From the Department of Pathology, Northwestern University Medical
School, Chicago, Illinois 60611-3008
 |
INTRODUCTION |
Peroxisomes are single membrane-bound organelles present in most
eukaryotic cells. They participate in a variety of metabolic processes
such as lipid metabolism, H2O2-based
respiration, production of bile acids and plasmalogens (membrane
phospholipids), synthesis of cholesterol, and catabolism of purines,
polyamines, and amino acids (1-3). Peroxisomes in liver parenchymal
cells can be stimulated to proliferate by the administration of
nonmutagenic chemicals designated as peroxisome proliferators (4).
These include certain phthalate ester plasticizers, industrial
solvents, herbicides, leukotriene D4 antagonists, the
adrenal steroid dehydroepiandrosterone, as well as amphipathic
carboxylates such as the hypolipidemic drugs, clofibrate, and
ciprofibrate (5). Peroxisome proliferation induced by these
structurally diverse agents is associated with transcriptional
activation of genes encoding for the peroxisomal
-oxidation system
(6, 7) and the cytochrome P450 CYP4A isoforms (8), among others (9,
10). The induction of peroxisome proliferation is mediated by
peroxisome proliferator-activated receptors
(PPARs)1 that form a complex
with the common heterodimeric partner, retinoid X receptor (RXR) (11,
12). The PPAR·RXR complex binds to peroxisome proliferator-responsive
element (PPRE), a region consisting of a degenerate direct repeat of
the canonical AGGTCA sequence separated by 1 base pair (DR1), present
in the 5'-flanking region of target genes (9, 10, 12). Three isotypes
of PPARs, namely PPAR
, PPAR
(also known as PPAR
, NUC-1), and
PPAR
, have been identified as products of separate genes from
Xenopus, rodents and humans (11, 13-16). These isotypes
exhibit distinct patterns of tissue distribution and differ
considerably in their ligand binding domains, suggesting that they
possibly perform different functions in different cell types (15-24).
Indeed, of the three isotypes, PPAR
expression is relatively high in
hepatocytes, enterocytes, and the proximal convoluted tubular
epithelium of kidney (17, 18), and evidence derived from mice with
PPAR
gene disruption indicates that this receptor is essential for
the pleiotropic responses induced by peroxisome proliferators (25).
Sustained induction of PPAR
-mediated peroxisome proliferation leads
to the development of liver tumors in rats and mice exposed chronically
to peroxisome proliferators despite their nonmutagenic nature (5, 26).
It has been postulated that H2O2 overproduced by the sustained transcriptional activation of peroxisomal
-oxidation system and concomitant cell proliferation contribute to
hepatocarcinogenesis in livers with peroxisome proliferation (5,
26-29). The peroxisomal
-oxidation system consists of three
enzymes, namely H2O2-generating fatty acyl-CoA
oxidase (AOX), enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase
multifunctional protein (MFP), and 3-ketoacyl-CoA thiolase (THL) (3, 6,
30). Although peroxisomes and mitochondria catalyze similar reactions,
the long chain and very long chain fatty acids (VLCFAs) are oxidized
predominantly, if not exclusively, by the peroxisomal
-oxidation
system (3, 30-32). To investigate the role of peroxisomal
-oxidation in hepatocarcinogenesis and to assess the long range
implications of disturbing VLCFA metabolism, we generated an animal
model of AOX deficiency by inactivating the gene encoding the AOX (33,
34), the first enzyme of the peroxisomal
-oxidation system, which
converts acyl-CoA to enoyl-CoA (30). Analysis of young
AOX-deficient mice revealed extensive microvesicular fatty
metamorphosis of liver parenchymal cells and inflammatory reaction
(34). Hepatic steatosis begins to dissipate gradually, resulting in a
liver consisting entirely of regenerative hepatocytes that display
massive spontaneous peroxisome proliferation indicative of PPAR
activation. Liver tumors develop in AOX-deficient mice by 15 months of
age. Our findings implicate AOX as a key regulator of PPAR
function
and acyl-CoAs (other possible natural substrates of AOX) as potent
biological ligands responsible for the transcriptional activation of
PPAR
in vivo.
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EXPERIMENTAL PROCEDURES |
Animals--
We utilized the fatty acyl-CoA oxidase null
(AOX
/
) mice as described (34). Heterozygous (AOX±) siblings were
mated to obtain AOX
/
mice because of reduced fertility of
homozygous males and females (34). Genotypes of mice were determined by Southern blot analysis using DNA isolated from tail tip (34). Age-matched wild-type (AOX +/+) siblings served as controls as needed.
