The Effects Of CD36 Palmitoylation On Metabolic Inflammation And Insulin Sensitivity In Adipose Tissue

OBJECTIVE: Fatty acid translocase CD36(FAT/CD36) is a transmembrane glycoprotein and is closely associated with Long chain fatty acids transshipment,which is extremely important in lipid and glucose metabolism. Our previous studies have shown that high fatty diet can increase the expression of CD36 and inflammatory cytokines and decreased insulin sensitivity. Although inhibition of CD36 expression can improve insulin sensitivity, it also may lead to abnormal lipid metabolism in vivo steady-state. Thus, we have to think out another way to mediate inflammation and insulin resistance caused by CD36. S-palmitoylation, a common and the only reversible post-translational protein modification, is recently recognized as a fine-tuning of regulating membrane protein localizations and its functions. Here we investigate the effects of CD36 palmitoylation on metabolic inflammatory and insulin resistance in adipose tissue, which provided experiment foundation for our future depth study.METHODS:1. C57BL/6J mice were choosed randomly and divided into two groups, respectively fed normal diet(NCD) and high fat diet(HFD).2. C57BL/6J mice were randomly divided into three groups,then lentiviral which constructs empty vector(NC), wide-type CD36 and AA-SS CD36 respectively were injected into mice(2×105 pfu/mice), all mice fed a high-fat diet(HFD). After 8 weeks, the CD36 palmitoylation degree was detected by IP-ABE in both groups, expression of CD36, inflammatory factor TNF-α,JNK,IL-6,F4/80,NF-κB, AKT and IRS-1 protein which were involved in insulin signaling pathway were also measured in mice adipose tissue by RT-PCR and western blot. HE staining in WAT sections was used to abserve the adipose tissue.3. 3T3-L1 cells were transfected by lentiviral which constructs which constructs empty vector(NC), wide-type CD36 and AA-SS CD36 respectively, and selected by puromycin. 3T3-L1 cells were treated with PA or with palmitoylation inhibitor 2-Bromopalmitate(2-BP) and Cerulenin(CER). The palmitoylation of CD36 was determined by ABE. Lipid rafts, endoplasmic reticulum and mitochondria were isolated by ultracentrifugation, western blot detected the efficiency of 3 groups of CD36 expression level and CD36 into lipid rafts. Lipid droplets stained with BODIPY and immune co-precipitation method was used to extract lines CD36/TLR4 cpolymers with western blot detection.RESULTS:1. The palmitoylation of CD36 and expression of TNF-α, JNK was increased while insulin signaling pathway protein AKT and IRS-1 were decreased in HFD fed.2. In mice adipose tissue, the expression of CD36 in WT-CD36 and AA-SS group both increased significantly, while the palmitoylation of CD36 was reduced in AA-SS group. An increasing body weight was observed in WT-CD36 group accompanied by glucose intolerance, insulin insensitivity, inflammation factors(TNF-α, IL- 6, F4/80, JNK, nf-kappa B) increasing and insulin signaling pathway phosphorylated protein AKT and IRS-1 reduced. AA-SS group showed obviously improvement both in the weight gain, insulin sensitivity, glucose intolerance, inflammation factors(TNF-α, IL- 6, F4/80, JNK, nf-kappa B) and the expression of p-AKT and p-IRS1 protein 3. In cell lines, the efficiency of CD36 into lipid rafts was increased,alone with the formation of lipid droplets and CD36/TLR4 copolymer, while these were redused in AA-SS cells. In 3T3-L1 cells, palmitic acid increased the efficiency of CD36 into lipid rafts, while it decreased when cells treated with palmitoylation inhibitor 2-BP and CER. Besides, cells treated with palmitic acid combine inhibitor 2-BP and CER redused the formation of lipid droplets and increased the expression of CD36 in mitochondria and endoplasmic reticulum.CONCLUSION: Our study suggests that high fat diet contributes to the development of metabolic inflammation, insulin sensitivity and CD36 palmitoylation in adipose tissue. Inhibition of CD36 palmitoylation reduces metabolic inflammation and improves insulin sensitivity both in adipose tissue and 3T3-L1 cells, suggesting that the palmitoylation of CD36 promotes inflammation and decreases insulin sensitivity.