Carnitine esters of fatty acids support mitochondrial fatty acid synthesis

Vol. 38, No. 1, 1970

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CARNITINE

ESTERS OF FATTY ACIDS SUPPORT

MITOCHONDRIAL FATTY ACID SYNTHESIS Joseph

B. Warshaw

and Robert

E. Kimura,

The Children's Service, Massachusetts General Hospital, Shriners Burns Institute and the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02114.

Received October 27, 1969 SUMMARY. Acylcarnitines are active primers for mitochondrial fatty synthesis. It is suggested that another metabolic fate of carnitine of fatty acids in addition to their role in fatty acid oxidation is vide substrates for mitochondrial fatty acid elongation.

It

has been

12 carbon

units

clearly or greater

esters

of the fatty

brane.

Acylcarnitine

transferase

as is

involves

chondrial inner

the

there

is

in the

following

capacity

of camitine

synthesis.

of 2 carbon

show that

elongation

acid

camitine

by guinea

the

pig

inner

mitochondrial

mem-

enzyme camitine

for

palmityl-

transport prior

of this

+ CoA transport

presumably

CoA ester

takes

acids

synthesis

carnitine

to support

esters

of fatty

heart

mitochondria.

EQ

in the acids

place

the

(palmitylAlthough

pathway

as it

relates

studies

of the

mitochondrial

fatty

involves

the addition

This

communication

system. are

the mito-

within

to $-oxidation.

by mitochondria

present

into

intermediate

have been no previous

of fatty

acids

of

reaction.

reaction

there

esters

to fatty

by the

acids

of acylcarnitine

palmitylcarnitine

where

(1,2),

Fatty units

catalyzed

investigation

oxidation

in the

of palmitylcarnitine

to the

of fatty

on the formation

v

The reverse

has been extensive acid

will

is

reconverted

the oxidation

are permeable

synthesis

membrane

to fatty

acid

which

formation

mitochondrial

that

dependent

+ camitine

membrane.

camitine

is

acids

seen

palmityl-CoA This

established

acid esters to pro-

active

primers

for

fatty

acid

Vol. 38, No. 1,197O

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Heart

METHODS AND MATERIALS. pigs

by a modification

ments were

carried

by sonic

oscillation.

equipped

with

out with

1.5 1-1moles

in the

method

of fatty

and bovine

Fatty

acid

surface

oxidation

of the

reaction

at 70'

acids

were

for

30 minutes.

extracted

scintillation

vial.

Extracted

lipids

chromatography

on silica

gel

developing

solvent

gifts

from

the Otsuka

were

obtained

from

primer were acids

heart

was present required

for

may reflect

using

commercial

in

the assay

optimal a low

one hour. over

are

the

A in the medium.

the

with

concen-

reaction

HCl,

the

mix-

fatty

in a liquid

in a Beckman scintillation

ether,

acids

ethyl

by thin

ether,

Osaka,

layer

acetic

and 1-palmitylcamitine

Factory,

acid

were Other

Japan.

generous

reagents

sources. incorporation was negligible medium.

acetyl-CoA level

at 3S" for

as fatty

a petroleum

Acetyl-CoA

mitochondria

to the

to dryness

identified

Pharmaceutical

assays

of KOH to a final

counted

L-camitine

(9O:lO:l).

RESULTS AND DISCUSSION. pig

were

in the

out by heating

was then

phos-

of 1.0 ml.

of nitrogen

acidification

50 mu

(20 m&Mole),

according

of antimycin

addition

was

protein,

volume used

flow

and evaporated

The material

acids

of potassium

was defatted

was carried

pentane

spectrometer.

by guinea

by the

sonifier of suspended

fatty

14C-CoA

was incubated

Following

with

chain

albumin

and by the presence

Saponification

a Branson

in a final

by a gentle

was terminated

of 15%.

ture

mixture

was inhibited

The reaction tration

serum albumin

The assay

long

CPM of acetyl-l-

A and water

of Chen (7).

disrupted

in a 1.0 ml volume

of NADPH, 50 1~ moles

acids

Experi-

1.0 mg of mitochondrial

30,000

Bovine

using

guinea

(5,6).

and mitochondria

into

1 ug of antimycin

tables.

from adult

and Hagihara

1 minute

containing

containing

The concentrations given

for

of NADH, 1.1 p moles

pH 6.5,

of Chance

of acetyl-CoA

medium

isolated

was accomplished

immersed

in an assay

were

mitochondria

Disruption

of acetyl-CoA

phate,

intact

Incorporation

determined moles

of the method

a microtip

mitochondria.

mitochondria

of those

into unless

In addition,

incorporation. substrates 59

long

chain

a fatty

fatty

acid

acids

substrate

NADH, NADPH, and ATP The requirement in the

guinea

pig

for heart.

fatty

Vol. 38, No. 1,197O

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I

Acetyl-CoA Incorporation Sonically Disrupted

into Intact Mitochondria.

mn Moles Fatty

Acid

Primer

Intact

Acetyl

l-14C-CoA

Mitochondria

None Palmitic Acid Palmityl-CoA Palmitylcamitine Capric Acid Caprinylcamitine

and

Disrupted

1 compares

sonically

disrupted

mers were

added

rates

acetyl-CoA

0 0.22 0.89 0.77 0.84 0.11

to the assay. were

The level

the value

seen with

The addition

incorporation

mitochondria

of incorporation

the primer.

