Absence of Spontaneous Peroxisome Proliferation in Enoyl-CoA
Hydratase/L-3-Hydroxyacyl-CoA Dehydrogenase-deficient
Mouse Liver
FURTHER SUPPORT FOR THE ROLE OF FATTY ACYL CoA OXIDASE IN
PPAR
LIGAND METABOLISM*
Chao
Qi
,
Yijun
Zhu
,
Jie
Pan
,
Nobuteru
Usuda
,
Nobuyo
Maeda§,
Anjana V.
Yeldandi
,
M. Sambasiva
Rao
,
Takashi
Hashimoto
, and
Janardan K.
Reddy
¶
From the
Department of Pathology, Northwestern
University Medical School, Chicago, Illinois 60611-3008 and the
§ Department of Pathology, University of North Carolina,
Chapel Hill, North Carolina, 27599
 |
ABSTRACT |
Peroxisomes contain a classical
L-hydroxy-specific peroxisome proliferator-inducible
-oxidation system and also a second noninducible
D-hydroxy-specific
-oxidation system. We previously generated mice lacking fatty acyl-CoA oxidase (AOX), the first enzyme
of the L-hydroxy-specific classical
-oxidation system; these AOX
/
mice exhibited sustained activation of
peroxisome proliferator-activated receptor
(PPAR
), resulting in
profound spontaneous peroxisome proliferation in liver cells. These
observations implied that AOX is responsible for the metabolic
degradation of PPAR
ligands. In this study, the function of
enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase
(L-PBE), the second enzyme of this peroxisomal
-oxidation system, was investigated by disrupting its gene. Mutant
mice (L-PBE
/
) were viable and fertile and
exhibited no detectable gross phenotypic defects.
L-PBE
/
mice showed no hepatic steatosis and
manifested no spontaneous peroxisome proliferation, unlike that
encountered in livers of mice deficient in AOX. These results indicate
that disruption of classical peroxisomal fatty acid
-oxidation
system distal to AOX step does not interfere with the inactivation of
endogenous ligands of PPAR
, further confirming that the AOX gene is
indispensable for the physiological regulation of this receptor. The
absence of appreciable changes in lipid metabolism also indicates that enoyl-CoAs, generated in the classical system in
L-PBE
/
mice are diverted to
D-hydroxy-specific system for metabolism by
D-PBE. When challenged with a peroxisome proliferator,
L-PBE
/
mice showed increases in the levels
of hepatic mRNAs and proteins that are regulated by PPAR
except
for appreciable blunting of peroxisome proliferative response as
compared with that observed in hepatocytes of wild type mice similarly
treated. This blunting of peroxisome proliferative response is
attributed to the absence of L-PBE protein in
L-PBE
/
mouse liver, because all other
proteins are induced essentially to the same extent in both wild type
and L-PBE
/
mice.
 |
INTRODUCTION |
In animal cells, mitochondria, as well as peroxisomes, oxidize
fatty acids via
-oxidation, with long chain and very long chain
fatty acids being preferentially oxidized by peroxisomes (1-3).
