Fatty acids and retinoids control lipid metabolism through activation of PPAR/RXR heterodimers

Full text

Proc.
Natl.
Acad.
Sci.
USA
Vol.
90,
pp.
2160-2164,
March
1993
Biochemistry
Fatty
acids
and
retinoids
control
lipid
metabolism through
activation
of
peroxisome
proliferator-activated
receptor-retinoid
X
receptor
heterodimers
(peroxisome
proliferator-activated
receptor
response
element/retinoid
X
receptor
response
element/acyl-CoA
oxidase
gene/
nuclear
hormone
receptors)
HANSJ6RG
KELLER*,
CHRISTINE
DREYERt,
JEFFREY
MEDINt,
ABDERRAHIM
MAHFOUDI*,
KEIKO
OZATO:,
AND
WALTER
WAHLI*§
*Institut
de
Biologie
animale,
Universite
de
Lausanne,
Batiment
de
Biologie,
CH-1015
Lausanne,
Switzerland;
tMax-Planck-Institut
fMr
Entwicklungsbiologie,
D-7400
Tubingen,
Germany;
and
tLaboratory
of
Molecular
Growth
Regulation,
National
Institute
of
Child
Health
and
Human
Development,
National
Institutes
of
Health,
Bethesda,
MD
20892
Communicated
by
Igor
Dawid,
November
30,
1992
ABSTRACT
The
nuclear
hormone
receptors
called
PPARs
(peroxisome
proliferator-activated
receptors
a,
(3,
and
v)
regulate
the
peroxisomal
(3-oxidation
of
fatty
acids
by
induction
of
the
acyl-CoA
oxidase
gene
that
encodes
the
rate-limiting
enzyme
of
the
pathway.
Gel
retardation
and
cotransfection
assays
revealed
that
PPARa
heterodimerizes
with
retinoid
X
receptor
(3
(RXR,B;
RXR
is
the
receptor
for
9-cis-retinoic
acid)
and
that
the
two
receptors
cooperate
for
the
activation
of
the
acyl-CoA
oxidase
gene
promoter.
The
strongest
stimulation
of
this
promoter
was
obtained
when
both
receptors
were
exposed
simultaneously
to
their
cognate
activators.
Furthermore,
we
show
that
natural
fatty
acids,
and
especially
polyunsaturated
fatty
acids,
activate
PPARs
as
potently
as
does
the
hypolipid-
emic
drug
Wy
14,643,
the
most
effective
activator
known
so
far.
Moreover,
we
discovered
that
the
synthetic
arachidonic
acid
analogue
5,8,11,14-eicosatetraynoic
acid
is
100
times
more
effective
than
Wy
14,643
in
the
activation
of
PPARa.
In
conclusion,
our
data
demonstrate
a
convergence
of
the
PPAR
and
RXR
signaling
pathways
in
the
regulation
of
the
peroxi-
somal
(3-oxidation
of
fatty
acids
by
fatty
acids
and
retinoids.
Peroxisome
proliferator-activated
receptors
(PPAR)
are
nu-
clear
hormone
receptors
activated
by
substances
including
fibrate
hypolipidemic
drugs,
phthalate
ester
plasticizers,
and
herbicides
that
cause
peroxisome
proliferation in
the
liver
(1,
2).
So
far
three
PPAR
receptors
(a,
B,
and
y)
have
been
described
in
Xenopus
(2),
one
in
mouse
(1),
and
one
in
rat
(3).
These
receptors
are
transcription
factors
that
control
the
peroxisomal
(3-oxidation
pathway
of
fatty
acids
through
regulation
of
the
acyl-CoA
oxidase
gene
that
encodes
the
rate-limiting
enzyme
of
the
pathway
(2,
4).
Thus,
PPARs
play
an
important
role
in
lipid
metabolism.
Structural
analysis
of
the
PPARs
revealed
that
they
belong
to
the
nuclear
hormone
receptor
subgroup,
which
comprises
receptors
for
all-trans-retinoic
acid
(RAR),
9-cis-retinoic
acid
(retinoid
X
receptor;
RXR),
thyroid
hormone,
vitamin
D,
and
several
orphan
receptors.
All
of
these
receptors
recognize
the
canonical
DNA
response
sequence
AGGTCA
and
accord-
ingly
possess
the
same
P-box
amino
acid
sequence
in
the
first
zinc
finger
of
their
DNA-binding
domain
(5).
We
and
others
have
identified
a
PRAR
response
element
(PPRE)
in
the
acyl-CoA
oxidase
promoter
(2, 4).
This
response
element
contains
a
direct
repeat
of
the
AGGTCA
motif
with
one
intervening
nucleotide,
which
is
called
DR-1.
Interestingly,
the
RXR
response
element
(RXRE)
in
the
promoter
of
the
cellular
retinol-binding
protein
type
II
gene
contains
also
DR-1
elements
(6).
Thus,
the
convergence
of
the
PPAR
and
RXR
signaling
pathways
in
the
transcriptional
regulation
of
the
acyl-CoA
oxidase
gene
was
an
interesting
hypothesis
to
test.
