The Impact Of Inflammatory Stress On FAT/CD36Expression And Lipid Accumulation In Livers

Part one The impact of inflammatory cytokines on FAT/CD36expression and lipid accumulation in HepG2cellsObjectives: Metabolism diseases including non alcoholic fatty liverdisease (NAFLD) and insulin resistance has a deep connection with chronicinflammation. Fatty acid translocase (FAT/CD36) plays an important role incellular fatty acid transport. Whether inflammation results in fatty acidtransport into the cells through influencing the FAT/CD36expression is notclear. In vitro study, inflammatory factors tumor necrosis factor-α (TNF-α)and interleukin-6(IL-6) were used to treat human hepatocarcinoma cell lineHepG2cells loaded by fatty acids, then we investigated the impact ofinflammation on the expression of FAT/CD36and lipid accumulation andwhether there was cell damage in cells.Methods: HepG2cells were cultured with palmitate of0、0.04、0.08、0.16or0.32mmol/L for24hours. The protein and mRNAexpression of FAT/CD36were detected by Western blot and RT-PCR, respectively. The intracellular lipid droplet formation was determined byOil red O staining. Then the HepG2cells were cultured and treated withpalmitate at concentration of0.04mmol/L combined with TNF-α (25ng/ml)or IL-6(20ng/ml) for24hours. The effect of inflammatory cytokines onthe protein levels of FAT/CD36in HepG2cells was also investigated. Oilred O staining was used to determine the intracellular lipid dropletformation. The intracellular triglyceride (TG) and free fatty acid (FFA)were measured by enzymatic assay and ELISA, respectively. For furtherstudy, we detected the levels of endoplasmic reticulum stress (ERS) andoxidative stress in HepG2cells.Results: Palmitate loading dose-dependently increased the protein andmRNA expression of FAT/CD36and intracellular lipid levels. Theinflammatory cytokines further increased the protein expression ofFAT/CD36in HepG2cells loaded by palmitate. Oil red O staining, TGdetection and FFA assay showed that the inflammation raised intracellularlipid accumulation in HepG2cells. Inflammatory stress up-regulated themRNA expression of genes related to ERS and raised the contents of H2O2and MDA in HepG2cells.Conclusions: Inflammatory cytokines up-regulated the expression ofFAT/CD36in HepG2cells loaded by palmitate and promoted the lipidaccumulation in cells. Inflammatory stress also resulted in ERS andoxidative stress of HepG2cells. Part two The impact of inflammation on FAT/CD36expression andlipids accumulation in livers of miceObjectives: Chronic systemic inflammation has a deep connectionwith metabolism diseases and excessive lipids deposit in tissues. In vivostudy, we established a chronic systemic inflammation model throughcontinuous subcutaneous injection of10%casein to C57BL/6J mice in14weeks. Then we observed the levels of FAT/CD36expression and lipidaccumulation in livers and levels of endoplasmic reticulum stress (ERS) andoxidative stress in livers under inflammatory state, to explore the impact ofinflammation on FAT/CD36expression and lipid accumulation in liversand liver damage.Methods: Twenty-four male C57BL/6J mice aged eight weeks wererandomly divided into four groups:(1) N group: normal diet plus salineinjection;(2) NC group: normal diet plus0.5ml10%casein infection;(3) Hgroup: western diet (60%calorie from fat) plus saline injection;(4) HCgroup: western diet (60%calorie from fat) plus subcutaneous infections of0.5ml10%casein. Mice were killed after fourteen weeks and serum ortissues were collected. Then we detected the levels of serum amyloid A (SAA), TNF-α and FFA by ELISA assays. Real-time quantitative PCR(RT-PCR) was used to detect the mRNA expression of TNF-α andmonocyte chemoattractant protein-1(MCP-1) in livers. Then we usedWestern blot and immunohistochemistry staining to observe the proteinexpression of FAT/CD36in livers under inflammatory state. The contents ofTG and FFA in livers were measured by enzymatic and ELISA method,respectively. For further study, we detected the mRNA expression of genesrelated to ERS and contents of hydrogen peroxide (H2O2) andmalondialdehyde (MDA) in livers.Results:(1) Casein injection increased serum levels of SAA andTNF-α with or without high-fat diet.(2) Both casein injection and high-fatdiet increased serum levels of FFA.(3) In livers, the mRNA expression ofinflammatory factor TNF-α and MCP-1in NC group, H group and HCgroup was up-regulated.(4) Inflammation up-regulated the proteinexpression of FAT/CD36in livers significantly.(5) Inflammation promotedthe lipid accumulation in livers as well.(6) The mRNA expression of genesrelated to ERS and contents of H2O2and MDA in livers were allup-regulated under inflammation plus high-fat diet.Conclusions: Inflammation of C57BL/6J mice was produced bysubcutaneous injection of10%casein fourteen weeks, and it means that theinflammatory model is successfully established. Inflammation raised FFAlevel in serum compared with normal diet but not compared with high-fat diet. We supposed that in inflammatory state, the mice had extremely lipidmetabolic dysfunction which resulted in lipids ectopic deposition, so theserum FFA increase was not obvious, on the contrary. Systemic chronicinflammation caused the lipid metabolism accumulation in liversup-regulating the expression of FAT/CD36expression. The abnormal lipiddeposit in livers caused ER stress and oxidative stress.