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

 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) Methyl Group Metabolism

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

Methyl Group Metabolism Choline Phosphatidylcholine VLDL packaging phospholipids for cell membranes Phosphatidylethanolamine Methionine BetaineDimethylglycine Hcy Cysteine glycineglutathione SAH SAM phospholipase D BHMT MAT SAHH MS 5-MTHF THF CH3 White

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 Choline, mM dL Met, mM  Design notes:  Mimics in vivo concentrations  Concentration and methyl group equivalents Chandler et al., 2015

Bovine Methyl Group Metabolism Choline, mM dL Met, mM  Does this mimic the transition cow state? Chandler et al., 2015

Bovine Methyl Group Metabolism Choline, mM dL Met, mM Choline, mM dL Met, mM 1 mM Fatty Acid Cocktail Chandler et al., 2015

Main Effects vs. Interactions Chandler et al., 2015 [met], mM Arbitrary units [choline], mM No interactions, main effects shown

Methionine Regeneration Choline Methionine BetaineDimethylglycine Hcy Cysteine glycineglutathione BHMT MS 5-MTHF THF Chandler et al., 2015

BHMT Methionine BetaineDimethylglycine Hcy BHMT arbitrary units Chandler et al., 2015

Methionine Synthase (MS) Methionine Hcy MS 5-MTHF THF arbitrary units Chandler et al., 2015

BHMT During Fatty Acid Challenge arbitrary units Chandler et al., 2015

MS During Fatty Acid Challenge FA P = arbitrary units Chandler et al., 2015

Methionine Regeneration Choline Methionine BetaineDimethylglycine Hcy Cysteine glycineglutathione BHMT MS 5-MTHF THF  Increased dL 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

PEMT Choline Phosphatidylcholine VLDL packaging Phosphatidylethanolamine Methionine PEMT  No change in PEMT with dL Met arbitrary units Chandler et al., 2015

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 ApoB100 ApoC

VLDL Export McCourt et al., 2015 P = P = VLDL packaging and export

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 mM choline had lowest ROS  dL Met did not change ROS Chandler et al., 2015

Hepatocyte Response Summary  Increasing dL 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

Questions?