Further
indications
of
a
coupling
between
PPARs
and
RXRs
came
from
the
recently
demonstrated
induction
of
the
acyl-CoA
oxidase
gene
by
retinoic
acid
in
cultured
rat
hepatocytes
(7)
and
the
observation
of
heterodimerization
of
RXR
with
other
members
of
the
nuclear
hormone
receptor
superfamily
(8-15).
f-Oxidation
of
long-chain
fatty
acids
is
an
essential
process
in
lipid
metabolism.
Its
disruption,
which
occurs
in
disorders
such
as
Zellweger
syndrome
and
adrenoleukodystrophy
(16),
leads
to
a
lethal
accumulation
of
very
long-chain
fatty
acids
in
the
blood.
To
further
our
understanding
of
the
hormonal
control
of
the
peroxisomal
(3-oxidation
by
PPARs
and
pos-
sibly
by
RXRs,
we
searched
for physiologically
occurring
activators
of
PPARa.
Fatty
acids
were
possible
candidates
for
this
role,
since
high-fat
diets
have
been
reported
to
stimulate
(-oxidation
(17).
In
this
paper,
we
show
that
PPARa
and
RXR(3
heterodimerize
and
that
they
coopera-
tively
stimulate
the
acyl-CoA
oxidase
gene
promoter.
Fur-
thermore,
we
show
that
physiological
concentrations
of
fatty
acids,
and
especially
polyunsaturated
fatty
acids
(PUFA),
activate
Xenopus
laevis
PPARa
(xPPARa)
to
the
same
extent
as
the
xenobiotic
peroxisome
proliferator
Wy
14,643.
Finally,
the
synthetic
arachidonic
acid
(AA)
analogue
5,8,11,14-
eicosatetraynoic
acid
(ETYA)
was
found
to
fully
activate
xPPARa
at
a
concentration
1/100th
that
of
Wy
14,643.
MATERIALS
AND
METHODS
Immunoprecipitations.
Nuclear
extracts
containing
bacu-
lovirus
recombinant
mouse
RXR,B
(mRXRf3)
were
prepared
from
infected
Sf9
cells
as
described
(18),
except
for
the
additional
inclusion
of
protease
inhibitors.
In
vitro
translated
and
35S-labeled
xPPARa
was
combined
with
1
ug
of
mRXR,B
or
control
baculovirus
extract
and
anti-mRXR(3
antiserum
(19)
in
100
,ul
of
buffer
A
[20
mM
Hepes,
pH
7.9/50
mM
NaCl/l
mM
EDTA/5%
(vol/vol)
glycerol/0.05%
Triton
X-100]
and
allowed
to
associate
overnight
at
4°C.
Samples
were
then
added
to
prewashed
protein
A-agarose
beads
(Boehringer
Mannheim)
and
incubated
at
4°C
for
2
hr
with
rocking.
The
beads
were
collected
by
centrifugation
and
Abbreviations:
AA,
arachidonic
acid;
ETYA,
5,8,11,14-eicosatet-
raynoic
acid;
NDGA,
nordihydroguaiaretic
acid;
PPAR,
peroxisome
proliferator-activated
receptor;
PPRE,
PPAR
response
element;
PUFA,
polyunsaturated
fatty
acid(s);
RAR,
all-trans-retinoic
acid
receptor;
RXR,
retinoid
X
receptor
(for
9-cis-retinoic
acid);
RXRE,
RXR
response
element;
mRXR,8,
mouse
RXRi,;
xPPARa,
Xenopus
laevis
PPARa;
CAT,
chloramphenicol
acetyltransferase.
§To
whom
reprint
requests
should
be
addressed.
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.
2160

Proc.
Natl.
Acad.
Sci.
USA
90
(1993)
2161
washed
twice
with
buffer
A
containing
0.05%
bovine
serum
albumin
followed
by
a
single
washing
in
buffer
A
alone.
The
beads
were
collected
and
boiled
in
50
,ul
of
SDS
loading
buffer
for
2
min
and
pelleted
by
centrifugation
for
4
min,
and
the
resulting
supematant
was
analyzed
by
SDS/PAGE.
Gel
Retardation
Assays.
Five
microliters
of
in
vitro
trans-
lated
xPPARa
and
2
ul
of
nuclear
extract
containing
bacu-
lovirus-expressed
recombinant
mRXR,B
(see
above)
or
mock
controls
were
incubated
on
ice
for
15
min
in
buffer
containing
10
mM
Tris-HCl
(pH
8.0),
40
mM
KCl,
0.05%
(vol/vol)
Nonidet
P-40,
5%
glycerol,
1
mM
dithiothreitol,
and
0.1
jig
of
poly(dl-dC)
(Pharmacia).
For
competition
experiments,
40
ng
of
ACO-A
or
ACO-B
double-stranded
oligonucleotides
(2)
were
also
included
during
preincubation.
