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INTRODUCTION |
The liver coordinates synthesis of fatty acids, esterification of
triacylglycerols, and their packaging into very low density lipoproteins for export during fed conditions, while in fasting it
controls the rates of
-oxidation and ketogenesis. By balancing these
processes, the liver handles large amounts of fat without accumulating
triacylglycerol. Many homeostatic responses of the hepatocyte to free
fatty acids (FFA)1 are
modulated by peroxisome proliferator-activated receptor
(PPAR
).
FFA are endogenous ligands for PPAR
(1-4), and numerous genes
involved in fat metabolism contain peroxisome proliferator response
elements (PPREs) in their promoters. It has been suggested that this
constitutes a feedback control system: elevated FFA activate PPAR
,
inducing a battery of enzymes (peroxisomal
-oxidation, mitochondrial
-oxidation, and microsomal fatty acid hydroxylation (which initiates
-oxidation)) involved in FFA oxidation (5, 6), which serve to reduce
the level of FFA. However, in certain forms of liver disease, this fine
balance is disrupted, and elevated levels of hepatocellular FFA
and triacylglycerol occur.
The most common liver disease in which fatty acid metabolism is
deranged is alcoholic liver disease. Alcohol metabolism alters the
intramitochondrial redox potential via generation of NADH by alcohol
dehydrogenase. This impairs
-oxidation and tricarboxylic acid cycle
activity (7), resulting in elevated FFA, increased formation of
triacylglycerol, and increased rates of very low density lipoprotein
synthesis and secretion (8, 9). Paradoxically, the fatty liver persists
despite attenuation of the altered redox state after chronic ethanol
administration (10). Fatty liver is not necessarily benign; the
development of liver injury in a rat model is clearly dependent upon
the amount and type of fat in the diet (11, 12), and a disproportionate
elevation of liver FFA after ethanol administration may contribute to
the susceptibility of women to alcoholic liver disease (13).
Genetically obese mice and rats with hepatic steatosis are unusually
sensitive to the effects of endotoxin (14). Furthermore, a number of
other compounds, including valproic acid, amiodarone, and perhexilene, are postulated to cause liver injury by way of inhibition of
-oxidation (15, 16). Thus, several lines of evidence suggest that
liver injury may occur when fatty acid oxidation or esterification and export are inadequate.
One would predict that ethanol consumption would induce the PPAR
battery of proteins by elevating intracellular fatty acid levels.
Although alcohol consumption resulted in peroxisomal proliferation in
humans (17) and alcohol feeding of rats induced cytochrome P450 4A1
(lauryl
-hydroxylase (13)) and liver fatty acid binding protein
(18), other typical responses to peroxisome proliferators were impaired
by ethanol. The excretion of dicarboxylic fatty acids was increased in
alcohol-fed animals (13), due to increased lauryl hydroxylase activity
but failure of induction of acyl-CoA oxidase (19). Medium chain
acyl-CoA dehydrogenase activity, the gene for which has a PPRE in its
proximal promoter (20), was reported to be decreased by ethanol feeding
(21). Thus, chronic ethanol feeding apparently does not activate a full
PPAR
response. One group has reported that PPAR
mRNA was
decreased in the livers of rats chronically fed alcohol via gastric
lavage and that several PPAR-inducible enzymes were not increased in these animals (22). It is noteworthy that fatty liver and
steatohepatitis, hallmarks of alcoholic liver injury, are also observed
in both PPAR
(23) and acyl-CoA oxidase (24) knockout mice. We
therefore examined the effect of ethanol on the function of PPAR
in
transfected cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Most chemicals were purchased from Sigma. Trypsin
and tissue culture media were purchased from Life Technologies, Inc.
Fetal bovine serum charcoal-stripped of lipids was purchased from
Hyclone Laboratories (Logan, UT). All radioisotopes were purchased from PerkinElmer Life Sciences. PPRE3-tk-luciferase (containing
three copies of the PPRE from the acyl-CoA oxidase gene ligated to a herpes simplex thymidine kinase promoter upstream of the luciferase gene (25)) and the expression plasmids for murine PPAR
, -
, and
-
were the kind gifts of Dr. Ronald Evans (Salk Institute) (26).
pALDH3'-BLCAT was used as an hepatocyte nuclear factor 4 (HNF-4)-responsive reporter and was described previously (27). Expression plasmids for apolipoprotein regulatory protein 1 (ARP-1) and
chicken ovalbumin upstream promoter transcription factor (COUP-TF) were
from Dr. H. Nakshatri (Indiana University), and that for HNF-4 was from
Dr. Frances Sladek (University of California, Irvine).
