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 methylationPrevention of oxidative stressPrevention of apoptosisEnergy metabolismProtein 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., 2012Rinella et al., 2008

Choline Methionine Deficient Diet in Rodents

Methyl Group Metabolism

Choline

Phosphatidylcholine

VLDL packagin

gphospholipids

for cell membranes

Phosphatidylethanolamine

Methionine

BetaineDimethylglycine

HcyCysteine

glycineglutathione

SAH

SAM

phosp

holip

ase

D

BHMT

MATSAHH

MS

5-MTHFTHF

CH3

White

Cell Culture Model

Allows for testing several concentrations and combinations

Eliminates confounders of other changesPrimary Hepatocytes Culture

Chandler et al., 2015

Bovine Methyl Group Metabolism

0.033 0.1 2 4.50.0160.0300.1000.300

Choline, mM

dL M

et, m

M

Design notes:Mimics in vivo concentrationsConcentration and methyl group equivalents

Chandler et al., 2015

Bovine Methyl Group Metabolism

0.033 0.1 2 4.50.0160.0300.1000.300

Choline, mM

dL M

et, m

M

Does this mimic the transition cow state?

Chandler et al., 2015

Bovine Methyl Group Metabolism

0.033 0.1 2 4.50.0160.0300.1000.300

Choline, mM

dL M

et, m

M

0.033 0.1 2 4.50.0160.0300.1000.300

Choline, mM

dL M

et, m

M

1 mM Fatty Acid Cocktail

Chandler et al., 2015

Main Effects vs. Interactions

Chandler et al., 2015

0

0.2

0.4

0.6

0.8

1

1.2

1.4

[met], mM

Arb

itra

ry u

nit

s

[choline], mM 0.033 0.1 2 4.5

No interactions, main effects shown

Methionine Regeneration

Choline

Methionine

BetaineDimethylglycine

HcyCysteine

glycineglutathione

BHMT

MS

5-MTHFTHF

Chandler et al., 2015

BHMT

0.033 0.1 2 4.50.58

0.60.620.640.660.68

0.70.720.74

[choline], mM

P = 0.8654

0.016 0.03 0.1 0.30

0.10.20.30.40.50.60.70.80.9

[methionine], mM

P = 0.3603

Methionine

BetaineDimethylglycine

Hcy

BHMT

arbi

trar

y un

its

arbi

trar

y un

its

Chandler et al., 2015

Methionine Synthase (MS)

0.033 0.1 2 4.50

0.2

0.4

0.6

0.8

1

1.2

[choline], mM

P = 0.0152Linear: P = 0.0037

0.016 0.03 0.1 0.30

0.2

0.4

0.6

0.8

1

1.2

[methionine], mM

P = 0.0026Linear: P = 0.0021

MethionineHcyMS

5-MTHFTHF

arbi

trar

y un

its

arbi

trar

y un

its

Chandler et al., 2015

BHMT During Fatty Acid Challenge

0.033 0.1 2 4.50

0.10.20.30.40.50.60.70.80.9

no FA FA

[choline], mM

FA: P = 0.0462

0.016 0.03 0.1 0.30

0.10.20.30.40.50.60.70.80.9

no FA FA

[methinonine], mM

FA: P = 0.0462

arbi

trar

y un

its

arbi

trar

y un

its

Chandler et al., 2015

MS During Fatty Acid Challenge

0.033 0.1 2 4.50

0.2

0.4

0.6

0.8

1

1.2

no FA FA

[choline], mM

0.0160.03 0.1 0.3

-2.22044604925031E-160.20.40.60.8

11.21.4

no FA FA

[methinonine], mM

FA P = 0.0001 FA P = 0.0001

arbi

trar

y un

its

arbi

trar

y un

its

Chandler et al., 2015

Methionine Regeneration

Choline

Methionine

BetaineDimethylglycine

HcyCysteine

glycineglutathione

BHMT

MS

5-MTHFTHF

Increased dL met decreases MS Increased choline increases MS

Choline may serve a role in methionine regenerationFatty acid load increases BHMT and decreases MS

Preference for pathway that produces antioxidants

Chandler et al., 2015

PEMT

Choline

Phosphatidylcholine

VLDL packagin

g

Phosphatidylethanolamine

Methionine

PEMT

No change in PEMT with dL Met

0.016 0.03 0.1 0.30

0.5

1

1.5

2

2.5

3

[methionine], mM

P = 0.7012

arbi

trar

y un

its

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

ApoB100ApoC

VLDL Export

McCourt et al., 2015

0.016 0.03 0.1 0.30.75

0.8

0.85

0.9

0.95

1

[methionine], mM

arbi

trar

y un

its

0.033 0.1 2 4.50

0.2

0.4

0.6

0.8

1

1.2

[choline], mM

arbi

trar

y un

its

P = 0.7039

P = 0.0258

VLDL packagin

g 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 MetDecreases endogenous Met regenerationDid not increase PEMT and does not change VLDL exportDid not change ROS

Increasing Choline Increases endogenous Met regenerationTended 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 levelLack of interaction supports separate roles for Met

and Choline Increased NEFA may shift pathways of Met

regenerationCellular Met need can be met by dietary Met or by

endogenous regenerationMet regeneration requires methyl donation

Choline can provide the methyl groupChandler et al.,

2015

Questions?