Date Published: February 2, 2017
Publisher: Public Library of Science
Author(s): Tawny L. Chandler, Heather M. White, Salvatore V. Pizzo.
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.
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; however, extensive rumen microbial degradation limits hepatic supply of these in functional ruminants [1,2]. Rumen-protection and gastro-intestinal administration of choline and methionine increase arterial concentrations of choline metabolites [3,4] and portal vein concentrations of methionine . Given that metabolism of both choline and methionine provides methyl groups, pathways of their catabolism and endogenous remethylation intersect. Exogenous sources or endogenously remethylated methionine are required for the methylation of S-adenosylmethionine (SAM), the universal biological methyl donor  in the transmethylation pathway (Fig 1). Although 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]. Methionine supply is further challenged by its use for the de novo synthesis of choline when choline is unavailable. In lactating goats, an equivalent of 28% of absorbable methionine is involved in choline synthesis . While choline recycled from phosphatidylcholine (PC) can supply labile methyl groups for the regeneration of methionine (Fig 1) , 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].
No interactions were detected between CC, DLM and FA treatment for any of the genes examined, therefore the 2- and 3-way interactions are not shown or discussed. Because quadratic effects were not significant, only linear effects of CC and DLM treatment for genes associated with methyl carbon metabolism are presented in Figs 2 & 3. Fatty acid treatment differentially affected the genes examined and the main effect of FA is shown in Fig 4.
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; however, these confounding factors are difficult to elucidate in vivo. For this reason, a well-established primary bovine hepatocyte in vitro model using hepatocytes isolated from neonatal [25,30–32] or weaned eight or ten week old calves [33,34] was used in the current work to directly examine the role of methionine and choline on hepatocyte metabolism. Primary bovine hepatocytes in the current research were derived from neonatal calves and the hepatocytes were subsequently incubated in media that reflects the long-chain fatty acid composition found in the plasma of ruminating animals, in order to examine the effect of fatty acids, choline, and methionine on methyl group metabolism and ROS responses. Treatment concentrations were selected guided by information available regarding bovine arterial concentrations; however, it is important to note that nutrient concentrations can be 50% greater in the portal vein than in arteries . Plasma concentrations of methionine have been consistently reported between 15 and 25 μmol/L in lactating cows [5,36–38], but range from 30 to 40 μM [5,37,39] and can increase up to 144 μmol/L  when supplemental rumen-protected methionine is fed. Circulating choline is present in many different forms including free choline, phosphocholine, PC, lysophosphatidylcholine, glycerophosphocholine, and direct products including betaine and sphingomyelin; most of which can be used to supply choline or choline-derived methyl groups to the liver . Uptake kinetics and metabolism of choline in perfused rat liver and isolated rat hepatocytes revealed that the cells were responsive to concentrations that ranged from 20 to 4500 μmol/L [40,41]. Recent characterization of plasma and milk choline metabolites in dairy cattle during different stages of lactation demonstrated that total plasma choline metabolite concentrations range from 1,305 to 14,241 μmol/L . Reflectively, at 4×4×2 factorial design of 16, 30, 100, 300 μmol/L DLM and 61, 128, 2028, and 4529 μmol/L CC with or without the addition of the 1 mmol/L fatty acid cocktail was used. The linear responses of gene expression to these CC and DLM doses did not appear to plateau within the range of concentrations examined here.
While both choline and methionine supplementation influence production and growth of dairy cattle, the lack of interaction in the present study supports preferential uses for choline and methionine in bovine hepatocytes. Increasing supply of choline appeared to support methionine regeneration from the folate methyl pool, while methionine supply decreased the need for homocysteine remethylation. This offers evidence that choline could support the transmethylation pathway, sparing methionine from catabolism for its preferential use in protein synthesis and later anabolic output. In addition, choline uniquely supported hepatic lipid metabolism by increasing VLDL export and limiting the accumulation of ROS. The data discussed here supports that the mechanism of choline to decrease hepatic TG is by increasing the synthesis and export of VLDL.