Functional Investigation Of Propionate On Hepatic Gluconeogenesis In Dairy Cows Via MTOR

Improving the efficiency of milk production is an important means to promote the development of dairy industry in China,and meeting the glucose demand of dairy cows is an important guarantee to improve the efficiency of milk production.In dairy cows,part of glucose is directly absorbed by the small intestine,and most of glucose is obtained by hepatic gluconeogenesis using propionic acid as a substrate.Therefore,improving the efficiency of hepatic gluconeogenesis is an effective method to improve the efficiency of milk production in dairy cows.During the perinatal period,dairy cows are in a state of negative energy balance due to insufficient feed intake,and the increase of palmitic acid content in blood increases the energy burden of liver.Therefore,it is necessary to improve the efficiency of liver gluconeogenesis to protect the health of dairy cows.Dairy cows and non-ruminants have different phylogenetic characteristics in liver gluconeogenesis,which is one of the entry points to find ways to improve liver gluconeogenesis efficiency in dairy cows.At the same time,mTOR signaling pathway is the center of cellular anabolism and plays an important role in the biosynthesis of milk protein and milk fat.Is mTOR signaling involved in the regulation of the efficient utilization of propionate by ruminant liver for gluconeogenesis? Based on this,this study aimed to explore the mechanism of mTOR signaling in hepatic gluconeogenesis in dairy cows,and to provide a theoretical basis for improving milk yield and ensuring the health of dairy cows.Experiment 1 Analysis of differentially expressed genes in dairy and pig/human liver transcriptomesBy comparing the liver transcriptomes of dairy cows and non-ruminant pigs/humans and combining with molecular experiments,this experiment aimed to explore the mechanism of efficient utilization of propionic acid by dairy liver for gluconeogenesis.The results showed that:(1)There were 6923 and 8435 differentially expressed genes between cow and pig/human liver transcriptome.(2)The liver of dairy cows highly expresses acyl-Co A synthetase shortchain family member 1(ACSS1),propionyl-coa carboxylase alpha chain(PCCA),methylmalonyl-Co A epimerase(MCEE),methylmalonyl-Co A mutase,(MMUT)and succinate-Co A ligase(SUCLG2)genes at the transcriptional level.In addition,the expressions of gluconeogenesis rate-limiting enzymes phosphoenolpyruvate carboxy kinase(PCK)and fructose 1,6-bisphosphatase(FBP)were also higher than those in pig/human.(3)The positive regulators of mTORC1 pathway,LAMTOR2,LAMTOR3,LAMTOR4 and USP4,were upregulated,while the negative regulators,DEPTOR,NPRL3,SESN2,SZT2,RNF152 and TSC1,were down-regulated at m RNA level.Western Blot analysis showed that the expression of p T389-S6 K and p-S6 in dairy cow liver were higher than that in pig liver.The present study demonstrated that propionate and gluconeogenesis metabolic pathways in dairy cow liver were higher than those in pig/human liver at the transcriptional level,and mTORC1 signaling pathway was highly activated in dairy cow liver compared with pig liver.Experiment 2 mTORC1 signaling regulates hepatic gluconeogenic gene expression in dairy cowsBased on the results of experiment 1,to explore the role of mTORC1 in hepatic gluconeogenesis in dairy cows,primary calf hepatocytes were isolated from calves and cultured.The expression of gluconeogenic genes was detected under conditions of activating or inhibiting mTORC1 activity.The results showed that:(1)After 24 h of treatment with mTORC1 inhibitor Rapamycin,the m RNA levels of SUCLG2,MMUT,PCCA,FBP1,FBP2,PCK1 and PCK2 in primary calf hepatocytes were significantly decreased(P < 0.05);However,after overexpression of Myc-Rheb Q64 L plasmid(mTORC1 signaling pathway activation),the expression levels of the above genes were increased(P < 0.05).(2)Knockdown of Raptor(mTORC1 signaling inhibition)reduced the m RNA expression of PCK1 and FBP1(P < 0.05),but did not reduce the expression of PCK1 and FBP1 genes in ZLN005 cells treated with PGC-1α activator(P > 0.05);Addition of MHY1485(mTORC1 signaling activation)enhanced intracellular PCK1 and FBP1 expression(P < 0.05),but did not enhance the expression of PCK1 and FBP1 in the cells treated with PGC-1α inhibitor SR18292(P > 0.05).These results suggest that the highly activated mTORC1 activity in the liver of dairy cows detected in experiment 1 can promote the high level of hepatic gluconeogenesis in dairy cows,and the PGC-1α plays an important role in this process.Experiment 3 Propionic acid promotes hepatic gluconeogenesis in dairy cows via mTOR signalingIn order to explore the mechanism of mTOR signaling pathway in hepatic gluconeogenesis in dairy cows,we investigated whether propionic acid has regulatory effect on mTOR signaling pathway in primary calf hepatocytes.The results showed as following:(1)Western Blot analysis showed that sodium propionate increased the expression of p T389-S6 K,p-S6 and p-4EBP1 and decreased the expression of p S473-AKT and p-SIN1 in primary calf hepatocytes(P < 0.05).(2)The glucose production of primary calf hepatocytes increased after propionate supplementation(P < 0.05),and gluconeogenesis gene expression FBP1、FBP2,PCK1,PCK2,G6 PC,ACSS1,SUCLG2,MCEE,HNF4 A,FOXO1,CREB and PGC-1α was significantly increased(P < 0.05).The PGC-1α protein level was also significantly increased(P < 0.05).(3)Propionic acid combined with Rapamycin,MHY1485 or SR18292 could not promote the expression of FBP1,PCK1 and G6PC(P > 0.05).(4)The expression of p T389-S6 K and p-S6 in primary calf hepatocytes treated with palmitic acid(PA)of 0,100,200,400μM decreased(P < 0.05),while the expression of p S473-AKT and p-Sin1 increased(P < 0.05).PA also inhibited the expression of FBP1,FBP2,PCK1,PCK2,G6 PC,ACSS1,SUCLG2,MCEE and MMUT in primary calf hepatocytes.After PA combined with Na P treatment,Na P restored the effect of PA on the decreased expression of p T389-S6 K,p-S6 and gluconeogenic genes,reduced the expression of p S473-AKT and p-SIN1,and restored hepatic gluconeogenesis.These results indicated that propionic acid promoted gluconeogenesis in primary calf hepatocytes by regulating mTOR signaling pathway and alleviated PA-induced hepatic gluconeogenesis reduction.In conclusion,propionate metabolism and gluconeogenesis genes were highly expressed in the liver of dairy cows compared to pig/human,and the highly activated mTORC1 signaling pathway promoted gluconeogenesis gene expression through PGC-1α.Propionic acid promoted hepatic gluconeogenesis through mTOR signaling pathway and alleviated PAinduced decrease in hepatic gluconeogenesis capacity in dairy cows.