A role for fatty acids and liver fatty acid binding protein in peroxisome proliferation?

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Biochemical Society Transactions
824
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Keceived
24
June
1002
IC
A
role for fatty acids and liver fatty acid binding protein in peroxisome
proliferation?
Isabelle Issemann, Rebecca Prince, Jonathan
Tugwood
and Stephen Green*
:I
PLC, Central Toxicology Laboratory, Cell and Molecular Biology Section, Alderley Park, Macclesfield, Cheshire
SK
I0
4TJ,
U.K.
Introduction
I’eroxisome proliferators are
a
diverse group of
chemicals which when administered to rats and
mice produce liver hyperplasia and hypertrophy,
the latter being predominantly due to an increase in
the size and number
of
hepatic peroxisomes
[l].
Interest in these chemicals stems from the observa-
tion that they are rodent liver carcinogens. In par-
Abbreviations used:
I’I’AK.
peroxisomc proliferator
acti-
vated receptor;
I’PKE.
peroxisonie proliferator response
clement;
1;ARI’.
fatty acid binding protein.
‘To whom correspondence should be addressed.
ticular, they are not directly mutagenic and appear
to use
a
novel carcinogenic mechanism. There are
therefore two important research questions: how
do peroxisome proliferators produce liver tumours
in rodents such as rats and mice and what is the
relevance of these findings to man?
The most potent peroxisome proliferators are
hypolipidaemic drugs that were developed for
the
treatment of coronary heart disease
[2].
These
drugs lower circulating levels of cholesterol but are
more effective at lowering triglycerides. They are
therefore used to treat patients with type
IV
hyper-
lipidaemia who have high levels of circulating tri-
Volume
20

Lipid-Binding Proteins
glycerides. The administration of peroxisome
proliferators to rats and mice increases the level of
several hepatic enzymes. These include the peroxi-
soma1 /?-oxidation enzymes such
as
acyl CoA
oxidase, that is responsible for the metabolism of
long chain fatty acids, and the microsomal cyto-
chrome P450 IVA1 that possesses lauric acid
hydroxylase activity. Of particular interest
is
the
finding that the induction of these enzymes
is
regu-
lation, at least in part, at the transcriptional level
We recently identified a member of the
steroid hormone receptor superfamily that can be
activated by peroxisome proliferators
[
51.
We there-
fore termed this the peroxisome proliferator acti-
vated receptor (PPAR). Such receptors are
ligand-activated transcription factors and our more
recent results have demonstrated that PPAR can
bind to a specific DNA sequence (termed the per-
oxisome proliferator response element,
PPRFC)
located upstream
of
the rat acyl CoA oxidase gene
[6].
Therefore PPAR appears
to
mediate the tran-
scriptional effects of peroxisome proliferators. Some
of the questions that remain to be resolved, how-
ever, are how do peroxisome proliferators activate
PPAR and what
is
its natural ligand?
We
present
here some preliminary data indicating a role for
fatty acids in the regulation of PPAR activity and of
PPAR in the regulation of liver fatty acid binding
protein (FABP) gene expression. These data
suggest that one action of peroxisome proliferators
~3~41.
may be to displace fatty acids from FABP, leading
to the activation of PPAR.
Results
Fatty
acids
activate
PPAR
Certain high fat diets induce peroxisome prolifera-
tion in rats [7]. We were therefore interested to
determine whether fatty acids could activate PPAR.
Hepal cells were transfected with the PPAR
expression vector and the ACO(
-
12731
-
471)G.CAT reporter gene that contains the regu-
latory sequences of the rat peroxisomal acyl CoA
oxidase gene upstream of the rabbit /?-globin pro-
moter and the CAT (chloramphenicol transferase)
coding sequence [6]. As shown previously, the per-
oxisome proliferator Wy- 14,643 strongly activated
PPAR at low concentrations producing an increase
in CAT enzyme activity from the ACO-G.CAT
reporter gene (Fig. 1). A variety of fatty acids were
next tested by adding them every 24 h for a total
period of 48 h. As shown in Fig.
