Abstract
Intersections in hepatic methyl group metabolism pathways highlights potential competition or compensation of methyl donors. The objective of this experiment was to examine the expression of genes related to methyl group transfer and lipid metabolism in response to increasing concentrations of choline chloride (CC) and DL-methionine (DLM) in primary neonatal hepatocytes that were or were not exposed to fatty acids (FA). Primary hepatocytes isolated from 4 neonatal Holstein calves were maintained as monolayer cultures for 24 h before treatment with CC (61, 128, 2028, and 4528 μmol/L) and DLM (16, 30, 100, 300 μmol/L), with or without a 1 mmol/L FA cocktail in a factorial arrangement. After 24 h of treatment, media was collected for quantification of reactive oxygen species (ROS) and very low-density lipoprotein (VLDL), and cell lysates were collected for quantification of gene expression. No interactions were detected between CC, DLM, or FA. Both CC and DLM decreased the expression of methionine adenosyltransferase 1A (MAT1A). Increasing CC did not alter betaine-homocysteine S-methyltranferase (BHMT) but did increase 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) and methylenetetrahydrofolate reductase (MTHFR) expression. Increasing DLM decreased expression of BHMT and MTR, but did not affect MTHFR. Expression of both phosphatidylethanolamine N-methyltransferase (PEMT) and microsomal triglyceride transfer protein (MTTP) were decreased by increasing CC and DLM, while carnitine palmitoyltransferase 1A (CPT1A) was unaffected by either. Treatment with FA decreased the expression of MAT1A, MTR, MTHFR and tended to decrease PEMT but did not affect BHMT and MTTP. Treatment with FA increased CPT1A expression. Increasing CC increased secretion of VLDL and decreased the accumulation of ROS in media. Within neonatal bovine hepatocytes, choline and methionine differentially regulate methyl carbon pathways and suggest that choline may play a critical role in donating methyl groups to support methionine regeneration. Stimulating VLDL export and decreasing ROS accumulation suggests that increasing CC is hepato-protective.
Highlights
Labile methyl groups are required to support hepatic metabolism and are involved in pathways of transmethylation, transsulfuration, the folate cycle, and synthesis of methylated compounds
Expression of methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) was linearly increased (P < 0.05) as choline chloride (CC) increased in the media
Utilization of choline and methionine as nutrients to support the anabolic output of production in transition dairy cows may be confounded by requirements to fulfill biological roles to support maintenance metabolism, such as methyl donation and protein synthesis, prior to fulfilling the demands associated with the anabolic output of growth and production; these confounding factors are difficult to elucidate in vivo
Summary
Labile methyl groups are required to support hepatic metabolism and are involved in pathways of transmethylation, transsulfuration, the folate cycle, and synthesis of methylated compounds. Methyl donors such as betaine, choline after oxidation to betaine, methionine, and folic acid can supply methyl groups; extensive rumen microbial degradation limits hepatic supply of these in functional ruminants [1,2]. Microbial protein supplies methionine for intestinal absorption in ruminants, this microbial protein has to meet the requirements of methionine as a source of methyl groups and a potential limiting amino acid for net protein synthesis to support ruminant growth and production [7,8]. While choline recycled from phosphatidylcholine (PC) can supply labile methyl groups for the regeneration of methionine (Fig 1) [10], this potential drain of PC may impair lipid membrane integrity and very-low density lipoprotein (VLDL) secretion by the liver, as PC is integral to both [11,12]
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