Hepatic gluconeogenic enzymes are differentially altered by methyl-donors choline and methionine in bovine primary hepatocytes

Abstract Text: Tricarboxylic acid cycle (TCA) and gluconeogenic carbon flux are controlled by balances of pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK). The lipotropic action of choline and methionine may alter fatty acid (FA) oxidation and gluoneogenic carbon availability. The objective of this experiment was to examine regulation of genes controlling gluconeogenesis in response to increasing concentrations of choline chloride (CC), DL-methionine (DLM), and added FA. Primary hepatocytes isolated from 4 Holstein calves were maintained as monolayer cultures for 24h in media containing optimal concentrations of essential amino acids and 1.25 mM pyruvic acid. Treatments of physiologically relevant concentrations of CC (33, 100, 2000, 4500 μM) and DLM (16, 30, 100, 300 μM), with or without a 1 mM FA cocktail, were added to a methionine-free media in a factorial design. After 24h of treatment, cells were harvested for RNA isolation, cDNA generation, and quantification of gene expression by quantitative PCR. Abundance of mRNA was normalized to the geometric mean of three reference genes. Data were analyzed using PROC MIXED of SAS 9.4 with linear and quadratic contrasts in a model accounting for fixed effect of treatment and random effect of calf and reported as least squares means ± SE. Expression of PC tended to be linearly increased (P=0.06) by CC (1.28, 1.42, 1.43, 1.50 ± 0.21 arbitrary units (AU)) and was unaffected (P>0.15) by DLM (1.39, 1.51, 1.38, 1.35 ± 0.21 AU). Although, mitochondrial PEPCK (PEPCKm) expression was unaffected (P≥0.15) by CC (1.64, 1.58, 1.60, 1.59 ± 0.5 AU) or DLM (1.49, 1.57, 1.65, 1.69 ± AU), cytosolic PEPCK (PEPCKc) tended to be linearly increased (P=0.11) by CC (1.03, 1.19, 1.30, 1.57 ± 0.32 AU) and decreased (P=0.08) by DLM (1.60, 1.31, 1.21, 0.97 ± 0.32). Expression of glucose 6-phosphatase (G6P) was quadratically affected (P=0.009) by CC (1.14, 1.42, 0.99, 1.13 ± 0.30) and unaffected (P>0.15) by DLM (1.14, 1.17, 1.19, 1.17 ± 0.30). Treatment with FA increased (P<0.001) expression of PC (1.11 vs. 1.70 ± 0.20 AU), PEPCKc (0.55 vs. 2.0 ± 0.27 AU), PEPCKm (1.36 vs. 1.84 ± 0.48 AU), and G6P (0.93 vs. 1.41 ± 0.29 AU). Coordinated increases in PC and PEPCKc with increasing CC suggests increased capacity for gluconeogenesis. Conversely, decreased PEPCKc without altered PCmay indicate that DLM may increase TCA capacity but not gluconeogenic capacity. Choline and methionine appear to differentially regulate TCA cycle and gluconeogenesis.

Keywords: Methyl-donors, gluconeogenesis, primary hepatocytes