The effect of 4-pentenoic acid on fatty acid oxidation

Vol. 34, No. 3, 1969

BIOCHEMKAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE EFFECT OF 4-PENTENOIC Klaus

ACID ON FATTY ACID OXIDATION.1

Brendel?

Carlos F. Corredor3, and Rubin Bressler Departments of Physiology and Pharmacology, Biochemistry, and Medicine Duke University Medical Center Durham, North Carolina 27706

Received January 7, 1969 4-Pentenoic carbon

acid

precursors

to inhibit

both

fatty

munications

from

gluconeogenesis tissue

important

cofactors

communication,

with

oxidation

this

laboratory,

acid

with liver

data

results and acetyl

inhibition

of fatty

acid

by the addition

2 Established

that

acid

the

its

oxidation,

amounts

Universidad

de1 Valle,

340

6),

(7,

of

due to

which

are

8, 9).

is

of fatty

In this

liver

in

of pigeon

liver

homogenates

augmented acid

a decrease

4-pentenoic

the

of CoA and

of the American

com-

and the accumulation (d)

in pigeon

In recent

homogenates

with

the incubation

shown

4-pentenoate-induced

the inhibition

(c)

been

were

(5,

liver

associated

three

the inhibitions

processes (a)

carnitines,

2).

oxida-

the levels homogenates of 4-

acid-induced can be prevented

(-)-carnitine.

was supported by grants HE 07061 from the American Heart Association.

Investigator

3Present Address: Colombia

(b) is

oxidation

of appropriate

investigation AHA 67-904

show: in pigeon

in

(1,

suggested

inhibited

from

It has also

A and (-)-carnitine

which

homogenates

acid

(l-4).

by 4-pentenoic

the inhibitor,

acryloyl

Cali,

oxidation

oxidation

gluconeogenesis

and liver

was

of these

pentenoyl,

This USPHS and

it

A and (-)-carnitine,

4-pentenoic

1

in heart

of coenzyme

in both

of fatty

to inhibit

and --in vivo

acid

we present

in pigeon

of coenzyme

acid

levels

by preincubation tion

--in vitro

and fatty

depressed

inhibition

has been found

Heart

and AM 12706

from

Association.

Faculdad

de Medicina,

Bioquimica,

the

BIOCHEMICAL

Vol. 34, No. 3, 1969

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALSANDMETHODS Two to three-month old white pigeons obtained from the Palmetto Pigeon Farms of South Carolina were fasted for 50 hours prior animals received water ad lib decapitated and the liver

to sacrifice.

during the fasting period.

immediately excised, chilled

The

The animals were in ice cold calcium-

free Krebs-Ringer Phosphate buffer

pH 7.4, and homogenized in 9 volumes of the

samesolution which had previously

been gassed with oxygen.

of Cl402 production from palmitate-l-C14, Cl4 were carried out essentially

as previously

Concentrations of free carnitine determined by termination centrifugation diethyl

ether.

pentanoate-l-Cl4

and 4-pentenoate-l-

described (10).

in pigeon liver

homogenateswere

of the incubation with trichloroacetic

to obtain a supernatant, The aqueous extract

made of the dried residue.

Determination

which was exhaustively

was lyophilised

The alcoholic

extract

acid and extracted with

and a methanolic extract was evaporated to dryness and

this residue taken up in water and used in the acyl coenzyme A-carnitineacetyltransferase

assay by the procedure of Tubbs, Chase and Pearson (11).

CoenzymeA-carnitine-acetyltransferase

was obtained commercially (Boehringer).

CoenzymeA and acyl CoA levels were determined by a procedure to be described in more detail

in a forthcoming publication

coenzyme A-pantetheine-H' taining

were added to pigeon liver

1 umole/ml reduced glutathione.

addition

4

.

Tracer amounts of homogenate incubates con-

The incubation was terminated by the

of methanol to the reaction mixture.

The reaction mixture was centri-

fuged and the supernatant passed through 0.2 cm2 x 3 cm columns of a mercuriated phenol-formaldehyde resin coated on silica (12) -

The effluents

material,

gel similar

to a procedure of Miles

from these columns charged with sulfhydryl

group specific

and the column washings were combined and evaporated to dryness.

residue was taken up in methanol-water added to toluene-triton

x 100 scin-

4 K. Brendel, C.F. Corredor and R. Bressler, Manuscript submitted for publication in J. Biol. Chem.

341

The

Vol. 34, No. 3, 1969

tillation ratio

cocktail of counts

counts present ratio

BIOCHEMKAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and counted in a liquid held on the column to counts

in the zero time incubation

of the labeled 4-Pentenoic

acryloyl

free and labeled

acid-l-C14,

carnitine

were synthesized

after

as previously

the procedure

prepared Carnitine using

(1, 2, 13).

