| Ketosis is a metabolic disorder frequently occurring in perinatal dairy cows.According to the concentration of blood β-hydroxybutyrate acid(BHBA),ketosis is divided into clinical(BHBA ≥ 3.0 m M)and subclinical ketosis(1.2 m M ≤ BHBA <3.0 m M),and its pathological basis is excessive lipolysis of adipose tissue caused by negative energy balance(NEB).As a degradation and recycling mechanism in eukaryotic cells in response to stress such as energy deficiency,autophagy-lysosomal pathway(ALP)is crucial for the maintenance of cellular energy metabolism homeostasis.Transcription factor EB(TFEB)is a key transcription factor in the regulation of autophagy and lysosome related genes,and the activated ALP contribute to lipid droplet degradation.However,whether TFEB regulates lipid catabolism in adipocytes of ketotic cows via ALP is unknown.Therefore,the aim of the present study was to ascertain the changes in TFEB transcriptional activity and ALP status in adipose tissue of dairy cows with subclinical ketosis,and further explore the role of TFEB and ALP in lipolysis of adipocytes.In this experiment,adipose tissues of subclinical ketosis and healthy cows were collected to detect the lipolysis status,TFEB transcriptional activity and ALP status.Compared with healthy cows,dairy cows with subclinical ketosis had smaller adipocytes,accompanied by greater phosphorylation level of hormone-sensitive lipase(HSL)and protein abundance of adipose triacylglycerol lipase(ATGL),and lower protein abundance of perilipin 1(PLIN1)and cell death-inducing DNA fragmentation factor-α-like effector c(CIDEC).Moreover,nuclear translocation and total protein abundance of TFEB,and m RNA abundance of TFEB and its downstream target gene peroxisome proliferator-activated receptor γ coactivator 1 α(PPARGC1A)were increased in adipose tissue of dairy cows with subclinical ketosis,while phosphorylation level of TFEB was decreased.Furthermore,m RNA abundance of autophagy-related gene(ATG)5,ATG7 and microtubule associated protein 1 light chain 3 β(MAP1LC3B),protein abundance of lysosome associated membrane protein 1(LAMP1)and cathepsin D(CTSD),and activity of β-Nacetylglucosaminidase(NAG)were increased,whereas protein abundance of sequestosome-1(p62)was decreased in adipose tissue of dairy cows with subclinical ketosis.These results indicate that lipolysis is enhanced and TFEB transcriptional activity and ALP are overactivated in adipose tissue of dairy cows with subclinical ketosis.To confirm the relationship between TFEB and ALP and lipolysis,mature bovine adipocytes were isolated and cultured in vitro and treated with lipolysis inducer isoproterenol(ISO)for different times,and TFEB agonist Torin1,TFEB small interfering RNA(si TFEB)and lysosomal function inhibitor leupeptin were used to detect changes in TFEB transcriptional activity and ALP status under lipolysis conditions.The results showed that ISO treatment increased phosphorylation level of HSL,protein abundance of ATGL and glycerol(GC)content of supernatant,but reduced protein abundance of PLIN1 and CIDEC,as well as intracellular contents of triacylglycerol(TG)and lipid drops,indicating that ISO could effectively promote lipolysis of adipocytes.Moreover,nuclear translocation and total protein abundance of TFEB were increased,phosphorylation of TFEB was decreased,m RNA abundance of TFEB,PPARGC1 A,ATG5,ATG7 and MAP1LC3 B were upregulated,protein abundance of LAMP1 and CTSD were increased,protein abundance of p62 were decreased,and activity of NAG was enhanced in ISO-treated adipocytes.These results suggest that ISO increases lipolysis while also enhancing TFEB transcriptional activity and activating ALP.In addition,TFEB agonist Torin1 upregulated m RNA abundance of ATG5,ATG7 and MAP1LC3 B,increased protein abundance of LAMP1 and CTSD,reduced protein abundance of p62,enhanced NAG activity,increased phosphorylation level of HSL,protein abundance of ATGL and GC content of supernatant,and reduced protein abundance of PLIN1 and CIDEC as well as intracellular contents of TG and lipid droplets,suggesting that TFEB activate ALP and increase lipolysis of adipocytes.Conversely,knockdown of TFEB using si RNA attenuated lipolysis and ALP activation induced by ISO,as indicated by decreased phosphorylation of HSL,protein abundance of ATGL and GC content of supernatant in adipocytes,while increased protein abundance of p62,PLIN1 and CIDEC,as well as intracellular contents of TG and lipid droplets.Similarly,lysosomal function inhibitor leupeptin also increased protein abundance of p62,PLIN1 and CIDEC and intracellular content of TG,but reduced GC content in supernatant.These results suggest that the TFEB-mediated ALP is involved in the regulation of lipid catabolism in bovine adipocytes.The NEB in perinatal dairy cows is often manifested as hypoglycemia,which is also a metabolic characteristic of dairy cows with subclinical ketosis.In order to simulate the metabolic status in dairy cows with subclinical ketosis,adipocytes were treated with glucose starvation to determine lipolysis status and TFEB-mediated ALP.The results showed that glucose starvation increased phosphorylation level of HSL,protein abundance of ATGL and GC content of supernatant,but reduced protein abundance of PLIN1 and CIDEC and intracellular content of TG.Moreover,glucose starvation decreased phosphorylation of TFEB,increased total protein abundance of TFEB,and upregulated m RNA abundance of TFEB and PPARGC1 A.Furthermore,m RNA abundance of ATG5,ATG7 and MAP1LC3 B,protein abundance of LAMP1 and CTSD,and activity of NAG were increased in glucose-starved adipocytes,while protein abundance of p62 was decreased.These results indicate that glucose starvation activates TFEB-mediated ALP and increases lipolysis of adipocytes.In conclusion,NEB activates TFEB-mediated ALP and promotes lipolysis of adipocytes in dairy cows with subclinical ketosis. |