Transfection of Tissue Culture Cells--
All cells were grown
in modified Eagle's medium supplemented with 10% fetal bovine serum,
100 µg/ml streptomycin, and 63 µg/ml penicillin G. The day before
transfection, the cells were plated at 106 cells/100-mm
dish. For studies on PPAR
and -
, the cells were transfected with
10 µg of reporter plasmid (PPRE3-tk-luciferase), 20 µg
of receptor expression plasmid, and 5 µg of pSV2CAT (as an internal control for transfection efficiency) by calcium phosphate precipitation (28). For studies on HNF-4, the reporter contained four copies of an HNF-4 response element from the aldehyde
dehydrogenase 2 promoter cloned in pBL2CAT (28) and the internal
control was SV40-luciferase. For studies on ARP-1 and COUP-TF, the
reporter was SV40-luciferase (which is activated by these two orphan
receptors (29)), and because of problems with effects of these
receptors on other promoters, the activity was expressed per µg of
cell extract protein. Four hours later, the cells were exposed to
phosphate-buffered saline containing 15% glycerol for 3 min. The cells
were rinsed twice with phosphate-buffered saline, and fresh modified
Eagle's medium with 10% charcoal-stripped fetal bovine serum was
added. Twenty-four to forty-eight hours after transfection, cells were washed twice with phosphate-buffered saline and lysed in 150 µl of a
buffer containing 25 mM Tris, pH 7.8, 2 mM
EDTA, 20 mM dithiothreitol, 10% glycerol, and 1% Triton
X-100. Fifty microliters of cell extract was incubated with luciferase
assay reagent based on the original protocol of deWet (30).
Chloramphenicol acetyltransferase activity was measured as described
previously (31). The conversion of chloramphenicol to its acetylated
products was quantified on an AMBIS
-scanner.
Primary hepatocyte suspensions were isolated from male Harlan
Sprague-Dawley rats (Harlan Laboratory Animals for Research, Indianapolis, IN) as described previously (32, 33). Briefly, rats were
anesthetized with pentobarbital (50-100 mg/kg body weight), their
portal veins were cannulated with a 16-gauge catheter, and the livers
were perfused with Ca2+-, Mg2+-free Hanks' A
solution, followed by Hanks' B solution containing Ca2+,
Mg2+, and 0.05% collagenase (Roche Molecular
Biochemicals). Livers were then excised, minced, and passed through
nylon mesh filters, and the resultant hepatocytes were suspended in
culture medium. Viability of the hepatocytes exceeded 90% by trypan
blue exclusion. These cells were cultured in Dulbecco's modified
Eagle's medium with 2.4 g/liter of sodium bicarbonate, 10 mM glucose, 1 µM each dexamethasone and
thyroxine, 1 nM insulin, and 10% fetal bovine serum. They
were transfected 4 h after plating by calcium phosphate precipitation according to the method of Ginot (34). Twenty-four hours
later, the cells were treated with 100 µM WY14,643 for an additional 24 h before they were harvested for assay of
reporter enzymes as described above.
Isolation of Nuclear Protein Extracts--
Nuclear proteins were
isolated from cultured cells based on a micropreparation method (35).
The nuclear extract was suspended in 20 mM HEPES (pH 7.9),
420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol,
and 0.2 mM phenylmethylsulfonyl fluoride, and aliquots were
frozen in liquid nitrogen and stored at
70 °C.
In Vitro Synthesis of Receptor Proteins--
PPARs and RXR
were synthesized using a rabbit reticulocyte lysate system (Promega
in vitro transcription/translation kit). The production of
protein of the expected molecular weight was monitored by labeling with
[35S]methionine and autoradiography. Aliquots of the
translation reaction were used without further purification.
DNA Binding Assays--
Electrophoretic mobility shift assays
(EMSAs) were performed by radiolabeling double-stranded
oligonucleotides corresponding to the peroxisome proliferator response
element of the acyl-CoA oxidase gene. Nuclear extracts (3-5 µg) or
reticulocyte lysates (1-2 µl) were incubated with 1-2 µg of
nonspecific competitor DNA (poly(dIC)) in binding buffer containing 10 mM Hepes, pH 7.9, 60 mM KCl, 1 mM
EDTA, and 7% (v/v) glycerol on ice for 15 min. Where indicated,
specific competitor oligonucleotides were added before the addition of
labeled probe and incubated for 15 min on ice. For supershift assays,
antibodies were added, and the mixture was incubated an additional 1-2
h. Labeled probe (20,000 cpm) was added last, and the reaction was
incubated an additional 15 min on ice. Reaction mixtures were
electrophoresed on a nondenaturing 4% acrylamide gel and subjected to
autoradiography. Anti-PPAR
antibody was from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA), and anti-RXR was donated by Drs.
