Regulation of hepatic betaine-homocysteine methyltransferase by dietary methionine

Vol. 108, No. 1, 1982 September

8lOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

16, 1982

Pages

REGULATION

James

D.

Veterans

OF HEPATIC

Finkelstein,

Barbara

Administration School

Received

August

BETAINE-HOMOCYSTEINE DIETARY METHIONINE J.

Harris,

Medical Medicine,

of

METHYLTRANSFERASE

John

Center and Washington,

J.

Martin

George D.C.

and

BY

Walter

Washington 20422

344-348

E.

Kyle

University

2, 1982

SUMMARY: The hepatic activity of betaine-homocysteine methyltransferase mpcomplex function of the content of methionine in the diet. Enzyme levels are lower in the livers of rats fed a 0.3% methionine diet than in livers of animals maintained on either methionine-free or excessivemethionine (1.0%) rations. The finding that activities are increased at both extremes of the spectrum of dietary methionine intake suggests the possibility that the betaine-homocysteine methyltransferase reaction may function both to maintain tissue concentrations of methionine when intake of this amino acid is limited and to remove homocysteine when methionine intake is excessive.

INTRODUCTION Betaine-homocysteine reaction In

essential

addition

for

this

methionine

the

betaine

may The

committed

resynthesis

liver by

of that was

the

injection

injection

and

in of

the

the

folate

to

homocysteine enzyme

transsulfuration

the

two

homocysteine

be

was more

0006-291X/82/170344-05$01.00/0 Copyrighr 0 1982 by Academic Press, Inc. AN rights of reproduction in any form reserved.

defined.

by

locus remethylation

methylases In

to

a previous

diet both

reduced

the

the

and

to

be

involved

(2)

in was

induced

methionine

hepatic

content We in

the

the

study

methyltransferase

contrast,

(I).

pathway.

methyltransferase. likely

4.2.1.22)

2.l.l.l3),

regulatory

conserved

a high-protein

feeding

of

a

the

fed

high-protein

constitute

to

animals

In

regulation

(EC

be

betaine-horrocysteine

methionine.

tissues.

(EC

may

of

5-methyltetrahydrofolate that

of remains

level

increased

the

to

irreversibly

methionine

a

mammalian

methyltranferase

methionine,

contributions

the

catalyzes

cystathionine-B-synthase

appears from

in in

homocysteine

relative

found

with

2.1.1.5)

choline

significant

Together

derived

be

of

be

methyltransferase

Homocysteine,

(EC

catabolism may

metabolism.

5-methyltetrahydrofolate

we

the

reaction

and

or

methyltransferase

of

concluded maintenance

rat

BIOCHEMICAL

108, No. 1, 1982

Vol.

of

tissue

this

levels

of

acid

was

amino

appeared

to

reduce

a more

tration

of

free reason

which to

to

level

recent

of

any

diminished

enzyme

homocysteine

only

0.3%

dietary

intake

of

methyltransferase choline

noted

that rats

methionine

impairment

we

certain

the

for

in

of

the

availability

protein,

under

we

significantly

catabolism

and

be

nutritional

fed

hepatic

to

concen-

a purified

(3).

Since

cholinethere

was

or

possibility

essential

reduction

that for

no

reaction

homocysteine the

may

the

methyltetrahydrofolate

of

considered

methyltransferase

methionine

a means

however,

fell

postulate

when

homocysteine.

study,

contained

either

as of

methionine

diet

due

content

periods

RESEARCH COMMUNICATIONS

Betaine-homocysteine

primarily

tissue

BIOPHYSICAL

during

restricted.

function

the In

methionine

AND

the

in

the

betaine

conservation

of

conditions.

MATERIALS

AND

METHODS

Animals and Diets. We used male, Sprague-Dawley rats weighing 150-250 grams.---” The purrfled amino acid diet of Rogers and Newberne was the basis for our preparations (4). The latter were isocaloric and the contents of vitamins, lipids, salts and cystine (0.5%) were constant. In individual experiments we varied the concentrations of methionine, choline and non-sulfur containing amino acids. Assays. We have described our methods for the determinations of betaine and choline in rat liver (3,5). The assay for betaine-homocysteine methyltransferase was a modification of the published procedure (6). The concentration of betaine was increased to 2 mM. We defined one unit of enzyme activity as the amount which catalyzes the methylation of 1 nmole of homocysteine in 60 min. Protein was determined by the Bio-Rad Method (Bio-Rad Laboratories, Richmond, CA.). Expression deviation t-test between

--Effect

of Results. Data from the mean for for unpaired samples groups.

of

Dietary

experiments than

in

methionine

Both ingestion

increase

in

chol

The

ine.

which and

respectively. table,

--Amino

of the

we

RESULTS

AND

Acids.

