From the Departments of Medicine and Biochemistry and Molecular Biology, Indiana University School of Medicine and Richard Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202
Received for publication, February 28, 2003 , and in revised form, April 24, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Peroxisome proliferator-activated receptor (PPAR
), a member
of the nuclear hormone receptor superfamily and a receptor for FFA
(7), has been identified as a
key transcriptional regulator of many genes involved in FFA oxidation systems
in liver (8). Studies on
PPAR
null mice have shown that the function of the PPAR
battery
is essential for constitutive mitochondrial fatty acid catabolism
(9,
10). Fasted PPAR
null
mice suffer from a severe impairment in hepatic FFA oxidation, resulting in a
phenotype characterized by hypoglycemia, hypothermia, hypoketonemia, elevated
plasma levels of FFAs, and fatty liver
(11). On the other hand,
constitutive peroxisomal
-oxidation is quite independent of PPAR
,
but the receptor is required for induction of this system by peroxisome
proliferators (9). Recent
observations
(1114)
point out the critical importance of PPAR
in determining the severity
of hepatic steatosis in fasting, in diabetes
(14), and in animals fed a
high fat diet (11).
Because PPAR coordinates fatty acid metabolism in the liver, and
PPAR
knock-out mice develop fatty liver when fasted
(1214),
it is obvious to ask whether PPAR
is involved in the pathogenesis of
alcoholic fatty liver. Fatty acid levels are increased in the liver during the
metabolism of ethanol; therefore, the PPAR
battery of proteins should
be induced by alcohol consumption. Although a subset of the PPAR
responsive genes were reported to be induced by ethanol, such as cytochrome
P450 4A1 (lauryl
-hydroxylase)
(15) and liver fatty
acid-binding protein (16),
many others did not change or even decreased
(17). Increased generation of
dicarboxylic fatty acids because of enhanced lauryl
-hydroxylase
activity and the failure of ethanol to induce acyl-CoA oxidase
(1820),
the first step in peroxisomal
-oxidation, led to augmented excretion of
dicarboxylic fatty acids in the urine in alcohol-fed rats
(15) and in alcoholic men
(21). Medium chain acyl-CoA
dehydrogenase activity (22)
and mRNA level (20) were
decreased by ethanol feeding.
These reports suggest that the PPAR battery of fat-metabolizing
enzymes is not fully induced during ethanol feeding and suggest the
possibility that activation of this system would ameliorate some of the toxic
effects of ethanol. Indeed, a recent study
(23) showed that fenofibrate,
a PPAR
ligand, ameliorated the fatty liver and hepatomegaly of ethanol
feeding in three of five rats and decreased serum triacylglycerol level in
alcoholic patients despite the fact that they continued drinking during the
fenofibrate administration. Similarly, an early study indicated that feeding
rats clofibrate ameliorated the fatty liver and hepatomegaly resulting from
ethanol feeding (24).
Recent data from our laboratory
(25) showed that ethanol and
its metabolite, acetaldehyde, inhibited the transcriptional and DNA binding
activity of the PPAR receptor, inhibited PPAR
activation of a
reporter plasmid in hepatoma cells, and reduced the ability of Wy14,643, a
potent PPAR
agonist, to activate the reporter in hepatoma cells or
cultured rat hepatocytes. This effect of ethanol was abolished by the alcohol
dehydrogenase inhibitor 4-methylpyrazole and augmented by the aldehyde
dehydrogenase inhibitor cyanamide, indicating that acetaldehyde was likely
responsible for the action of ethanol. Furthermore, in vitro
translated PPAR
exposed to acetaldehyde failed to bind DNA
(26). Because some
PPAR
-regulated genes mentioned above are induced with ethanol
treatment, it is uncertain to what extent the effect of ethanol on the
expression of a simple reporter construct in a hepatoma cell can be
extrapolated to the expression of hepatic genes under the control of complex
promoters or what the potential contribution of PPAR
dysfunction to the
multiple effects of ethanol on the liver might be. Therefore, in the present
paper we study the effect of chronic ethanol feeding on PPAR
and
RXR
levels, PPAR/RXR binding activity, levels of PPAR-regulated genes,
measures of fat metabolism in mice fed alcohol chronically, and the ability of
the PPAR
agonist WY14,643 to reverse the effects of ethanol.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Assays from BloodAfter animals were anesthetized, blood was
collected by heart puncture. Plasma was separated by centrifugation at 4
°C and stored at 20 °C. Plasma triacylglycerol and glucose were
determined using kits from Roche Diagnostics. -Hydroxybutyrate
(
-OHB) was measured using a KetoSite kit (GDS Diagnostics, Elkhart, IN).
