The Study On The Negative Regulation Of Fatty Acid Translocase CD36 On Lipophagy And Its Molecular Mechanism In Hepatocytes

Objective Non-alcoholic fatty liver disease(NAFLD)is the most common chronic liver diseases in the world.It is a metabolic stress liver injury characterized by abnormal accumulation of lipid in liver tissue.Lipophagy is an autophagic process that selectively recognizes lipid,degrades lipid,produces free fatty acids and promotes β-oxidation.It is an important process to regulate lipid metabolism and prevent lipid over-accumulation.Inhibition of autophagy weakens lipid degradation in the liver,significantly increases triglyceride content in hepatocytes,and promotes the occurrence and development of hepatic steatosis.Cluster of differentiation 36(CD36),also named fatty acid translocatase(FAT),is a transmembrane transport glycoprotein facilitating the uptake of long-chain fatty acid and involved in lipid metabolism.CD36 also plays an important role in the regulation of intracellular signal transduction,and is involved in the storage and utilization of fatty acid,glucose metabolism and metabolic inflammation.Recent studies have found that CD36 can affect the fatty acid β-oxidation and regulate the triglyceride content in cells through AMPK signaling pathway.However,little is known about the relationship between CD36 and lipophagy.This study aimed to investigate whether CD36 regulates lipophagy in hepatocytes,thereby affecting the development of NAFLD,and its possible molecular mechanism.Methods Male C57BL/6J mice aged 6-8 weeks with similar body weight were randomly assigned into normal chow diet(NCD)group and high-fat diet(HFD)group,six mice in each group.Mice were fed with NCD and HFD for 14 weeks respectively to obtain NAFLD mouse model.The lipid deposition in liver tissue was detected by haematoxylin & eosin(HE)staining.Triglyceride and free fatty acid(FFA)contents in liver tissue were detected.The protein expressions of CD36,LC3Ⅱ and p62 in liver tissue were detected by western blotting.HepG2 and Huh7 cells were transfected with small interfering RNA(siRNA)to knockdown CD36 expression,or infected with lentivirus expressing CD36 to obtain CD36 overexpression stable cell line.Autophagy was detected by western blotting,monodansylcadaverine(MDC)staining and immunofluorescence staining after changing CD36 expression.The effect of CD36 expression on autophagic flux was detected after treatment with autophagy inhibitor chloroquine or transfection with autophagic double-labeled adenovirus(mRFP-GFP-LC3).Wild-type(WT)mice and CD36 knockout(CD36-/-)mice created on a C57BL/6J background aged 6-8 weeks were randomly assigned into NCD and HFD group respectively,six mice in each group.Mice were fed with NCD and HFD for 14 weeks respectively.Autophagy in liver tissue was detected.CD36-/-mice aged 6-8 weeks were injected with GV341 lentivirus empty vector or GV341 lentivirus expressing CD36 gene via tail vein and kept on HFD for 8 weeks.The autophagic vacuoles in liver tissue were observed by transmission electron microscopy.Lipophagy of hepatocytes after knocking down CD36 expression was observed with the colocalization of lipid with LC3 or lysosome.Triglyceride content was measured by enzymatic assay.Oxygen consumption rate was detected with the cell mito stress test.Carnitine palmitoyltransferase 1a(CPT1a)expression was detected by western blotting and quantitative PCR(qPCR).AMPK activation in hepatocytes and liver tissue were tested by western blotting after down-regulating CD36 expression.Autophagy in hepatocytes was detected and intracellular lipid was observed by bodipy staining after treatment with AMPK inhibitor compound C.Several autophagy-related genes(ATGs)were screened by qPCR.The total protein and phosphorylation levels of ULK1 and Beclin1 were detected by western blotting.Results Compared with NCD group,the hepatic steatosis was obvious in HFD-treated mouse liver,and the contents of triglyceride and FFA in liver tissue were significantly increased.Increased CD36 expression was coupled with a decreased LC3Ⅱ and an accumulation of p62 in the mouse livers of HFD group.Autophagy was induced after knocking down CD36 expression in HepG2 and Huh7 cells and was inhibited after CD36 overexpression.Autophagic flux was confirmed to be increased after knocking down CD36 expression by treatment with chloroquine and autophagic double-labeled adenovirus transfection experiments.In in vivo experiment,deficiency of CD36 in mice significantly increased autophagy in liver tissue,and reconstruction of CD36 expression in CD36-/-mice reduced autophagy in liver tissue.CD36 knockdown in hepatocytes increased lipophagy and β-oxidation,reduced triglyceride content,and promoted CPT1 a expression.Lipophagy and β-oxidation were attenuated after inhibiting autophagy.AMPK activation in hepatocytes or liver tissue was increased after down-regulating CD36 expression.Autophagy was decreased and lipid content was increased again after inhibiting AMPK.The knockdown of CD36 in hepatocytes enhanced the protein levels of ULK1,p-ULK1(Ser555),Beclin1 and p-Beclin1(Ser93),decreased the expression of FOXO1 and enhanced the level of p-FOXO1,while the levels of mTOR,p-mTOR and ATG7 did not change significantly.ULK1 and p-ULK1(Ser555)levels were decreased after inhibiting AMPK.Conclusion CD36 expression negatively regulates autophagy in vivo and in vitro.Knockdown of CD36 in hepatocytes increases lipophagy and β-oxidation through an AMPK-ULK1/Beclin1 pathway,which contributes to the steatosis alleviation.This suggests that attenuating CD36 expression,which enhances clearance of fatty acid by induction of lipophagy in addition to the reduction of fatty acid uptake,could be a potential therapeutic strategy for fatty liver diseases,as well as other metabolic disorders.