[ACO
is
a
reporter
plasmid
containing
the
5'
flanking
region
of
the
acyl-CoA
oxidase
gene
in
front
of
the
chloramphenicol
acetyltrans-
ferase
(CAT)
gene;
ACO-A
and
ACO-B
are
synthetic
oligo-
nucleotides
with
sequences
of
the
two
enhancer
regions
of
the
acyl-CoA
oxidase
gene
promoter.]
Then,
1
,ul
of
ACO-A
double-stranded
oligonucleotide
(1
ng/,ul),
labeled
with
32p
by
fill-in
with
Klenow
polymerase,
was
added,
and
the
incubation
was
continued
for
10
min
at
room
temperature
before
samples
were
electrophoresed
on
a
5%
polyacrylam-
ide
gel
in
0.5
x
TBE
buffer
(45
mM
Tris/45
mM
boric
acid/l
mM
EDTA)
at
4°C.
For
antibody
supershift
assays,
anti-
xPPARa
(unpublished
data)
or
preimmune
serum
as
a
control
was
added
to
the
samples
after
the
incubation
with
the
ACO-A
probe,
and
incubation
was
continued
for
another
10
min
at
room
temperature
followed
by
gel
electrophoresis
on
a
3.5%
polyacrylamide
gel.
Transfections.
The
xPPARa
expression
vector
and
the
reporter
plasmids
ACO-A.TK.CAT,
ACO-G.CAT,
and
G.CAT,
as
well
as
the
mRXR,B
expression
vector
pRSV-H-
2RIIBP
have
been
described
(2,
20);
G
refers
to
the
,3-globin
gene
promoter
and
TK
refers
to
the
thymidine
kinase
gene
promoter.
Cotransfections
of
CV-1
cells
were
performed
as
described
for
HeLa
cells
(2),
except
for
the
application
of
a
glycerol
shock
(15%)
for
2
min
before
the
activators
were
added.
As
an
internal
control,
a
luciferase
expression
plasmid
(21),
which
does
not
respond
to
the
activators
used,
was
also
cotransfected,
and
luciferase
activities
were
used
to
normal-
ize
CAT
activities.
Charcoal-treated
serum,
which
was
de-
pleted
of
fatty
acids
(22),
was
used
in
transfection
experi-
ments.
Activators
were
added
to
the
cell
culture
medium
either
in
ethanol
solutions
in
the
case
of
free
acids
or
in
10%
ethanol/0.2%
NaHCO3
in
the
case
of
sodium
salts.
Final
ethanol
concentrations
in
the
cell
culture
medium
were
<0.1%
to
avoid
negative
effects
on
the
cells.
All
fatty
acids
used
were
from
Sigma
and
were
stored
at
-20°C
under
argon.
9-cis-Retinoic
acid
was
obtained
from
M.
Klaus
at
Hoff-
mann-La
Roche
(Basel).
RESULTS
Heterodimerization
of
PPARa
with
RXRI3.
Using
a
coim-
munoprecipitation
assay,
we
observed
that
35S-labeled
PPARa,
when
mixed
with
unlabeled
RXR,3,
is
specifically
precipitated
by
anti-mRXRp6
serum
(Fig.
1,
lane
2).
No
coimmunoprecipitation
of
PPARa
was
observed
with
preim-
mune
serum
in
the
presence
of
RXRI3
(lane
3)
or
with
anti-RXR,6
serum
in
the
absence
of
RXR,3
(lane
4),
confirm-
ing
the
specificity
of
the
interaction
between
xPPARa
and
mRXR,B.
In
agreement
with
this
result,
chemical
cross-
linking
experiments
also
revealed
specific
heterodimer
for-
mation
between
xPPARa
and
mRXR3
(data
not
shown),
demonstrating
that
xPPARa
binds
to
mRXR/3
in
solution.
With
this
evidence
for
an
association
between
xPPARa
and
mRXR,3
in solution,
we
next
performed
gel
mobility-shift
experiments
to
analyze
whether
xPPARa-mRXRS
het-
erodimers
bind
to
the
recently
identified
PPRE
of
the
acyl-
CoA
oxidase
gene
(2, 4).
mRXRp8
and
xPPARa
alone
did
not
1
2
3
4
xPPARa-'
Anti-mRXR,B
serum:
mRXR,B:
35S-xPPARa:
±
+±
FIG.
1.
Formation
of
PPARa-
RXRRp
heterodimers:
coimmuno-
precipitation.
An
equal
amount
of
in
vitro
translated
and
35S-
labeled
xPPARa
(35S-xPPARa)
as
shown
in
lane
1,
was
incubated
with
baculovirus-expressed
mRXR,B
(lanes
2
and
3)
or
control
nuclear
extract
(lane
4)
and
was
subjected
to
coimmunoprecipita-
tion
with
anti-mRXR,B
serum
(lanes
2
and
4)
or
preimmune
serum
(lane
3)
followed
by
SDS/
PAGE
analysis
of
the
precipi-
tated
material.
bind
significantly
to
the
PPRE
within
the
ACO-A
probe
(Fig.
2,
lanes
3
and
4).