(13).

anhydride

and acylcarnitines

chloroform-methanol-ammonia-water

was a gift versity

of Dr. P. Majeras

and

was prewas

coenzyme A was synthetized coenzyme A was

of Simon and Shemin (15).

on Al203 plates

(12:7:1:1).

of the Department

carnitine

(-)-Carnitine

Acryloyl

by the procedure

were separated

mixtures.

4-pentenoyl

hydrogenation. 4-Pentenoyl

for

a measure of the

Pentanoate-l-Cl4

of Wieland and Koeppe (14).

from acrylic

gives

acid-4,5-H3,

The

corrected

acyl CoA in the incubation

by catalytic

described

spectrometer.

in the effluent, effluent,

4-pentenoic

pared from 4-pentenoate-l-Cl4 prepared

scintillation

(Brockman

IV),

Coenzyme A-pantetheine-H3

of Medicine,

Washington

Uni-

of St. Louis.

RESULTS Figure liver

1 shows the conversion

homogenates after

without

additions

rates acid.

oxidation

as the incubations were higher

containing

to the reaction prevention

oxidation. the period The addition

of palmitate-l-C14

of the inhibitory

time course

incubated effects

with

4-pentenoic

The magnitude

oxidation. with

4-pentenoic

of 4-pantenoate

342

of palmi-

resulted Addition

the

of pentanoic

acid showed a of the depression

of the preincubation

pentanoate

acid.

acid was added, whereas

of

and reached approxj

of CoA and (-)-carnitine

acid or conta!ining

mixtures

4-pentenoic

in the absence than in the presence

with

15 minutes.

no 5-carbon

pentanoic

added to pigeon

of the homogenates

acid or with

show a similar

had been preincubated

varied

tudes of stimulation

pentanoic

to which

in palmitate-l-Cl4

oxidation 80% after

with

of preincubation

endogenous substrate

Homogenates which

palmitate mately

with

of oxidation

marked decrease

periods

(endogenous),

The incubations tate

varying

to Cl402 of palmitate-l-Cl4

to incubates

in similar

magni-

of CoA and carnitine

acid resulted on palmitate-l-Cl4

in partial oxidation.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 34, No. 3, 1969

Palmitate Oxidation

00

, , ,

Endogenous + CoA + car

Inmoles C1407 zt liver x 15 min 16(

12(

8(

4t \

l\

l -•

-*-+2*c-*

I

I

3

27

15

4-Pentenoate b

39 Minutes of Incubation

Figure 1. Palmitic Acid Oxidation by Pigeon Liver Homogenatein Presence of 5-Carbon Acids. The incubations contained 2 ml 10%pigeon liver homogenate in calcium free KRPbuffer of pH 7.4. No additions were made, or 1 umole of 4pentenoate or lumole of pentanoate and where indicated 200 mumolescoenzyme A and 400 ml,auoles(-)-carnitine were added at the beginning of the 55-minute, 40' incubation. 200 mpoles palmitic acid-l-c l4 (200,000 cpm) was added at 3, 15, 27, 39 minutes after the beginning of the experiment and C1402was collected for 15 minutes. Figure 2 shows that 4-pentenoic acid acid oxidation,

but also depressed its

tenoic acid-l-Cl4

to pigeon liver

not only

own oxidation.

homogenates after

inhibited

long-chain fatty

The Addition

of 4-pen-

varying periods of pre-

incubation with non-labeled 4-pentenoic acid resulted in a time dependent inhibition

of 4-pentenoate-l-Cl4

oxidation

was found after

depression after preincubation

oxidation

to CL402. About 40%depression of

15 minutes of preincubation

30 minutes.

and approximately

An analogue experiment in which the effect

with non-labeled pentanoic on pentanoic acid-l-Cl4

to C1402 was carried out and revealed no depression of oxidation.

343

80% of

conversion The addi-

Vol. 34, No. 3, 1969

tion

of optimal

stimulated the

amounts

of CoA and carnitine

the oxidation

inhibitory

results

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

effects

are

oxidation

of 4-pentenoic

compared

acid-l-Cl4

of 4-pentenoic with

is more sensitive

those

at the start

acid

of Figure

incubation

and completely

on its 1, it

to inhibition

of the

prevented

own catabolism. is

apparent

If

that

by 4-pentenoate

than

these

palmitate is

4-pentenoate

oxidation.

