Choline and methionine differentially alter methyl carbon metabolism in bovine neonatal hepatocytes

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.

Fig 1. Intersection between pathways of choline and methionine metabolism in the transmethylation cycle and key enzymes that control methyl group transfer: methionine adenosyltransferase 1A (MAT1A), betaine-homocysteine S-methyltranferase (BHMT), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), methylenetetrahydrofolate reductase (MTHFR), microsomal triglyceride transfer protein (MTTP), S-adenosylhomocysteine hydrolase (SAHH), glycine-N-methyltransferase (GNMT), guanidinoacetate N-methyltransferase (GAMT), phosphatidylethanolamine N-methyltransferase (PEMT), vitamin B₁₂ (B₁₂), dimethylglycine (DMG), glutathione (GSH), homocysteine (HCY), S-adenosylmethionine (SAM), S-adenocylhomocysteine (SAH), tetrahydrofolate (THF), phosphatidylethanolamine (PE), phosphatidylcholine (PC), very low-density lipoprotein (VLDL).

Fig 2. Expression of genes related to choline and methionine metabolism in the transmethylation pathway in neonatal bovine hepatocytes exposed to increasing concentrations of choline chloride (CC) and DL-methionine (DLM).

The P-values for linear effects of CC and DLM are shown; there were no interactions between CC and DLM. Values are least squares means, with SE represented by vertical bars. Methionine adenosyltransferase 1A (MAT1A), betaine-homocysteine S-methyltranferase (BHMT), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), methylenetetrahydrofolate reductase (MTHFR).

Fig 3. Expression of genes related to lipid metabolism and oxidation in neonatal bovine hepatocytes exposed to increasing concentrations of choline chloride (CC) and DL-methionine (DLM); there were no interactions between CC and DLM.

Values are least squares means, with SE represented by vertical bars. Phosphatidylethanolamine N-methyltransferase (PEMT), microsomal triglyceride transfer protein (MTTP), carnitine palmitoyltransferase 1A (CPT1A).

Fig 4. Expression of genes related to pathways of metabolism in transmethylation, lipid metabolism, and lipid oxidation in neonatal bovine hepatocytes exposed to increasing concentrations of choline chloride and DL-methionine in the presence (closed bars) or absence (open bars) of a 1 mmol/L fatty acid cocktail.

The main effects of fatty acid (FA) treatment are shown; there were no interactions between the methyl donor or FA treatment. Values are least squares means, with SE represented by vertical bars. Effects are denoted as tendency (P ≤ 0.10, †) and significant (P ≤ 0.05, *; P ≤ 0.001, **). Methionine adenosyltransferase 1A (MAT1A), betaine-homocysteine S-methyltranferase (BHMT), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), methylenetetrahydrofolate reductase (MTHFR), phosphatidylethanolamine N-methyltransferase (PEMT), microsomal triglyceride transfer protein (MTTP), carnitine palmitoyltransferase 1A (CPT1A).

Fig 5. Relative concentration of VLDL in cell culture media that incubated neonatal hepatocytes treated with increasing concentrations of choline chloride (CC) and DL-methionine (DLM) and a 1 mmol/L fatty acid cocktail for 24 h.

Concentration of VLDL was normalized to the lowest CC and DLM treatment within cell preparation. Increasing CC linearly increased (P = 0.03) VLDL release from neonatal hepatocytes and treatment with DLM had no effect (P ≥ 0.10); there was no interaction between CC and DLM.

Fig 6. Relative concentration of reactive oxygen species (ROS) in media that incubated neonatal hepatocytes treated with increasing concentrations of choline chloride (CC) and DL-methionine (DLM) and a 1 mmol/L fatty acid cocktail for 24 h.

Concentration was normalized to controls and expressed as the ratio of treatment with FA to treatment without FA. Increasing CC tended to linearly decrease (P = 0.08) the accumulation of ROS and DLM had no effect (P ≥ 0.10); there was no interaction between CC and DLM.