Metabolic Characteristics And Effects On Cell Functions Of Different Forms Of Methionine Sources In Different Pig Cell Lines

Methionine?Met?is not only one of the substrates for protein synthesis,but also participates in many other biological events through its metabolism.The metabolism of Met mainly includes transmethylation,aminopropyl transfer,transsulfuration and remethylation processes.S-adenosylmethionine?SAM?,a transmethyl metabolite of Met,provides methyl donors for methylation modification of DNA,RNA and histones;cysteine?Cys?,glutathione?GSH?,taurine?Tau?are all metabolites of Met transsulfuration,which can play an antioxidant role;spermine,spermidine and putrescine in aminopropyl transfer metabolites have important regulatory effects on cell proliferation,and5-methylthioadenosine?MTA?generated by this process can be used as the substrate for Met resynthesis;and the remethylation can use the intermediate metabolite homocysteine?Hcy?to resynthesize Met,so as to regulate the relative balance of Met content in cells.Met is an important limiting amino acid for pigs.Met supplements commonly used in feed mainly include DL-Methionine?DL-Met?,DL-2-hydroxy-4-?methylthio?butyric acid?DL-HMTBA?and DL-Methionine-Mehthionine?DL-MM?.But it is unclear whether there are differences in the ability of different Met sources to be converted to L-Met in tissues and metabolized to produce metabolites.Therefore,revealing the metabolic differences of different Met sources in different tissues in pigs and clarifying the relationship between metabolic differences and the effects of different Met sources on tissue function are of great significance for understanding the different nutritional mechanisms of different Met sources,and it is also of great guiding value to the selection of appropriate Met additive sources in production.In this study,an LC-MS/MS method for simultaneously detecting the contents of Met metabolites and cofactors in different pig cells was established;the metabolic changes and rules of different Met sources in various pig cell lines were determined;the effects of different Met sources on cell function in the intestinal porcine epithelial cell line?IPEC-J2?,pig iliac artery endothelial cell line?PIEC?and porcine trophectoderm cell line?pTr2?were revealed;and the differences in metabolism of different Met sources and their effects on pig cell functions and RNA m⁶A modification level were explored from the perspective of methylation modification.The main research contents and results are as follows:1. Establishment of LC-MS/MS method for simultaneous detection of Met metabolites and cofactors in pig cellsIn this study,IPEC-J2 cells and PIEC cells were used as materials to establish a LC-MS/MS method for simultaneous detecting Met metabolites and cofactors.By optimizing the mobile phase and the reducing agent,we chosed 10 mmol/L ammonium acetate as the mobile phase A,methanol and acetonitrile mixture at the ratio of 4:1 as mobile phase B,and TCEP as the reducing agent.The results showed that the quantitative limit of each analyte is 0.02-0.91 ng/10⁶ cells,the recovery range is 74.1%-117.4%,the accuracy range is 93.5%-123.4%.Except for MTA and VB12,the RSD of the intra-day and inter-day detection values for other analytes are less than 15%.Except for Hcy,the matrix effect of each analyte is less than 20%.The above results indicate that the detection method is reliable.2. Metabolic characteristics of different Met sources in various pig cell linesUsing the established LC-MS/MS detection method,the contents of Met metabolites and cofactors in cells with L-Met,DL-Met,DL-HMTBA,and DL-MM as Met sources were detected to reveal the metabolic characteristics of different Met sources in IPEC-J2,pig kidney cell line?PK-15?,PIEC,pTr2 cell and the primary hepatocytes of pigs.The main results are as follows:?1?When DL-HMTBA and DL-MM were used as Met sources,the content of Met in IPEC-J2 and pTr2 cells was significantly lower than that in the other Met source groups?P<0.01?;while in the primary hepatocytes of pigs,PIEC and PK-15 cells,there was no significant difference in Met content in each group.By applying the Transwell model and measuring the contents of the Met sources in the lower chamber,it was found that a large amount of DL-HMTBA escaped the metabolism of IPEC-J2 cells and was transported out of IPEC-J2 cells in the form of DL-HMTBA;while some DL-MM were transported out of IPEC-J2 cells in the form of dipeptide and free Met,resulting in higher extracellular supply of Met sources by DL-HMTBA and DL-MM rather than other Met source groups.?2?For the transmethylation,when DL-HMTBA was used as the Met source,the mRNA levels of MAT2A in these five kinds of cells were significantly increased,but in IPEC-J2 and pTr2 cells,the product SAM was still significantly lower than that in the other Met source group?P<0.01?;while only in IPEC-J2 cells,The SAM transmethylated product SAH is still lower,and is significantly higher than that in the other Met source groups in the other cells;except for the primary hepatocytes of pigs,when DL-HMTBA was used as Met source,the ratio of SAM to SAH was significantly lower than that in the other Met source groups?P<0.01?.The impact of DL-MM on SAM and SAH is similar to DL-HMTBA,but the impact is smaller.The above results indicate that DL-HMTBA promotes the transmethylation of Met,and this change may be related to the increased expression of the metabolic enzyme MAT2A.?3?For the transsulfuration,as a substrate of transsulfur metabolism,in IPEC-J2 cells,the intracellular Hcy content in the DL-HMTBA group was significantly lower than that in the other Met source groups,but significantly higher in the primary