The Changes Of Hepatic Mitochondrial Function In Mild Fatty Liver And Clinical Ketotic Cows And The Underlying Regulation Mechanism

Decreased dry matter intake and increased energy requirement induce negative energy balance and further enhance blood concentration of non-esterified fatty acids(NEFA).Liver can uptake and complete oxidize NEFA to provide energy;however,excessive NEFA can be incompletely oxidized to ketone bodies,including β-hydroxybutyrate(BHBA),acetoacetate(AA)and acetone,or re-esterified into triglyceride(TG)and further induce ketosis and fatty liver.Besides,high concentrations of NEFA and ketone bodies display lipotoxicity and result in pathological damage and lipid metabolism dysfunction in the liver.NEFA is oxidized in mitochondrion to generate ATP;therefore,mitochondrion plays a crucial role in ameliorating energy deficient and metabolizing excessive NEFA.Studies has shown that dairy cow with mild fatty liver can make an uneventful transition during transition period;however,the underlying mechanism was largely unknown.Moreover,increased morbidity of clinical ketosis may relate to hepatic mitochondrial dysfunction and pathological damage;nevertheless,the involved signaling pathway remained unknown.Therefore,the objective of this study was to investigate the changes of hepatic mitonchondrial function in mild fatty liver and clinical ketotic cows,the effects of mitochondrial function on dairy cow with metabolic disorder diseases and the underlying mechanism.Dairy cows with mild fatty liver displayed hepatic lipid accumulation.The protein levels of sterol regulatory element-binding protein 1c(SREBP-1c)and peroxisome proliferator-activated receptor α(PPARα)were significantly higher in dairy cows with mild fatty liver than those in control cows.The hepatic mitochondrial content,mRNA levels of oxidative phosphorylation(OXPHOS)complexes,protein levels of mitochondrial marker and function regulator,and adenosine triphosphate(ATP)content were all markedly increased in the liver of dairy cows with mild fatty liver compared with healthy cows.Besides,PA treatment significantly increased lipid accumulation,protein levels of SREBP-1c and PPARα in calf hepatocytes.Moreover,the mtDNA content,mRNA levels of CO I-V,protein levels of COX IV,VDAC1,PGC-1α,NRF1 and ATP content were significantly increased in PA-treated hepatocytes compared with control hepatocytes.The protein level of mitofusin-2(MFN2)was significantly decreased in PA-treated groups.In conclusion,lipid synthesis and oxidation,number of mitochondria and ATP production were increased in the liver of dairy cows with mild fatty liver and PA-treated calf hepatocytes.Ketotic cows displayed hepatic fat accumulation,mitochondrial dysfunction and oxidative stress.The hepatic malondialdehyde(MDA)content was significantly increased,but the activities of catalase(CAT),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),and expression of mitochondrial function regulator,OXPHOS complexes and ATP content were markedly decreased in ketotic cows compared with control cows.The number of TUNEL-positive cells in the liver of ketotic cows was 2.61-fold higher than the values observed in control cows.Besides,Caspase 3 activity was significantly increased in the liver of ketosis cows.Importantly,the levels of phosphorylated c-Jun N-terminal kinase(JNK)and p38 mitogen-activated protein kinase(p38MAPK)were significantly increased but the level of phosphorylated extracellular signal-regulated kinase1/2(ERK1/2)was markedly decreased,which further promoted tumor protein 53(p53)expression and inhibited nuclear factor E2-related factor 2(Nrf2)expression.The apoptosis-related molecules p21,MDM2,Caspase 3,Caspase 9 and Bax were expressed at significantly higher levels in ketotic cows than in healthy cows,whereas the anti-apoptosis molecule Bcl-2 was expressed at significantly lower levels.Based on these results,ketotic cows display severe hepatic mitochondrial dysfunction and oxidative stress.The hepatic MAPKs-p53-Nrf2 apoptotic pathway is over induced and partially mediated apoptotic damage to the liver.Moreover,the extent of hepatic damage and oxidative stress had a positive relationship with the AA levels.In vitro,AA treatment impaired mitochondrial function and increased reactive oxygen species(ROS)content and further induced oxidative stress and apoptosis of bovine hepatocytes.In this process,AA treatment increased the phosphorylation levels of JNK and p38 MAPK and decreased the phosphorylation level of ERK,which could increase p53 and inhibit nuclear factor E2-related factor 2(Nrf2)expression,nuclear localization and DNA-binding affinity,thereby inducing the overexpression of pro-apoptotic molecules Bax,Caspase 3,Caspase 9,PARP and inhibition of anti-apoptotic molecule Bcl-2.Antioxidant Nacetylcysteine(NAC)treatment or interference of MAPKs pathway could attenuate the hepatocytes apoptosis induced by AA.Collectively,these results indicate that AA triggers hepatocytes apoptosis via the ROS-mediated MAPKs pathway in ketotic cows.Besides,we also found that the hepatic protein levels of p-STAT3(Tyr705)and pSTAT3(Ser727)were higher and lower in the mild fatty liver and clinical ketotic cows,respectively.Moreover,100 and 200 μM PA increased and 400 μM PA,BHBA(0.6,1.2,2.4 and 3.6 mM)or AA(4.8 mM)decreased protein levels of pSTAT3(Tyr705)and p-STAT3(Ser727)in dairy cow hepatocytes.In addition,inhibiton of STAT3 impaired mitochondrial function and caused oxidative stress,and overexpression of STAT3 ameliorated PA,BHBA and AA induced oxidative stress.Taken together,dairy cow with mild fatty liver displayed enhanced mitochondrial function,increased lipid synthesis and oxidation,which contributed to improve the adaptation capacity and make an uneventful transition;dairy cow with ketosis displayed dysfunctional mitochondria,which produced excessive ROS and further induced apoptosis of hepatocytes;AA induced oxidative stress,futher overactived MAPKs-p53/Nrf2 pathway and increased apoptosis in dairy cow hepatocyte;overexpression of STAT3 contributed to alleviate the oxidative stress.