FFA was determined with a NEFA C-Test (Wako Chemicals, Richmond, VA), and
cholesterol was determined with Infinity cholesterol reagent (Sigma).
Tissue Lipid DeterminationFrozen sections of the liver (10 µm) were stained with Oil Red O and counterstained with hematoxylin and eosin for histology. Samples of liver were homogenized in 0.25% sucrose containing 1 mM EDTA. Lipids were extracted using chloroform/methanol (2:1, v/v) and evaporated in a Speedvac, and the pellets were dissolved in 5% fatty acid-free bovine serum albumin dissolved in water. Protein in the homogenate was assayed using protein assay reagent (Bio-Rad) to normalize the amount of lipid extracted. Colorimetric cholesterol and triacylglycerol assays were carried out as described above.
Fatty Acid -Oxidation ActivityFatty acid
-oxidation activity was measured according to Aoyama et al.
(9). Briefly, fresh livers were
homogenized and incubated with the assay medium containing 50 µM
[1-14C]palmitic acid (American Radiolabeled Chemicals, St. Louis,
MO) for 30 min. The reaction was stopped by adding 0.2 M perchloric
acid. The mixture was centrifuged at 2000 x g for 10 min, and
the unmetabolized fatty acids were removed by three extractions using 2 ml of
n-hexane. Radioactive degradation products in the water phase were
counted. Fatty acid
-oxidation activity was expressed as pmol
[14C]palmitate oxidized/mg protein/min.
Preparation and Analysis of Nuclear ExtractsFresh livers
were homogenized in 10 mM Hepes (pH 7.9), 25 mM KCl, 10
mM NaF, 0.15 mM spermine, 1 mM EDTA, 10%
glycerol, 2 M sucrose, 2% protease inhibitor (Roche Diagnostics),
and 0.5 mM dithiothreitol, and nuclear proteins were extracted as
described by Neish et al.
(28) except that 1
mM NaF and 2% protease inhibitor were added to all buffers. Western
blotting was performed by fractionating 60 µg of nuclear extract protein on
10% SDS-PAGE gels. After electrotransfer onto nitrocellulose membranes, blots
were blocked with Tris-buffered saline containing 0.1% Tween 20 and 5% dry
milk for 1 h at room temperature and incubated overnight at 4 °C with the
following polyclonal rabbit antibodies: anti-PPAR and anti-PPAR
(Geneka Biotechnology, Montreal, Quebec, Canada) and anti-RXR
(Santa
Cruz Biotechnology, Inc., Santa Cruz, CA). Immune complexes were detected
using the ECL Plus kit (Amersham Biosciences). Immunoreactive bands were
quantified by Fluorimager (Amersham Biosciences). DNA binding assays were
performed using the Gelshift kit according to the manufacturer's instructions
(Geneka Biotechnology, Montreal, Quebec, Canada). The following probes were
used: PPAR response element (PPRE),
5'-GAACTAGGTCAAAGGTCATCCCCT-3'; and mutant PPRE,
5'-GGAACTAGAACAAAGAACATCCCCT-3'. For supershift assays,
anti-PPAR
antibody was added (Geneka Biotechnology). Reaction mixtures
containing 5-µg nuclear extracts were separated by electrophoresis
(constant 210 V) on a 5% nondenaturing PAGE in 1x Tris/glycine
electrophoresis buffer at 4 °C. The shifted bands were quantified using a
PhosphorImager and ImageQuant (Amersham Biosciences) software analysis.
RNA Isolation and Relative Quantitative RT-PCRTotal RNA was
prepared from frozen liver using an RNeasyTM Total RNA kit (Qiagen).