Animal care and experiments were carried out in accordance with both
institutional and federal animal care regulations.
Histology and Immunohistology--
For routine histology,
tissues were fixed in 10% phosphate-buffered formalin (pH 7.4)
dehydrated in 100% ethanol and embedded in paraffin at 58 °C using
standard procedures. Sections (4 µm thick) were cut and stained with
hematoxylin and eosin. For immunohistochemical localization of AOX,
MFP, THL, catalase, and proliferating cell nuclear antigen (PCNA),
tissues fixed in 10% formalin or 70% ethanol were processed using
monospecific polyclonal antibodies. Immunostaining was performed by an
avidin-biotinylated peroxidase complex (ABC kit, Vector Laboratories)
or by the peroxidase-anti-peroxidase method. Negative controls
consisted of staining with normal rabbit serum instead of specific
antibodies or by omitting the primary antibodies. The slides were
counterstained with either hematoxylin or methyl green.
Electron Microscopy and Immunocytochemistry--
For routine
transmission electron microscopy, samples of liver were fixed with 2%
paraformaldehyde and 2.5% glutaraldehyde in 0.05 M
cacodylate buffer (pH 7.2, 4 °C) for 4 h. They were washed
overnight in cacodylate buffer, post-fixed in 1% osmium tetroxide in
cacodylate buffer (pH 7.4) for 1 h at 4 °C, and embedded in
Epon. For cytochemical localization of catalase, tissue samples were
fixed in 1.5% glutaraldehyde in 0.1 M sodium cacodylate
buffer (pH 7.4) for 4 h at 4 °C, washed overnight with 0.1%
cacodylate buffer (pH 7.4), and cut into 60-µm sections. The sections
were stained for catalase with alkaline 3,3'-diaminobenzidine substrate and post-fixed with 1% osmium tetroxide in 0.1 M
cacodylate buffer (pH 7.4). Semithin sections with or without toluidine
blue counterstain were examined by light microscopy. Ultrathin sections
for electron microscopy were contrasted with uranyl acetate and lead
citrate. For immunogold localization of AOX, MFP, THL, and urate
oxidase, tissues were fixed for 24 h by immersion in 4%
paraformaldehyde, 0.1% glutaraldehyde in 0.1 M sodium
phosphate buffer (pH 7.4) at 4 °C. The sections were rinsed for
3 h in 0.1 M sodium phosphate, pH 7.4, 0.15 M NaCl, 0.1 M lysine, dehydrated in graded
series of cold ethanol, and embedded in Lowicryl K4M at -20 °C.
Ultrathin sections were stained with each antibody by the protein
A-gold technique. The polyclonal antibodies used in these studies were raised in rabbits against rat catalase, rat AOX, rat MFP, rat THL, and
rat urate oxidase (35).
Northern Blots--
Total cellular RNA was isolated from fresh
liver or liver frozen at -80 °C using the acid guanidinium
thiocyanate-phenol-chloroform extraction method. RNA was glyoxylated,
separated on 0.8% agarose gel, and transferred to a nylon membrane.
cDNA probes used for Northern blotting included AOX, catalase MFP,
THL, 70-kDa peroxisomal membrane protein (PMP70), urate oxidase (UOX),
CYP4A1, CYP4A3, liver FABP, fatty acid synthase (FAS), fatty acyl-CoA
synthetase (ACS), and ribosomal RNA (28 S). Changes in mRNA levels
were estimated by densitometric scanning of autoradiograms.
H2O2 Measurement--
Samples of liver
were homogenized (1% w/v) in lysis buffer (0.2 M Tris-HCl,
pH 8.0, 0.1 M EDTA, 2% SDS containing 20 mM
NaN3 to inhibit endogenous catalase), and
H2O2 was measured using the phenol red method
(36).
 |
RESULTS |
Histological Changes in Liver of AOX
/
Mice--
Information on
the generation of AOX
/
mice has been presented previously (34).
Homozygous AOX
/
mice lacked the expression of AOX protein,
accumulated VLCFAs in blood, and exhibited growth retardation
during the first six months of age when compared with wild-type
(AOX+/+) littermates (34). Both male and female AOX
/
mice at four
months are infertile, possibly due to retarded growth and development
(34). For this study, homozygous (AOX
/
) animals were obtained by
mating heterozygous (AOX±) mice. At 2 months of age, AOX
/
mice
exhibited severe and diffuse fatty metamorphosis of liver, reflecting
acute fatty acid hepatotoxicity in younger animals. Scattered
hepatocyte death and numerous foci of steatohepatitis are encountered
during the first 4 months of age (Fig.