1
a
high concentra-
tion
of
palmitic acid stimulated CAT activity indi-
cating that
it
could activate PPAR. We next tested a
thio-substituted fatty acid (tetrathio decanoic acid
[8])
that is structurally similar
to
palmitic acid
except that
it
has a sulphur atom located between
the
/?
and
y
carbon atoms and
is
therefore not
a
substrate for the /?-oxidation pathway. This com-
pound was far more effective than palmitic acid at
activating PPAR suggesting that palmitic acid
is
a
Fig.
I
Fatty acids activate
PPAR
The PPAR expression vector and the acyl CoA oxidase reporter gene were transiently
transfected in Hepal cells in the presence
of
increasing concentrations of palmitic acid,
tetrathio decanoic acid (TTA) or the peroxisome proliferator Wy-
14.643.
The amount of
induced CAT enzyme was measured and expressed as the amoaunt
of
chloramphenicol
acetylated by the cellular extract. Technical details can be found in
[S].
50
C
2
30-
f!
0
-
5
20
~
V
x
zl
10
-
I
E-9
I
E-8
I
E-7
I
E-6
I
E-5 I
E-4
I
E-3
Concentration
(M)
825
I992

Biochemical Society Transactions
826
poor PPAR activator because
it
is rapidly meta-
bolized.
A
consensus
PPRE
in the
FABP
promoter
Both FABP enzyme activity and mRNA levels are
upregulated in primary hepatocyte cultures by per-
oxisome proliferators
[9].
We were interested to
determine whether FABP was regulated at the tran-
scriptional level and in particular whether the
promoter of the FABP gene contained a PPRE. As
seen in Fig. 2, inspection of the
5'
flanking sequence
of the FABP gene
[
101
reveals an imperfect direct
repeat element between
-68
bp and
-56
bp
(S'TGACCTA
TGGCCT
3') that closely resembles
the PPRE (5'TGACC??'TGTCCT 3') identified in
the rat acyl CoA oxidase gene
[6].
To determine
whether this sequence had any role to play in
mediating the effects of peroxisome proliferators we
cloned a region of the rat liver FABP promoter
(-
565
to
+
21) using the polymerase chain reac-
tion and placed this upstream of the coding
sequence of the bacterial enzyme CAT gene. This
reporter gene, pFABP(
-
56.51
+
21)CAT was trans-
fected into a mouse hepatoma cell line (Hepal) in
the absence or presence of a PPAR expression
vector (pSG5-PPAR) and the potent peroxisome
proliferator Wy- 14,643. Disappointingly, the
presence of PPAR and Wy-14,643 had no effect
upon CAT enzyme activity indicating that the
receptor was unable
to
stimulate the transcription of
the FABP promoter.
To
investigate whether this
was because the PPRE-like sequence was unable to
respond to PPAR or whether
it
was due
to
the con-
text of this sequence in the FABP promoter, we
constructed a second reporter gene pFABP(
-
5651
-5O)G.CAT in which the FABP sequences from
-
565
bp to
-
50
bp were placed upstream of the
heterologous rabbit /3-globin promoter (Fig. 2).
Using this reporter gene in transfection assays
demonstrated that
it
was responsive to the presence
Fig.
2
FABP
reporter
genes
The
5'
regulatory sequences of the rat liver fatty acid binding protein gene are shown schematically. The start of transcription
is
indicated with an arrow at
+
I
and the PPRE-like sequence is underlined between -68bp and -56bp. Two reporter gene
constructs were created using the rabbit /3-globin promoter
(
-
I
10
to
+
20)
and two using the natural FABP promoter. These were
tested in transient transfection assays using Hepal cells in the absence or presence
of
the PPAR receptor expression vector (R) and
the peroxisome proliferator Wy-
14,643
(Wy). The values on the right indicate the percentage
of
chloramphenicol acetylated by the
cell extract. Technical details can be found in [6]. Abbreviation used: TTA, tetrathio decanoic acid.