Quantitative RT-PCR was performed using reverse transcription and
co-amplification of 18 S ribosomal RNA as internal control (QuantumRNA 18 S
internal standard kit; Ambion, Austin, TX) according to the manufacturer's
instructions. 5 µg of RNA was mixed with 2 µl of random primers (50
µM; Ambion) and RNase-free water up to 13.5 µl of volume,
denatured at 80 °C for 3 min, and rapidly cooled on ice for 5 min. 1 µl
of dNTP mix (10 mM), 0.5 µl of RNasin ribonuclease inhibitor
(Promega), 4 µl of avian myeloblastosis virus reverse transcriptase
5x buffer (Promega), and 1 µl of avian myeloblastosis virus RT enzyme
(Promega) were added to each sample. The mixtures were incubated for 1 h at 42
°C. PCR was performed in duplicate using 2 µl of the RT product in a
reaction mixture containing 5 µl of 10x PCR buffer (Promega), 2
mM MgCl2, 1 µl of dNTPs (10 mM), 2 µl
of gene-specific primer pair (5 µM each), 4 µl of 18 S
Classic Primer and Competimer pair (Ambion), 0.25 µl of Taq DNA
polymerase, 0.5 µl of [-32P]dATP (10 µCi/µl), and
nuclease-free water up to 50 µl. The optimal ratio of 18 S Classic
Primer:Competimer was determined for each target gene individually. PCR was
performed using the following general cycle profile: 94 °C for 5 min (94
°C for 30 s, 5660 °C for 30 s, 72 °C for 30 s) x
cycle number, followed by a 72 °C for 7-min extension. PCR primer pairs
were designed from mouse- and rat-specific sequence data (GenBankTM)
except for LCAD (29). PCR
cycle numbers were determined for each primer pair by extensive pretesting to
optimize the conditions and that the product is with in the linear range of
the PCR amplification (see Table
I). Annealing temperature was 60 °C except for the LCAD primer
pair (56 °C). Optimum MgCl2 concentration was 2 mM.
40 µl of the PCR products were separated on a 6% polyacrylamide gel and
quantified with a PhosphorImager and ImageQuant software (Amersham
Biosciences). Data were expressed as ratio of the signals of the mRNA of
interest to that of the internal control.
|
Data AnalysisAll data are expressed as mean ± S.D. for the experiments, which included between four and eight animals in each group. The paired t test was used to evaluate statistical differences between ethanol- and pair-fed mice. A p value < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histological analysis of the livers with Oil Red O staining revealed prominent lipid accumulation in the livers of ethanol-fed animals (Fig. 1B) whereas lipid droplets were rare in the livers of the control group (Fig. 1A). Liver sections of pair-fed mice given Wy14,643 treatment looked similar to those of controls (Fig. 1C). When the ethanol-fed animals were co-treated with Wy14,643 for the last 2 weeks of the experiment, the Oil Red O staining showed no sign of hepatic steatosis (Fig. 1D).
|
Quantification of liver lipids was concordant with the histologic findings
(Fig. 2A). Ethanol
feeding increased the liver triacylglycerol content by 4-fold. Wy14,643
treatment alone did not alter the amount of triacylglycerol in the liver. In
striking contrast, when Wy14,643 was co-administered for the last 2 weeks of
the ethanol feeding, the triacylglycerol content was significantly decreased
to the level of control animals. Cholesterol content was induced by 2-fold in
the livers of ethanol-fed animals (Fig.
2B), possibly a result of activation of SREBP-1
(31). Wy14,643 itself
increased liver cholesterol content by about 2-fold. Gemfibrozil, another
PPAR agonist, was reported recently
(32) to increase the
transcriptional activity of SREBP-2, a major regulator of cholesterol
biosynthesis, and strongly induced 3-hydroxy-3-methylglutaryl-CoA reductase
mRNA and activity. This effect of the PPAR
activator may have resulted
in enhanced cholesterol production in the liver. Animals fed ethanol plus
Wy14,643 had lower cholesterol in liver than those W14,643.
|
Effects of Chronic Ethanol Administration and Wy14,643 on Plasma Lipids
and Hepatic Fatty Acid -OxidationWe measured plasma
lipids to better understand the mechanisms by which ethanol and WY14,643 might
influence hepatic steatosis. Because the liquid diets were withdrawn
5 h
before the blood samples were taken, the plasma FFA most likely reflects the
balance between peripheral lipolysis and hepatic fat oxidation, as the liver
is the major utilizer of circulating FFAs in the early postabsorptive phase.
