The Molecular Mechanisms Of CD36 Palmitoylation In Hepatocyte Apoptosis

Objective: Non-alcoholic fatty liver disease(NAFLD) is the most common form of liver disease all over the world. Progression to more aggressive forms of liver injury, such as nonalcoholic steatohepatitis(NASH) and cirrhosis, occurs in less than a third of affected subjects.However, the mechanisms are still being elucidated. Apoptosis is a common feature of viral, cholestatic, fatty, and alcoholic liver disease,human data and both in vivo and in vitro models demonstrate that cell death, particularly apoptosis, is increased in NAFLD and NASH patients,suggesting that it is crucial in disease progression. CD36 also named fatty acid translocase(FAT), belongs to the scavenger receptor family, functions as an important fatty acid transporter and involved in NASH. The expression of CD36 in liver is increased and positively related to the pathologic degree of NASH, but the dificiency of CD36 also promotes the development of NASH. Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid,palmitate, to cysteine residues of substrate proteins through a labile thioester bond, and it has been reported to be influenced cell apoptosis.CD36 also can be palmitoylated, but there was no report whether CD36 palmitoylation is related to cell apoptosis. This study was undertaken to investigate whether CD36 palmitoylation is invoved in hepatocyte apoptosis, as well as explore the molecular mechanism preliminarily.Methods: In Part one, male C57BL/6J mice were fed with nornal chow diet(NCD) or high fat diet(HFD) for 22 weeks to establish NASH model. In Part two, male C57BL/6J mice were all fed with high fat diet and administrated with lentivirus via tail injection to overexpression wild-type CD36(wt-CD36) and palmitoylation sites mutant CD36(AA-SS). Hep G2 cells were transfectioned with the same lentivirus to establish cell lines,which stably expressed wt-CD36 and AA-SS. 2-bromopalmitate(2BP) was used to inhibit CD36 palmitoylation.HE stain and Oil Red O stain were used to evaluate the inflammation grade and sclerosis grade.TUNEL stain and Caspase-3 detection were used to test apoptosis grade. CD36 palmitoylation was measured by Acyl-Biotinyl exchange(ABE) assay. The expression of Bax and Bcl-2 was determined by real-time PCR.,Phosphorylated and total protein levels of Jun N-terminal kinase(JNK) and TNF-α was detected by Western blotting.CD36-TLR4 copolymer formation was detected by co-immunoprecipitation(CO-IP). Lipid rafts were isolated by DRMs method and then subjected to western blot to analysis the ratio of CD36 in DRMs and non-DRMs.Results: HE and Oil Red O stain showed that mice in HFD exhibited much more ballooning degeneration, inflammatory infiltration and lipid accumulation in liver compared with NCD. TUNEL positive cells and Caspase-3 activity were increased in HFD, and the palmitoylation of CD36 was increased too(P<0.05). Western blotting showed that, TNF-α and p-JNK/JNK were increased in HFD. While the employment of lentivirus(AA-SS) and palmitoylation inhibitor 2BP changed the circumstances.Compared with wt-CD36/PA+DMSO, in AA-SS/PA+2BP group there were less TUNEL positive cells and lower Caspase-3 activity; the expression of Bcl-2 was upregulated; less CD36 was located in lipid raft. Secretion of TNF-α, phosphorylation of JNK and CD36-TLR4 copolymer were significantly decreased(P<0.05). All this contributed to improve liver ballooning degeneration and lipid accumulation.Conclusion: High-fat-diet increased hepatocyte apoptosis and CD36 palmitoylation. Inhibition palmitoylation of CD36 decreased CD36 located in lipid raft, CD36-TLR4 copolymer formation as well as JNK phosphorylation and TNF-αsecretion, all contributed to reduce apoptosis and protect mice from NASH.