The effect of increasing concentrations of dl-methionine and 2-hydroxy-4-(methylthio) butanoic acid on hepatic genes controlling methionine regeneration and gluconeogenesis
2016
Abstract Metabolizable methionine (Met) concentrations can be increased by feeding rumen-protected dl-Met or the isopropyl ester of 2-hydroxy-4-(methylthio) butanoic acid (HMBi). Hepatic responses to increasing concentrations of metabolizable Met as a result of supplementation of different Met sources have not been comparatively examined. The objective of this experiment was to examine the regulation of key genes for Met metabolism, gluconeogenesis, and fatty acid oxidation in response to increasing concentrations of dl-Met or 2-hydroxy-4-(methylthio) butanoic acid (HMB) in bovine primary hepatocytes. Hepatocytes isolated from 4 Holstein calves less than 7d old were maintained as monolayer cultures for 24h before addition of treatments. Cells were then exposed to treatments of dl-Met or HMB (0, 10, 20, 40, or 60 µ M ) in Met-free medium for 24h and collected for RNA isolation and quantification of gene expression by quantitative PCR. Expression of betaine-homocysteine methyltransferase ( BHMT ), 5-methyltetrahydrofolate-homocysteine methyltransferase ( MTR ), and 5,10 methylenetetrahydrofolate reductase ( MTHFR ) genes, which catalyze regeneration of Met from betaine and homocysteine, decreased linearly with increasing dl-Met concentration. We observed similar effects with increasing HMB concentration, except expression of MTHFR, which was not altered. Expression of Met adenosyltransferase 1A ( MAT1A ), which catalyzes the first step of Met metabolism to generate S-adenosylmethionine (SAM), a primary methyl donor, was decreased with increasing dl-Met or HMB concentration. Expression of S-adenosylhomocysteine hydrolase ( SAHH ) was decreased linearly with increasing HMB concentration, but not altered by dl-Met. Increasing concentrations of dl-Met and HMB decreased cytosolic phosphoenolpyruvate carboxykinase ( PCK1 ) expression, but did not alter the expression of mitochondrial phosphoenolpyruvate carboxykinase ( PCK2 ) or pyruvate carboxylase ( PC ). Expression of glucose-6-phosphatase( G6PC ) decreased linearly with increasing HMB concentration, but not altered by dl-Met. Neither dl-Met nor HMB altered the expression of carnitine palmitoyltransferase 1A( CPT1a ). These findings demonstrate reduced necessity for Met regeneration with increased Met concentrations in the medium, regardless of the Met source. The lack of upregulation of gluconeogenesis indicates that increased dl-Met or HMB is not prioritized for glucose synthesis in primary bovine hepatocytes.
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