C. Rochette-Egly and P. Chambon.
Western Blotting--
Nuclear extracts (20 µg of protein) were
fractionated in an 8% SDS-PAGE gel and electroblotted to
nitrocellulose filters. PPAR
was visualized using antiserum from
Santa Cruz Biotechnology. Detection of the protein bands was performed
using the Amersham ECL kit.
 |
RESULTS |
Effects of Ethanol on Transcriptional Activation by PPAR
in
Cells with and without the Enzymatic Capacity for Ethanol
Oxidation--
The activity of a PPAR
-responsive reporter gene
(PPRE3-tk-luciferase, containing three copies of the PPRE
from the acyl-CoA oxidase gene) was used as an index of PPAR
function in CV-1 and H4IIEC3 hepatoma cells with or without
co-transfected PPAR
(Table I). These
cells contain low amounts of PPAR
protein on Western blots (Fig.
2B, lane 2), but both cell lines
contained immunoreactive retinoid X receptor (RXR; Ref. 36), the
required dimerization partner for PPAR
(25). An important difference
between CV-1 and H4IIEC3 cells was the presence of enzymes capable of
oxidizing ethanol in the latter cells (37, 38). Responses of the
reporter were relatively small (no more than 2-fold induction) in the
absence of co-transfected PPAR
. Clofibrate markedly induced the
reporter activity in CV-1 cells transfected with PPAR
. In the
hepatoma cells, the reporter activity was much less dependent on the
presence of clofibrate (36). Ethanol at a physiologically relevant
concentration of 20 mM inhibited clofibrate-independent and
-stimulated activity of PPRE3-tk-luciferase by PPAR
by
over 50% in the hepatoma cells (Table I). Ethanol had no effect on
basal or clofibrate-stimulated PPAR
action in the CV-1 cells, either
in the presence or absence of transfected PPAR
.
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Table I
Effect of ethanol on PPAR -induced reporter activity in CV-1 and
H4IIEC3 cells
The cells were transfected using the calcium phosphate procedure with
the reporter, PPRE3-tk-luciferase (26), the chloramphenicol
acetyltransferase expression vector pSV2CAT as an internal
control of transfection efficiency, and, where indicated by columns
labeled +PPAR , the PPAR expression plasmid (26). The cells were
exposed to 20 mM ethanol and/or 1 mM clofibrate
(dissolved in Me2SO as vehicle) from 24 to 48 h after
transfection. Control cells were treated with vehicle alone. The
concentration of ethanol in the medium was maintained by culturing the
cells in an incubator containing a reservoir of 20 mM
ethanol in water. Forty-eight hours after transfection, the cells were
harvested for luciferase and chloramphenical acetyltransferase assays.
In each experiment, duplicate plates were transfected, and the reporter
activities were averaged. The results are shown as percentage of
luciferase reporter activity in control cells (transfected with the
reporter but not the PPAR expression plasmid) after correction for
chloramphenical acetyltransferase activity of the internal control
vector (means ± S.D. for three or four replications of each
condition).
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The effect of ethanol on the ability of the more potent and specific
PPAR
agonist WY14,643 was also tested. In duplicate experiments with
H4IIEC3 cells transfected with the PPAR
expression plasmid and
reporter, WY14,643 at 100 µM increased reporter activity by 591 ± 49%. Ethanol (20 mM) reduced the basal
activity of the reporter to 45 ± 4% and decreased the
WY14,643-stimulated activity to 216 ± 134% of the control level
(means ± S.E.). We also tested the effect of WY14,643 on primary
hepatocyte cultures that were transfected with the PPAR reporter
plasmid to see if ethanol also inhibited the activity of the endogenous
rat PPAR
. WY14,643 stimulated reporter activity by 433 ± 107. Ethanol (20 mM) reduced basal activity to 57 ± 3%
and WY14,643-stimulated activity to 128 ± 32% of control levels
(means ± S.E. for four replicate experiments). Thus, ethanol
reduced the activity of the reporter by about 50% in both the basal
and WY-14,643-stimulated cells, similar to the magnitude of the effect
on clofibrate-stimulated activity. Further, the effect was also seen in
primary cultures of hepatocytes, indicating that the rat PPAR
is
also sensitive to ethanol.