1A). During the first 2 to 4 months of age, the inflammatory response consisted mostly of
polymorphonuclear neutrophils, lipogranulomas, and clusters of foamy
macrophages. Death of single hepatocytes (apoptosis) and inflammatory
response served as stimuli for progressive liver cell regeneration,
resulting in the appearance of multiple clusters of hepatocytes with
hypertrophic granular cytoplasm. These regenerated hepatocytes are
resistant to fatty change and contained no lipid vacuoles. Few cells
with fatty change are sometimes present at the periphery of these
newly formed regenerating hepatocyte foci. As the regeneration
extended, eosinophilic hepatocyte foci coalesced to form confluent
areas of liver regeneration. Thus, between 6 and 8 months of age,
almost all steatotic hepatocytes in AOX
/
mice were replaced by
hepatocytes devoid of steatosis (Fig. 1B). Few areas of
inflammatory cell collections and large lipid-laden macrophages, some
of which are multinucleated, are seen as remnants in a liver that was
once steatotic (Fig. 1B, arrows).

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Fig. 1.
Hepatocellular alterations in liver of
AOX / mice. The liver of a 3-month-old (A) and an
8-month-old (B) AOX / mouse stained with
hematoxylin-eosin. Fatty metamorphosis of hepatocytes and aggregates of
inflammatory cells (arrows) are common in young
AOX-deficient mice (A). In older AOX / mice, liver is
composed predominantly of cells with abundant eosinophilic granular
cytoplasm with few large foamy lipid-laden macrophages
(arrows) and residual inflammation (B).
C-F, light microscopic appearance of liver as revealed in
semithin sections of tissues that were processed for the cytochemical
localization of peroxisomal catalase using the alkaline
3,3'-diaminobenzidine substrate. These sections are intended to
demonstrate the magnitude of spontaneous peroxisome proliferation in
the liver parenchymal cells of older AOX / mice. C, liver
of a 3-month-old AOX / mouse similar to that in (A) shows
a single cell (arrow) with numerous peroxisomes (brown
granules) and other hepatocytes display abundant lipid droplets
and few or no detectable peroxisomes. D and E,
liver of an 8-month-old (D) and 12-month-old (E)
AOX / mouse reveals a profound increase in the number of peroxisomes
(brown granules) in all hepatocytes. A macrophage
(D, *) with lipid debri similar to that shown in panel
B (arrows) has no peroxisome. F,
wild-type (AOX+/+) mouse liver shows few peroxisomes
(arrows). Erythrocytes in sinusoids (C, E, and
F) show intense reaction due to the peroxidatic activity of
hemoglobin.
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|
Spontaneous Hepatic Peroxisome
Proliferation--
Peroxisomes can be identified at the light
microscopic level in cytochemically stained sections by the alkaline
3,3-diaminobenzidine procedure for the peroxisomal marker enzyme
catalase (1). In hepatocytes of AOX+/+ mice, peroxisomes are few,
randomly distributed in the cytoplasm, and appeared as diaminobenzidine
positive, dark brown granules (Fig. 1F). Evaluation of 2-to
4-month-old AOX
/
mice revealed a conspicuous absence or paucity of
recognizable diaminobenzidine-positive organelles in diffusely
steatotic hepatocytes (Fig. 1C). Since steatosis is
extensive in young AOX
/
mice, recognizable peroxisomes are found
only in a rare hepatocyte. An occasional hepatocyte with no fatty
change displayed an abundance of peroxisomes (Fig. 1C). As
hepatocellular regeneration ensued, the number of hepatocytes lacking
steatosis increased, and such cells exhibited conspicuous peroxisome
proliferation. As steatosis dissipated in animals six months and older,
repopulation of liver occurred with hepatocytes containing eosinophilic
cytoplasm (Fig. 1B). These cells displayed massive
spontaneous peroxisome proliferation (Fig. 1, D and
E). Ultrastructural analysis of the liver of AOX
/
mice
confirmed the increase in the number of peroxisomes in hepatic parenchymal cells (Fig. 2). The magnitude
of peroxisome proliferation encountered in these AOX-deficient mice is
comparable with that induced in wild-type mice by exogenous peroxisome
proliferators such as hypolipidemic drugs Wy-14, 643, clofibrate, and
ciprofibrate (4, 5). Many peroxisomes in hepatocytes of AOX
/
mice
revealed the presence of prominent UOX-containing crystalloid cores
(Fig. 2A).

