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,
REFERENCES ElovsOn, F. (1965) Biochim. Biophys. Acta 106, 480-494. Goransson, G. and Olivercrona, T. (1965) Acta Physiol. Stand. 63, 121-127. Heimberg, M., Weinstein, I. and Kohout, M. (1969) J. Biol. Chem. 244, 5131-5139. Ockner, R. R., Manning, J. A., Poppenhausen, R. B. and Ho, W.K.L. (1972) Science 177, 56-58. Mishkin, S., Stein, L., Gatmaitan 2. and Arias, I. M. ( 1972) Biochem. Biophys. Res. Commun. 47, 997-1003. Ockner, R. K. and Manning, J. A. (1974) J. Clin. Invest. 54, 326-338. Mishkin, S.. Stein, L., Fleishner, G., Gatmaitan, 2. and Arias, I. M. (1973) Gastroenterology 64, 154, Berry, M.N. and Friend, D. S. (1969) J. Cell. Biol. 43, 506-520. Zahlten, R. N., Stratman, F. W. and Lardy, H. A. (1973) Proc. Nat. Acad. Sci. U. S. A. 70, 3213-3218. Dole, V. J. (1956) J. Clin. Invest. 35, 150-154. Borgstrom, B. (1952) Acta Physiol. Stand. 25, 111-119. Biochem. 1, 279-285. Bray, G. A. (1960) Anal. Homey, C. J. and Margolis, S. (1973) J. Lipid Res. 14, 678-687. Schneider, W. C. and Hogeboom, G. H. (1950) J. Biol. Chem. 183, 123-128. Samuel, D. and Ailhand, G. P. (1969) FEBS Lett. 2, 213-216. Lowry, 0. H., Rosebrough, N. J. and Randall, R. J. (1951) J. Biol. Chem. 265-275.
816