The Role Of Endoplasmic Reticulum Stress In Hepatic Steatosis In Dairy Cows With Fatty Liver

Fatty liver is a major metabolic disorder in dairy cows,with a prevalence of 25% to 50% in perinatal dairy cows.Fatty liver develops when the triglycerides(TG)over accumulated in hepatocytes.The endoplasmic reticulum(ER),as a place for lipid synthesis and settlement of a variety of lipid metabolizing enzymes,the balance of its internal environment is essential for maintaining lipid homeostasis.The disruption of ER homeostasis,namely endoplasmic reticulum stress(ERS),can disrupt lipid homeostasis and promote lipogenesis.Sterol regulatory element binding protein 1c(SREBP-1c)is a key transcription factor regulating lipogenesis,which is synthesized on the ER.Target genes regulated by SREBP-1c include acetyl-Co A carboxylase-alpha(ACACA),fatty acid synthase(FASN),and diacylgycerol acyltransferase(DGAT),etc.,these enzymes are directly involved in lipid synthesis.The newly synthesized SREBP-1c is an inactive precursor SREBP-1c(p SREBP-1c),which must undergo a series of processing to form an active nuclear SREBP-1c(n SREBP-1).In humans and non-ruminants,ERS can up-regulate the expression and maturation of SREBP-1c,thereby promoting lipid accumulation.However,the role of ERS in hepatic steatosis in dairy cows with fatty liver are still unclear.The expression of ERS marker molecules,p SREBP-1c,n SREBP-1c and key genes related to lipogenesis in the livers of severe fatty liver and healthy cows were detected to determine the ERS and lipogenesis status in the liver of dairy cows with severe fatty liver.The results showed that protein kinase RNA-like ER kinase(PERK),activating transcription factor 6(ATF6),and inositol require enzyme 1α(IRE1α)was activated,and the m RNA expressions of glucose-regulated protein 78 k D(GRP78),activating transcription factor 6(ATF4)and spliced-X-box binding protein(s XBP1)were significantly higher in dairy cows with severe fatty liver than those in healthy cows,confirming the occurrence of ERS in dairy cows with severe fatty liver.Besides,the protein expressions of p SREBP-1c and n SREBP-1c and the m RNA expressions of ACACA,FASN,and DGAT1 were significantly higher in the fatty liver group than in the healthy group,indicating that hepatic lipogenic pathways were activated in severe fatty liver cows.To clarify the role of ERS in free fatty acid(FFA)-induced hepatic lipid accumulation in dairy cows,the FFA and/or ERS inhibitor taurodeoxycholic acid(TUDCA)were added to calf primary hepatocytes,and the relevant indicators were detected.The present results showed that FFA could induce ERS,promote the expression of p SREBP-1c,n SREBP-1c and its target genes,and lipid accumulation in calf hepatocytes,and inhibit ERS can alleviate these effects,indicating that FFA-induced ERS favors the expression and maturation of SREBP-1c,thereby promoting the expression of lipogenic genes and exacerbating lipid accumulation in calf hepatocytes.Considering that hepatic steatosis in cows with severe fatty liver is accompanied by high concentrations of fatty acids,ERS,and increased expression of p SREBP-1c and n SREBP-1c,we speculate that ERS participates in the development of fatty liver in dairy cows by promoting the expression and maturation of SREBP-1c.The process of p SREBP-1c hydrolysis and maturation is regulated by the ER membrane protein insulin-induced gene 1(Insig-1)and sterol regulatory element binding protein cleavage-activating protein(SCAP).Studies in non-ruminant animals have shown that ERS can regulate the hydrolytic maturation of SREBP-1c by degrading the Insig-1 protein.To determine whether Insig-SCAP-SREBP-1c is involved in ERS-induced lipid accumulation in dairy cows in vitro,the ERS classic inducer tunicamycin(TM)and/or Insig-1 overexpression plasmids was added in calf hepatocytes,and the relevant indicators were detected.The results showed that TM down-regulated the protein expression of Insig-1,and up-regulated the protein expression of SCAP,p SREBP-1c and n SREBP-1c,and TG content in calf hepatocytes,while overexpression of Insig-1 inhibited TM up-regulated protein expression of n SREBP-1c and m RNA expression of lipogenic genes and TG content.These results indicate that ERS can regulate the transport of SCAP-SREBP-1c complex and the maturation of SREBP-1c by down-regulating the protein expression of Insig-1,thereby promoting the expression of lipogenic genes and ultimately induce lipid accumulation in calf hepatocytes.The main cause of ERS in fatty liver cows is high concentration of FFA.Therefore,in order to define whether ERS promotes the development of fatty liver through Insig-1-SCAP-SREBP-1c in dairy cows,the verification was performed in FFA-treated dairy cow liver cells.The results indicate that FFA down-regulate the protein expression of Insig-1 by inducing ERS,thereby promoting the maturation of SREBP-1c and the expression of lipogenic genes,eventually induce lipid accumulation in calf hepatocytes.Interestingly,overexpression of Insig-1 did not affect the expression of p SREBP-1c up-regulated by either TM or FFA,suggesting that the up-regulation effects of ERS/FFA on SREBP-1c at the transcriptional levels does not depend on Insig-1 in calf hepatocytes.Finally,the expression of Insig-1 and SCAP were detected in the livers of dairy cows with severe fatty liver and healthy cows.The results showed that Insig-1 was low-expressed and SCAP was high-expressed in severe fatty liver cows.Taken together,the present results showed that the ERS-activated Insig-1-SCAP-SREBP-1c transport system is involved in FFA-induced lipid accumulation in calf hepatocytes.Besides,the hepatic steatosis is accompanied by high concentrations of FFA,ERS,and activated Insig-1-SCAP-SREBP-1c transport system in cows with severe fatty liver.Therefore,we speculate that high concentrations of FFA in the blood of perinatal cows can induce hepatic ERS,which promotes the transport of SCAP-SREBP-1c complex by down-regulating the protein expression of Insig-1,thereby increasing the maturation of SREBP-1c and eventually leading to lipid accumulation in the liver of dairy cows.However,the hypothesis that ERS contributes to the development of fatty liver through the Insig-1-SCAP-SREBP-1c pathway in dairy cows still needs more in vivo pieces of evidence to support.