The role of fatty acid binding protein on the metabolism of fatty acids in isolated rat hepatocytes

BIOCHEMICAL

Vol. 71, No. 3,1976

The Role Metabolism Maria

of Fatty of

Acid

June

Binding

Acids

Y. C. Wu-Rideout,

Departments University Received

Fatty

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Protein

in Isolated Charles

of Nutritional of Wisconsin,

on the

Rat Hepatocytes

Elson

and Earl

Shrago

Sciences and Medicine, Madison, Wisconsin 53706

1,1976

In isolated rat hepatocytes flavaspidic acid, a SUMMARY. competitor with free fatty acids for the fatty-acid-bindingprotein, decreased the uptake of oleic acid and triglyceride synthesis but stimulated the formation of COs and ketone bodies from oleic acid. Flavaspidic acid had no effect on the utilization of octanoic acid. Stimulation of the microsomal fatty-acidactivating enzyme by the fatty-acid-binding protein was reversed by flavaspidic acid. In contrast, the binding protein inhibited the mitochondrial fatty-acid-activating enzyme. Flavaspidic acid not only prevented this inhibition but actually stimulated the enzyme activity. The results indicate that the cytosol fatty-acid-binding protein directs the metabolism of long chain fatty acids toward esterification as well as enhancing their cellular uptake. The hepatic involves

their

into

the

tissue

its

plasma

trations

acids

other

tissues,

cytosol.

diet

Copyright All rights

PABP is

(6) which

ABBREVIATIONS:

acid

Until

the

increased

suggests FFA, free protein

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

the

fatty

the

membrane

is dependent

recent for

long

809

of a

chain

mechanics

as in of

the

environment

of

subsequent

The concentration are

fed a high

FABP may be involved acids;

at concen-

as well

aqueous

unclear.

upon

discovery

or FABP in

when animals

that

plasma

the

Z-protein is

the

and intestine

FFA into

metabolism

rapid

occurring

protein

was known regarding

of the

extremely

uptake

of liver

hydrophobic

is

FFA uptake

maximal

binding

cytosol

The role

fatty

intestinal

(4,5)

across

of

3 mM (3).

little

of the

albumin

with

weight in the

FFA which

The rate

concentration

fatty

hepatic

from

(1,2).

approaching

transfer

of plasma

transfer

low molecular

the

uptake

FABP,

in

fatty-acid-binding

of fat the

Vol. 71, No. 3,1976

partition

BIOCHEMICAL

of cellular

venting

their

FFA towards

accumulation

Flavaspidic

strong

compete

evidence

that

identical. the

In

esterification

isolated only

through

long-chain

between

and

has recently

quite

acid

is

oxidation

similar

been

if

was not

is not

shown to decrease of olsic

of octanoate

B-oxidation

the

fatty-acid

microsomes of the

on the

were therefore

stimulate

microsomal

mitochondrial

acid

in

which

precedes

affected

by

also fatty

effects

these

acid

are distrib-

mitochondrial

membrane,

membrane-activating

enzymes

of FABP and flavaspidic fatty

investigated.

acid

activating

FABP was shown to

activation

activation, of

enzymes

outer

and mitochondrial

fatty-acid these

and the

The influences

microsomal

ensyes

activating

FABP with

be anticipated.

reversed

bilirubin

to FABP (7) which

are

the

Metabolism

mitochondrial

an association

acid

binding

flavaspidic

and increase

pre-

acid.

Since

might

FFA for

with

Z-protein,

two proteins

report

hepatocytes.

flavaspidic

uted

with

to

thereby

cell.

compete

binding

the

this

the

known to for

also

esterification

within

acid,

sulfobromophthalein shown to

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

while

and to inhibit flavaspidic

acid

FABP.

MATERIALS

AND METHODS

Male rats (250 g Holtsman) fed ad libidum were used for these studies. Hepatocytes were isolated from liver DerfUSed with collagenase according to the method of Berry and-Friend (8) The cells were suspended in a as modified by Zahlten --et a1.19). calcium-free Krebs bicarbonate buffer, pH 7.4, with 5 mM glucose and 1 ml suspensions of the cells were preincubated in rubber shaker (100 cycles/min) capped 2.5 x 5 cm vials at 37O on a Dubnoff under an atmosphere of 95% Os-5% COs. The reactiP~~~=~t~~:iated 3% albumin with 3 mMpAfter 17 min, 1 ml of the reaction mixture Alisolvents according to Dole (10). quots of the upper phase were washed twice with 50% ethanolic KOH (ll), mixed with Bray'8 solution (12) and the radioactivity In agreein the esterified fatty--acid fraction was determined. fatty acids ment with other reports (e.g. 13), no esterified were present in the incubation medium after the cells were removed by centrifugation. Residual free fatty acids in the

