| Ketosis in dairy cows is a commonly disease occurring at perinatal period,which experiences negative energy balance(NEB)states with increased energy requirements and reduced nutrient intake,and manifested as metabolic disorders with hyperketonemia and high free fatty acidemia.Overproduction of non-esterified fatty acid(NEFA)and ketone bodies display lipotoxicity,which subsequently promote hepatic lipid deposition,and then contributes to hepatic pathological damage.Conversely,hepatic pathological damage can further induce hepatic lipid deposition,resulting in a vicious spiral that ultimately accelerate the occurrence of ketosis in dairy cows.Thus,hepatic lipidosis is one of the key pathogenic processes complicating the treatment of ketosis in dairy cows.Autophagy and endoplasmic reticulum stress(ERS)have been put forward as two important biological process in the maintaince of metabolic homeostasis,such as lipid metabolism.Studies in humans and mice have indicated that both impaired autophagic activity and ERS are crucial contributors to liver lipid accumulation.Althogh previous studies in our lab have observed impaired autophagosome degradation and activated ERS in the liver of perinatal high-yield dairy cows,whether those two signals mediate hepatic lipidosis in clinical ketosis has not been addressed yet.Therefore,the aim of our current experiment is to investigate the state of autophagy and ERS in the liver of cilinical ketotic cows,and further explore the role of autophagy and ERS in the development of hepatic lipidosis.Collected blood and liver samples from healthy and clinically ketotic cows to measure the status of lipid metabolism,autophagy and ERS in the liver.Compared with healthy cows,cows with clinical ketosis had increased hepatic lipid deposition and TG content,accompanied by higher m RNA abundance of genes involved in lipid biosynthesis,sterol regulatory element binding protein-1C(SREBP-1c),fatty acid synthase(FASN)and diacylglycerol O-acyltransferase 1(DGAT1),and lower m RNA abundance of genes related to lipid oxidation,peroxisome proliferator–activated receptor α(PPARα),carnitine palmitoyltransferase 1(CPT1),and carnitine palmitoyltransferase 2(CPT2).Furthermore,the m RNA abundance of sequestosome-1(SQSTM1,also called p62)and microtubule-associated protein 1light chain 3(MAP1LC3,also called LC3)were markedly decreased in the liver of clinical ketosis,while the protein expression levels of p62 and LC3 II were significantly increased.Moreover,we also found up-regulated m RNA abundance of hepatic 78 k Da glucose-regulated protein(GRP78)and spliced X-box binding protein1(s XBP-1),and markedly increased phosphorylation levels of hepatic protein kinase RNA-like ER kinase(PERK)and inositol requiring protein-1α(IRE1α)proteins and the cleavage level of hepatic activating transcription factor-6(ATF6)protein in the liver of clinical ketosis.These results indicate that clinical ketotic cows displayed lipid deposition,impaired autophagic activity and ERS in the liver.High levels of blood NEFA are know as the major trigger of liver pathological changes in clinical ketosis.We detected the effects of different concentrations of NEFA on autophagy,ERS and lipid deposition in cultured calf hepatocytes,and then explored its internal mechanism using autophagy inhibitors,chloroquine(CQ)and bafilomycin A1(Baf A1).Results showed that NEFA treatment significantly increased the protein levels of p62 and LC3 II while concomitantly highering the levels of ATF6 cleavage,PERK and IRE1α phosphorylation,and up-regulating the GRP78 and s XBP-1 m RNA abundance.Moreover,the m RNA expression levels of genes involved in lipid synthesis(SREBP-1c,FASN and DGAT1)were substantially increased,whereas the m RNA expression levels of genes involved in lipid oxidation(PPARα,CPT1 and CPT2)were markedly decreased under NEFA treatment condition,resulting in hepatocytic lipid deposition and TG accumulation.Importantly,compared with control group,CQ and Baf A1 both promote hepatocytic lipid accumulation by promoting the expression of lipid synthesis genes(SREBP-1c,FASN and DGAT1)and inhibiting the expression of lipid oxidation genes(PPARα,CPT1 and CPT2).Notably,this process is accompanied by activated ERS.These results indicate that NEFA-induced hepatocytic lipid deposition is mediated by impaired autophagy and ERS.To further validate the above conclusion,we added rapamycin(Rap,autophagy activator)and GRP78 overexpression plasmids to isolated primary calf hepatocytes.The results showed that supplemention with Rap ameliorated NEFA-induced ERS by down-regulating m RNA abundance of hepatic GRP78 and s XBP-1,phosphorylation levels of PERK and IRE1α proteins,and the cleavage level of ATF6 protein.Meanwhile,Rap supplement also relieved NEFA-increased hepatocytic lipid deposition and TG content by inhibiting the expression of lipid synthesis genes(SREBP-1c,FASN and DGAT1)and promoting the expression of lipid oxidation genes(PPARα,CPT1 and CPT2).Importantly,no matter under CQ or NEFA treatment conditions,overexpression of GRP78 attenuated hepatocytic lipid deposition and TG accumulation by reducing the expression of lipid synthesis genes(SREBP-1c,FASN and DGAT1)and increasing the expression of lipid oxidation genes(PPARα,CPT1 and CPT2),suggesting ERS caused by impaired autophagy mediates NEFA-induced hepatocytic lipid deposition.Overall,we provided evidence that high levels of blood NEFA in clinical ketosis can block hepatic autophagy,which further activated ERS,thereby promoting lipid synthesis and inhibiting lipid oxidation,and ultimately leading to lipid accumulation in the liver of clinical ketotic cows. |