68
-56
Rat
FABP
gene
and
promoter
CAATCACTWCCTATCATATTT
-6
-565
FABP
-50
Globin
CAT
1% 10%
1% 1%
-565
68
+21
pFABP(-565/+21)CAT
J
665
68
+21
e
pFABP(-565/+21 PPRE)CAT
CAATCACTTCTACiATCATATATTT
-
--
1%
1%
1% 1%
Volume
20

Lipid-Binding Proteins
of PPAR and Wy-14,643 (Fig. 2). In addition, a
third reporter gene, pFABP(
-
565/
-
68)G.CAT,
which does not contain the PPRE-like sequence
was not stimulated by PPAR in the presence of
Wy-14,643. These data therefore suggest that
PPAR can recognize and activate the PPRE-like
sequence present in the FABP promoter but that
this
sequence
is
not functional in the context of the
natural promoter when transfected into Hepa 1 cells.
Discussion
A number of potent peroxisome proliferators are
hypolipidaemic drugs and lower the level of circulat-
ing triglycerides in man. These chemicals induce
enzymes that are important in fatty acid metabolism
such as peroxisomal acyl CoA oxidase and cyto-
chrome P450
IVA1.
Furthermore, high fat diets can
induce peroxisome proliferation in rats and per-
oxisome proliferators can elevate the level of rat
liver FABP. Taken together, therefore, these data
suggest an important role of PPAR in regulating
fatty acid homeostasis.
Examination of the
5’
regulatory sequences of
the rat liver FABP indicated the presence of a
sequence that
is
almost identical to the PPRE iden-
tified in the
5’
regulatory sequences of the rat acyl
CoA oxidase gene. This putative response element
appears to be inactive in the context of the natural
FABP promoter but is active when placed upstream
of the rabbit B-globin promoter (Fig. 2). One reason
for the failure of PPAR to activate the FABP-CAT
reporter gene may be due to TATA binding pro-
teins that bind to the adjacent TATA box sequence
interfering with PPAR binding to the
PPRE.
Alter-
natively,
it
may be that Hepal cells lack a co-factor
required for PPAR activity when the PPRE
is
present in the context of the natural FABP promo-
ter. Clearly, further work
is
required to determine
how peroxisome proliferators regulate FABP
expression.
Of particular interest was the finding that fatty
acids are weak PPAR activators ([ 113, Fig. 1). One
explanation for this weak activity is that fatty acids
are metabolized rapidly by the /?-oxidation path-
way. This
is
supported by the observation that the
thio-substituted fatty acid that is blocked for
/?-
oxidation is a much better PPAR activator (Fig. 1).
We and others have found that a wide range of fatty
acids are able to activate PPAR ([ 113, I. Issemann
and R. Prince, unpublished results). An important
question, therefore, is whether any or all of these
fatty acids can bind directly to PPAR or alterna-
tively whether they are metabolized to the ligand or
induce the ligand. These data also raise the question
of how synthetic peroxisome proliferators activate
PPAR. One possibility is that they bind directly to
the receptor in the same way that antioestrogens
such as tamoxifen bind to the oestrogen receptor.
However, thus far we have been unable to
827
demonstrate any interaction between PPAR and the
peroxisome proliferator nafenopin
[
51.
An interest-
ing alternative mechanism for peroxisome prolifera-
tor action is that these chemicals bind to FABP
displacing fatty acids that activate PPAR. Others
[12] have demonstrated that a number of per-
oxisome proliferators can bind to FABP and dis-
place the oleic acid used to monitor the purification
of FABP. It will therefore be important to determine
whether peroxisome proliferators act by simply
competing with fatty acids for binding to FABP
leading to an elevation of fatty acids that activate
PPAR, or alternatively whether they and fatty acids
can bind directly to PPAR. These experiments and
others that determine the true nature of the PPAR
ligand will be important in understanding the role of
peroxisome proliferators and PPAR in hypo-
lipidaemia and rodent liver cancer.
We thank Jon Bremer
for
providing the tetrathio
decanoic acid used in these studies.
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Received 25 June 1992
I992