However,
incubation
of
the
ACO-A
probe
with
a
mixture
of
PPARa
and
RXR,3
resulted
in
a
prominent
complex
(lane
5).
The
specificity
of
this
complex
was
demon-
strated
by
competition
with
a
40-fold
excess
of
unlabeled
ACO-A
oligonucleotide
(lane
6),
whereas
a
40-fold
excess
of
ACO-B
oligonucleotide,
which
does
not
contain
a
PPRE
(2),
did
not
lead
to
disappearance
of
the
complex
(lane
7).
The
1
23456
7
xPPARa-
mRXR,B
*
-_0
Free
probe
...
8
910
.4
-Supershift
_
xPPARa-
'P
0
'
mRXR/3
..
.Fre
.pr
E
.I
Free
probe
-
ACO-A
probe:
CCCGAACGTGACCTTTGTCCTGGTCC
FIG.
2.
Formation
of
PPARa-RXR,3
heterodimers:
gel
retarda-
tion
assay.
In
vitro
synthesized
xPPARa
and
baculovirus-expressed
mRXR,3
or
mock
controls
were
incubated
in
the
presence
of
the
PPRE-containing
32P-labeled
ACO-A
probe,
and
protein-DNA
com-
plexes
were
analyzed
by
electrophoresis
on
a
5%
polyacrylamide
gel.
In
the
case
of
antibody-induced
supershifts,
samples
were
analyzed
by
electrophoresis
on
a
3.5%
polyacrylamide
gel.
Lanes:
1,
free
probe;
2,
mock
baculovirus
wild-type
nuclear
extract
(bac-WT)
and
mock
reticulocyte
lysate
(RL);
3,
baculovirus-expressed
mRXR,B
and
RL;
4,
bac-WT
and
in
vitro
translated
xPPARa;
5,
baculovirus-
expressed
mRXR,3
and
in
vitro
translated
xPPARa;
6,
competition
of
the
mRXR,8-xPPARa
complex
as
in
lane
5
with
a
40-fold
excess
of
unlabeled
ACO-A
oligonucleotide;
7,
same
as
lane
6,
but
competition
with
ACO-B
oligonucleotide
which
does
not
contain
a
PPRE;
8-10,
antibody
supershift
assay
of
the
mRXRf3-xPPARa
complex:
mRXR/3-xPPARa
complex
as
in
lane
5
(lane
8),
supershift
of
the
mRXR,B-xPPARa
complex
with
anti-xPPARa
(lane
9),
but
not
with
preimmune
serum
(lane
10).
Biochemistry:
Keller
et
al.

Proc.
Natl.
Acad.
Sci.
USA
90
(1993)
nature
of
the
minor
band
indicated
by
an
asterisk
in
lane
4
is
unknown.
It
could
represent
weak
binding
of
xPPARa
to
the
probe
as
a
monomer,
a
homodimer,
or
a
heterodimer
with
an
insect
cell
nuclear
protein
such
as
Usp,
the
homologue
of
RXR.
The
presence
of
PPARa
in
the
PPARa-RXRf8
complex
was
confirmed
by
a
specific
anti-xPPARa-induced
supershift
(lane
9),
whereas
preimmune
serum
had
no
effect
(lane
10).
In
agreement
with
the
fact
that
nuclear
hormone
receptors
bind
as
dimers
to
response elements
consisting
of
two
half
sites
(5),
we
conclude
that
PPARa
and
RXR,8
heterodimerize
in
solu-
tion
and
bind
synergistically
as
heterodimers
to
the
PPRE.
This observation
and
the
similarity
of
the
PPRE
and
the
RXRE
of
the
cellular
retinol-binding
protein
type
II
gene
promoter
(8)
led
us
to
examine
whether
there
is
a
functional
interaction
of
PPARa
and
RXR,
in
transcriptional
activation
of
the
PPRE-containing
ACO-G.CAT
reporter
gene.
The
ACO-G.CAT
plasmid
contains
the
acyl-CoA
oxidase
gene
promoter
sequence
from
-471
to
-1273
in
front
of
the
rabbit
,B-globin
basal
promoter-controlled
CAT
gene.
The
PPRE
is
located
between
-578
and
-553
within
this
promoter
region
(2,
4).
PPARa
and
RXRf3
expression
plasmids,
either
alone
or
combined,
were
cotransfected
with
the
ACO-G.CAT
reporter
plasmid
into
CV-1
cells,
and
CAT
assays
were
performed
after
induction
in
the
presence
or
absence
of
the
specific
activator
for
each
receptor
(100
AM
Wy
14,643
for
PPARa
and
1
,uM
9-cis-retinoic
acid
for
RXRJ3).
The
highest
stimulation
of
the
ACO-G.CAT
reporter
plasmid
was
ob-
served
by
cotransfection
of
the
PPARa
and
RXRj3
expression
plasmids
in
the
presence
of
the
two
activators,
indicating
that
optimal
cooperation
between
both
signaling
pathways
de-
pends
on
the
simultaneous
activation
of
both
receptors
(Fig.
3).