Chronic ethanol feeding significantly increased the serum levels of FFA,
triacylglycerol, and
-OHB as shown in
Table II. Wy14,643 entirely
prevented the elevation of FFA and triacylglycerol caused by ethanol, and its
administration was accompanied by an even higher level of
-OHB than
ethanol alone. Chronic ethanol feeding had no effect on serum cholesterol
level, whereas Wy14,643 treatment alone and in combination with ethanol
feeding increased the level of cholesterol.
|
Total hepatic fatty acid -oxidation capacity was assessed by using
14C-labeled palmitic acid (Fig.
3). This substrate was chosen, because studies on PPAR
null
mice showed defective mitochondrial catabolism of long chain fatty acids
(9). The rate of fatty acid
oxidation was similar in control and ethanol-fed mice. Wy14,643 caused
significant induction in palmitic acid oxidation compared with the control and
ethanol-fed groups. These results are consistent with the hypothesis that
despite the elevation of plasma and presumably hepatic FFA in the ethanol-fed
animals, there was no induction of fatty acid oxidizing capacity. As a result,
some FFA are converted to ketone bodies, but the remainder are esterified to
triacylglycerol and either stored as fat droplets or secreted as
triacylglycerol-rich lipoproteins. WY14,643 was able to induce fatty acid
oxidizing capacity in the ethanol-fed animals sufficiently to dispose of them
as ketone bodies or complete oxidation and therefore normalize plasma FFA and
triacylglycerol levels and prevent fatty liver. This suggested that ethanol
feeding subverted the normal homeostatic role of PPAR
. Therefore the
expression and functional capacity of PPAR
were studied.
|
Effect of Ethanol Feeding on Expression of PPAR and Its
Heterodimerization Partner, RXR
, in Liver and DNA Binding
Ability of PPAR
/RXR
HeterodimersThe level of PPAR
mRNA
(Table III) and protein were
not altered by ethanol (Fig.
4A). Wy14,643 increased PPAR
mRNA expression by
5-fold (Table III) and
significantly increased the amount of PPAR
protein
(Fig. 4, A and
B). However, RXR
mRNA content decreased by 20%
(Table III), and the level of
immunoreactive RXR
protein was reduced by 60% in alcohol-fed mice,
which could not be prevented by Wy14,643
(Fig. 4, A and
B). Protein content of PPAR
, a receptor
structurally related to PPAR
that also recognizes DR-1 promoter
elements, was unchanged by ethanol feeding. We attempted to quantify the
levels of RXR
and RXR
but were unable to detect these proteins
with available antibodies.
|
|
Previously, we found that the DNA binding ability of PPAR/RXR
was impaired by ethanol in cultured cells
(26). To investigate whether
this in vitro observation could be extrapolated to in vivo
conditions, we studied the ability of nuclear extracts from liver to bind a
PPAR response element (Fig.
5A). Ethanol feeding reduced the ability of
PPAR
/RXR
heterodimer to bind a PPRE by 40% compared with nuclear
extracts from control animals. Wy14,643 treatment enhanced this binding
activity by 2-fold. When ethanol-fed mice were given Wy14,643, the relative
binding ability of PPAR
/RXR
was 3-fold higher than in
ethanol-fed animals, which restored it to higher levels than the controls
(Fig. 5A), although
still significantly less than observed in mice treated with Wy14,643 alone.