To determine if this effect of ethanol was restricted to PPAR
,
additional transfection assays were performed using PPAR
, HNF-4,
ARP-1, or COUP-TF. These receptors are structurally related to PPAR
,
and each recognizes DR-1 promoter elements. Compared with hepatoma
cells transfected with PPAR
alone (n = 5, means ± S.E.), clofibrate increased activity of
PPRE3-tk-luciferase by 189 ± 20%, while ethanol
reduced activity to 60 ± 3% and reduced the
clofibrate-stimulated activity to 154 ± 21%. These small
differences were statistically significant. Transfection of the H4IIEC3
cells with an HNF-4 expression plasmid stimulated its reporter plasmid expression (pALDH3'-BLCAT containing four copies of an HNF-4 response element from the aldehyde dehydrogenase promoter) by 956 ± 159% in the absence and 1034 ± 109% in the presence of ethanol
(n = 3, not significant). ARP-1 stimulated its reporter
plasmid expression (SV40-luciferase (29)) by 8222 ± 1776% in the
absence and 12,203 ± 6875% in the presence of ethanol
(n = 4, not significant). COUP-TF stimulated its
reporter plasmid expression (SV40-luciferase) by 10,408 ± 1092%
in the absence and 9312 ± 3432% in the presence of ethanol (not
significant, n = 4). The large errors observed in the
transfections with ARP-1 and COUP-TF were related to the use of
cellular protein for normalizing the data rather than an internal
control plasmid (29). Thus, the effect of ethanol was relatively
specific for PPAR
, although the small effect on the
isoform was
studied further with in vitro translated receptor (see below).
Effects of Inhibitors of Ethanol Metabolism and Acetaldehyde on
PPAR
Function--
The alcohol dehydrogenase inhibitor
4-methylpyrazole and the aldehyde dehydrogenase inhibitor cyanamide
were then used to determine if the effect of ethanol on PPAR
was
dependent on its metabolism (Table II).
Neither compound affected PPRE3-tk-luciferase activity in
the hepatoma cells in control experiments. However, 4-methylpyrazole
completely prevented the effect of ethanol on PPAR
function, while
cyanamide augmented the effect. This suggested that acetaldehyde
generated from ethanol was responsible for the inhibition of PPAR
action, and indeed, low levels of exogenous acetaldehyde (50-150
µM) inhibited PPRE3-tk-luciferase reporter activity, both in H4IIEC3 cells and CV-1 cells (Table
III). There was no visible evidence of
toxicity to the cells of these doses of acetaldehyde. Because many
biological effects of acetaldehyde have been attributed to modification
of proteins (39, 40), we examined the effect of pyridoxal phosphate on
ethanol inhibition of PPAR
induction of the reporter. Pyridoxal
phosphate has been reported to protect proteins from formation of
Schiff bases with acetaldehyde by reversibly blocking lysyl residues
(41), even in whole cell models (42), and thus is useful in
understanding mechanisms of acetaldehyde effects. Treatment of the
cells with 10 mM pyridoxal phosphate completely prevented
the effect of ethanol (control activity = 970 ± 10, ethanol-treated cells = 454 ± 43, ethanol plus pyridoxal
phosphate = 830 ± 49; activity represents percentage
increase over the untransfected control cells as described for Table I;
means ± S.E. for three replications), indicating that
acetaldehyde-protein adduct formation may explain the inhibitory effect
of ethanol metabolism.
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Table II
Effect of inhibitors of alcohol and aldehyde dehydrogenase on the
effect of ethanol on PPAR -induced reporter activity in H4IIEC3 cells
The cells were transfected as described in Table 1 with the PPAR
expression plasmid, internal control plasmid, and the
PPRE3-tk-luciferase reporter; the inhibitors were added
24 h later, and the cells were exposed to ethanol (20 mM) where indicated, beginning at the same time. The cells
were harvested for assay of the reporter enzymes at 48 h; data are
reported as in Table 1. The data are normalized to control cells
analyzed in each replication that were not transfected with the PPAR
expression plasmid.
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Table III
Effect of acetaldehyde on PPAR -induced reporter activity in H4IIEC3
and CV-1 cells
The cells were transfected with the PPAR expression plasmid,
internal control, and the PPRE3-tk-luciferase reporter as
in Table I; acetaldehyde was added to the medium 24 hours later and the
culture dishes were sealed to reduce evaporative losses. In the
hepatoma cells, 4-methylpyrazole and cyanamide were present at 0.1 mM
to slow the metabolism of the added acetaldehyde. The cells were
harvested for assay of the reporter enzymes at 48 hours. Data are
reported as in Table I.