Table

varied

cystine diets the

hepatic concentration

is presented each group of when we tested

the which

content

0.2%

summarizes

amino 0.3%

As

resulted

protein 345

at

choline.

but

results

of

maintained

betaine

soluble

the

content

acid-diet of

of

I dietary

contained

the standard We used the of differences

DISCUSSION

were

5% amino

as the mean and determinations. the significance

no

change

declined

acids and

shown in

of other

0.5% in

the

a significant in from

the 108

level to

of

BIOCHEMICAL

vol. 108, No. 1, 1982

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I EFFECT OF DIETARY AMINO ACID CONTENT ON BETAINE BETAINE-HOMOCYSTEINE METHYLASE IN RAT LIVER Diet

5% AA 22% AA

Choline nmol/g

Betaine Ilmol/g

lJ/mg

156t39

3.1t1.0

39.6k6.3

3480f557

170+37

12e39

l.lk1.3

40.9+9.0

42545745

212-c43

c.05

< .02

P

89 mg/g

consisted The diets

methyltransferase

the in

activity

the

per

more

fed

diets

was

the

content

marked

of

dietary

Dietarv

basal

of

diets

ine.

As

each

content

of

the

of

methionine

the

activities, diets

the

hepatic The

activity

weight.

acid

-70% in

as The

and

liver.

rats

fed

a

biphasic.

in

change

of

the

basal

72%

amino

346

acid

the -0.2%

explanation

experiments. changes

amino

in

and/or of the

of fall

5% amino choline

dietary betaine-

0.3% in

significant

diets

changes.

of

addition

may

we

acids

as

or

the

which

activity

liver,

contents

the

possible

pattern

was U/g

fed

rats

a marked

decrease

animals

compared

in

the

the

in

which

current

The

resulted

a decrease with

decreased in

in

contrast

consistent

changes

protein,

of

One

of

change

acids

of

content

of

-41%

no

experiments

magnitude

choline from

U/mg

was

effect

of

The

there effects

the

betaine

diet

rat

the

was

were

in

varied to

the

increased.

enzyme

body amino

there

a methionine-free

enzyme

units/g

series

II,

total

(2). in

their

and

studies

casein

a series in

Table

three

to

expressed

differed

in

in

tested

liver

amino

content

in

methyltransferase

methionine of

which

of

methionine

methionine

U/g

1 imited

noted

amounts

We

shown

homocysteine

level

the

we

diet methionine

betaine-homocysteine

containing

which

w

content

used chol

of

the

These

non-sulfur

varying

in

decreased,

enzyme.

differences

constancy

were

U/l iver/gBW

were fed the designated both contained 0.3%

While

weight

the of

and

decrease

specific.

of

which

the

g body

containing

Effect

not

activity

dietary

the

In

was

specific

of ten rats were isocaloric

Consequently

(P
hepatic

Methyltransferase U/g liver

prot

< .Ol

Each group five days. 0.2% choline.

for and

Betaine

AND

the

when total be

we

hepatic

a function

and,

for

acid

the -0.2%

diets.

of specific choline In

vol.

BIOCHEMICAL

108, No. 1, 1982

AND

BIOPHYSICAL

TABLE EFFECT

Diet AA

OF DIETARY

: 22 22 22

0 0 0

0.3

22 22 22

0.2 0.2 0.2

Each diet

group consisted for seven days.

0

the

additional homocysteine

Ia

1.0

3.6+1.8 1.1co.8c 0.5+0.5

0 0.3 1.0

17.5t7.7 4.5_+0.7b 0.5f0.8‘ of

comparison b P
at with

results

as

well

changes

were

by

seen

contained

22%

calculted

as

in

amino

226+70 156+8b

65+7 28+6a 43*vb

6339tlO71

2799+97Vba 84V8tl456

236?29 145+14b 288&71b

I 93+24c

11537t2845 3208+690a 405&4EOC

five

next

rats

lowest

of

which

473Al41 162+35b 209c 14b

were

methionine

fed

the

designated

intake:

3.3% methionine

the

resulted

in The

the

the the

an

selected

composition

of

livers

of The

protein,

U/g

the

the

change

in

enzyme

expression diet.