810

Vol. 71, No. 3,1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

incubation medium were isolated according to Dole (10). Watersoluble material (ketone bodies) were measured after protein precipitation by the addition of 1 ml 30% perchloric acid to the medium (13). The pR was adjusted to 8.5 with 20% KOB and after centrifugation, an aliquot of the supernatant fluid was added to Bray's solution (12) and radioactivity determined. At the end of the incubation period, COs was displaced from the cells and medium by the addition of 0.5 ml 6 N HsSO4. The radioactive Cop was collected in 0.2 ml hyamine hydroxide injected into a plasticsuspended through the rubber cap of the vial. After shaking for 1 hr the cup was placed in scintillation vials containing Bray's solution (12) and radioactivity was determined. FABP was prepared from rat liver as described by Ockner et al. (4). Mitochondrial and microsomal fractions were xt&ed by differential centrifugation (14) of rat liver homogenates. The assay for the acyl CoA synthetase was a modification of the method of Samuel and Ailhaud (151, which is based on the insolubility of acyl CoA in diethyl ether. The standard system ing constituents which were added in the ZZne&t$ z+& palmitate, 0.4 mg of aqueous solution of Triton WR-133 By, 350 mM his-HCl (pH 7.4), 10 mM ATP, 4 mM MgSO4, 1.2 mM CoA, 5 mM dithiothreitol and water in a final volume of 0.4 ml. Control experiments without CoA and ATP were systematically included in each series of incubations. Reactions started by the addition of the enzyme were continued for 5 min at 370. Reactions were terminated by the addition of 250 ~1 of 0.5 M HsSOq and 250 ~1 of water was added to dissolve any KsSO4 formed at the termination of the reaction. The remaining free fatty acids were extracted four times with 5 ml diethyl ether. The aqueous phase was then mixed with Bray's solution (12) and directly transferred to counting vials. Radioactivity was measured in a Packard Tricarb scintillation spectrometer and all measurements were corrected by use of internal standards. Protein was determined by the Dowry procedure (16). Collagenase (CDS II) was purchased from Worthington Biochemical Corporation; 8-flavaspidic acid-n-methyl glut infxe was e r usly provided by Esa Aho, Turku,Finland; - C octanoic, pamitic acid and Pl- ri4 C3 oleic acid were purchased rom New "e2 pl- w and unlabeled fatty acids were obtained from England Nuclear, Sigma Chemical Company. RESULTS AND DISCUSSION Hepatocytes,

isolated

oleic

acid

fatty

acids

was towards

label

taken

up by control

lar

lipid

eliciteda bound into

under

the

fraction. 13.#

oleic the

ester

conditions

fed rats

Within

fraction

were

shown on Table

esterification;527% cells

in the the

incubated 1.

of

cell,

was decreased 811

uptake the

into

of

incorporation slightly

of

radioactive

1 mM flavaspidic

cellular

with

The flow

of the

was incorporated

The addition

decrease

acid.

from

with

the

cellu-

acid the

albumin of label a concomitant

Flavaspidic

Acid

on Uptake

added

uotake,

*% of

Experimental - % of added dose, % of added dose, Control Experimental - % of uptake, Control % of uptake of control

dose,

to CO=

of

1%

Oxidation % uptake % control2

Acids

x loo

Control

x loo

3.8

59.7

as Esterified

Incorporation % uptake % control2

Fatty

33.2

to Water-Soluble

Conversion % uptake % control2

Products

66.4

Control

6.2 +64.0

54.2 - 9.2

34.7 + 4.5

57.7 -13.1

Flavaspidic 1mM

- 3 mM ~-14(Jolea~lues 24 mg protein.

and Metabolism

ml liver cells and 1.0 ml of 3% albumin Each vial contained for 17 min at 37O. of duplicate assays.

of

1

Total Uptake % added dose % of control%

Supensions containing 1.0 (839810 dpm) were incubated presented are the averages

Effects

Table

18.9 +397.0

43.0 -27.9

47.0 +41.6

40.3 -30.3

Acid 10 mM

Vol. 71, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effects

of Flavaspidic

C1Octanoate cl-14

2

Acid

on the

in

Isolated

uptake

and Metabolism

Liver

Incubations and calculations were carried Table 1. Radioactivity added was 800,000 average of duplicate vials.

soluble

Cells

out as discussed dpm. Each value

% of Uptake % of added dose

of

in is the

uptake

Water products

Esterified Fatty Acids

&

Control

25

81

17

1

Flavaspidic Acid (1 mM1

25

78

18

1

Flavaspidic Acid (10 mM)

24

77

19

2

increase fatty

in acid

spidic the

the

appeared soluble

stimulatory

ratios

ester

observed

of oleic

bodies).

effects

of

(0.75

significantly

influence

the

microsomal

and mitochondrial

mW),

increase

partitioning fractions.

of the

10 mM flava-

only

the

43.0%

remainder

of

in

To determine acid,

A four-fold

in ketone

body

was inhibited

was added.

