Insufficient supply of Met and choline (Chol) around parturition could compromise hepatic metabolism and milk protein synthesis in dairy cows. Mechanistic responses associated with supply of Met or Chol in primary liver cells enriched with hepatocytes (PHEP) from cows have not been thoroughly ascertained. Objectives were to isolate and culture PHEP to examine abundance of genes and proteins related to transmethylation, transsulfuration, and cytidine 5′-diphosphocholine (CDP-choline) pathways in response to Met or Chol. The PHEP were isolated from liver biopsies of Holstein cows (160 d in lactation). More than 90% of isolated cells stained positively for the hepatocyte marker cytokeratin 18. Cytochrome P450 (CYP1A1) mRNA abundance was only detectable in the PHEP and liver tissue compared with mammary tissue. Furthermore, in response to exogenous Met (80 μM vs. control) PHEP secreted greater amounts of albumin and urea. Subsequently, PHEP were cultured with Met (40 μM) or Chol (80 mg/dL) for 24 h. Compared with control or Chol, mRNA and protein abundance of methionine adenosyltransferase 1A (MAT1A) and phosphatidylethanolamine methyltransferase (PEMT) were greater in PHEP treated with Met. The mRNA abundance of S-adenosylhomocysteine hydrolase (SAHH), betaine-homocysteine methyltransferase (BHMT), and sarcosine dehydrogenase (SARDH) was greater in Met-treated PHEP compared with control or Chol. Compared with control, greater expression of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), betaine aldehyde dehydrogenase (BADH), and choline dehydrogenase (CHDH) was observed in cells supplemented with Met and Chol. However, Chol led to the greatest mRNA abundance of CHDH. Abundance of choline kinase α (CHKA), choline kinase β (CHKB), phosphate cytidylyltransferase 1 α (PCYT1A), and choline/ethanolamine phosphotransferase 1 (CEPT1) in the CDP-choline pathway was greater in PHEP treated with Chol compared with control or Met. In the transsulfuration pathway, mRNA and protein abundance of cystathionine β-synthase (CBS) was greater in PHEP treated with Met compared with control or Chol. Similarly, abundance of cysteine sulfinic acid decarboxylase (CSAD), glutamate-cysteine ligase, catalytic subunit (GCLC), and glutathione reductase (GSR) was greater in response to Met compared with control or Chol. Overall, these findings suggest that transmethylation and transsulfuration in dairy cow primary liver cells are more responsive to Met supply, whereas the CDP-choline pathway is more responsive to Chol supply. The relevance of these data in vivo merit further study.

分娩期间Met和胆碱（Chol）的供应不足可能会损害奶牛的肝代谢和牛奶蛋白合成。尚未充分确定与奶牛中富含肝细胞（PHEP）的原代肝细胞中Met或Chol的供应相关的机械反应。目的是分离和培养PHEP，以检查与Met或Chol反应的转甲基化，转硫和胞苷5'-二磷酸胆碱（CDP-胆碱）途径相关的基因和蛋白质的丰富性。从荷斯坦奶牛（泌乳期160 d）的肝活检中分离出PHEP。超过90％的分离细胞对肝细胞标记物细胞角蛋白18染色呈阳性。细胞色素P450（ CYP1A1）与乳腺组织相比，仅在PHEP和肝组织中可检测到mRNA的丰度。此外，响应于外源性的Met（80μ中号相对于对照）PHEP分泌更大量的白蛋白和尿素。随后，PHEP用的Met（40μ培养中号）或澈（80毫克/分升）24小时。与对照组或Chol相比，Met处理的PHEP中蛋氨酸腺苷基转移酶1A（MAT1A ）和磷脂酰乙醇胺甲基转移酶（PEMT）的mRNA和蛋白质丰度更高。S-腺苷同型半胱氨酸水解酶（SAHH），甜菜碱-同型半胱氨酸甲基转移酶（BHMT）和肌氨酸脱氢酶（SARDH）的mRNA丰度与对照组或Chol相比，经Met处理的PHEP中的）更高。与对照相比，在补充了Met和Chol的细胞中，观察到了5-甲基四氢叶酸-高半胱氨酸甲基转移酶（MTR ），甜菜碱醛脱氢酶（BADH）和胆碱脱氢酶（CHDH）的更高表达。但是，Chol导致CHDH的最大mRNA丰度。胆碱激酶α（CHKA），胆碱激酶β（CHKB），磷酸胞苷转移酶1α（PCYT1A）和胆碱/乙醇胺磷酸转移酶1（CEPT1）的丰度与对照或Met相比，用Chol处理的PHEP中CDP-胆碱途径的CDP胆碱途径更大。在转硫途径中，与对照或Chol相比，用Met处理的PHEP中胱硫醚β-合酶（CBS）的mRNA和蛋白丰度更高。类似地，与对照或Chol相比，对Met响应的半胱氨酸亚磺酸脱羧酶（ CSAD），谷氨酸-半胱氨酸连接酶，催化亚基（GCLC ）和谷胱甘肽还原酶（GSR）的丰度更高。总体而言，这些发现表明，奶牛原代肝细胞中的甲基化和转硫反应对Met的供应更敏感，而CDP-胆碱途径对Chol的供应更敏感。这些数据在体内的相关性值得进一步研究。