+75%,

total

an betaine-

of The

the

most

choline-free

were and

hepatic

the

the

with

in

basal

fed

increases liver,

of for

rats

diets

increase

magnitude

means

acids.

U/mg

65892 1752 3372k26Bb 4156~815

P
by

as

BW

c

methionine

affected

U/liver/g

68t.1 40+3 l 51+11

least

methyltransferase. was

Methyltransferase U/g liver

protein

110+27 33t7a 43f6=

supplementation

0.7%

activity

o.v*o.

0

P
contrast,

2.8t0.3~

1.0

for

U/mg

11.5k3.5

0.3

P value a

Betaine

Betaine umol/g

0.2 0.2 0.2

II

METHIONINE ON BETAINE AND BETAINE-HOMOCYSTEINE METHYLASE IN RAT LIVER

Components Choline Met P

5

RESEARCH COMMUNICATIONS

marked

diet

+201%

hepatic

and

which +99%

units/g

when

body

weight. The

data

in

Table

from

the

changes

in

have

found

that

the

II betaine

ne We have

of

meth

both

methionine

the

content.

In of

is

not

mediated defined

not

which diet.

are Betaine

and

by the

changes

a change

in

mechanism

for

by

the

homocysteine

in

recent, does

the the

the eve1

effect of

of

this

347

study

of

this

we

dietary

metabolite.

in amino is

studies

activity

hepatic

differences

leve

However

i shed

increase

methyltransferase (7).

enzyme

unpubl

Clearly

determined

dimethylglycine

a

betaine

methyltransferase.

onine

antecedent

dissociate

administration

betaine-homocysteine methion

also

with

the

effect

acid inhibited dialyzed

in

the by

BIOCHEMICAL

Vol. 108, No. 1, 1982 extracts

failed

existence

of

present

in

by

the

of

rats

no

evidence

to different

the

of

fed

(1.0%)

for

different

forms

this

the

such

the

may

be

diets.

in of

of

the

homocysteine

which

cystathionine

is

(and

observed

fall

homocysteine

to diet

sustain

also of

excessive

subnormal

our

previous

enzyme

the

in

low

animals the

this

with

function

explain

rats

on Km for

that

ability in

of

maintained

of

the

of

with

than

would

of

catabolism

consistent

less

growth

found

conservation

with

impairment

and

have

we

the

animals

consistent

(3)

livers

intake

for

is

magnitude

the

of

betaine

of

in

betaine-homocysteine

role

deprivation,

methionine only

the

livers

Finally,

betaine)

hepatic

the

orders

(1).

therefore in

two

is

date

However, of

repressed

methyltransferase.

a means

This in

To

the of

as

function

it

synthase

choline

both

methionine.

Furthermore,

of

be

induced

a revision

homocysteine.

enzyme

be

warrant

periods

serve

One,

could

diets.

role

essential

inadequate level

methionine

an

might

The

explanation.

methionine,

studies

During

removal

inhibitors.

betaine-hcmocysteine

metabolic

may

an

methionine of

RESEARCH COMMUNICATIONS

soluble

provide

A second

present

enzyme

for

methionine

increased

the

of

dietary

acid.

reaction.

and

diets

amino

concerning

methionine,

fed

adequate

excessive

of

presence might

the

methyltransferase

choline

of

the

results

conclusion

the

isoenzymes

absence

feeding

The

demonstrate

AND BIOPHYSICAL

both of

fed

by the

folate a

+

low

(8). ACKNOWLEDGEMENT

grant

This work AM-13048

was from

supported by the National

the Veterans Institutes

of

Administration Health.

and

by

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Finkelstein, J.D. (1978) Transmethylation, Usdin, E., Borchardt, R.T., and Creveling, C.R., eds., pp 49-58, Elsevier/North Holland, New York. Finkelstein, J.D., Kyle, W.E. and Harris, B.J. (1971) Arch. Biochem. Biophys. 146, 84-92. Finkelstein, J.D., Martin, J.J., Harris, B.J., and Kyle, W.E. Arch. Biochem. Biophys. (in press). Rodgers, A.E. and Newberne, P.M. (1973) Amer. J. Path. 73, 817-820. Martin, J.J. and Finkelstein, J.D. (1981) Analytical Biochem. 111, 72-76. Finkelstein, J.D. and Mudd, S.H. (1967) J. Biol. Chem., 242, 873-880. Finkelstein, J.D., Harris, B.J. and Kyle, W.E. (1972) Arch. Biochem. Biophys. 153, 320-324. Bennett, Y.A. (1950) J. Biol. Chem. 187, 751-756. 348