813

cell,

calculated.

flavaspidic

of

flavaspidic

esterification acid

acid

with

(ketone

and 41.6%

10 mM flavaspidic

The uptake

the presence in the

were

while

to COP.

fraction

products

% distributions

were

27.9%.when tration

the

in CO* production

formation

in

radioactivity

and inhibitory

of the

increase

in

acids

by 30.3%

and of the

COs and water the

fatty

was inhibited

acid, label

oxidation

At a lower acid

did

of FFA between

by concen-

not the

BIOCHEMICAL

Vol. 71, No. 3,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effect

of

3

Flavaspidic

Microsomal

Acid

Palmitate

and FABP on

Activating

Enzyme(s)

The radioactive assay procedure a 8 cribed in Methods was employed with 3 kg protein, 15 ~J.M potassium palmitate Id (2472 dpm/nmole). The results represent the mean + S.E.M. of three experiments.

Enzyme Activity nmole/min/mg microsomal protein

Flavaspidic Acid (1 mm

Additions None None

% of control

102.60

+ 3.10

100.0

26.31

t 0.43

25.6

+

FABP (1.2

IIIIIOle)

119.7

+ 3.3

116

FABP (4.0

MlOle)

140.2

+ 1.13

136

FABP (1.2

nmole)

t

90.27

+ 0.93

88.0

FABP (4.0

nmole)

+

95.6

2 2.85

93.2

103.9

+ 0.95

101

90.3

2 1.17

88

Albumin

(4.0

nmole)

Albumin

(4.0

nmole)

The more

soluble

oxidized

and not

contrast

to its

without

effect Fatty

acid

was stimulated 4.0

nmoles

usually effect

chain

fatty

esterified

to

in oleic

on octanoate

by hepatic

serves

functions believed

3).

other

to compete

effective

which

than

inhibitor

of the

814

completely

triglycerides.

(Table

In acid

was

2).

microsomes

suggests

FFA for

are

flavaspidic

from

additions

added

the binding

with

form

respective

Albumin,

no effect

acids

metabolism,

metabolism

activation

FABP (Table elicited

was a most

short

16 and 36% by the

trations

acid,

+

1.2

at equimolar that

of FFA. binding microsomal

the

fed rats and

concen-

FABP also

Flavaspidic sites fatty

on FABP acid

Vol. 71, No. 3,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effects

of

4

Flavaspidic

Acid

Mitocho&lPalmitate

and FABP on

Activating

Enzyme(s)

The radioactive assay procedure as employed with 3 wg protein, 15 @l The results represent (2978 dpm/nmole). three experiments.

Flavaspidic Acid (1 mM)

Additions

the

of

Enzyme Activity nmole/min/mg

None None

mean + S.E.M.

+

% of control

80.0

+ 1.21

100

105.3

5 4.06

131

FABP (1.2

nmole)

69.3

2 3.4

86

FABP (4.0

nmole)

49.3

+ 0.76

61.0

FABP (4.0

NnOle)

102.1

+ 2.7

+

Albumin

(4 nmole)

Albumin

(4 nmole)

+

activating

enzyme

in the

presence

of either

FABP or albumin,

effective

inhibitor.

Thus # the

FABP and albumin In

all

contrast

isolated

to results

low as 1.2 reverse

fatty

fashion

nmoles

failed

acid.

Albumin These

to offset

data

did are

not

the exert

interpreted

96

74.0

2 1.5

93

3).

In the

flavaspidic

acid

was not

the

enzyme,

sites

flavaspidic

experiments

effectively

(Table

case

815

on the

indicate

at levels acted

FABP,

effect

an effect to

acid

activation.

stimulatory

involving inhibit

4) even

flavaspidic

the

of

an

acid.

from

activation

by increasing

+ 8

FABP (Table

FABP was shown to acid

77.3

binding

obtained

In this

nmoles.

of

accommodate

microsomes,

mitochondrial

absence

127

of

in

even

as a at 4.0

flavaspidic

enzyme. that

FABP increases

Vol. 71, No. 3,1976

the

uptake

fatty

acids

glyceride fatty

of

BIOCHEMICAL’ AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FFA and specifically

toward

microsomal

formation. acids

are

directed

When the

enhances esterification binding

principally

the

flow

3. 4. 5. 6. 7. 8. 9.

12. 13. 14. 15. 16.

long

and subsequent sites

toward

on FABP are mitochondrial

This work was supported in part ACKNOWLEDGEMENTS. Collegecf Agricultural and Life Sciences, University Madison and by NIH Grant AM-15893.

1. 2.

of

chain tri-

filled, oxidation.

by the of Wisconsin,

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