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Fig. 2.
Electron micrograph of a hepatocyte from the
liver of an 8-month-old AOX / mouse (A), compared with
that of a wild-type (AOX+/+) mouse (B). Liver was
processed for the cytochemical localization of catalase using
3,3'-diaminobenzidine medium as described in Fig. 1. Note the presence
of numerous catalase positive peroxisomes (P) with urate
oxidase crystalloid inclusions in AOX / mouse hepatocytes. In
contrast, hepatocytes in wild-type mice (A)
reveal the presence of a few peroxisomes (P).
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|
Up-regulation of PPAR
Target Genes--
PPAR
plays
a crucial role in the peroxisome proliferation induced in liver by
peroxisome proliferators such as clofibrate and Wy-14,643 (25, 37).
Mice deficient in PPAR
(PPAR
/
) contain a normal complement of
peroxisomes in liver parenchymal cells, but these animals do not
exhibit the typical predictable pleiotropic responses, including the
development of liver tumors, when treated with peroxisome proliferators
(25, 37). The massive spontaneous peroxisome proliferation observed in
AOX-deficient mice in the present study is reminiscent of that
encountered in mice exposed to potent peroxisome proliferators such
as methyl clofenapate, Wy-14, 643, and ciprofibrate (5, 27).
This magnitude of spontaneous peroxisome proliferation clearly suggests
that PPAR
is transcriptionally activated in AOX
/
mice due to
increases in the levels of biological (natural) ligands (agonists) of
this transcription factor. To further affirm PPAR
activation, the structural findings are extended by analysis of the mRNA levels of
selected genes in liver that are regulated by PPAR
(for review see
Refs. 9, 10, and 38). As shown in Fig. 3,
the remaining two genes (down-stream of AOX) of the peroxisomal
-oxidation system, namely MFP and THL, are up-regulated
(~10-30-fold) in AOX
/
mice. The MFP mRNA level showed a
remarkable increase in the liver of 2-8-month-old AOX
/
mice (Fig.
3, lanes 3-6) compared with wild-type controls (Fig. 3,
lanes 1 and 2). This may be due to the presence
of four imperfect TGACCT motifs in the MFP gene promoter, constituting
a unique hyper-responsive PPAR-RXR heterodimer binding site (39).
Significant increases in hepatic CYP4A1 and CYP4A3 mRNA levels are
also discerned in AOX
/
mice (Fig. 3). These two members of the
CYP4A subfamily encode microsomal fatty acid
-hydroxylases, and an
increase in the activity of this enzyme system is a component of the
peroxisome proliferator-induced pleiotropic responses in liver (7, 8).
Peroxisomal membranes contain proteins, including PMP70 and X-linked
adrenoleukodystrophy protein, that possibly serve as carriers for
VLCFAs (40, 41). In addition ACS, which converts inactive fatty acids
into active acyl-CoA derivatives, also plays a key role in peroxisomal
fatty acid metabolism (42). In the liver of animals exposed to
peroxisome proliferators, increases in PMP70 and ACS mRNA levels
occur to reflect increases in peroxisomal membrane profiles (40, 42,
43). The mRNA levels of both ACS and PMP70 increased markedly in
the liver of AOX
/
mice, with spontaneous peroxisome proliferation
(Fig. 3). The mRNA level of FAS, which catalyzes seven reactions
involved in the conversion of acetyl-CoA and malonyl-CoA to palmitate
(44), is also increased in the liver of 2-to-8-month-old AOX
/
mice (Fig. 3). This increase is most marked in 6- and 8-month-old mice. Modest increases in liver FABP (~3-4-fold increase), UOX
(~2-4-fold), and catalase (~ 2-fold) mRNA levels occurred in
the liver of AOX
/
mice (Fig. 3). The basal levels of these three
mRNAs in wild-type mice are relatively high when compared with low
basal expression of genes encoding the
-oxidation system, CYP4A
subfamily, PMP70, ACS, and FAS (Fig. 3).

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Fig. 3.
Northern blot analysis of total RNA extracted
from the liver of wild-type (AOX+/+) and homozygous AOX / mice.
Lanes 1 and 2 represent control wild-type mice,
and lanes 3, 4, 5, and 6 represent, respectively, 2-, 4-, 6-, and 8-month-old AOX / mice.