The interpretation of this experiment was complicated by the large number of
transcription factors that can bind DR-1 elements; therefore, supershift
assays were performed. The amount of supershifted complexes was decreased in
ethanol-fed mice. Wy14,643 co-administration increased the abundance of the
supershifted complex by 2.5-fold in animals fed ethanol compared with controls
(Fig. 5B). Our in
vitro data suggested that the effect of ethanol on PPAR
was not a
nonspecific, toxic effect of alcohol, because several other members of the
nuclear receptor family were not affected by ethanol
(26). To see whether other
transcription factors were affected in vivo we checked the DNA
binding ability of HIF1
(hypoxia inducible
factor-1) in the liver and found it to be unaltered by
ethanol feeding (data shown).
|
Effects of Ethanol Feeding on Expression of PPAR Target
GenesTo determine whether the decreased DNA binding ability of
PPAR
/RXR
heterodimer resulted in altered gene expression,
hepatic mRNA levels of several PPAR
targets were analyzed by RT-PCR
(Table III). mRNAs for
mitochondrial LCAD and medium chain acyl-CoA dehydrogenase (MCAD) were
decreased by 30 and 40%, respectively, in the ethanol-fed group. Levels of
mRNAs encoding acyl-CoA oxidase (AOX), liver carnitine palmitoyl-CoA
transferase (LCPT), very long chain acyl-CoA synthetase (VLACS), and very long
chain acyl-CoA dehydrogenase (VLCAD) were unchanged between ethanol-fed and
control animals. Liver fatty acid-binding protein (LFABP) was the only target
gene that was induced by ethanol feeding, consistent with earlier reports
(16). Fourteen days of
Wy14,643 treatment increased the mRNA level of the PPAR
target genes
1.57-fold except for LCPT and VLCAD. In ethanol-fed animals, Wy14,643
effectively induced mRNAs of LCAD, MCAD, AOX, VLACS, and LFABP to levels
significantly higher than that in ethanol-treated mice.
Although acetyl-CoA carboxylase (ACC) is not usually considered a
PPAR-regulated gene, we also analyzed its mRNA level. ACC not only
catalyzes the pace-setting step of fatty acid synthesis
(34) but also plays a crucial
role in the control of mitochondrial fatty acid
-oxidation through its
product, malonyl-CoA (35).
Ethanol feeding induced the level of ACC mRNA by 2.5-fold, and Wy14,643
prevented this induction (Table
III).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There have been clues to this effect of ethanol in the fatty liver
literature. Although a major effect of ethanol metabolism is an increased
NADH/NAD+ ratio in the liver as demonstrated by numerous short term
studies, this perturbation appears to abate with chronic ethanol consumption,
as estimated by the lactate/pyruvate ratio in hepatic vein samples from
ethanol-fed baboons (36).
Because fatty liver persisted in these animals, alternative mechanisms were
needed to explain this. One such mechanism is the increase in lipid synthesis
driven by activation of SREBP-1 that was reported recently
(31). Previous data have
suggested that ethanol may impair the ability of the liver to respond to an
increased level of FFA (15,
21), and we have summarized
effects of ethanol on PPAR-regulated genes
(17). Furthermore, we have
presented evidence for ethanol-inhibition of PPAR
action in
vitro (26). It was
crucial, therefore, to carry out experiments to directly test this
hypothesis.
We have utilized mice in these experiments, as they present the opportunity for using genetically modified animals in the future to further dissect the abnormal response of the animals to ethanol. Ethanol was accepted in the diet without short term health effects, and the ethanol-fed animals developed histological and biochemical fatty liver (see Figs. 1 and 2). Thus, they provide a model for the early effects of chronic nol consumption.
We found that chronic alcohol feeding inhibited DNA binding activity of
PPAR/RXR heterodimer in liver nuclear extracts, consistent with in
vitro findings (26). We
saw only a modest effect of ethanol on the mRNA for these factors (a 20%
decrease in the level of mRNA for RXR
, similar to that reported in rats
studied using the Tsukamoto-French model
(37), and no effect on
PPAR
mRNA), but there was a substantial decrease in RXR
protein
level. It has been reported that endotoxin can cause such a decrease in mRNA
and protein levels of RXR
(38). Because ethanol feeding
is reported to increase endotoxin in mice
(39) and in rats
(40), this may explain the
decrease. Although mRNAs for RXR
or RXR
were not altered by
alcohol feeding (19), it will
be necessary to determine whether the protein levels were affected, because
protein and mRNA levels were not correlated for RXR
.