Compared
with
the
effect
of
the
two
individual
receptors,
the
combined
effect
of
both
receptors
on
transcriptional
induction
was
additive.
It
is
noteworthy
that
induction
by
PPARa
transfected
alone
occurred
also
with
the
RXR
ligand
9-cis-retinoic
acid,
and
conversely,
stimulation
by
RXRB,
albeit
weaker,
was
observed
with
Wy
14,643.
These
results
are
compatible
with
the
involvement
of
endogenous
CV-1
cell
RXR
and
PPAR
activities
through
heterodimerization
with
the
introduced
PPARa
and
RXRI
receptors,
respectively.
The
presence
of
a
low
level
of
endogenous
receptors
in
these
cells
was
further
supported
by
a
2-fold
receptor-independent,
but
activator-dependent,
stimulation
of
ACO.G-CAT,
but
not
of
G-CAT,
as
observed
previously
in
HeLa
cells
(2).
Furthermore,
expression
of
the
two
receptors
alone
or
in
combination
in
the
absence
of
inducers
led
to
an
increase
in
activity,
indicating
a
low
level
of
constitutive
receptor
activity
or
the
presence
of
a
weak
unidentified
endogenous
activator.
Effects
similar
to
those
seen
with
the
ACO.G-CAT
reporter
gene
were
observed
with
the
ACO-A.TK.CAT
reporter
gene
containing
one
copy
of
the
PPRE
in
front
of
the
thymidine
kinase
gene
promoter
(2),
indicating
that
the
functional
interaction
between
PPARa
and
RXRf3
does
indeed
occur
through
the
PPRE
(data
not
shown).
Taken
together,
these
results
show
that
the
PPAR
and
RXR
signaling
pathways
converge
in
the
regulation
of
the
acyl-CoA
oxidase
promoter.
Fatty
Acids
Activate
PPARa.
In
our
search
for
endogenous
activators
of
xPPARa,
two
reasons
prompted
us
to
test
whether
fatty
acids
activate
PPARa:
(i)
known
potential
ligands
of
PPAR,
such
as
fibrate
hypolipidemic
drugs,
present
an
amphipathic
structure
similar
to
fatty
acids-e.g.,
having
a
free
carboxyl
group
and
a
lipophilic
moiety-and
(ii)
high
dietary
fat
intake
and
certain
fatty
acid
analogues
induce
the
peroxisomal
p-oxidation
of
fatty
acids
(23, 24).
For
these
experiments,
HeLa
cells
were
cotransfected
with
the
xPPARa
expression
plasmid
and
the
ACO-A.TK.CAT
reporter
plas-
mid.
Subsequently,
various
fatty
acids
were
added
to
the
culture
medium
to
a
final
concentration
of
50
,uM,
and
CAT
activities
were
determined.
All
of
the
PUFAs
tested
activated
Relative
CAT
activitY
0
100
200
300
G.CAT
[
ACO-G.CAT
G.CAT
[
ACO-G.CAT
G.CAT
[
ACO-G.CAT
Reporter
plasmid
xPPARa
mRXRA
XPPARainRXRp
xPPARa
mRxR"
xPPARa/mRXRO
+
xPPARa
mRXRa
xPPARaWmRxap
_
9-cis-Retinoic
acid
I__
-________
Wy
14,643
9-cls-Retinoic
acid
+
Wy
14,643
Expression
plasmid
Activator
FIG.
3.
Transcriptional
activation
of
the
acyl-CoA
gene
promoter
by
xPPARa
and
mRXR,B.
CV-1
cells
were
cotransfected
with
the
expression
vectors
for
xPPARa
and
mRXR,3
and
the
reporter
plasmids
ACO-G.CAT
and
G.CAT
as
indicated.
ACO-G.CAT
con-
tains
the
acyl-CoA
oxidase
gene
promoter
from
-471
to
-1273
in
front
of
the
rabbit
(-globin
basal
promoter-driven
CAT
gene,
whereas
G.CAT,
which
is
used
as
control,
is
the
same
construct
without
the
acyl-CoA
oxidase
gene
promoter
sequences
(2,
4).
After
treatment
with
the
indicated
activators
(+)-Wy
14,643
(100
,uM)
or
9-cis-retinoic
acid
(1
,uM)
or
both-or
with
solvent
(ethanol)
as
a
control
(-),
CAT
assays
were
performed,
and
the
results
were
normalized
arbitrarily
to
the
activity
observed
by
PPARa
in
the
presence
of 100
ItM
Wy
14,643,
which
was
taken
as
100%t.
The
mean
values
of
three
independent
experiments
with
the
corresponding
standard
deviations
are
shown.
PPARa
by
4-
to
8-fold
(Fig.
4-i.e.,
to
the
same
extent as
Wy
14,643,
which
is
the
most
potent
activator
known
so
far
(1,
2).
No
significant
difference
in
the
activation
of
xPPARa
was
observed
between
the
c--6
fatty
acids
(AA
and
linoleic
acid)
and
cw-3
fatty
acids
(docosahexaenoic,
eicosapentaenoic,
and
linolenic
acids),
which
represent
the
two
classes
of
essential
PUFAs
(25).