hepatocytes of pigs and PK-15 cells?P<0.01?,and there was no significant difference in PIEC cells.In these five kinds of cells,DL-HMTBA increased the content of intracellular transsulfur metabolites Cys or/and GSH or/and H₂S.This metabolic difference may be related to DL-HMTBA increasing intracellular CBS or/and CTH mRNA levels?P<0.01?.It indicates that DL-HMTBA may promote the transsulfation metabolism of Met by up-regulating the expression of CBS or/and CTH.?4?For the aminopropyl transfer and remethylation,in IPEC-J2,the primary hepatocytes of pigs,PK-15 and pTr2 cells,DL-HMTBA increased the content of 5-MTHF or/and MTA,and significantly increased the mRNA levels of MTHFR or/and MTR in IPEC-J2,the primary hepatocytes of pigs and pTr2 cells?P<0.01?,which means that DL-HMTBA can promote Met remethylation or/and resynthesis.Therefore,DL-HMTBA significantly increased the transmethylation,transsulfuration and remethylation of Met,and this metabolic difference may be related to the increased expression of related metabolic enzymes by DL-HMTBA.The metabolic differences between DL-HMTBA and other Met sources mainly manifested in IPEC-J2 and pTr2 cells,followed by PIEC cells.3. The effects of different ratios of L-Met and DL-HMTBA on Met metabolism in IPEC-J2,PIEC and pTr2 cellsThe metabolic changes of Met in IPEC-J2,PIEC and pTr2 cells were studied when different ratios of DL-HMTBA were used as the Met source by setting different ratios?100%and 0%,85%and 15%,70%and 30%,40%and 60%,0%and 100%?of L-Met and DL-HMTBA in cell culture medium.The main results are as follows:?1?For the transsulfuration,compared with the 100%L-Met group,when the ratio of L-Met to DL-HMTBA is?85%:15%,the ratio of SAM to SAH in these cells decreased significantly;but only in IPEC-J2 cells,the mRNA level of MAT2A was significantly higher?P<0.01?,indicating that the effect of DL-HMTBA on Met transmethylation metabolites is more sensitive than the effects on metabolic enzymes.?2?For the transsulfuration,compared with the 100%L-Met group,when the ratio of L-Met to DL-HMTBA is?85%:15%,there was no significant difference or significant increase in the content of the transsulfuration metabolites Cys,GSH or/and H₂S in these cells;When the DL-HMTBA is further improved so that the ratio of L-Met to DL-HMTBA?40%:60%?IPEC-J2 cells?or only 100%DL-HMTBA?PIEC and pTr2 cells?,the mRNA levels of metabolic enzymes CBS and CTH were significantly increased?P<0.01?.The above results indicate that the effect of DL-HMTBA on Met transmethylation metabolites is more sensitive than the effects on metabolic enzymes.?3?For the remethylation,compared with the 100%L-Met group,when the ratio of L-Met to DL-HMTBA is?40%:60%,the Met content was significantly lower and5-MTHF content was significantly higher only in IPEC-J2 cells,while only 100%DL-HMTBA was present,the mRNA levels of MTHFR and MTR were significantly increased?P<0.01?.It showed that only when the intracellular Met is significantly deficient,DL-HMTBA could significantly increase the content of metabolic enzymes and metabolites related to the Met remethylation.4. Effects of different ratios of L-Met and DL-HMTBA on the function of PIEC,pTr2and IPEC-J2 cells and the possible mechanisms?1?Compared with the 100%L-Met group,when the ratio of L-Met to DL-HMTBA is?40%:60%,it will significantly affect the cell functions,for example,significantly reduced the number of nodes,tubes and the total tube length of PIEC cells;significantly increased the contents of extracellular leucine,isoleucine and lysine transported to the outside of pTr2 cells;significantly inhibited the increase in the permeability of IPEC-J2monolayer cells after H₂O₂treatment?P<0.05?.?2?The intracellular SAM content and the ratio of SAM to SAH were significantly positively correlated with angiogenesis and VEGF164 mRNA levels in PIEC cell;significantly negatively correlated with mRNA levels of amino acid transporter CAT1 and SNAT2 in pTr2 cell,significantly negatively correlated with the mRNA expression levels of the tight junction protein ZO-1,Claudin and Occludin?P<0.05?.This suggested that the effect of DL-HMTBA on cell function may be related to the change of intracellular SAM content and the ratio of SAM to SAH,as well as the change of mRNA level of key genes related to the corresponding function.?3?In IPEC-J2 cells,when the ratio of L-Met to DL-HMTBA is?70%:30%,the mRNA level of the mRNA levels of RNA transmethylase METTL3,RNA demethylase FTO and recognition protein YTHDF2 were significantly higher than those in the 100%L-Met group?P<0.01?;DL-HMTBA significantly reduced the m⁶A modification levels of MAT2A and ZO-1,and significantly improved the mRNA stability of MAT2A and ZO-1?P<0.05?.It showed that DL-HMTBA increases the expression of tight junction proteins in IPEC-J2 cells,which may be related to the reduction of m⁶A modification of intracellular mRNA.In summary,the main conclusions of this study are:?1?DL-HMTBA has different metabolic characteristics from other Met sources,and significantly increased the transmethylation,transsulfation and remethylation levels in IPEC-J2 and pTr2 cells,and this difference may be related to the increased expression of related metabolic enzymes by DL-HMTBA.?2?In PIEC,pTr2 and IPEC-J2 cells,when DL-HMTBA accounts for more than 15%of the total Met sources,it will significantly affect the metabolite contents of transmethylation and transsulfuration,reduce the ratio of SAM to SAH;and when further increased to more than 30%and 60%,the expression levels of mRNA will be changed.In IPEC-J2 cells,DL-HMTBA reduces the m⁶A modification of ZO-1,improves the mRNA stability and the mRNA and protein levels of ZO-1,thus improving the cell barrier function.