Study On The Effect Of Hepatic FAT/CD36 Palmitoylation On Its Localization And Function In Mitochondria

Objective: Nonalcoholic fatty liver disease(NAFLD)is a clinicopathological syndrome characterized by excessive accumulation of fat in hepatocytes except for alcohol and other definite liver injury factors.It has high morbidity and does great harm to human health,and there is no effective treatment at present.During the pathogenesis of NAFLD,the imbalance of fatty acid metabolism homeostasis in liver is the most critical point.There are three main sources for fatty acids in the liver: food intake,adipose tissue breakdown,and de novo synthesis.At the same time,there are four ways for fatty acids in the liver: β-oxidation,synthesis of very low density lipoprotein(VLDL)and secretion outside the liver,synthesis of triglyceride(TG)for storage,and synthesis of other lipids.Impaired mitochondrial fatty acid oxidation(FAO)in hepatocytes leads to excessive accumulation of lipids and production of reactive oxygen species(ROS)and oxidative damage,which is an important mechanism for the formation of NAFLD.Fatty acid translocase(cluster of differentiation 36,FAT/CD36)is a transmembrane glycoprotein that promotes the uptake of long chain fatty acids(LCFAs)and is expressed on the surface of many cells.The expression of FAT/CD36 in liver was positively correlated with the occurrence and development of NAFLD.Recent studies have found that FAT/CD36 is also involved in fatty acid oxidation,beyond its function of LCFA uptake.The function of FAT/CD36 is related to its localization in cells.Studies have found that FAT/CD36 also exists in the mitochondria of skeletal muscle cells and is related to muscle energy consumption during exercise.Protein palmitoylation is a reversible post-translational lipid modification that is generally thought to regulate the subcellular localization of proteins.Our previous study found that palmitoylation of FAT/CD36 can increase the distribution of FAT/CD36 in the cell membrane of hepatocytes and promote fatty acid uptake,resulting in hepatic steatosis.De-palmitoylation of FAT/CD36 can reduce fatty acid uptake on the one hand,and promote mitochondrial fatty acid oxidation on the other hand,but the specific mechanism needs to be further studied.In this study,we aimed to investigate the effect of FAT/CD36 palmitoylation on its localization in mitochondria and its role in fatty acid oxidation in hepatocytes,so as to provide a theoretical basis for the prevention and treatment of NAFLD.Methods: Part 1: The relationship between palmitoylation of FAT/CD36 and NAFLD.(1)Eight-week-old male C57BL/6J mice were fed a high-fat diet(HFD)or normal chow diet(NCD)for 16 weeks to construct a NAFLD mouse model.The pathological changes of mouse liver were observed by hematoxylin & eosin(HE)staining,and the content of TG in mouse liver was detected by kit.The expression levels of FAT/CD36 in mouse liver were detected by immunohistochemistry(IHC)and western blotting(WB).Resin-assisted capture of S-acylated proteins(Acyl-RAC)combined with immunoblotting was used to assess the level of palmitoylation of FAT/CD36 in the livers of NCD or HFD mice.(2)FAT/CD36-KO mice were injected with Adreno-Associated Virus(AAV2/8)empty vectors(NC),AAV2/8 vectors containing wild-type FAT/CD36(WT-CD36),or AAV2/8vectors containing palmitoylation site-mutated FAT/CD36(AA-SS)in the tail vein and kept on a HFD for 8 weeks.HE staining was used to observe the pathological changes of the mouse liver,and a kit was used to detect the TG content of the mouse liver.Lentiviral vectors containing wild-type FAT/CD36(WT-CD36)and palmitoylation site-mutated FAT/CD36(AA-SS)were transfected to Hep G2 and Huh7 cells for the creation of stable cell lines.The accumulation of lipid droplets in cells was observed by Bodipy staining.(3)The ratio of reduced glutathione(GSH)to oxidized glutathione(GSSG)in mouse liver(GSH/GSSG)and the content of GSH were detected by kits.Levels of reactive oxygen species(ROS)and the activity of mitochondrial respiratory chain complexes I-IV in cells were detected with kits.Part 2: Effects of FAT/CD36 palmitoylation on its localization and function in mitochondria of hepatocytes.(1)The expression of CPT1 A in cells was analyzed by western blot.CPT1 activity and FAS activity in cell homogenates were detected spectrophotometrically.The m RNA levels of ACC and FAS were detected by real-time PCR.The content of malonyl-Co A(malonyl Co A)in mouse liver was detected by LC-MS.(2)Mitochondrial proteins and total proteins were isolated.The content of FAT/CD36 in mitochondria was analyzed by immunoblotting,and the expression of total FAT/CD36 in each group was detected.Immunofluorescence staining combined with confocal microscopy was used to observe the co-localization of FAT/CD36 and mitochondria.FAT/CD36 was labeled by colloidal gold,and the localization of FAT/CD36 in mitochondria was observed under electron microscope.