Regulation Of Choline On Hepatic Glycometabolism In Dairy Cows With Negative Energy Balance

Glucose is one of the main nutrients for maintain lactation and body health of dairy cows in the important source of energy.Due to the unique digestive system of ruminants,there is less exogenous glucose in the feed that can enter the small intestine and be absorbed by the small intestine.However,in order to meet the needs of production and survival,dairy cows will generate endogenous glucose through the hepatic gluconeogenesis pathway to complete the supply.When dairy cows are in the perinatal period,their demand for glucose will be higher,and the energy intake and supply can’t meet the output of energy,so they will simply get into negative energy balance（NEB）and result in the production of non-esterified fatty acids（NEFA）,which further leads to the metabolic imbalance in liver.The studies have reported that choline could improve liver function,exert an impact on glucose metabolism,support postpartum lactation and increase milk production,which is crucial for alleviating NEB.The aim of this study was to systematically reveal the regulation of choline on hepatic glucose metabolism of NEB dairy cows,and find potential signaling targets to provide the scientific basis for preventing or improving bovine NEB state in the era of banning antibiotics.Experiment 1 Effects of different concentrations of NEFA on glucose metabolism in bovine hepatocytesAfter the bovine hepatocytes have been isolated and cultured,the experiment treated them with 0,0.6,1.2,2.4 mmol/L NEFA（n=3 per group）,then the optimal concentration of NEFA was chosen to simulate the hepatic glucose metabolism of NEB dairy cows.The results showed:（1）With the increase of NEFA concentration,the activities of aspartate aminotransferase（AST）and alanine aminotransferase（ALT）were enhanced（P<0.05）.When NEFA reached 1.2 mmol/L,it had a tendency to increase alkaline phosphatase（AKP）（P<0.1）,and when it reached 2.4 mmol/L,it would significantly increase the activity of AKP（P<0.05）;（2）In the 1.2 and 2.4 mmol/L NEFA groups,the intracellular glucose content was significantly increased（P<0.05）,the glycogen content was significantly decreased（P<0.05）,and the addition of NEFA had no effect on the glucose content in the cell culture medium（P>0.05）;（3）When the concentration of NEFA was above 1.2mmol/L,it would increase the transcription levels of glycolysis rate-limiting enzymes HK1,GCK（P<0.05）,and decrease the transcription level of glucose transporter SLGT1 and glycogen synthase GYS2（P<0.05）,but had no effect on GLUT2（P>0.05）,which is also a glucose transporter.In addition,with the increase of NEFA concentration,the m RNA expressions of gluconeogenesis rate-limiting enzymes PC,PEPCK,FBP1,and G6PC were all enhanced in a gradient（P<0.05）,the glycogenolytic enzyme PYGL showed an upward tendency（P<0.1）.This experiment showed that 1.2 mmol/L NEFA was the optimal concentration,which would aggravate the damage of bovine hepatocytes,inhibit the transport of extracellular glucose,increase the level of intracellular glucose,gluconeogenesis and glycolysis,and reduce content of glycogen.These would make bovine hepatocytes into the unbalanced glucose metabolism state of NEB dairy cows.Experiment 2 Effects of different concentrations of choline on glucose metabolism in bovine hepatocytes treated with NEFAIn order to explore the regulatory effects of choline on hepatic glucose metabolism in dairy cows under NEB state,on the basis of 1.2 mmol/L NEFA chosen in Experiment 1,bovine hepatocytes were treated with 0,25,50,75,100,150μmol/L choline chloride（n=3per group）.The results showed:（1）Choline could inhibit the activities of AST（P<0.05）,but had no effect on the AKP（P>0.05）.When choline reached more than 25μmol/L,it inhibited the activity of ALT（P<0.05）;（2）When choline reached more than 75μmol/L,it could increase the intracellular glucose content（P<0.05）.The content of glycogen was increased in 50,75 and 100μmol/L choline groups（P<0.05）,and returned to the same level as the control group in 150μmol/L choline group（P>0.05）;（3）When choline respectively reached more than 50,75,100μmol/L,the expression of PC,SGLT1,PCPEK m RNA increased significantly（P<0.05）.When choline was lower than 75μmol/L,there were no significant difference in the m RNA levels of glycolysis rate-limiting enzymes HK1,GCK,and PK（P>0.05）.The m RNA expression of glycogen synthase GYS2 was increased in 25,75,and 100μmol/L choline groups（P<0.05）.This experiment showed that choline could prevent liver damage,promote gluconeogenesis and glycogen synthesis without changing the process of glycolysis.Experiment 3 Analysis of the regulatory effects of NEFA and choline on glucose metabolism in bovine hepatocytes based on transcriptomic sequencingIn order to dig deeper into the changes of hepatic glucose metabolism signaling pathway under NEB state of dairy cows and find out the way that choline is involved in regulation,this experiment was based on Experiments 1 and 2 to isolate and culture bovine hepatocytes,and designed 3 treatments,namely the blank control group（T₀）,the 1.2mmol/L NEFA group（T_N）and the mixed addition group（1.2 mmol/L NEFA+75μmol/L choline chloride）(T_N+C).The samples were collected for 2 different experiments（n=5 per group for transcriptome;n=3 per group for RT-q PCR validation）.The results showed:There were 2075（T_N vs T₀）,1412(T_N+C vs T₀),657(T_N+C vs T_N)differentially expressed genes（DEGs）in differential comparison combinations,respectively.Combining the results of DEGs and enrichment analysis of GO and KEGG,SOCS3-JAK-STAT pathway,IGF system,BCL2 and PEKFB4 genes were successfully focused.After adding NEFA and NEFA+choline,the m RNA of SOCS3,IGFBP1,IGFBP4 and IGFBP7 all appeared a significant decrease and then returned to the original level.The experiment showed that NEFA and choline might exert an impact on the signal transductions of downstream JAK-STAT and IGF through SOCS3 and IGFBP,then acted on hepatic glucose metabolism.To sum up,the study successfully used NEFA in bovine hepatocytes to simulate the hepatic glucose metabolism imbalance of NEB dairy cows,revealed the positive regulation of choline on the hepatic glucose metabolism of NEB dairy cows,and explored signal pathways of potential glucose metabolism that might play a mediating role.The research results would be expected to provide theoretical and technical supports for choline to prevent and improve NEB state of perinatal dairy cow.