Twenty µg of total RNA was glyoxylated, electrophoresed on a 0.8%
agarose gel, blotted onto a nylon membrane, and probed with different
random-primed 32P-labeled cDNA probes as shown. MFP,
the second gene of the peroxisomal -oxidation system; THL, the third
gene of the -oxidation system. The 28 S RNA is for loading
control.
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To determine whether alterations in mRNA levels of MFP, THL, PMP70,
and UOX represent changes in protein concentration at the peroxisome
level, localization of these proteins was investigated by
immunoelectron microscopy using the protein A-gold procedure (35). The
matrix of peroxisomes proliferating spontaneously in AOX
/
mouse
liver revealed numerous gold particles when stained with antibodies
against MFP and THL, whereas hepatic peroxisomes of normal wild-type
mice displayed only a few gold particles (data not shown). UOX and
PMP70 staining is localized exclusively to the crystalloid cores and to
the peroxisome membranes, respectively (not illustrated).
Catalase has been localized to the matrix of all peroxisomes in
hepatocytes of AOX
/
mice (not shown). Immunoblot analysis of liver
homogenates confirmed increases in the content of MFP, THL, UOX, and
PMP70 in AOX
/
mouse liver with spontaneous peroxisome proliferation
(results not shown).
Liver Tumor Development in AOX
/
Mice--
Hepatocellular
adenomas and hepatocellular carcinomas developed in AOX
/
mice
between 10 and 15 months of age (Fig. 4,
A and B). By 15 months of age all AOX
/
mice
developed hepatocellular carcinomas with solid, trabecular, acinar, or
fibrolamellar histological patterns. No metastases to lungs or other
sites were detected in AOX
/
mice killed by 15 months. Hepatic
adenomas generally preceded the appearance of hepatocellular
carcinomas. These adenomas and carcinomas showed reduced staining for
MFP, THL, and catalase when analyzed immunohistochemically (Fig.
5). The liver parenchyma surrounding the
tumors revealed a strong staining pattern for these three peroxisomal
proteins. In particular, the intensity of staining was much stronger
for MFP and THL (Fig. 5, C-F), the two enzymes of the
-oxidation system that are downstream of AOX. As expected, AOX is
absent in liver and tumors in AOX
/
mice (Fig. 5A).

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Fig. 4.
Liver tumors in AOX / mice. The gross
appearance of liver with tumors in a 14-month-old AOX / mice
(A). Histological pattern (hematoxylin-eosin-stained) of a
typical hepatocellular carcinoma in AOX / mice (B).
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Fig. 5.
Representative immunohistology of liver of a
12-month-old AOX / mouse with a hepatic adenoma (A-D)
and hepatocellular carcinoma (E). Immunoperoxidase staining for
A, AOX; B, catalase; C and
E, peroxisomal multifunctional protein; D,
3-ketoacyl-CoA thiolase. F, wild-type mouse (AOX+/+) stained
for multifunctional protein for comparison. As expected, AOX is
negative in the liver, whereas the staining for the two downstream
proteins of the -oxidation system is intense in nontumorous AOX /
mouse livers. Adenomas (AD) and hepatocellular carcinomas
(HC) show minimal or no staining in comparison to the
surrounding liver
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Increased Hepatic H2O2 Levels and
Hepatocellular Regeneration--
To determine whether pronounced
inflammatory response and increases in the expression of CYP4A1,
CYP4A3, and possibly other enzymes in the liver of AOX
/
mice
generates increased production of reactive oxygen species, the levels
of H2O2 in liver homogenates were measured
using the phenol red method (36). In 2-month-old AOX
/
, the level of
hepatic H2O2 was significantly higher than that
present in age-matched wild-type (AOX+/+) mice (Fig.
6A). Hepatic
H2O2 level was also high in 4-month-old
AOX
/
mice. In 6- to13-month-old AOX
/
, the levels of
H2O2 were elevated when compared with 2- and
13-month-old controls but not as high as in 2- to 4-month-old
AOX-deficient mice (Fig. 6A). Because of the inflammatory
response and isolated hepatocyte death in younger AOX
/
mice, it
appeared necessary to evaluate hepatocellular proliferation in 2- to
13-month-old mice using immunohistochemical staining to localize PCNA.