(3)The contents of fatty acid metabolism intermediates acyl-Co A and acylcarnitine in Hep G2 cells were detected by liquid Chromatograph Mass Spectrometer(LC-MS).(4)Using XFe24 extracellular flux analyzer(seahorse)to detect mitochondrial oxygen consumption in hepatocytes when different substrates(palmitate or palmitoyl-Co A)were administered.Part 3: The mechanism by which reduction of FAT/CD36 palmitoylation promotes fatty acid oxidation in hepatocytes.(1)Total intracellular and mitochondrial proteins were isolated and the expression of each isoform of long-chain acyl-Co A synthetases(ACSL)was analyzed by immunoblotting.ACSL1 was labeled with colloidal gold,and the localization and content of mitochondrial ACSL1 in mouse liver were observed under electron microscope.The transcriptional level of intracellular ACSL1 was detected by real-time PCR.WT-CD36 and AA-SS-Hep G2 cells were treated with cycloheximide(CHX)(50 μg/ml)for various times(0,6,12,18 and 24 hours)and total cell lysates were harvested and analyzed by immunoblotting.Hep G2 cells were transfected with ACSL1-si RNA and the mitochondrial oxygen consumption of palmitate in the cells was detected.(2)Immunofluorescence staining combined with confocal microscopy was used to observe the co-localization of FAT/CD36 and ACSL1.Coimmunoprecipitation(CO-IP)was used to analyze the interaction between FAT/CD36 and ACSL1.In situ proximity ligation assay(PLA)was used to further verify the interaction between FAT/CD36 and ACSL1.(3)Cells were pulsed with green BODIPY C16 to track the localization of fatty acids to mitochondria.The AA-SS-K164 A mutant(the lysine residue 164(Lys-164)of AA-SS-CD36 was replaced by alanine,so that CD36 loses fatty acid binding activity)was transfected into Hep G2 cells,and the total and mitochondrial expression of FAT/CD36 was detected.The mitochondrial oxygen consumption in cells was detected with palmitate as the substrate.Results: Part 1: The relationship between palmitoylation of FAT/CD36 and NAFLD.(1)Compared with normal chow diet(NCD)-fed mice,C57BL/6J mice fed with high-fat diet(HFD)developed typical hepatocyte ballooning and increased hepatic TG content.FAT/CD36 expression levels were significantly increased in the liver of HFD-fed mice.Resin-assisted capture of S-acylated protein(Acyl-RAC)combined with WB showed that the level of FAT/CD36 palmitoylation was increased in HFD-fed mice.(2)Compared with WT-CD36 mice,hepatic lipid accumulation and TG content was significantly reduced in AA-SS mice.Furthermore,palmitate-induced lipid droplet accumulation was reduced in AA-SS-Hep G2 cells compared to WT-CD36 mice.Treatment with the palmitoylation inhibitor2-bromopalmitate(2BP)also reduced lipid droplet accumulation in Hep G2 cells.(3)Compared with NCD-fed mice,the ratio of GSH/GSSG and the content of reduced glutathione were decreased in the liver of HFD-fed mice.Compared with WT-CD36 mice,the GSH/GSSG ratio and the content of reduced glutathione in the liver were increased in AA-SS mice.Furthermore,compared with WT-CD36-Hep G2 cells,ROS production was reduced in AA-SS-Hep G2 cells and the activity of mitochondrial respiratory chain complexes I-IV was increased.Part 2: Effects of FAT/CD36 palmitoylation on its localization and function in mitochondria of hepatocytes.(1)The protein expression of fatty acid oxidation rate-limiting enzyme CPT1 A did not change in WT-CD36 and AA-SS group,while the activity of CPT1 A increased in AA-SS cells.The m RNA levels of ACC and FAS in the liver were decreased in AA-SS mice compared with WT-CD36 mice.FAS activity was also reduced in AA-SS-Hep G2 cells.In addition,the content of malonyl-Co A was also decreased in the liver of AA-SS mice.These data suggest that reduction of FAT/CD36 palmitoylation upregulates hepatic fatty acid oxidation capacity and reduces hepatic lipogenesis.(2)The mitochondrial localization of FAT/CD36 was increased in FAT/CD36 palmitoylation site-mutated cells(AA-SS)and in cells treated with2-bromopalmitate(2BP),an inhibitor of palmitoylation,while the total expression of FAT/CD36 remained comparable in the respective groups.Furthermore,confocal microscopy analysis showed that the co-localization of FAT/CD36 and mitochondria was significantly increased in the AA-SS group of Hep G2 and Huh7 cells compared with the WT-CD36 group.Likewise,in AA-SS mice,the co-localization of FAT/CD36 and mitochondria was significantly increased compared to the WT-CD36 group.Immunogold labeling of FAT/CD36 visualized via electron microscopy analysis confirmed that FAT/CD36 accumulated on the mitochondrial membrane of AA-SS-Hep G2 cells.These data suggest that FAT/CD36 is present on the mitochondrial membrane in hepatocytes and that reduction of FAT/CD36 palmitoylation significantly promotes the localization of FAT/CD36 to mitochondria.