Cd36lko mice compared with Cd36fl/fl mice.These suggest that CD36 deficiency may reduce hepatic lipogenesis.In vitro cell experiments also found that CD36 overexpression stimulated insulin-induced cellular lipid accumulation and activation of the SREBP1 pathway,while the CD36 deficiency showed the opposite effects.These data suggest that CD36 promotes insulin-mediated lipogenesis,which contributes to lipid deposition in liver cells.PART Ⅲ:The liver endoplasmic reticulum and Golgi components were isolated from Cd36fl/fl and Cd36lko mice.In the endoplasmic reticulum fractions,the protein levels of SREBP1 were similar between Cd36fl/fl and Cd36lko mice,but in the Golgi fractions,there was a decrease of SREBP1 in Cd36lko mice compared with that in the Cd36fl/fl mice.Isolation of nuclear and cytoplasmic proteins revealed that nuclear SREBP1 was significantly increased in CD36OE cells.Immunofluorescence staining showed that the nuclear and Golgi localization of SREBP1 and the Golgi localization of SCAP were decreased in primary hepatocytes from Cd36lko mice,while CD36OE showed the opposite effects.To directly confirm CD36 regulates SREBP1 cleavage,we introduced exogenous HA-FL-SREBP1 into CD36OE and NC cells.Western blotting showed that FL-SREBP1 was similarly overexpressed in CD36OE and NC cells,but the expression of mature form of SREBP1 was significantly increased in CD36OE cells.These suggest that CD36 is involved in regulating the proteolytic processing of SREBP1.Co-immunoprecipitation and PLA demonstrated that CD36 interacted with INSIG2 in CD36OE cells.Furthermore,PLA signals for SCAP-INSIG2 interaction were reduced in CD36OE cells,indicating that CD36 competitively inhibited the binding of SCAP to INSIG2.CD36 knockdown inhibited the activation of SREBP1 pathway in HepG2 cells,while INSIG2 knockdown relieved the inhibitory effect of CD36 knockdown on SREBP1 pathway.Likewise,SREBP1/SCAP complex inhibitors 25-HC or betulin reversed CD36 overexpression-induced SREBP1 cleavage and lipogenic gene expression.CONCLUSION:CD36 is up-regulated by nutritional signals,and promotes hepatic de novo lipogenesis through mediating SREBP1 processing by interacting with INSIG2,CD36 is up-regulated by nutritional signals,and by binding to INSIG2,thereby promoting the occurrence and development of NAFLD.Targeting hepatocyte CD36 may be a potential strategy for the treatment of NAFLD.