It is unlikely that the reduction in the ability of PPAR/RXR
to bind DNA in the liver extracts was solely because of the reduction in
RXR
, because treatment of the animals with WY14,643 restored the
binding ability without inducing RXR
. Work utilizing a ligand-induced
complex formation assay has shown that PPAR
ligands induce formation of
PPAR
/RXR
complexes
(25). Because RXR
was
decreased with chronic ethanol feeding, we suggest that one of the effects of
Wy14,643 in vivo was to increase the ability of PPAR
to
compete for limited amounts of RXR
. However, it is not clear why this
increased formation of dimer would be observed in the nuclear extracts of the
WY14,643-treated animals. Unless there was very tight binding of the drug to
the receptor heterodimer that survived extraction, the finding suggests that
the presence of WY14,643 prevented the effect of ethanol on the receptor,
which we hypothesize is a post-translational modification. Several
possibilities can be suggested: binding of WY14,643 or formation of the
heterodimer might alter the conformation of the receptor and alter its
susceptibility to modification. We suggested that ethylation of the receptor
at lysyl residues near the zinc finger domain could be a mechanism for the
effect of acetaldehyde (26);
conceivably activation of the receptor by Wy14,643 increased the fraction that
was DNA-bound and inaccessible to acetaldehyde.
Presumably, the endogenous PPAR ligands, FFAs, do not bind with
sufficient affinity to prevent the effect of ethanol. Forman et al.
(25) showed in the
ligand-induced complex formation assay that 30 µM palmitate or
linoleic acid induced complex formation only 3040%, as well as 5
µM WY14,643
(25). Thus, weaker ligands
like FFA may not be able to prevent the actions of ethanol on the receptor,
whereas high affinity ligands like WY14,643 retain that ability. This will
require additional study of receptor purified from cells exposed to ethanol or
acetaldehyde, in the presence or absence of WY14, 643.
To assess the functional implications of the reduced DNA binding of the
PPAR/RXR complex, we measured the levels of a number of mRNAs known to be
regulated by this transcription factor. PPAR/RXR
affects genes
in two ways; some mRNAs, such as LCAD and VLCAD, are strongly dependent on
PPAR
for constitutive expression, as judged by the reduction in mRNA in
PPAR
/ animals
(9). mRNAs of LCAD was
decreased in the ethanol-fed animals as well
(Table III). There are also a
number of genes for which PPAR
/RXR
is not required for basal
expression, but rather for induction by peroxisome proliferators, and
presumably by fatty acids. These include AOX, LCPT, LFABP, VLACS, and MCAD,
which were not decreased in PPAR
null mice but also not inducible by
peroxisome proliferators when analyzed by immunoblotting
(9). Although one would predict
that they would be induced by the elevated FFA levels in the alcohol-fed
livers, with the exception of fatty acid-binding protein, they were not.
Moreover, mRNA of MCAD was decreased. This disparate behavior of these mRNAs
presumably reflects the influence of ethanol on other control regions of their
promoters or on mRNA stability. Treatment of the ethanol-fed mice with
Wy14,643 resulted in induction of mRNAs of many PPAR
target genes
(Table III), indicating that
the increased ability of PPAR/RXR to bind DNA in gel shift experiments was
paralleled by induction of a number of PPAR-regulated genes.
This failure of full induction of critical mRNAs for FFA metabolism during
ethanol feeding was functionally reflected in the plasma lipid profile and the
rate of palmitate oxidation in liver homogenates. In the ethanol-fed animals,
the plasma triacylglycerols and FFA were increased, consistent with much
previous work
(4143).