In
contrast,
the
monounsaturated
fatty
acids
tested
displayed
a
wide
range
of
effectiveness
in
the
activation
of
PPARa.
Whereas
petroselinic
acid
activated
PPARa
with
a
similar
efficiency
as
PUFAs,
oleic
acid
and
elaidic
acid
were
less
potent,
and
the
very
long-chain
fatty
acids
erucic
acid
and
nervonic
acid
did
not
activate
PPARa.
Since
most
of
the
naturally
occurring
fatty
acids
have
double
bonds
in
the
cis
configuration,
it
is
interesting
that
elaidic
acid,
which
has
a
trans
double
bond,
activated
PPARa
to
the
same
level
(about
2.5-fold)
as
did
its
natural
cis
homologue
oleic
acid.
Moreover,
the
saturated
fatty
acid
lauric
acid
activated
PPARa
only
weakly,
and
the
dicarboxylic
fatty
acid
dodecanedioic
acid
did
not
activate
PPARa.
As
a
control,
triiodothyroacetic
acid,
which
is
also
an
amphipathic
molecule
but
not
a
fatty
acid
and
+rF
--
4
2162
Biochemistry:
Keller
et
al.
.

Proc.
Natl.
Acad.
Sci.
USA
90
(1993)
2163
non-induced
Wy
14,643
3.3'.5
Trliiodothyroacetic
acid
Polyunsaturated
fatty
acids:
Docosahexaenoic
acid
Elcosapentaenoic
acid
LAnolenic
acid
LAnoleic
acid
Arachidonic
acid
Monounsaturated
fatty
acids:
Petroselinic
acid
Oleic
acid
Elaidic
acid
Erucic
acid
Nervonic
acid
Saturated
fatty
acids:
Lauric
acid
1,12
Dodecanedioic
acid
C22:6&o3
C20:5co3
C18:3w3
C18:2w6
C20:4e6
C18:1(ol2
C18:1w9
C18:1h9
trn
C22:
lco9
C24:lco9
C12
C12
%
CAT
activity
100
200
Standard
-4
which
activates
the
thyroid
hormone
receptor,
also
did
not
activate
PPARa.
Furthermore,
activation
of
the
ACO-
A.TK.CAT
reporter
plasmid
by
fatty
acids
was
dependent
upon
the
presence
of
PPARa
and
a
reporter
plasmid
without
the
PPRE
was
not
induced
by
fatty
acid-activated
PPARs
(data
not
shown).
The
activator-independent
transcriptional
activity
of
xPPARa
(see
above)
was
not
due
to
residual
fatty
acids
possibly
present
in
the
cell
culture
medium,
since
this
activity
was
also
observed
in
the
absence
of
10%
fetal
calf
serum
in
the
culture
medium.
ETYA,
a
Synthetic
AA
Analogue,
Is
a
100-fold
More
Potent
Activator of
xPPARa
Than
AA
or
Wy
14,643.
Since
AA
is
the
precursor
for
the
synthesis
of
eicosanoids
such
as
prosta-
glandins,
thromboxanes,
lipoxins,
and
leukotrienes,
which
are
implicated
in
various
cell-specific
signaling
events,
we
tested
whether
the
activation
of
xPPARa
by
AA
was
due
to
AA
itself
or
to
an
AA
metabolite.
Three
pathways
are
involved
in
the
production
of
the
eicosanoids
mentioned
above,
the
cyclooxygenase,
lipoxygenase,
and
epoxygenase
pathway
(26).
Commonly
used
specific
blockers
of
these
pathways
are
aspirin
and
indomethacin
for
the
cycloxygenase
pathway,
nordihydroguaiaretic
acid
(NDGA)
for
the
lipoxy-
genase
pathway,
and
metyrapone
for
the
epoxygenase
path-
way
(27).
Activation
of
xPPARa
by
10
,uM
AA
in
transfection
experiments
(data
not
shown)
was
not
blocked
or
signifi-
cantly
inhibited
by
100
,uM
aspirin,
10
,uM
indomethacin,
10
,uM
NDGA,
or
10
,uM
metyrapone.
Consistently,
the
pros-
taglandins
PGD2,
PGE2,
and
PGF2a
(10
,uM
each),
and
the
hydroperoxyeicosatetraenoic
acids
(HPETE)
5-,
8-,
12-
and
15-HPETE
(0.6
,uM
each)
did
not
activate
xPPARa
(data
not
shown).
Surprisingly,
ETYA,
a
blocker
of
lipoxygenases
and
cyclooxygenases,
fully
activated
xPPARa
at
a
concentration
of
only
1
,uM,
and
the
dose-response
curve
revealed
an
ED50
of
200
nM,
which
is
lower
by
a
factor
of
about
100
than
those
for
Wy
14,643
and
AA
(Fig.
5).
DISCUSSION
Interaction
of
PPARa
and
RXRI3
Signaling
Pathways.
