The Mechanism Of NEFA-induced PERK-eIF2α Signaling Pathway To Regulate Lipid Metabolism In Bovine Hepatocytes

High levels of NEFA in the blood induced by NEB at the beginning of lactation is characteristic of dairy cows and can be used by other tissues for energy supply.However,elevated NEFA is also considered a critical pathogenic factor of metabolic diseases,including fatty liver and ketosis.Some researches showed that high plasma NEFA was a very critical inducement of the activation of ERS in early lactation.Emerging evidence also indicates that high concentrations of fatty acids can induce ERS in the bovine hepatocytes in vitro.Moreover,the protein kinase R-like endoplasmic reticulum kinase(PERK)branch of the endoplasmic reticulum stress(ERS)response plays an important role in lipid metabolism in hepatocytes.However,so far,the effects of activated PERK signaling on the lipid metabolism of hepatocytes in dairy cows remains unclear.Therefore,we hypothesized that the PERK branch may play a role in NEFA-induced lipid accumulation.Different concentrations of NEFA or GSK2656157(a novel catalytic inhibitor of PERK)were used to treat hepatocytes isolated from calves.The present study aimed to investigate this possibility and identify the underlying mechanism from multiple aspects of lipid metabolism(including lipid synthesis,lipid oxidation,and lipid transport).1.The optimal time point for NEFA to induce phosphorylation of PERK was 5 h.NEFA treatment increased the phosphorylation levels of PERK and e IF2α and promoted the protein and m RNA levels of Grp78,ATF4 and CHOP,indicating that NEFA can activate the PERKe IF2α signaling pathway.2.NEFA can also promoted TG accumulation in hepatocytes.Besides,the treatment with GSK2656157 significantly increased the NEFA-induced lipid accumulation,indicating that the activation of the PERK branch could effectively alleviate the accumulation of TG in hepatocytes induced by NEFA.3.NEFA treatment increased the protein and m RNA levels of SREBP-1c,SCD1,FASN,ACC,and ACL.Pretreatment with GSK2656157 alleviated the NEFA-induced increase in the protein abundance of SREBP-1c and the m RNA levels of SREBP-1c,SCD1,FASN,ACC,and ACL.These results indicate that the NEFA-induced PERK-e IF2α signaling pathway mediates an increase in lipid synthesis.4.NEFA treatment increased the protein and m RNA levels of PPARα,CPT1 A,CPT2,ACOX1,SIRT1,and PGC-1α.Pretreatment with GSK2656157 alleviated the NEFA-induced increase in the protein and m RNA levels of PPARα,CPT1 A,CPT2,and SIRT1.These results demonstrated that the PERK-e IF2α signaling pathway mediates the NEFA-induced increase in lipid oxidation.5.NEFA treatment increased the protein and m RNA levels of APOB,APOE,MTTP,CD36,LDLR,and L-FABP.GSK2656157 pretreatment increased the protein abundance of APOB and the VLDL content in supernatant.These results demonstrated that the NEFAmediated PERK-e IF2α signaling pathway can inhibit the assembly and secretion of VLDL.In conclusion,this study provided evidence that the NEFA-mediated PERK-e IF2αsignaling pathway can increase lipid de novo synthesis,stimulate lipolysis,but inhibit the assembly and secretion of VLDL,thereby regulating hepatocyte lipid metabolism on treatment with high NEFA concentrations.