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Effect of Ethanol and Acetaldehyde on DNA Binding Ability of
PPAR
--
To understand how ethanol metabolism impaired PPAR
function, it was of interest to study the effect of ethanol on the
ability of nuclear factors to bind the PPRE in EMSA. In preliminary
experiments, ethanol was found to have no effect on the level of
endogenous RXR
in the hepatoma cells. Nuclear extracts from
untransfected H4IIEC3 cells contained proteins that retarded the
mobility of the PPRE oligonucleotide (Fig.
1). The major and minor bands appeared to
be specific, in that they were competed with unlabeled oligonucleotide (lanes 3-5). These bands were not PPAR
, since
the hepatoma cells contain very low levels of PPAR
(Fig.
2B, lane
2), and the bands could not be shifted with antibody to
PPAR
(not shown). The bands probably represent other nuclear factors
present in H4IIEC3 cells that can bind to DR-1 elements, such as HNF-4.
When the cells were exposed to ethanol, alone or in the presence of
inhibitors of its metabolism, there was no change in the intensity of
the bands.

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Fig. 1.
Effect of exposure of H4IIEC3 hepatoma cells
to ethanol on binding of endogenous nuclear factors to bind DNA.
EMSAs were performed using nuclear extracts from hepatoma cells exposed
to ethanol and inhibitors of ethanol or acetaldehyde metabolism. The
probe was a double-stranded oligonucleotide containing a copy of the
acyl-CoA oxidase PPAR response element (26). Nuclear extract was
incubated with 20,000 cpm of the probe and then analyzed by
electrophoresis through a 4% PAGE gel and autoradiography.
Lane 1 represents the probe alone; all other
lanes contained the nuclear extract. Cold
Competitor indicates the addition of unlabeled
oligonucleotide at the noted molar excess. In lanes
6-13, the cells had been pretreated with the indicated
compounds for 24 h before the cells were harvested for nuclear
extraction. 4-Methylpyrazole and cyanamide were added at 0.1 mM, and ethanol was added at 20 mM.
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Fig. 2.
Effect of exposure of H4IIEC3 hepatoma cells
to ethanol on the ability of PPAR to bind
DNA. A, EMSAs were performed using nuclear extracts
from hepatoma cells transfected with a PPAR expression plasmid as
described the legend to Fig. 1. Lane 1 represents
the labeled oligonucleotide in the absence of added nuclear proteins,
and lane 2 represents extract from cells that had
not been transfected. The Cold Competitor
lanes indicate the molar excess of unlabeled oligonucleotide
added to the binding reaction. Where noted, ethanol had been present in
the medium at 20 mM, cyanamide at 0.1 mM, and
4-methylpyrazole at 0.1 mM for 24 h before harvesting
the cells. B, Western blots of the nuclear extracts used in
A were performed using anti-PPAR to confirm the presence
of similar amounts of PPAR in each lane.
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We then analyzed nuclear extracts from H4IIEC3 cells that were
transfected with the PPAR
expression plasmid (Fig. 2, A
and B). The intensity of the major band was markedly
increased in the transfected cells, which contained large amounts of
PPAR
seen by Western blotting (Fig. 2, A and
B, lane 3), and there was a prominent
shift induced with anti-PPAR
. Binding was again competed with
unlabeled competitor oligonucleotides (lanes
4-6). The major band could also be shifted with antibody to
RXR (not shown). The more slowly migrating band was also more intense
in the transfected cells. The identity of this band is uncertain; however, it might represent nuclear receptors bound to other factors such as NRBF-1 (43) or PBP165 (44). These factors interact with a
number of nuclear receptors including PPAR
and HNF-4.
The effect of 24 h of treatment with ethanol on PPRE binding
activity in extracts of hepatoma cells transfected with the PPAR
expression plasmid was then evaluated (Figs. 2 and
3). The Western blots for PPAR
protein
demonstrated that similar amounts of the receptor were present in all
of the nuclear extracts and that ethanol metabolism did not impair
synthesis or nuclear localization of the receptors (Fig.