Peroxisomal
-oxidation process is carried out by two distinct groups
of enzymes; the classical first group utilizes straight chain saturated
fatty acyl-CoAs as substrates, whereas the recently discovered second
group acts on branched chain acyl-CoAs (3, 4). In the
L-hydroxy-specific classical
-oxidation spiral,
dehydrogenation of acyl-CoA esters to their corresponding trans-2-enoyl-CoAs is catalyzed by fatty acyl-CoA oxidase
(AOX),1 whereas the second
and third reactions, hydration and dehydrogenation of enoyl-CoA esters
to 3-ketoacyl-CoA, are carried out by a single enzyme, enoyl-CoA
hydratase/L-3-hydroxyacyl-CoA dehydrogenase (L-bifunctional enzyme (L-PBE)) (3). The third
enzyme of this classical system, 3-ketoacyl-CoA thiolase (PTL) cleaves
3-ketoacyl-CoAs to acetyl-CoA, and an acyl-CoA that is two carbon atoms
shorter than the original molecule can re-enter the
-oxidation
spiral (1, 2). In the second D-hydroxy-specific
-oxidation pathway, dehydrogenation of acyl-CoA esters to their
corresponding trans-2-enoyl-CoAs is catalyzed by the
branched chain acyl-CoA oxidase (2), with the recently identified
D-3-hydroxyacyl-CoA
dehydratase/D-3-hydroxyacl-CoA dehydrogenase
(D-bifunctional enzyme (D-PBE)), converting
enoyl-CoAs to 3-ketoacyl-CoAs via D-3-hydroxyacyl-CoAs (3,
4). The third enzyme of this second system is designated as sterol
carrier protein x (SCPx), which possesses 3-ketoacyl-CoA thiolase
activity (5).
Of these two peroxisomal
-oxidation systems, the enzymes belonging
to the classical group are markedly induced in the liver of rats and
mice in conjunction with profound proliferation of peroxisomes by a
group of structurally diverse agents designated as peroxisome
proliferators (6). They exert their pleiotropic effects by activating a
nuclear receptor called peroxisome proliferator-activated receptor
(PPAR
) (7). Sustained induction of PPAR
-mediated peroxisome
proliferation and transcriptional activation of classical
-oxidation
system genes lead to the development of liver tumors in rats and mice
(8). It is postulated that H2O2 overproduced by
the induction of this
-oxidation system and cell proliferation contribute to hepatocarcinogenesis in livers with peroxisome
proliferation (8, 9). To investigate the functional implications of
PPAR
-regulated L-hydroxy-specific
-oxidation system,
we recently generated mice deficient in AOX, the first enzyme of this
inducible system, and found that they exhibit steatohepatitis and
spontaneous peroxisome proliferation in liver cells (10). These results
suggested that straight chain acyl-CoAs and other putative substrates
for classical AOX serve as natural ligands for PPAR
, and this enzyme
is indispensable for the physiological regulation of PPAR
(10).
Here, we describe the generation of mice homozygous for a disruption of
the L-PBE gene, which encodes the second enzyme of this
-oxidation spiral and report that these mice do not exhibit
phenotypic alterations such as those found in AOX null mice, further
confirming that disruption of this classical
-oxidation pathway
distal to AOX does not affect the metabolism of natural ligands of
PPAR
.
 |
MATERIALS AND METHODS |
Gene Targeting--
P1 genomic clone (6346) containing the
L-PBE gene was obtained by screening a mouse 129/Sv P1
bacteriophage library (Genome Systems, St. Louis, MO) using polymerase
chain reaction with primers 5'-GTGCTGATATCCATGGCTTTAGTG-3'and
5'-GATAGTGACAGCCCAAGGCCAGCTC-3' (11). The targeting construct was
assembled in the pPNT targeting vector with the 2.5-kb XbaI
fragment from the SacI subclone and 4-kb
XbaI/ApaI fragment from the ApaI
subclone serving as the 5' and 3' homologous regions, respectively, of
the phosphoglycerate kinase promoter/neomycin-resistance gene (Neo)
(Fig. 1A). The targeting vector also contained the herpes
simplex thymidine kinase (hsv-tk) gene, which allowed the
use of a positive-negative selection scheme. The final construct,
designated pPNT-L-PBE, is illustrated in Fig.
1A.
Generation of L-PBE Mutant
Mice--
NotI-linearized targeting vector (30 µg) was
electroporated into BK4 embryonic stem (ES) cells and selected in 200 µg/ml G418 and 2 µM ganciclovir, and resistant colonies
were subjected to Southern analysis. Two positive ES clones were
injected into 3.5-day-old C57BL/6J blastocysts and transferred into
pseudopregnant CBAF1 foster female recipients. The resulting chimeras
were mated with C57BL/6J mice, and germ-line transmission was
ascertained by coat color and confirmed by Southern analysis of genomic
DNA (5 µg) isolated from tails, transferred to nitrocellulose
membrane, and hybridized with probe 1 and probe 2 (Fig. 1A)
at 42 °C in hybridization solution containing 50% formamide (12).
