Regulation Of DGAT2 In Lipolysis And Autophagy Of Adipose Cells Of Dairy Cows With Clinical Ketosis

Ketosis is an important nutritional and metabolic disease with high incidence in dairy cows during the perinatal period.The incidence of ketosis in dairy herds around the world remains high.The susceptibility of dairy cows to other diseases will increase,and the reproductive performance will be greatly reduced.The aquaculture and dairy industries have caused huge economic losses.As the postpartum dry matter intake(DMI)of dairy cows decreases,while milk production and nutritional requirements increase,the body enters a state of negative energy balance(NEB).To maintain homeostasis,the body mobilizes fat.Excessive lipolysis of adipose tissue leads to elevated concentrations of β-hydroxybutyrate(BHB)and free fatty acid(FFA)in blood and milk,leading to adipose tissue inflammation and immune cell infiltration,ultimately affecting lactation performance and increasing disease susceptibility.Therefore,the metabolic adaptation of adipose tissue is crucial for the successful perinatal period of dairy cows.Diacylglycerol acyltransferase(DGAT)is a key enzyme that catalyzes the combination of diacylglycerol and fatty acid acyl to generate triglyceride(TAG),including two isoforms of DGAT1 and DGAT2,and plays an important role in the regulation of lipid metabolism.Autophagy is an important adaptive mechanism that maintains cellular homeostasis and adapts to changes and stimuli brought about by changes in the intracellular or surrounding environment,and is also considered a key cytoprotective pathway.Dysregulation of autophagy in adipose tissue affects the accumulation of body fat.Our previous study demonstrated that DGAT2 was lowly expressed in the adipose tissue of cows with ketosis.However,the regulatory role of DGAT2 on autophagy during perinatal dairy cow fat mobilization is still unclear.Therefore,we put forward the scientific hypothesis that DGAT2 may be involved in the regulation of lipid metabolism and autophagy in adipose tissue of cows with ketosis.We intend to further explore the regulatory effect of DGAT2 on adipose tissue lipolysis and its effect on the activation of adipose tissue autophagy signaling pathway,so as to clarify DGAT2 Whether there is a regulatory effect on autophagy during severe lipolysis of adipose tissue in ketotic cows.This provides new ideas for regulating the occurrence of autophagy in adipose tissue of cows with ketosis.In this study,we performed in vivo and in vitro experiments to explore the role of DGAT2 in adipose tissue autophagy in ketotic cows.In the in vivo experiments,10 healthy and 10 ketotic cows were selected.Adipose tissue samples were collected,and the adipose tissue was stained by HE staining technique to observe the pathological changes of adipose tissue in ketosis cows.The protein abundances of DGAT2,lipolysis-related molecules and autophagy-related molecules were detected.In in vitro experiments,primary bovine adipocytes were first isolated and cultured,and the differentiation and maturation of bovine adipocytes were identified by oil red staining.Isoproterenol(ISO)is a catecholamine hormone that promotes fat mobilization and significantly increases the rate of lipolysis.Therefore,in this study,ISO was utilized as a lipolytic agonist.After the cells differentiated into mature adipocytes,they were divided into 7 groups according to different treatments: control group,ISO group,transfection overexpressing DGAT2 adenovirus group(Ad-DGAT2),adding ISO and transfecting DGAT2 overexpressing adenovirus group(Ad-DGAT2).Ad-DGAT2+ISO),adding DGAT2 inhibitor PF-06424439 group(PF-06424439),adding ISO and DGAT2 inhibitor PF-06424439 group(PF-06424439+ISO).After the treatment,the cells were collected,and the protein expression levels of lipid metabolismrelated molecules HSL,p-HSL and ATGL and autophagy-related molecules Beclin1,SQSTM1,LC3,PI3 K,AKT and m TOR in the cells were detected.Through in vivo and in vitro experiments,it was determined whether DGAT2 has a regulatory effect on adipose tissue and autophagy in ketosis cows.The results of in vivo experiments showed that compared with healthy cows,the protein expression levels of lipid metabolism-related molecules HSL,p-HSL and ATGL,and autophagyrelated molecules Beclin1,SQSTM1,LC3 and PI3 K in the adipose tissue of cows with ketosis were significantly increased(P < 0.01),while the protein expression levels of DGAT2 and negatively regulated autophagy-related molecules AKT and m TOR were significantly decreased(P < 0.01).The above results indicate that the adipose tissue of ketosis cows has severe lipolysis and more active autophagy.The results of in vitro experiments showed that ISO could up-regulate the protein expression levels of lipolysis-related molecules HSL,p-HSL and ATGL and autophagy-related molecules Beclin1,SQSTM1,LC3 and PI3 K in bovine adipocytes.ISO can reduce the protein expression levels of negatively regulated autophagy-related molecules AKT and m TOR in adipocytes.Bovine adipocytes were transfected with DGAT2 adenovirus or added with a DGAT2 inhibitor to determine the regulatory effect of DGAT2 on lipolysis and autophagy in bovine adipocytes.When the adenovirus overexpressed DGAT2 was transfected for 12 h or the DGAT2 inhibitor PF-06424439 was added for 0.5 h,ISO was added for stimulation for 3 h,and it was found that overexpression of DGAT2 could inhibit ISO-induced lipolysis-related proteins HSL,Protein expression of p-HSL and ATGL and autophagy-related molecules Beclin1,SQSTM1,LC3 and PI3 K.Conversely,inhibition of DGAT2 expression promoted ISO-induced protein expression of lipolysis and autophagy-related molecules.In conclusion,in vivo and in vitro experiments showed that DGAT2 protein abundance was lower and autophagy was more active in adipose tissue when NEB-induced adipose tissue lipolysis in ketosis cows was aggravated.Furthermore,DGAT2 negatively regulates autophagy in adipose tissue of cows with ketosis.