2B). Compared with control transfected cells
(lane 7), ethanol treatment reduced the ability
of PPAR
to bind the oligonucleotide (lane 8);
binding was further reduced if cyanamide had been present during the
ethanol treatment (lane 9). The band was not
completely eliminated by ethanol, as expected from the existence of
PPRE-binding proteins in the untransfected cells (Fig. 1). DNA binding
activity of the PPAR
from cells treated with ethanol plus
4-methylpyrazole was normal (lane 10);
4-methylpyrazole or cyanamide treatment in the absence of ethanol had
no effect on the ability of the PPAR
to bind DNA (lanes
11 and 12).

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Fig. 3.
Effect of acetaldehyde on the ability of
PPAR to bind DNA. A, nuclear
extracts were prepared as in Fig. 1 from hepatoma cells transfected
with PPAR that had been incubated with the noted concentrations of
acetaldehyde for 24 h prior to harvest. The culture medium also
contained 0.1 mM 4-methylpyrazole and 0.1 mM
cyanamide to retard the reductive or oxidative metabolism of added
acetaldehyde. B, Western blots of the nuclear extracts for
PPAR
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Exposure of the cells to progressively increasing concentrations of
acetaldehyde (in the range of those that can be achieved in the liver
during ethanol metabolism (45)) reduced the ability of extracted
PPAR
to bind DNA (Fig. 3A). This treatment of the cells
did not reduce the amount of immunoreactive PPAR
present in the
nuclear extracts (Fig. 3B). Acetaldehyde also reduced the intensity of the more slowly moving band. Inclusion of pyridoxal phosphate in the culture medium prevented the inhibitory effects of
ethanol and acetaldehyde on DNA binding activity (not shown).
To further document that the nuclear factor binding that was reduced by
ethanol and acetaldehyde was PPAR
, antibody against this factor was
used to supershift the binding complex (Fig.
4). This autoradiogram was exposed for a
shorter time than Figs. 1 and 2 to allow better resolution of the
shifted bands, and the binding activity in the untransfected cells is
therefore less prominent. The major band of binding activity was
shifted with anti-PPAR
antibody (lane 4). The
addition of ethanol in the presence of cyanamide markedly reduced the
intensity of the nonshifted and the shifted bands (lanes
7 and 8). Similarly, acetaldehyde at 50 or 150 µM reduced the intensity of both the major and the antibody-shifted band (lanes 9-12). Thus, the
ethanol, by way of acetaldehyde, dramatically reduces the ability of
PPAR
to bind to DNA.

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Fig. 4.
Effect of ethanol and acetaldehyde on
PPAR binding to DNA. H4IIEC3 cells were
treated with ethanol (20 mM), ethanol plus cyanamide (0.1 mM), or acetaldehyde (50 or 150 µM in the
presence of cyanamide plus 4-methylpyrazole) for 24 h before
preparation of nuclear extracts, and EMSAs were performed as described
for Figs. 1-3. In lanes 4, 6,
8, 10, and 12, antibody to PPAR was
added to the binding reaction prior to the addition of radiolabeled DNA
and incubated for 1-2 h prior to electrophoresis. With this shorter
autoradiographic exposure, the major band is resolved to two bands,
both of which are shifted by anti-PPAR antibody.
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Effect of Acetaldehyde on DNA Binding Ability of in Vitro
Translated PPARs--
Because there is evidence that acetaldehyde can
alter protein function via covalent modification, the effect of
acetaldehyde on in vitro synthesized PPAR
and RXR
was
examined (Fig. 5A). The
receptors were incubated on ice (lanes 2-5), at
37 °C (lanes 6-10), or at 37 °C in the presence of 1 mM acetaldehyde (lanes 11-17; the
receptor incubated with acetaldehyde is indicated by the
dot) and then either tested individually or after mixing
PPAR
with RXR
for DNA binding ability. In the lanes
indicated by RXR/PPAR, the two receptors were mixed to form
heterodimers before the incubation. Under the binding conditions used
here, only the mixture of RXR and PPAR bound DNA. The incubation of
PPAR or RXR at 37° C reduced their ability to form a DNA-binding
complex (compare lanes 5 and 9). When the RXR and
PPAR were mixed before the incubation at 37° C, the complex was
stable (lane 10). Incubation of RXR with acetaldehyde before
mixing reduced the intensity of the binding complex somewhat
(lane 14). However, treatment of PPAR with
acetaldehyde abolished the band, as did treatment of the preformed
heterodimer (lane 17).

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Fig. 5.
Effect of pre-incubation of in
vitro synthesized PPAR and RXR
proteins with acetaldehyde on the ability of
PPAR ·RXR heterodimers to bind DNA.