It is interesting to note that ketone bodies (represented by -OHB) were
increased nearly 3-fold in the ethanol-fed animals. Given that rodents tend to
consume most of their diet early in the dark cycle, it is likely that they had
metabolized the ethanol taken in the diet many hours earlier. Therefore, the
increased level of
-OHB may represent acceleration of ketogenesis that
follows relief of the redox pressure of ethanol metabolism, similar to the
pathogenesis of alcoholic ketoacidosis
(44). This capacity for fatty
acid oxidation and ketogenesis was not, however, sufficient to dispose of
excess fat in the liver, possibly because the maximum rate of oxidation, as
assessed from the oxidation of palmitate in liver homogenates, was not
increased. Induction of the rate-limiting enzymes for fat oxidation is
apparently needed to handle the FFA load seen with ethanol feeding, as shown
by the effect of Wy14,643. This compound induced mRNAs for fatty acid
oxidation by mitochondrial and peroxisomal systems, increased the maximum rate
of palmitate oxidation, lowered plasma triglyceride and FFA levels, and
increased ketone body levels to over four times the control level. These
effects were observed even in the presence of ethanol in the diet, and thus
appeared to overcome the block in fat metabolism caused by ethanol. Thus, it
appears that fatty liver and the other disturbances of lipid metabolism seen
in the ethanol-fed mice resulted, in part, from an incomplete activation of
the PPAR
homeostatic system.
An additional interesting finding was that ethanol feeding induced ACC
mRNA, and Wy14,643 reduced this to normal. Recent data
(31) suggest that ethanol
feeding activates SREBP-1, a major regulator for ACC. Although ACC is not
known to have a PPRE in its promoter, there is some evidence that the enzyme
can be negatively regulated by PPAR agonists in short and long term
(45). The induction of ACC by
ethanol would not only increase the rate of fatty acid synthesis and
contribute to the accumulation of fatty acids and triacylglycerol in the liver
but would also increase the level of malonyl-CoA in the liver. Malonyl-CoA is
the major allosteric regulator of LCPT, and limiting the access of acyl-CoA
esters to the mitochondrial matrix space would be an additional mechanism by
which ethanol could inhibit FFA oxidation in liver.
These data suggest that treatment of individuals with alcoholic fatty liver
with activators of PPAR may reduce the degree of fat accumulation. The
fact that animals improved with WY14,643 treatment while still receiving
ethanol in the diet indicates that this effect is not simply prevention of
fatty liver. Thus, such therapy might be effective even in patients unable to
completely abstain. Because some of the hepatotoxicity of ethanol may be
because of the presence of fatty infiltration (e.g. sensitivity to
endotoxin, susceptibility to lipid peroxidation, or alterations in blood flow
to the centrilobular zone with resultant hypoxia), PPAR
agonist
treatment might even have a benefit in preventing or ameliorating more serious
forms of liver injury such as alcoholic hepatitis or fibrosis, but this will
clearly require additional study. Further, this study suggests mechanisms for
increased genetic risk for alcoholic liver injury. Several polymorphisms of
the human PPAR
gene have been described recently
(4648).
Of these, the L162V variant showed decreased non-ligand-dependent
transactivation activity and decreased sensitivity to low concentrations of
ligand in tissue culture experiments
(46). In human studies, it was
reported to be associated with elevated plasma low density lipoprotein
concentration and increased risk for atherosclerosis and ischemic heart
disease (33,
47,
48). Our study suggests that
carriers of this allele might be more susceptible to the toxic hepatic effects
of ethanol.
![]() |
FOOTNOTES |
---|
To whom correspondence should be addressed: Indiana University School of
Medicine, 545 Barnhill Dr., Emerson Hall 317, Indianapolis, IN 46202-5124.
Tel.: 317-274-8438; Fax: 317-274-1437; E-mail:
dcrabb{at}iupui.edu.
1 The abbreviations used are: FFA, free fatty acid; ACC, acetyl-CoA
carboxylase; AOX, acyl-CoA oxidase; LCAD, long chain acyl-CoA dehydrogenase;
LFABP, liver fatty acid-binding protein; LCPT, liver carnitine palmitoyl-CoA
transferase; MCAD, medium chain acyl-CoA dehydrogenase; PPAR, peroxisome
proliferator-activated receptor; RXR, retinoid X receptor; VLACS, very long
chain acyl-CoA synthetase; VLCAD, very long chain acyl-CoA dehydrogenase;
Wy14,643, pirinixic acid
([4-chloro-6-(2,3-xylindino)-2-pyrimidinyl-thiol]acetic acid); -OHB,
-hydroxybutyrate; RT, reverse transcription; PPRE, PPAR response
element; SREBP, sterol regulatory element-binding protein.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|