High Fat And Inflammation Increases Hepatic CD36Translational Efficiency Via Activation Of The Mtor Signalling Pathway

Objective: Non-alcoholic fatty liver disease (NAFLD) has becomeone of the major metabolic disorders that harm human’s health, but thepathogenesis is not fully understood. Both high fat and inflammation areindependent risk factors for the development of NAFLD. The class Bscavenger receptor CD36is known to facilitate long-chain fatty acid andoxidised low-density lipoprotein uptake. Hepatic CD36expression isclosely associated with hepatic steatosis. Mammalian target of rapamycin(mTOR) is a highly conserved serine/threonine kinase that plays a crucialrole in the regulation of cell growth, proliferation, and protein translation.This study was undertaken to investigate whether high fat and inflammationincreases hepatic CD36translational efficiency via activation of the mTORpathway, resulting in an increase of CD36protein expression and lipidaccumulation.Methods: Human hepatoblastoma HepG2cells were treated withpalmitate, tumour necrosis factor alpha (TNF-) and interleukin-6(IL-6) invitro and C57BL/6J mice were fed with high fat diet or treated with casein subcutaneous injection in vivo to induce hepatic steatosis or inflammation.HepG2cells were incubated for24hours in medium containing0.2%BSA(Ctrl1) or0.2%BSA plus0.08mmol/L palmitate (Palmitate) or0.2%BSAplus0.04mmol/L palmitate (Ctrl2) or0.2%BSA plus0.04mmol/Lpalmitate and25ng/mL TNF-(TNF-) or0.2%BSA plus0.04mmol/Lpalmitate and20ng/mL IL-6(IL-6). Male C57BL/6J mice were fed withnormal chow diet (NCD) or high fat diet (HFD) or normal chow diet plussubcutaneous injection of0.5mL10%casein (NCD+Casein) for14weeks.Lipid droplet accumulation was visulised using oil red O staining.Intracellular free fatty acids (FFA) and triglycerides (TG) were measuredusing ELISA kit and enzymic assay, respectively. The mRNA expression ofCD36was detected using real-time PCR. The protein expression of CD36,phosphorylated and total protein levels of mTOR and its downstreamtranslational regulator including p70ribosomal protein S6kinase (p70S6K),eukaryotic initiation factor4E-binding protein1(4E-BP1), and eukaryoticinitiation factor4E (eIF4E) were detected by western blotting. CD36proteinstability was investigated using protein degeneration assay. CD36translational efficiency was detected using polysomal analysis. We aslodeterminated the effect of rapamycin, an mTOR-specific inhibitor, on theseindices in HepG2cells (10ng/mL) and livers of C57BL/6J mice (2mg/kgbody weight) under conditions of high fat or inflammatory stress.Results: Oil red O staining, quantitative assay of FFA and TG showed that high fat and inflammation aggravated lipid accumulation in HepG2cells and C57BL/6J mice (P<0.05). Western blotting and real-time PCRshowed that high fat and inflammation increased hepatic CD36proteinexpression (P<0.05) but had no effect on mRNA expression (P>0.05).Protein degradation assay revealed that high fat and inflammation had noeffect on CD36protein stability (P>0.05). Polysomal analysis indicatedthat high fat and inflammation significantly increased CD36translationalefficiency (P<0.05). Western blotting further showed that high fat andinflammation enhanced the phosphorylation of mTOR and its downstreamtranslational regulators including p70S6K,4E-BP1, and eIF4E (P<0.05).Rapamycin reduced the phosphorylation of mTOR pathway (P<0.05) anddecreased the CD36translational efficiency (P<0.05) and proteinexpression (P<0.05), resulting in the alleviation of lipid accumulation(P<0.05) in HepG2cells and livers of C57BL/6J mice even under high fatand inflammation.Conclusions: High fat and inflammation increases hepatic CD36translational efficiency via activation of the mTOR pathway, resulting in anincrease of CD36protein expression, therefore aggravates lipidaccumulation in HepG2cells and livers of C57BL/6J mice.