Mitochondria

0 0.04 0.54 1.05 0.02 0.60

1.0 mg of mitochondria was used in the assay. of palmitic acid, palmityl-CoA and palmitylcarnitine concentration of capric acid and caprinylcarnitine conditions are as in the text.

Table

Incorporated

acids

by intact

and 16 carbon

substrate

mitochondria,

observed

when palmitylcamitine

consistently

with

palmitylcamitine

fatty

case of intact

of incorporation

of 1-camitine

into

when 10 carbon In the

The concentrations were 1.0 mM. The was 3.0 mM. Other

palmityl-CoA

and was very

to the assays

pri-

the highest was

was about

limited

and

with

50% of

palmitic

acid.

completely

inhibited

acetyl-CoA

increased

acetyl-CoA

incorporation

incorporation. Sonic

disruption

when palmitic

acid

permeability probably

enhanced

indicate

carnitine,

activity

the high

acid

membrane

substrate

was used

the membrane acids

more

value

fatty

observed

acid

primer

as a substrate

transport for

long primer,

exhibit

a

disruption

and other

readily.

as substrate

the

energy

As mitochondria

acids,

the fatty

palmitylcamitine

is an effective

When capric

by allowing

and approximated that

primers.

of fatty

mitochondrial

seen with

disruption

were

to CoA esters

the inner

of acetyl-CoA

data

and palmityl-CoA

barrier

to penetrate

after

of the mitochondria

substrates

The incorporation

primer with

was decreased

palmityl-CoA. intermediate,

chain

fatty

acetyl-CoA

These palmityl-

acid

elongation.

incorporation

Vol. 38, No. 1, 1970

was uniformly after the

BIOCHEMICAL

low in experiments

disruption. intact

These

With

were

unexpected.

(8,9)

are oxidized

at comparable

not

rate

activity

of capric

penetration

acid

has been

of acylcarnitines carnitine In the

mitochondria

but

remove

that

from

acid

oxidation

by intact

and its

carnitine

ester

mitochondrial

may also

mitochondria effect with

permeability the

increased

reflect

intact

effect

albumin

on the

of disrupted

inhibited

the data

regard

suggest

0.40 0.80 1.20 1.60

Intact

to the

intact

Mitochondria

0.47 0.72 0.62

0.53 0.65 0.57 0.62

1.0 mg of mitochondria camitine concentration as in the text.

Incorporated

Disrupted

Mitochondria 1.75

0.08

0.07 was used in the assay. Palmitylare was 1.0 mM. Other conditions

61

incorporthat

and effectively

Effect of Bovine-Albumin on Primer Activity of Palmitylcamitine.

mg BSA None

a 70%

concentration.

activity

effectively

l-14C-CoA

on palmitylmitochondria.

TABLE II

Acetyl

effect

in approximately

palmitylcarnitine With

reaction.

the detergent

of albumin

mitochondria

may bind

mu Moles

primer

enhanced

and disrupted

resulted

above 1 mg/ml disrupted

from of bovine

irrespective

had little

the

mitochondria.

of elongation.

albumin

albumin

in

disrupted

However,

disruption

incorporation,

of bovine substrate

that

2 shows the

concentrations

acid

high

was much greater

fatty

oxidation.

mitochondria,

albumin

and relatively

of sonically

with

in experiments

In the case of the

concentrations

acid sonic

Table

in acetyl-CoA

Up to 1 mg of bovine

that

capric

to the site

activity

case of intact

both

shown to protect

(9).

primer

decrease

ation.

after

mitochondria

indicating

capric

of acetyl-CoA

Albumin

that

rates

for

with

Experience

has shown

limiting

intact

incorporation

as compared

mitochondria

is

using

caprinylcarnitine

mitochondria

results

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mito-

higher

Vol. 38, No. 1, 1970

BIOCHEMICAL

one can postulate

chondria,

palmitylcarnitine

bound

finite

dissociation

course

of the reaction.

acids

mers

(Table

sonically

3).

(4,lO)

ATP requirement conditions

mitochondria

utilized

that

addition

of CoA esters

cluded

assay.

liver

and Wakil into

a variety

mitochondria

to the medium

(4)

could

to serve

as primers. of fatty

free

fatty

of fatty

acids.

be markedly as exogenous

rat

by the

have

acids.

enhanced primers

the

under

were

in-

incorporated

incorporation

by rat

of fatty

acetyl

units

were

acids added.