5-Carbon Oxidation mpmoles g wet liver

Acid

Pentanoate

Cl40 2 x 15 min

Pentanoate

+ CoA + car

4-Pentenoate

+ CoA +c

240

160

4-Pentenoate

, 3

15

27

and 4-Pentenoic Figure 2. Pentanoic Conditions were as in Figure 1, but l-Cl4 (150,000 cpm) and pentanoated-C substrates.

The data 4-pentenoic

acid

CoA and acyl carnitine acyl

of Table

1 show that

resulted

CoA's.

and CoA, whereas

carnitines

(data

it

incubation

acid resulted

not presented

b Minutes of Incubation

Acid Oxidation by Pigeon Liver Homogenate. instead of palmitate, 500 mumoles 4-pentenoatel4 (150,000 cpm) were added as radioactive

in marked

4-Pentenoic

39

changes caused

of pigeon in

the levels

significant

in increased here).

344

liver

Changes

homogenates

with

of (-)-carnitine, depressions

levels of this

of acyl magnitude

of

(-)-

CoA's,

and

in

CoA,

Vol. 34, No. 3, 1969

(-)-carnitine

BIOCHEMICAL

and their

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

acyl derivatives

were not produced by incubations with

pentanoic acid, the saturated analogue of 4-pentenoic acid or with endogenous substrate. TABLE 1. Carnitine and CoA Levels in Presence of &Carbon Acids. 10 ml of a 10%gigeon liver homogenate in calcium free KRP buffer pH 7.4 were incubated at 40 for 60 minutes in the presence of coenzyme A-pantetheine H3 (100,000 cpm) and in presence and absence of 5 pmoles 4-pentenoic acid or pentanoic acid. mumoles g wet liver (-)-carnitine

CoA-SH

Acyl CoA

Control

600 + 38

83 2 2%

17 + 2%

Pentanoic Acid

550 f. 46

79 + 3%

21 + 3%

4-Pentenoic Acid

350 + 33

54 f- 2%

46 + 2%

Addition

% of Total CoenzymeA

TABLE 2. Formation of Acyl Carnitines in Presence of 4-Pentenoate. The incubations contained 10 ml 10%pigeon liver homogenate in calcium free JXRPpH 7.4, 2.5 nmoles coenzyme A, 5 pmoles (-)-carnitine and12 pmoles sodium fumarate. Where appropriate 10 umoles 4- entenoic acid-l-C (1 x 106 cpm), 10 umoles 4-pentenoic acid -4,5-H3 (1 x 10% cpm) or 2 pmoles palmitic acid were added. Incubations were carried out at 40° for 30 minutes.

Additions

Acetyl Carnitine

Acryloyl Carnitine

4-Pentenoyl Carnitine

cpm x lo4 /g wet weight liver. 4-Pentenoic Acid+13

--

9

18

4-Pentenoic Acid-H3 + Palmitate

--

13

13

4-Pentenoic Acid-Cl4

11

--

16

4-Pentenoic Acid-Cl4 + Palmitate

10

--

8

345

Vol. 34, No. 3,1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Incubations

of pigeon liver

of CoA and (-)-carnitine resulted

in the production

acryloyl-2,3-H3 indicates

that

acid-l-C14.

acid-l-Cl4

acyl

in incubations

4-pentenoic

the ratio

acid-4,5-H3

The appearance 4-pentenoic

S-oxidation.

acid 4,5-El3

containing

was added to incubations

of acryloyl-2,3-H3

of

This is further

in incubations

palmitate

concentrations

or C-pentenoic

containing

14 carnttine

When non-radioactive

optimal

carnitines.

acid undergoes

of acetyl-1-C

4-pentenoate-4,5-H3, carnitine

4-pentenoic

of various

carnitine

by the appearance

carnitine

confirmed 4-pentenoic containing

to 4-pentenoyl-4,5-H3

was increased. Under the same conditions, but using 4-pentenoic 14 as the precursor, the ratio of 4-pentenoyl-l-C14 carnitine to acetyl-

acid-l-C l-Cl4

with

homogenates containing

carnitine

was decreased.

DISCUSSION The data presented

in this

both long and medium-chain a latent

period.

communication

fatty

The effect

acid oxidation

on long-chain

be more pronounced.