As shown in Fig. 6B, the most active cell proliferation
occurred in the liver of AOX
/
mice between 2 to 4 months of age
when compared with age-matched wild-type controls. For example, the
PCNA labeling index at 4 months in AOX
/
was 5.56 as compared with
0.35 in wild-type controls. In 8- and 13-month-old AOX
/
mice, the
PCNA labeling index in nontumorous areas of liver continued to be
higher than that observed in controls. These observations are
consistent with the proposed role of inflammatory changes,
H2O2 generation, and cell proliferation in
carcinogenesis (45). As expected, PCNA labeling was much higher in
neoplastic nodules and hepatocellular carcinomas developing in
13-month-old AOX
/
mice.

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Fig. 6.
H2O2 level and cell
proliferation index in the liver of AOX / mice. A,
H2O2 concentration in liver homogenates was
determined by phenol red method. Two- to 13-month old AOX / mice
(hatched bars) and age-matched wild-type (AOX+/+) mice
(plain bars) were killed. The values are expressed as
mean ± S.E. for 3 to 4 animals for each interval. B,
liver cell proliferation index was ascertained by immunoperoxidase
staining of liver sections to localize PCNA. Proliferation index
(percent labeling) was estimated by counting 2000 cells for each
animal
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 |
DISCUSSION |
A variety of structurally diverse peroxisome proliferators induce
predictable pleiotropic responses characterized by peroxisome proliferation and liver tumor development by a receptor-mediated cell-specific mechanism (4, 5, 9, 11). Recent assays based on in
vitro binding, conformation change, or receptor-coactivator transactivation indicate that these synthetic peroxisome proliferators act as direct ligands for PPAR
(21, 22, 24). These studies have also
shown that fatty acids, i.e 18:2 (n-6), 18:3 (n-3
and n-6), and 20:4 (n-6), as well as the
eicosanoids 8(S)-hydroxyeicosatetraenoic acid and
leukotriene B4, and prostaglandin I2 analogs
carbaprostacyclin and iloprost function as PPAR
ligands (21, 22,
24). Direct activation of PPARs by fatty acids and eicosanoids is an
attractive mechanism regulating lipid homeostasis. However, these
natural ligands do not induce peroxisome proliferation-associated
pleiotropic responses in liver cells to the same extent as those
induced by synthetic ligands, such as peroxisome proliferators and
5,8,11,14-eicosatetraynoic acid, a nonmetabolizable synthetic fatty
acid (24, 27). It is possible that natural fatty acids either function
as weak ligands for PPAR
in vivo or that liver cells have
a high capacity for
-oxidation, thereby preventing the accumulation
of these ligands to levels that effectively activate PPAR
.
The results obtained from the studies of AOX mutant mice unequivocally
establish the spontaneous induction of PPAR
-mediated pleiotropic
responses, including profound peroxisome proliferation and development
of liver tumors, reminiscent of those encountered in rats and mice
exposed to synthetic peroxisome proliferators. In the liver of these
AOX
/
mice, there is clear evidence of enhanced transcriptional
activity of PPAR
on PPAR
-regulated genes. Spontaneous increases
in liver mRNA levels in AOX
/
mice of MFP, THL, CYP4A1, CYP4A3,
ACS, and PMP70 genes that have PPRE-containing promoters are comparable
with those observed in the liver of wild-type mice exposed to synthetic
peroxisome proliferators. Accordingly, the results of this study
establish a fundamental role for AOX in the biological activities of
PPAR
(Fig. 7). AOX, the first enzyme
of the peroxisomal
-oxidation system, oxidizes the long chain
acyl-CoA (>C20) to 2-trans-enoyl-CoA for
further processing in the
-oxidation spiral (Fig. 7). VLCFAs,
metabolized almost exclusively by the peroxisomal
-oxidation,
must first be activated by ACS to acyl-CoA. AOX deficiency
imposes a block on long chain acyl-CoA to enter the
-oxidation
pathway. It is conceivable that unmetabolized long chain acyl-CoA then
functions as a biological ligand of PPAR
, leading to sustained
transcriptional enhancement of genes with PPRE-containing promoters
(Fig. 7).

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Fig. 7.
Model illustrating the role of AOX
vis-à-vis acyl-CoA and other putative substrates of
AOX in the activation of PPAR in normal (A),
PPAR / (B), and AOX / (C) mice.
FA represents long chain and very long chain fatty acids
(>C20) that are preferentially metabolized by peroxisomal
-oxidation system (31). Long chain ACS converts FA to acyl-CoA,
which enters the peroxisomal -oxidation spiral. Acyl-CoA is
desaturated to a trans-enoyl-CoA by AOX, the first enzyme of
the -oxidation system (3). Enoyl-CoA is metabolized to
hydroxyacyl-CoA and ketoacyl-CoA by the second multifunctional enzyme
(MFP) of the -oxidation system. Ketoacyl-CoA is thiolytically
cleaved by thiolase (THL), the third enzyme of the of the -oxidation
system, to release acyl-CoA (2 carbon atoms shorter than the original
molecule), which can re-enter the -oxidation spiral, and acetyl-CoA,
which can be converted in to a fatty acid by fatty acid synthase (30).