(3)Liquid chromatography–mass spectrometry(LC–MS)results showed significant differences in the content of intermediates of fatty acid oxidation between the WT-CD36 and AA-SS groups.The quantified total short/medium-chain acyl-Co A(C2-C10)levels were decreased,while the contents of total long-chain acyl-Co A(C12-C20)were increased in the AA-SS group.Moreover,the decrease in short/medium-chain acyl-Co A was dominated by C3,C4,C5 and C6acyl-Co A,and the increase of long-chain acyl-Co A was mainly due to C16,C18 and C20 acyl-Co A.Consistently,the contents of total acyl-carnitine were increased in AA-SS group,in which C16,C18 and C20 acyl-carnitine levels were significantly increased.These findings suggest that reduction of FAT/CD36 palmitoylation promotes the production of long-chain acyl-Co A and acyl-carnitine in hepatocytes.(4)Compared with the WT-CD36 group,the mitochondrial oxygen consumption(OCR),maximal respiration and ATP production were higher in the AA-SS group when palmitate was used as a substrate.When palmitoyl-Co A was used as the substrate,mitochondrial oxygen consumption(OCR),maximal respiration and ATP production did not change in the two groups.Reduction of FAT/CD36 palmitoylation increased palmitate-supported OCR but not palmitoyl-Co A-supported OCR,suggesting that the effect of depalmitoylated FAT/CD36 on fatty acidβ-oxidation is located upstream of acyl-Co A.Part 3: The mechanism by which reduction of FAT/CD36 palmitoylation promotes fatty acid oxidation in hepatocytes.(1)ACSL is a key enzyme for intracellular synthesis of long-chain acyl-Co A.Compared with the WT-CD36 group,the total intracellular content of ACSL1 was increased in the AA-SS-Hep G2 cells and AA-SS mice,while the levels of ACSL3-6 were comparable between the two groups.Likewise,the mitochondrial content of ACSL1 in AA-SS cells and mouse liver was increased compared with the WT-CD36 group,and the mitochondrial expression levels of ACSL3-6 were not significantly different between the two groups.The m RNA levels of ACSL1 did not differ between the WT-CD36 and AA-SS groups,but delayed degradation of ACSL1 protein was observed in AA-SS cells,suggesting that reduction of FAT/CD36 palmitoylation might regulate ACSL1 expression at the post-transcriptional level.Hep G2 cells was transfected with ACSL1-si RNA and the expression of ACSL1 was reduced.Knockdown of ACSL1 in AA-SS-Hep G2 cells significantly reduced palmitate-dependent mitochondrial oxygen consumption,maximal respiratory capacity and ATP production.This indicates that ACSL1 is necessary for the increased mitochondrial fatty acid β-oxidation induced by the reduction of FAT/CD36 palmitoylation.(2)Confocal microscopy analysis showed that the co-localization of FAT/CD36 and ACSL1 was increased in AA-SS cells compared with the WT-CD36 group.Co-immunoprecipitation analysis showed that reduction of FAT/CD36 palmitoylation promoted the interaction between FAT/CD36 and ACSL1.PLA results showed that AA-SS cells exhibited stronger positive fluorescent signals,further indicating that the reduction of FAT/CD36 palmitoylation promoted the interaction between FAT/CD36 and ACSL1.(3)We pulsed WT-CD36 and AA-SS cell lines with green BODIPY C16,a fatty acid analog bound with a BODIPY fluorophore,to track fatty acids in relation to mitochondria.The results showed that fatty acids seldom colocalized with mitochondria in WT-CD36 cells,while in AA-SS cells,fatty acids mostly colocalized with mitochondria,indicating that reduction of FAT/CD36 palmitoylation increased fatty acid transport to mitochondria.Lentiviral vectors containing AA-SS-K164 A mutant was transfected into Hep G2 cells.Compared with AA-SS cells,the expression of total intracellular FAT/CD36 and mitochondrial FAT/CD36 were unchanged in AA-SS-K164 A cells.Fatty acid trafficking to mitochondria was reduced in AA-SS-K164 A cells compared with AA-SS cells.Mitochondrial oxygen consumption,maximal respiration and ATP production in AA-SS-K164 A cells were lower than those in AA-SS cells.These data suggest that depalmitoylated FAT/CD36 on the mitochondria continued to function by transporting fatty acids and that Lys-164 of FAT/CD36 is involved in binding fatty acids.FAT/CD36 transports fatty acids to ACSL1.The enhanced interaction between depalmitoylated FAT/CD36 and ACSL1 contributes to fatty acid trafficking to mitochondria,thereby promoting fatty acid oxidation.Conclusion: Reduction of FAT/CD36 palmitoylation can promote fatty acid β-oxidation in hepatocyte and reduce lipid accumulation in the liver of NAFLD mice.Reduction of FAT/CD36 palmitoylation promotes the distribution of FAT/CD36 to mitochondria.The increased FAT/CD36 interacts with ACSL1 and acts as a molecular bridge to transport LCFA to ACSL1,thereby generating more acyl-Co A,and subsequently entering mitochondria for β-oxidation.Since palmitoylation controls the distribution and function of FAT/CD36,targeting the palmitoylation site of FAT/CD36 may be a potential strategy for the treatment of NAFLD and other metabolic diseases.