4-Pentenoic

which

of the short-chain

are,

are substrates therefore,

formation

in equilibrium

of 4-pentenoyl,

acryloyl

chemically. creased

The formation

levels

4-pentenoyl

as well

acyltransferase levels

liver

fatty

carnitine

as acryloyl

acyl CoA-carnitine their

respective

and acetyl studies

the addition

homogenates

same time decreases

increases

after seems to

CoA and acryloyl

acyltransferase,

CoA and

derivatives.

from 4-pentenoic

The acid

and these compounds have been identified is consonant

found in the 4-pentenoate carnitine

are substrates

the increased

levels

of palmitate the relative

incubates.

of 4-pentenoic

346

carnitine

moiety

the deThe fact

tha

of the acyl CoA-carnitine

across

acid.

It is

acid metabolizing

amounts of acryloyl

of the acryloyl

with

of acyl coenzyme A and decrease

to 4-pentenoic

the amounts of 4-pentenoyl

augments the translocation

however,

carnitine

carnitines

coenzyme A found in the presence

that

to acetyl

acid inhibits

homogenates

acid oxidation,

of these acyl carnitines

also explains

of free

noteworthy

of free

4-pentenoic

in pigeon liver

acid is S-oxidized

with

has been shown by way of tracer

show that

carnitine

suggesting

pigeon

and at the that palmitate

the mitochondrial

membran

BIOCHEMICAL

Vol. 34, No. 3, 1969

and competes results

with

of our

are

that partly

dation

of the

later

of fatty effects

an effect

acid, acid

on fatty

cannot

oxidation

of 4-pentenoic

effects

acid

of 4-pentenoic consonant

the

inhibition

completely

This

metabolites

acid

hypothesis.

of fatty

acid

oxi-

established

may be due to other

such as enzyme inhibition

or one of its

this

oxiand CoA.

on fatty

an already

acid.

acid

carnitine

with

The

in earlier

on fatty

acid

reverse

by 4-pentenoic

oxidation

acid

are

space.

advanced

of the cofactors

can prevent

they

acid

levels

(-)-carnitine

addition

an hypotheses

of 4-pentenoic

in the

of CoA and

by 4-pentenoic

with

effects

inhibitory

RESEARCH COMMUNICATIONS

in the extramitochondrial

in agreement

deleterious

CoA and carnitine

inhibition

activation

due to decreases

by addition

Whereas

are

the

The prevention oxidation

acid

experiments

communications dation

4-pentenoic

AND BlOPHYSlCAL

by acryloyl

on the electron

CoA or

transport

chain. REFERENCES (1)

Corredor,

C.,

Brendel,

K. and Bressler,

R.,

(1967)

(2)

Corredor,

C.,

Brendel,

K. and Bressler,

R.,

J.

(3)

Ruderman,

N.B.,

Shafrir,

E. and Bressler,

(4)

Ruderman,

N.B.,

Vreeland

I.

(5)

Brendel,

(6)

Corredor,

(7)

Sandler,

(8)

Fritz,

K., C., R., I.B.

(9) Delisle, (10)

Wittels,

(11)

Tubbs,

Corredor,

Thesis.

and Freinkel, (1967)

N.

I.B.

B. and Bressler, Pearson,

Life

R.

(1967)

J.

Biol.

(1967) R.

D.J.

(1968)

(1968)

Clin.

Nat.

Acad.

Sci.

Chem. (In

press).

Life

7~1083.

Sci.

Sci. (In

58:

press).

Pharmacologist

Duke University.

Perspectives

G. and Fritz,

P.K.,

E.

C. and Bressler,

Ph.D.

Biol.

R.,

and Shafrir,

Proc.

=:213.

1968. Investig.

45:1067.

Med. =:643.

Proc.

Nat.

(1964)

J. Clin.

and Chase,

J.F.

Acad.

Sci.

Investig. (1965)

58:790. 2:630.

Biochim.

Biophys.

Acta

g:162. (12)

Miles,

H.T.,

Stadtman,

E.R. and Kielley,

W.W. (1954)

J. Am. Chem. Sot. 2:

4041. (13)

Brendel,

K. and Bressler,

(14)

Wieland,

T. and Koeppe,

(15)

Simon,

E.J.

and Shemin,

R.

(1967)

Biochim.

H.

(1953)

Ann.

D.

(1953)

J.

Biophys.

Acta

Chem. 581:l. Am. Chem. Sot.

347

75:2520.

137:98.

2299.