In wild-type mice, PPAR is known to regulate ACS, AOX, MFP, THL, and
several other target genes that possess PPRE. Recent studies dealing
with transfection and transactivation indicate that fatty acids and
eicosanoids are ligands for PPAR (21, 22, 24), but it is well known
that fatty acids are not very efficient in inducing peroxisome
proliferation in vivo when compared with synthetic
peroxisome proliferators (27). Studies with PPAR / mice have
unequivocally established the crucial role for this receptor in
peroxisome proliferator-induced pleiotropic effects, as PPAR /
mice failed to respond to peroxisome proliferators (25, 35). The data
on the profound proliferation of peroxisomes and activation of several
PPAR -responsive genes that we presented in this paper on AOX /
mice as well as the failure of fatty acids to induce peroxisome
proliferation in liver to the same extent as peroxisome proliferators
strongly indicate that acyl-CoA and other natural substrates of AOX
serve as the biological ligands (agonists) for PPAR . Inability of
AOX / mice to metabolize acyl-CoA (most likely long chain and very
long chain fatty acyl-CoAs) and other putaive AOX ligands leads to
sustained activation of PPAR and up-regulation of genes that possess
PPRE.
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It is worth noting that long chain acyl-CoA was once considered a
metabolic message responsible for the induction of
-oxidation system
(46, 47). This raises the issue whether free fatty acids and
unmetabolized synthetic peroxisome proliferators act as direct ligands
of PPAR
in vivo or that activation of this receptor is
mediated by their CoA esters and other downstream derivatives resulting
from the
-oxidation. The sulfur-substituted fatty acid derivatives
and peroxisome proliferators of the fibrate class are activated to
their esters with CoA. Although these cannot enter the
-oxidation
spiral, they can still function efficiently as peroxisome proliferators
in vivo, implying that
-oxidation is not essential to
generate the PPAR
agonists (47, 48). In X-linked
adrenoleukodystrophy (X-ALD), a peroxisomal disorder with impaired
VLCFA metabolism, there is progressive VLCFA accumulation, resulting in
neurological abnormalities and death during childhood (32, 41). In
X-ALD patients, there is no report of spontaneous peroxisome
proliferation in liver parenchymal cells despite VLCFA accumulation
(32, 41). This lack of peroxisome proliferation in X-ALD patients may
not be attributable entirely to differences in the sensitivity of human
PPAR
(49), because there is no indication of the occurrence of
spontaneous peroxisome proliferation in mouse models for this disease,
developed recently by inactivating X-ALD gene (50, 51). In addition,
failure to induce peroxisome proliferation by dietary lipid overload
(27, 52) and by increased VLCFA levels in X-ALD (32, 41, 50, 51)
implies that, under in vivo conditions, fatty acids are not
effective inducers of PPAR
. In contrast, the remarkable induction of
spontaneous peroxisome proliferative response in AOX null mice raises
the possibility that the PPAR
signal-transducing event is distal to
the ACS-catalyzed fatty acid activation step (Fig. 7). In this context
it is important to note that fatty acyl-CoAs have been shown to act as
potent inhibitors of the nuclear thyroid hormone receptor and promoted dissociation of the hormone bound to the receptor (53). Long chain
fatty acids are activated by ACS to their corresponding acyl-CoAs,
which either participate in the synthesis of cellular lipids including
triacylglycerols, phospholipids, and cholesterol esters or enter the
peroxisomal
-oxidation spiral for degradation (3, 30). The strong
increases of hepatic ACS mRNA in AOX
/
mice (Fig. 3) suggests
efficient VLCFA activation to acyl-CoA, but the resulting acyl-CoA
cannot be
-oxidized by peroxisomes due to lack of AOX in these mice.