A, expression plasmids for the receptors were used to
prepare proteins using the Promega TNT in vitro
transcription/translation kit. The proteins were either incubated on
ice, incubated at 37 °C, or treated with 1 mM
acetaldehyde at 37 °C for 1 h. The RXR and PPAR proteins were
incubated individually or after mixing to form heterodimers (indicated
by RXR/PPAR). The receptors were then mixed to form dimers
and analyzed by EMSA as described for Fig. 1. Lane
1 represents the labeled probe in the absence of added
receptors. Lanes 2-4 show that neither the
in vitro translation mixture programmed with a luciferase
plasmid (( )IVT) nor RXR or PPAR alone bound the DNA probe,
while the mixture of RXR and PPAR bound. The 37 °C control
lanes show that incubation at this temperature reduced the
ability of the individually incubated receptors to form a DNA binding
complex but that the preformed heterodimer withstood the incubation
well. The receptor or mixture that was treated with acetaldehyde is
indicated by the solid dots. When RXR was treated
with acetaldehyde before mixing, there was still formation of a faint
binding complex. When the PPAR receptor or the preformed complex was
treated with acetaldehyde, DNA binding was abolished. B,
this experiment was performed as described for A, but with
PPAR and . RXR/ indicates the preformed
RXR·PPAR heterodimer, and RXR/ indicates the
preformed RXR·PPAR heterodimer. Treatment of the individual PPARs
or the preformed dimers with acetaldehyde substantially reduced DNA
binding.
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Because there was a modest reduction in the ability of PPAR
to
activate the reporter in transfection studies, we also studied the
effect of acetaldehyde on the ability of the PPAR
and -
isoforms
to bind DNA (Fig. 5B). This experiment was carried out as
described for PPAR
(Fig. 5A). The preformed heterodimers
(lanes 10 and 11) appeared to be
somewhat more stable than the receptor subunits incubated alone
(lanes 8 and 9), as was seen with
PPAR
. Treatment of the RXR with acetaldehyde did not dramatically
reduce its ability to form a DNA-binding complex with either PPAR
or -
(lanes 14 and 15). As with
PPAR
, both PPAR
and -
were sensitive to preincubation with
acetaldehyde (lanes 16 and 17). The
preformed heterodimers appeared to be somewhat more resistant to the
effect of acetaldehyde than the corresponding PPAR
·RXR
complex (lanes 19 and 20).
 |
DISCUSSION |
These studies reveal potentially important interactions between
ethanol metabolism and the function of PPAR
. The presence of ethanol
reduced the ability of transfected PPAR
to activate a reporter
construct in the H4IIEC3 hepatoma cell line. This effect was mediated
by acetaldehyde, since inhibition of ethanol oxidation by
4-methylpyrazole blocked the effect completely, while the aldehyde dehydrogenase inhibitor cyanamide enhanced the effect of ethanol. Furthermore, low concentrations of acetaldehyde added directly to the
medium inhibited PPAR
activity in both the hepatoma and CV-1 cells.
Although ethanol modestly reduced the ability of PPAR
to activate
the reporter plasmid, this effect of ethanol was not observed with
several other members of the nuclear receptor family (HNF-4, ARP-1, or
COUP-TF), further suggesting that it is not a nonspecific, toxic effect
of ethanol.
The effect of ethanol and acetaldehyde on expression of the PPAR
reporter gene was correlated with the ability of PPAR
·RXR extracted from the cells to bind to its response element. The H4IIEC3
cells contain factors that can bind the PPRE oligonucleotide used for
the EMSA. These factors have not been identified, but they could
include HNF-4, COUP-TF, ARP-1, or RXR, all of which are known to bind
DR-1 sequences. The major band observed is likely to correspond to the
dimeric form of these transcription factors, since each of these
factors is of similar molecular weight, and the mobility was close to
that of in vitro translated PPAR
·RXR. A minor band was
also observed whose identity is unknown but could represent DR-1
binding factors complexed with other nuclear proteins. The intensity of
these bands was not affected by treatment of the cells with ethanol,
again arguing against the effects of ethanol being nonspecific. Nuclear
extracts from H4IIEC3 cells transfected with a PPAR
expression
plasmid had more prominent shifted bands at both positions, and
treatment with ethanol, ethanol plus cyanamide, or acetaldehyde reduced
the intensity of both bands. That the reduced intensity of the major
band represented decreased the ability of PPAR
·RXR to bind the
PPRE was further demonstrated by the use of antibody to PPAR
. The
intensity of the shifted bands was decreased by ethanol, ethanol plus
cyanamide, and acetaldehyde. The reduction in intensity of the more
slowly moving complex cannot be fully interpreted at present.