TABLE III

Effect

of ATP on Acetyl-CoA mp Moles Intact

Palmitic Acid Palmitic Acid, ATP Palmityl-CoA Palmityl-CoA, ATP Palmitylcarnitine Palmitylcamitine, ATP

Incorporation. Acetyl

Mitochondria 0.02 0.05 0.08 0.62 0.11 1.26

l-14C-CoA

Incorporated

Disrupted

Mitochondria 0.06 0.34 0.19 0.89 0.09 0.82

1.0 mg of mitochondria were used in the assay. Concentrations acids and ATP were 1.0 mM and 3 mM, respectively. Other conditions in the text. 62

the

of acetyl-CoA

by the addition to which

reported

However,

mitochondria

Acetyl-CoA

ATP and

can replace

and palmitylcarnitine

liver

pri-

ATP gave

plus

acids

stimulation

palmityl-CoA

that

plus

Others

chain

substrate

of acetyl-CoA

was consistent

showed

were

the

long

when palmityl-CoA

ATP were

by ATP when both

into

of

and a

during

palmitylcamitine

plus

there

the albumin

Incorporation

with

pool

in the medium

of acetyl-CoA

was similar

incorporation

acetyl-CoA

from

mitochondria,

observed.

of our assays,

Harlan

present

and palmityl-CoA

seen in experiments

in the

albumin

the incorporation

intact

incorporation

palmitylcamitine

of an extra-mitochondrial

palmitylcamitine

In the

disrupted

previously

to the excess

enhanced

when both

the highest

the presence

of the palmitylcamitine

ATP markedly fatty

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

of fatty are as

BIOCHEMICAL

Vol. 38, No. 1,197O

Similar this

data elongation

Porter

(11)

brane

when free

similar

to data

chondrial the

elongation

it

Whereat,

ratios

elongation

acids

barrier,

the

is

suggest

into

substrates

mitochondria

(11)

in our

experi-

primers.

to disruption

This

is

of the mito-

better

access

inhibited

to

fatty

acid

of the acetyl-CoA

acetylcamitine

the

thought

rather

than fate

membrane,

depending

on metabolic

fatty

other

transferase

pathway.

to occur

by reversal

This work Institute.

than

The results $-oxidation

which

within high

of these

experi-

acids

may be to provide

fatty

acid

elongation

of B-oxidation either

with

of fatty

As fatty

may support of the

elongation

of mitochondria,

esters

elongation

demands

acid

oxidation.

as camitine

acylcamitine

chondrial

that

by the NADH:NAD ratio

mitochondria

ACKNOWLEDGEMENTS. the Shriners Burns

differed

of mitochondria

due to the conversion

have proposed

a metabolic

for is

mem-

NADPH inhibited

substrate

completely

and

reaction.

synthesis that

probably

have

activity

substrates

by mitochondrial

controlled

favoring

L-carnitine

results

Disruption

were

allowing

Dahlen

in an outer

in that

and may relate

thus,

(10). This

(3,4,10)

and palmityl-CoA

by others

reaction

is

transported acid

acid

et al

mitochondria

ments

fatty

increased

who localized

elongation Their

acids.

reported

from

mitochondria.

fatty

to acetylcamitine

removes

acid

into

activity.

substrate

fatty

(10)

membrane.

of acetyl-CoA

fatty

of the

mitochondrial

investigators

permeability

site

heart

et al

of other

oscillation

ments

inner

mitochondrial

from beef

incorporation

by Quagliariello,

to the

investigated

and those

by sonic

reported

activity

preparation

from ours the

have been

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

oxidation

by

the inner

mito-

or elongation

cell.

was supported

by USPHS Grant

#I-ID-03610-01

and

REFERENCES. 1. Fritz, I.B. and Marquis, N.R., Proc. N.A.S., 54, 1226 (1969). Compartmentalization and Control of Fatty Acid 2. Bremer, J., Cellular Metabolism, Academic Press, New York, 1967, p. 65. L.W. and Warshaw, J.B., J. Biol. Chem., 235, PC31 3. Wakil, S.J., McLain, (1960). 63

Vol. 38, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4. Harlan, W.R. and Wakil, S.J., J. Biol. Chem., 238, 3216 (1963). 5. Warshaw, J.B., J. Cell Biol., 41, 651 (1969). 6. Chance, B. and Hagihara, B., Proc. Intern. Congr. Biochem., 5th MOSCOW, 2, 3 (1961). Chem., 242, 173 (1967). 7. Chen, R.F., J. Biol. 8. Fritz, I.B., Am. J. Phys., 202, 117 (1962). 9. Warshaw, J.B. and Terry, M.L., J. Cell Biol., In Press. 10. Quagliariello, E., Landriscina, C., and CoratelliP., Biochim. Biophys. Acta, 164, 12 (1968). 11. Dahlen, J.V. and Porter, J.W., Arch. Biochem. Biophys., 127, 207 (1968). 12. Whereat, A.F., Hull, F.E., and Orishimo, M.W., J. Biol. Chem., 242,4013 (1967).

64