The observed increases in ACS mRNA and PPAR
activation in
AOX
/
mice do not support the possibility that ACS may inactivate
PPAR
ligands (21). However, quantitative data on the levels of
VLCFAs and fatty acyl-CoAs are necessary to provide direct evidence for
the efficient conversion of VLCFAs to acyl-CoAs in AOX
/
mouse
liver. It is also important to consider the possibility that long chain
acyl-CoA thioesterases may play an active role in cleaving fatty
acyl-CoA to the corresponding free fatty acids and CoASH (54, 55) and,
thus, modulate the levels of acyl-CoAs in AOX
/
mouse liver. Since
these thioester hydrolases in rat liver are inducible by peroxisome
proliferators (55), it would be important to determine the levels of
these enzymes in AOX
/
mouse liver to appreciate the relative
importance of free VLCFAs and their CoA derivatives in the activation
in vivo of PPAR
. Recently, the eicosanoids
8(S)-hydroxyeicosatetraenoic acid and leukotriene
B4 , which are derived from arachidonic acid, have also
been shown to function as ligands for PPAR
(21, 22, 24, 56). Since
leukotriene B4 is
-oxidized within the peroxisome (57,
58), there is a possibility that these endogenous ligands of PPAR
may also contribute to enhanced PPAR
activation in the liver of
AOX
/
mice. Nonetheless, the data presented here clearly establish
that a functional AOX gene is a key regulator of
PPAR
function by its ability to metabolize acyl-CoA vis
à vis fatty acids and thus keep the level of these and other
physiological PPAR
ligands in check. Thus, this mouse model of
peroxisomal AOX deficiency may provide helpful clues in the search for
the PPAR
agonists and in screening for the antagonists for this
receptor.
Massive increases in the levels of ACS, MFP, THL, CYP4A1, and
CYP4A3 mRNAs in the liver of AOX
/
mice reflect spontaneous induction of the PPAR
signal transduction pathway, affecting a
plethora of genes with PPRE-containing promoters (Fig. 7). The induction in AOX
/
mice of PPAR
-mediated pleiotropic responses, including the development of liver tumors, strongly implies that PPAR
functions as an oncogene in liver. The abrogation of peroxisome proliferator-induced pleiotropic responses including the development of
liver tumors in PPAR
/
mice supports this contention (37). This
interpretation is consistent with our proposal more than a decade ago
that the receptor-mediated activation of specific genes and peroxisome
proliferation-induced oxidative stress lead to oncogenesis in liver
(27, 28). We further propose that AOX gene
functions as a tumor suppressor gene under normal physiological conditions by metabolizing PPAR
ligands. Our data also indicate that
inactivation of tumor suppressor function of AOX gene leads to oncogenesis. The suggestion that normal AOX functions as a tumor
suppressor gene may appear paradoxical, because increased AOX activity
in rats and mice exposed to peroxisome proliferators results in excess
production of H2O2, leading to sustained
oxidative stress, thus contributing to liver tumor development (5, 27, 28). The precise mechanism by which AOX null mice, which show extensive
spontaneous peroxisome proliferation, develop liver tumors, remains to
be elucidated. It is important to point out that these animals, during
steatotic phase, exhibited marked inflammatory response, increased
hepatic levels of H2O2, and hepatocellular proliferation. Proinflammatory molecules such as leukotriene
B4 and 8(S)-hydroxyeicosatetraenoic acid are
normally degraded by the peroxisomal
-oxidation system, and the
ineffective degradation of these agents in AOX
/
mice may lead to
inflammatory changes. Inflammatory cell infiltrate, as well as
increased levels of CYP4A1 and CYP4A3 are known to generate reactive
oxygen species and can lead to oxidative DNA damage similar to that
observed in the livers of rats and mice exposed to peroxisome
proliferators (45). Cell proliferation occurring in these livers of
AOX
/
mice can facilitate the fixation of resulting mutations. In
AOX
/
mice, putative preneoplastic and neoplastic liver lesions
manifested between 10 and 15 months of age, which essentially
paralleled the changes observed in wild-type rats and mice
exposed to peroxisome proliferators (5, 26, 28). Our findings suggest
that AOX-deficient mice will be an invaluable model system for the
further unraveling of the molecular events in PPAR
-mediated signal
transduction, hepatitis, liver cell regeneration, and
oncogenesis.
We thank H. Sakuraba for assisting
with mouse colony, Sujatha Pulikuri for excellent technical assistance,
and Vanessa Jones for the preparation of illustrations. We are grateful
to Dr. J. I. Gordon for the liver FABP plasmid, to Dr. Frank J. Gonzalez for CYP4A1 and CYP4A3 plasmids, to Drs. Genevieve Martin and
Johan Auwerx for the ACS plasmid, to Dr. Takashi Hashimoto for PMP70 plasmid, and to Dr. D. B. Jump for the FAS plasmid.