An attractive explanation for the observed effects of ethanol was the
formation of acetaldehyde adducts with PPAR
. Acetaldehyde is known
to react with lysyl side chains in a number of proteins (40, 46-48)
and has been implicated in the dysfunction of liver microtubules in
alcohol-fed animals (49). This hypothesis is consistent with the
ability of pyridoxal phosphate pretreatment of the cells to prevent the
effects of ethanol and acetaldehyde on both PPAR
activation of the
reporter gene and on the ability of the receptor to bind DNA. It was
possible to show that exposure of in vitro synthesized
PPAR
to acetaldehyde for only 1 h interfered with DNA binding
by PPAR
·RXR. Although the concentration of acetaldehyde used in
the in vitro experiments was higher than can be achieved in vivo, exposure to this concentration had much less effect
on the ability of RXR
to form DNA-binding heterodimers with PPAR
. This suggests that PPAR
is unusually sensitive to acetaldehyde. The
DNA binding domain of PPAR
contains a number of conserved lysyl
residues that are predicted to be directly involved in DNA-protein interactions (50). The ethylation of these residues by acetaldehyde might be expected to dramatically alter the electrostatic interactions of these side chains with DNA. Other possible explanations for impaired
DNA binding could be the inability of acetaldehyde-treated PPAR
to
dimerize with RXR and impairment of the activation functions of the
receptor. However, additional studies are needed to determine if the
intracellular and in vitro effects of acetaldehyde on
PPAR
function involve similar mechanisms. It will also be important to examine the ability of other biologically occurring aldehydes (e.g. aldehydic products of lipid peroxidation or glucose)
to affect PPAR
function. We also observed sensitivity of PPAR
and -
to acetaldehyde in vitro, although the kinetics of
inactivation were not formally studied to allow quantitative
comparisons of the PPAR isoforms.
Earlier work has shown that exposure to ethanol increases the level of
fatty acids in hepatocytes (8, 9). The results of the present work show
that ethanol also can impair the function of PPAR
. The failure of
induction of PPAR
-controlled genes such as those for peroxisomal
-oxidation and medium chain acyl-CoA dehydrogenase could thus
contribute to the development of alcoholic fatty liver. This effect of
ethanol may be responsible for the persistence of fatty liver despite a
return of the redox state toward normal during chronic ethanol
administration (10). Inhibition of PPAR
function may also contribute
to more serious alcoholic hepatic injury. Indeed, this suggests that
pharmacologic or nutritional maneuvers that activate the PPAR
system
may ameliorate the hepatotoxicity of ethanol. However, ethanol feeding
does not uniformly inhibit expression of genes known to contain PPREs
(13, 18). This presumably results from the presence of multiple factors
controlling most promoters. In addition, the consensus PPRE is a direct
repeat with one spacer nucleotide (DR-1 element) that can also be bound by such factors as retinoic acid receptors, HNF-4, COUP-TF, and ARP-1
(51). We speculate that ethanol inhibition of DNA binding by PPAR
might permit retinoic acid receptors or HNF-4 to bind and activate
certain genes, explaining, for instance, the apparent induction of
apoAI and apoAII by ethanol (52). Conversely, binding of COUP-TF and
ARP-1 might actively repress certain genes. Such interactions among the
steroid receptor family of transcription factors could increase the
spectrum of biological actions of ethanol.
The actions of ethanol on PPAR
also deserve additional study. We
have shown that PPAR
may play an important role in the control of
proliferation of hepatic stellate cells (53). The expression of PPAR
decreases as the cells proliferate after being plated on plastic
substrate, and activated PPAR
antagonizes the actions of
platelet-derived growth factor, a major contributor to proliferation of
stellate cells. We hypothesize that the high intrahepatic levels of
acetaldehyde occurring during prolonged alcohol consumption inhibit
PPAR
and render the stellate cells more susceptible to activation.
This could contribute to the pathogenesis of alcoholic cirrhosis as
well as the increased risk of hepatic fibrosis in patients with
hepatitis C who drink heavily. Further, PPAR
is extremely important
for the differentiation of preadipocytes to adipocytes, in the control
of sensitivity to the actions of insulin, and in the pathogenesis of
atherosclerosis (54, 55). Although the multiple roles of this PPAR
isoform are still incompletely understood, inhibition of PPAR
function by heavy ethanol consumption might contribute to insulin
resistance, syndrome X, and accelerated cardiovascular disease. This
possibility deserves further study.