Transfection
experiments
with
xPPARa
and
RXR,B
demon-
strated
that
the
receptors
cooperatively
activate
the
acyl-
FIG.
4.
Activation
of
xPPARa
by
fatty
acids.
The
xPPARa
expression
vector
and
ACO-A.TK.CAT
reporter
plasmid
were
cotransfected
into
HeLa
cells.
Subsequently,
activators
or
solvent
as
a
control
was
added
to
the
cell
culture
medium,
and
CAT
activity
was
assayed;
1O0o
CAT
ac-
tivity
was
taken
arbitrarily
as
the
CAT
activity
observed
with
50
&M
AA
(all
additives
were
at
50
,uM).
Basal
level
of
CAT
activity
is
indi-
cated
by
the
dashed
line.
Experi-
ments
were
done
at
least
in
triplicate,
and
the
mean
values
with
the
corre-
sponding
standard
deviations
are
shown.
Fatty
acids
are
listed
by
their
trivial
name,
and
their
structure
is
indicated
by
the
w
nomenclature,
which
shows
from
left
to
right
the
number
of
carbon
atoms,
the
number
of
double
bonds,
and
the
location
of
the
first
double
bond
counting
from
the
w
(end)
carbon
of
the
carbohy-
drate
chain
(25).
Most
naturally
oc-
curring
fatty
acids
have
double
bonds
in
the
cis
configuration.
Thus,
the
only
exception,
elaidic
acid,
is
labeled
"trans."
CoA
oxidase
promoter
through
the
PPRE.
This
is
consistent
with
the
observation
that
retinoic
acid
(9),
most
likely
by
isomerization
to
9-cis-retinoic
acid
(28),
and
fatty
acids
induce
the
acyl-CoA
oxidase
gene
in
vivo.
While
it
is
known
that
9-cis-retinoic
acid
binds
to
RXR
and
thereby
converts
it
into
an
active
transcription
factor,
we
do
not
know
whether
fatty
acids
work
in
a
similar
way
and
bind
directly
to
PPARs.
Our
transfection
experiments
indicate
that
the
strongest
activation
of
the
acyl-CoA
oxidase
gene
requires
PPARa
and
RXRf3
in
the
activated
state.
However,
a
slight
cooperative
stimulation
was
also
observed
in
the
absence
of
activators.
Although
in
vitro
gel
retardation
assays
indicated
a
synergis-
tic
binding
of
PPARa-RXRI3
heterodimers
to
the
PPRE,
it
is
not
clear
from
the
transfection
experiments
whether
there
is
also
a
preferential
or
even
exclusive
formation
of
PPARa-
RXR,B
heterodimers
on
the
PPRE
in
vivo
because
the
tran-
scriptional
activation
observed
by
PPARa
and
RXRP
is
additive.
Furthermore,
the
fact
that
the
acyl-CoA
oxidase
i
100
1o-10
10-9
16-8
10-7
10-6
10-5
log
M
1o-4
10-3
FIG.
5.
Activation
of
xPPARa:
ETYA
(o),
AA
(e),
and
Wy
14,643
(o)
dose-response
curves.
Activation
of
xPPARa
by
increas-
ing
concentrations
of
ETYA,
AA,
or
Wy
14,643
was
assayed
in
cotransfection
experiments
as
described
in
Fig.
4.
Higher
concen-
trations
of
activators
than
those
shown
were
cytotoxic
or
led
to
complete
detachment
of
the
cells.
Mean
values
of
at
least
three
independent
experiments
are
shown.
a
I
M*
---4
I
i
4
Biochemistry:
Keller
et
al.
ir,
I
i

Proc.
Natl.
Acad.
Sci.
USA
90
(1993)
gene
promoter
is
activated
by
transiently
expressed
PPARa
and
RXR,3
alone
may
suggest
that
homodimers
are
also
transcriptionally
active.
However,
since
endogenous
PPAR
and
RXR
are
present
in
CV-1
cells,
as
is
indicated
by
the
low
level
of
activation
of
the
acyl-CoA
oxidase
gene
promoter
in
the
absence
of
transfected
receptors
and
by
the
apparently
ubiquitous
expression
of
the
two
receptors
(2,
30),
we
believe
that
heterodimers
are
in
fact
responsible
for
the
transcrip-
tional
activation
observed.
Ultimately,
transfection
experi-
ments
with
dominant
negative
PPAR
and
RXR
mutants
that
still
form
heterodimers
but
do
not
stimulate
transcription
or
transfection
experiments
with
cells
deficient
in
endogenous
PPAR
and
RXR
are
needed
to
answer
the
question
of
which
PPAR-RXR
species
are
functionally
relevant
in
vivo.
Regulation
of
the
Peroxisomal
,8-Oxidation
by
Fatty
Acids.
Based
on
structural
and
functional
considerations,
we
have
tested
several
natural
fatty
acids
for
activation
of
PPARa.
Stimulation
of
xPPARa
was
strongest
in
the
presence
of
PUFAs,
followed
by
monounsaturated
fatty
acids
and
saturated
fatty
acids.
