The Mechanism Of Choline Regulating Lipid And Energy Metabolism In Perinatal Cow Hepatocytes Via GNMT

Due to the continuous physiological process of pregnancy,calving and lactation,coupled with changes in diet,perinatal cows lead to the imbalance of energy intake and nutritional requirements of the body,which is prone to negative energy balance and induce a variety of metabolic diseases.Choline has the function of regulating lipid metabolism in the body.At the same time,it can participate in the carbon cycle as a methyl donor,slow down the occurrence of diseases in dairy cows,and improve milk yield and milk quality.The rumen protection products of choline have been widely used in the diet of perinatal dairy cows,but the molecular mechanism of choline’s regulation of lipid metabolism and energy balance in vivo remains unclear.Glycine-N-methyltransferase(GNMT),which can regulate the transformation of SAM and SAH,so as to participate in the carbon cycle,is used as a transcription factor to regulate the expression of target genes in the nucleus,affecting fat synthesis,energy metabolism,oxidative stress and other processes.However,it is not clear whether choline can regulate liver metabolism via GNMT.Therefore,by utilizing the model of fatty deposited constructed by non-esterified fatty acid(NEFA),we explore the mechanism of choline on liver metabolism in perinatal cows and illustrate the effect of choline’s regulation of lipid and energy metabolism through GNMT.AMPK inhibitors(Compund C)were further used to elucidate the potential molecular mechanism of GNMT in the presence of choline.The above studies indicated that choline can regulate fat and energy metabolism in hepatocytes under the action of NEFA through GNMT,and AMPK/c-Myc axis was its potential signaling pathway.The main research results were as follows.Experiment 1.The regulation mechanism of choline on fat metabolism based on transcriptomicsIn this experiment,primary calf hepatocytes and bovine liver cell lines were used as research objects.1.2 mmol/L NEFA was added to build a liver cell model of fatty deposition,and different concentrations of choline(0,25,50,75,100,150 μmol/L)were added to detect intracellular triglyceride(TG)levels.It was found that TG content in 50 μmol/L and 75μmol/L groups was significantly decreased(P < 0.01).Transcriptome sequencing was conducted in 0 μmol/L choline(N)group and 75 μmol/L choline(NC)group to explore the regulatory mechanism of choline on fat metabolism in adipose-deposited hepatocytes.The results showed that 480 differentially expressed genes were identified,249 of which were up-regulated and 231 of which were down-regulated.GO enrichment analysis was conducted on all differentially identified genes,and a total of 550 significantly different GO entries were annotated,mainly including vitamin biosynthetic process,response to reactive oxygen species,positive regulation of MAPK cascade,response to interferon-alpha,cellular response to oxidative stress,phosphate ion transport.KEGG pathway enrichment analysis was performed on the obtained differential genes,and a total of 38 differential pathways were enriched,including MAPK signaling pathway,TNF signaling pathway,fluid shear stress and atherosclerosis,Rap1 signaling pathway,Toll-like receptor signaling pathway,Ras signaling pathway,Fox O signaling pathway,biosynthesis of amino acids.The gene and protein expression levels of GNMT in the N group and the NC group were further determined,and the results showed that the gene and protein expression levels of GNMT were significantly increased after choline addition(P < 0.05).The results of this study indicate that choline can affect the fat metabolism process of hepatocytes under the add of NEFA,and GNMT may be a potential regulatory factor.Experiment 2.The effect of choline concentration on the gene and protein expression of GNMTThis part of the whole study,calf primary hepatocytes and human hepatocyte lines(LO2)were used as research objects to construct the perinatal bovine fatty liver model by adding 1.2mmol/L and 1.6 mmol/L NEFA,respectively,and adding different concentrations of choline(0,25,50,75,100 and 150 μmol/L),each group had 3 replicates.The gene and protein expression levels of GNMT was detected under different choline concertration.The results showed that,in calf primary hepatocytes,GNMT gene expression in 50 μmol/L group was significantly higher than that in 0,75,100,150 μmol/L groups(P < 0.05).In LO2 cells,the expression level of GNMT in 50 μmol/L group was significantly higher than that in 0,25,100 and 150 μmol/L groups(P < 0.05).Similarly,the gene and protein expression levels of GNMT showed a trend of first increasing and then decreasing in two types of liver cells.This experiment indicated that the change of choline concentration can affect the gene and protein expression level of GNMT,and GNMT is apparently concentration-dependent on choline.At the same time,50 μmol/L choline concentration was used for follow-up experiment.Experiment 3.The mechanism of choline regulating liver cell fat and energy metabolism through GNMTOn the basis of experiment 2,this study explored whether choline could continue to play a role after GNMT silence,and simply verified whether AMPK could regulate choline/GNMT axis.1.6 mmol/L NEFA and 50 μmol/L choline chloride were added in choline-free medium.GNMT of LO2 cell line was knocked down by si RNA interference technique and divided into NEFA group(N),NEFA+si-GNMT group(N-Si),choline group(NC),choline+si-GNMT(NC-Si).Subsequently,the expression levels of genes related to lipid synthesis,metabolism and transport(FAS,ACC,CPT1,Apo B100,PPARα),cholesterol synthesis and transport(HMGCR,SRB1),bile acid synthesis and transport(CYP7A1,CYP27A1,NTCP,BSEP),and energy metabolism(PGC1-α,FOXO1,ATP5F1 B,SDH)were detected.The results clarified that compared with the N group,the gene expression levels of FAS,CPT1,ACC,PGC1,FOXO1,ATP5F1 B,SDH,HMGCR,NTCP and BSEP in N-Si group were significantly increased(P < 0.05),the expression levels of Apo B100,PPAR,CYP7A1 and CYP27A1 were significantly decreased(P < 0.05).After the knockdown of GNMT,choline lost its regulatory effect on PGC1,FOXO1 and CPT1(P < 0.05),and its regulatory effect on ACC,MTTP,Apo B100,CYP7A1,CYP27A1 and BSEP was impaired(P < 0.05),but had no regulatory effect on ATP5F1 B,SDH,HMGCR and SRB1(P > 0.05).Compound C(AMPK inhibitor)was used to explore the regulatory effect of AMPK on GNMT,and the gene expression level of Myc,the negative regulator of GNMT,was also determined.It was found that adding Compound C could significantly improve the gene expression of Myc and reduce the protein expression level of GNMT.This experiment showed that GNMT could regulate liver fat metabolism,energy metabolism and bile acid metabolism under the effect of NEFA,and GNMT was an important factor in choline’s regulation of fat and energy metabolism,and AMPK/Myc/GNMT was its potential signaling pathway.In conclusion,Choline can reduce fat deposition in liver cells under the action of NEFA,regulate fat and energy metabolism and affect bile acid metabolism and transport in liver cells through GNMT.AMPK/Myc/GNMT is a potential signaling pathway for choline to regulate fat and energy metabolism in the liver of perinatal cows.