Palmitate Increase Lipid Uptake By Upgrating CD36 Level And Increasing Palmitoylation In HepG2 Cells

Objective With the popular trend of globalization of obesity and related metabolic syndrome, nonalcoholic fatty liver disease has become one of the most primary liver disease around the world to. Fatty acid translocase, as a cell membrane glycoprotein mediating long chain Fatty acids uptake, play an important role in the transmembrane transport of FFA. The aim of this article is to investigate the effects of palmitate on CD36 expression and modification after translation, and thus caused to intracellular lipid accumulation in HepG2 cells and explore the underlying mechanisms.Methods HepG2 cells were treated with different consentration of palmitate. The dual-luciferase assay was used in HepG2 cells to determine the activity of human CD36 promoter. Real-time PCR and western blotting were performed to examine the mRNA and protein levels of CD36. Polysome ribosome analysis were performed to detect CD36 CD36 translation efficiency. The rate of uptake of fluorescent-labeled fatty acid was examined using fluorescent microscope. Oil Red O staining and free fatty acids quantitative measurements were used to evaluate the lipid accumulation in cells. Furthermore, the siRNAs for human CD36 (CD36-siRNA) and NC-siRNA were transfected into cells to restrain CD36 expression, and the lipid accumulation in CD36-siRNA-HepG2 and NC-siRNA-HepG2 was analyzed. IP-ABE was performed to detect CD36 palmitoylation degree in normal control group and palmitate treated group. Rapid site mutation kit was used to constructed CD36 eukaryotic expression vector which palmitoylation modification sites were mutated, and they were packaged as lentiviruses. Empty vector and lentiviral expressing wide-type CD36, and AA-SS CD36 were incubated with HepG2 cells and the stable transfectants were selected with puromycin. The CD36 expression of cells were detected by flow cytometry and western bolt. CD36 palmitoylation degree were detected by IP-ABE. Western blot was used to detected CD36 level in cell membrane lipid rafts.The uptaking rate of fluorescent-labeled fatty acid was examined using fluorescent microscope and double-labled flow cytometry.Results The CD36 promoter luciferase reporter plasmid was successfully constructed. A relative high concentration of palmitate up-regulated CD36 promoter activities and CD36 mRNA, but low concentration of palmitate also promoted the expression of CD36 protein expression. Polysome ribosome analysis confirmed palmitate incrased CD36 translation efficiency. In palmitate-treated cells, more lipid droplets was observed and FFA contents were higher(240.17±19.83ng/mg)than that (144.60±53.52ng/mg) in control group (P<0.05). The rate of fatty acid uptake was also accelerated in palmitate-treated group. In addition, the mRNA and protein levels in CD36-siRNA-HepG2 were only 42% and 50% of NC-siRNA-HepG2 cells. In response to palmitate loading, CD36-siRNA-HepG2 showed less lipid droplets formation and lower levels of FFA contents (98.38±14.18ng/mg), compared with NC-siRNA-HepG2 (240.17±21.12ng/mg) (P<0.05). The rate of fatty acid uptake was also slower in CD36-siRNA-HepG2 than in NC group. We constructed CD36 palmitoylation site mutationed eukaryotic expression vector successfully and established wild-type CD36 and palmitoylation deficient CD36 stabled overexpressing cell model. Western blot and flow cytometry showed CD36 palmitoylated mutation did not impact its expression. While, IP-ABE confirmed CD36 palmacylated modification degree of AA-SS group reduced contrast with WT group, and CD36 in the cell membrane lipid rafts reduced, resulting in the alleviation of free fatty acid uptake and lipid accumulation.Conclusion Palmitate promotes the expression of CD36 at transcriptional and translational levels, resulting in excess intracellular lipid accumulation in HepG2 cells. Palmitoylation of CD36 affect its expression in subcellular organelles membranes and lipid accumulation. This provide clues to our treatment of non-alcoholic fatty liver disease.