Similar
observations
have
also
been
made
with
a
chimeric
receptor
containing
the
transactivation
and
DNA-
binding
domains
of
the
glucocorticoid
receptor
and
the
ligand-
binding
domain
of
the
rat
PPAR
(3).
However,
in
contrast
to
these
results,
we
found
that
PUFAs
activated the
genuine
xPPARa
receptor
as
efficiently
as
the
most
potent
peroxisome
proliferator,
Wy
14,643.
This
may
be
due
to
differences
between
the
full-length
and
the
artificial
chimeric
receptors,
to
the
different
transfection
assay
systems
applied,
or
to
species
differences.
Interestingly,
the
very
long-chain
monounsat-
urated
fatty
acids
nervonic
acid
and
erucic
acid,
which
exert
a
negative
effect
on
the
peroxisomal
,(oxidation
system
(31),
did
not
activate
xPPARa.
Dietary
fatty
acids
occur
in
a
great
variety
as
saturated
and
unsaturated
fatty
acids.
In
contrast
to
the
saturated
and
monounsaturated
fatty
acids,
PUFAs
are
absolutely
necessary
for
the
growth
and
health
of
animals
and
humans.
According
to
their
origin
from
linolenic
or
linoleic
acid,
PUFAs
are
classified
into
w-3
and
co-6
PUFAs
as
defined
by
the
location
of
the
first
double
bond
from
the
end
of
the
terminal
methyl
group
of
the
carbohydrate
chain
(25).
Great
interest
in
co-3
and
o-6
PUFAs
has
recently
arisen
because
of
their
beneficial
role
in
the
prevention
of
atherosclerosis
due
to
their
effect
on
lowering
triglyceride
and
cholesterol
plasma
concen-
trations
(32,
33).
We
show
now
that
w-3
and
o-6
PUFAs
are
potent
activators
of
xPPARa
and,
thus,
the
degradation
of
fatty
acids
via
peroxisomal
(-oxidation.
This
represents
a
positive
feedback
regulation
and
may
explain
the
hypolipidemic
effect
of
PUFAs
at
the
molecular
level.
ETYA
is
a
structural
analogue
of
AA
in
which
four
alkyne
bonds
replace
the
four
alkene
bonds
present
in
AA.
ETYA
has
been
synthesized
as
a
candidate
hypocholesterolemic
drug,
and,
indeed,
inhibition
of
cholesterol
biosynthesis
and
reduction
of
serum
cholesterol
concentration
has
been
ob-
served.
However,
ETYA
has
not
been
introduced
as
hypo-
cholesteremic
drug
because
of
side
effects
(29).
We
show
now
that
ETYA
is
a
potent
activator
of
xPPARa
and,
based
on
dose-response
curves,
that
it
is
100
times
more
effective
than
Wy
14,643
or
AA.
Comparison
of
the
ED5o
of
ETYA
with
the
ED50s
of
retinoids
activating
transiently
expressed
RAR
or
RXR
in
CV-1
cells
(28)
suggests
the
possibility
that
ETYA
may
be
a
high-affinity
ligand
of
xPPARa.
Alterna-
tively,
ETYA
could
induce
the
formation
or
release
of
endogenous
ligands
because
of
its
high
metabolic
stability.
Indeed,
metabolic
studies
of
ETYA
in
rats
revealed
only
partial
-
and
/3oxidation
of
this
compound,
and
all
of
the
triple
bonds
in
the
molecule
remained
intact
(29).
Along
the
same
line,
ETYA
blocks
several
AA-metabolizing
enzymes
such
as
lipoxygenase
and
cycloxygenase
by
acting
as
a
false
substrate
(27),
and
it
has
been
reported
that
1
'gtM
ETYA
led
to
total
inhibition
of
prostaglandin
release
from
isolated
perfused
rabbit
heart
(29).
Ultimately,
binding
studies
will
be
required
to
determine
whether
ETYA
is
a
high-affinity
ligand
of
xPPARa.
In
conclusion,
regulation
of
the
expression
of
genes
involved
in
lipid
metabolism
by
nutrients
such
as
PUFAs
is
of
great
physiological
and
clinical
importance,
and
it
will
require
the
identification
of
further
PPAR
activators
and
target
genes
to
elucidate
the
complete
role
of
PPARs
in
the
hormonal
control
of
lipid
metabolism.
We
thank
F.
Givel,
M.
Perroud,
and
B.
Glaser
for
expert
technical
help
and A.
Hihi
for
baculovirus
nuclear
extracts.
Special
thanks
go
to
M.
F.
Vesin
for
providing
prostaglandins,
S.
Gut
for
HPETEs,
and
M.
Klaus
for
9-cis-retinoic
acid,
and
to
S.
Child
and
N.
Mermod
for
critical
reading
of
the
manuscript.
The
work
was
supported
by
the
Swiss
National
Science
Foundation,
the
Etat
de
Vaud,
and
the
Deutsche
Forschungsgemeinschaft.
A.M.
was
supported
by
the
Association
pour
la
Recherche
sur
le
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