Methyldonors choline and methionine differentially alter hepatic carbon

Methyl-donors choline and methionine differentially alter hepatic carbon metabolism Tawny L. Chandler, Courtney L. Mc. Court, Sandra J. Bertics, Barbara A. Barton, and Heather M. White

Methyl Group Metabolism Methyl donation is needed for DNA methylation Prevention of oxidative stress Prevention of apoptosis Energy metabolism Protein synthesis Methyl group competition by pathways

Methyl Group Metabolism Methyl groups come from methyl donors methionine (1) choline (3) betaine (3) folate (5 -methyltetrahydrofolate; 1)

VLDL Packaging and Export Choline Methionine Deficient Diet in Rodents Rinella et al. , 2008 Cole et al. , 2012

Methyl Group Metabolism glycine Dimethylglycine Cysteine Hcy SAHH BHMT Betaine MS THF Choline Methionine 5 -MTHF phospholipase D glutathione MAT SAH SAM CH 3 Phosphatidylethanolamine phospholipids for cell membranes White Phosphatidylcholine VLDL packaging

Cell Culture Model Allows for testing several concentrations and combinations Eliminates confounders of other changes Primary Hepatocytes Culture Chandler et al. , 2015

Bovine Methyl Group Metabolism d. L Met, m. M 0. 033 Choline, m. M 0. 1 2 4. 5 0. 016 0. 030 0. 100 0. 300 Design notes: Mimics in vivo concentrations Concentration and methyl group equivalents Chandler et al. , 2015

Bovine Methyl Group Metabolism d. L Met, m. M 0. 033 Choline, m. M 0. 1 2 0. 016 0. 030 0. 100 0. 300 Does this mimic the transition cow state? Chandler et al. , 2015 4. 5

Bovine Methyl Group Metabolism d. L Met, m. M Choline, m. M 0. 033 0. 1 2 4. 5 0. 016 0. 030 0. 100 0. 300 d. L Met, m. M Choline, m. M 0. 033 0. 1 2 Chandler et al. , 2015 0. 016 0. 030 0. 100 0. 300 1 m. M Fatty Acid Cocktail 4. 5

Main Effects vs. Interactions No interactions, main effects shown 1. 6 1. 4 Arbitrary units 1. 2 1 0. 8 0. 6 0. 4 0. 2 [choline], m. M Chandler et al. , 2015 0. 033 0. 1 2 4. 5 3 0. 16 0. 03 0. 0 3 0. 1 0. 0 [met], m. M 16 0. 03 0

Methionine Regeneration glutathione glycine Dimethylglycine Cysteine BHMT MS Hcy THF Chandler et al. , 2015 Betaine Methionine 5 -MTHF Choline

BHMT Dimethylglycine P = 0. 3603 1 0. 8 BHMT Betaine Methionine Hcy 0. 6 0. 4 0. 2 1 0 0. 8 -0. 2 -0. 4 0. 016 0. 03 0. 1 [methionine], m. M 0. 3 arbitrary units 1. 2 P = 0. 8654 0. 6 0. 4 0. 2 0 -0. 2 Chandler et al. , 2015 -0. 4 0. 033 0. 1 2 [choline], m. M 4. 5

Methionine Synthase (MS) 1. 4 P = 0. 0026 Linear: P = 0. 0021 Hcy THF 1 Methionine 5 -MTHF 0. 8 0. 6 1. 4 0. 4 1. 2 P = 0. 0152 Linear: P = 0. 0037 1 0. 2 0 0. 016 0. 03 0. 1 0. 3 [methionine], m. M arbitrary units 1. 2 MS 0. 8 0. 6 0. 4 0. 2 0 -0. 2 -0. 4 Chandler et al. , 2015 0. 033 0. 1 2 [choline], m. M 4. 5

BHMT During Fatty Acid Challenge 1. 2 FA: P = 0. 0462 1 0. 8 arbitrary units 1 0. 6 0. 4 0. 2 0 -0. 2 -0. 4 FA: P = 0. 0462 0. 016 0. 03 0. 1 [methinonine], m. M no FA Chandler et al. , 2015 FA 0. 3 0. 8 0. 6 0. 4 0. 2 0 -0. 2 -0. 4 0. 033 0. 1 2 [choline], m. M no FA FA 4. 5

MS During Fatty Acid Challenge FA P = 0. 0001 1. 2 arbitrary units 1. 4 1 0. 8 0. 6 0. 4 0. 2 0 -0. 2 0. 016 0. 03 0. 1 0. 3 [methinonine], m. M no FA Chandler et al. , 2015 FA 1. 4 1. 2 1 0. 8 0. 6 0. 4 0. 2 0 -0. 2 -0. 4 FA P = 0. 0001 0. 033 0. 1 2 [choline], m. M no FA FA 4. 5

Methionine Regeneration glutathione glycine Dimethylglycine Cysteine BHMT Betaine MS Hcy THF Choline Methionine 5 -MTHF Increased d. L met decreases MS Increased choline increases MS Choline may serve a role in methionine regeneration Fatty acid load increases BHMT and decreases MS Preference for pathway that produces antioxidants Chandler et al. , 2015

arbitrary units PEMT 4 3. 5 3 2. 5 2 1. 5 1 0. 5 0 P = 0. 7012 Choline 0. 016 0. 03 0. 1 Methionine [methionine], m. M Phosphatidylethanolamine 0. 3 PEMT No change in PEMT with d. L Met Chandler et al. , 2015 Phosphatidylcholine VLDL packaging

VLDL Quantification is challenging due to differences in lipid profile of ruminant VLDL Dual antibody based ELISA that specifically identifies VLDL from LDL and HDL Apo. B 100 Apo. C

VLDL Export P = 0. 7039 1. 4 arbitrary units 1. 2 VLDL packaging and export 1 0. 8 0. 6 0. 4 0. 2 0 0. 03 0. 1 0. 3 [methionine], m. M P = 0. 0258 1. 4 1. 2 arbitrary units 0. 016 1 0. 8 0. 6 0. 4 0. 2 0 0. 033 Mc. Court et al. , 2015 0. 1 2 [choline], m. M 4. 5

Oxidative Stress ROS accumulation decreases hepatocyte function Fatty acid relative to no fatty acid for each treatment Increasing Choline concentration tended to decrease ROS • 2 m. M choline had lowest ROS d. L Met did not change ROS Chandler et al. , 2015

Hepatocyte Response Summary Increasing d. L Met Decreases endogenous Met regeneration Did not increase PEMT and does not change VLDL export Did not change ROS Increasing Choline Increases endogenous Met regeneration Tended to decrease ROS accumulation Increased VLDL packaging Chandler et al. , 2015

Hepatocyte Response Summary Suggests a biological priority for methyl donor use Whole-animal and cellular level Lack of interaction supports separate roles for Met and Choline Increased NEFA may shift pathways of Met regeneration Cellular Met need can be met by dietary Met or by endogenous regeneration Met regeneration requires methyl donation Choline can provide the methyl group Chandler et al. , 2015

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