2. 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

3.  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

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

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

6. 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

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

8. Bovine Methyl Group Metabolism 0.0330.124.5 0.016 0.030 0.100 0.300 Choline, mM dL Met, mM  Design notes:  Mimics in vivo concentrations  Concentration and methyl group equivalents Chandler et al., 2015

9. Bovine Methyl Group Metabolism 0.0330.124.5 0.016 0.030 0.100 0.300 Choline, mM dL Met, mM  Does this mimic the transition cow state? Chandler et al., 2015

10. Bovine Methyl Group Metabolism 0.0330.124.5 0.016 0.030 0.100 0.300 Choline, mM dL Met, mM 0.0330.124.5 0.016 0.030 0.100 0.300 Choline, mM dL Met, mM 1 mM Fatty Acid Cocktail Chandler et al., 2015

11. Main Effects vs. Interactions Chandler et al., 2015 [met], mM Arbitrary units [choline], mM 0.0330.124.5 No interactions, main effects shown

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

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

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

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

16. MS During Fatty Acid Challenge FA P = 0.0001 arbitrary units Chandler et al., 2015

17. 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

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

19. 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

20. VLDL Export McCourt et al., 2015 P = 0.7039 P = 0.0258 VLDL packaging and export

21. 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

22. 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

23. 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

24. Questions?