F1 heterozygous siblings for the disrupted PBE gene were then mated to
obtain homozygous PBE null mice.
Genotype of L-PBE Mutant Mice--
The offspring
from subsequent breeding were genotyped by polymerase chain reaction
amplification and confirmed by Southern analysis as needed. Two
primers, primers P1 (5'-GAGCTGGCCTTGGGCTGTCACTA-3') and P2
(5'-TAGAAGCTGCGTTCCTCTTGCACCA-3'), derived from exons 3 and 4 of
L-PBE gene, respectively, shown in Fig. 1A were
designed to detect wild type allele, and two primers, primers P3
(5'-TGAATGAACTGCAGGACGAGG-3') and P4 (5'-CCACAGTCGATGAATCCAGAA-3'),
from the neomycin cassette were used to detect the PBE gene-targeted allele.
Treatment with Peroxisome Proliferators and Morphological
Studies--
Mice were fed powdered diet with or without a PPAR
ligand, ciprofibrate (0.0125% w/w), or Wy-14,643 (0.1% w/w) for 4 to
14 days. For light microscopy, tissues were fixed in 10%
neutral-buffered formalin and embedded in paraffin using standard
procedures. Sections (4-µm thick) were cut and stained with
hematoxylin and eosin. For cytochemical localization of catalase (CTL),
tissues were processed and examined as described elsewhere (10).
Immunocytochemical localization of CTL and L-PBE was
performed using protein A-gold technique as described elsewhere (13).
To assess liver cell proliferation, some mice were given
bromodeoxyuridine (0.5 mg/ml) in drinking water for 3 days, and their
livers were processed for immunohistochemistry.
AOX
/
mice utilized in these studies were as
described (10).
Western Blot Analysis and Fatty Acid Oxidation--
Contents of
-oxidation enzymes and other proteins in liver were determined by
immunoblot analysis using rabbit polyclonal antibodies against rat
acyl-CoA synthetase, AOX, L-PBE, D-PBE, PTL,
carnitine octanoyltransferase, urate oxidase, short chain acyl-CoA
dehydrogenase, medium chain acyl-CoA dehydrogenase, very long chain and
long chain acyl-CoA dehydrogenases (VLCAD and LCAD, respectively),
SCPx, and CTL, as described (14). Fatty acid
-oxidation activity was
measured using radioactive fatty acids (ICN Pharmaceuticals) and
postnuclear fractions from livers as described (14).
Northern Hybridization--
Total RNA (20 µg) extracted from
the liver of wild type, L-PBE
/
, and
AOX
/
mice by Trizol reagent (Life Technologies, Inc.),
was glyoxylated, separated on 0.8% agarose gel, and transferred to
nylon membrane. Hybridization was performed at 42 °C in 50%
formamide hybridization solution (12) using cDNA probes acyl-CoA
synthetase, AOX, L-PBE, D-PBE, PTL, cytochrome
P450
-hydroxylases (CYP4A1, CYP4A3), fatty acid-binding protein, and
ribosomal RNA (28 S) as described (10). Changes in mRNA levels were
estimated by densitometric scanning of autoradiograms.
 |
RESULTS |
Generation of L-PBE
/
Mice--
The
targeting construct illustrated in Fig. 1
was used to disrupt the L-PBE gene in ES cells. Screening
of 100 G418 ganciclovir-resistant colonies by Southern blotting yielded
5 homologous recombinants for a targeting frequency of 5%. ES cells
from two different colonies were injected into C57BL/6J blastocysts to
generate chimeric animals. Chimeras from both lines were able to
transmit the mutant allele to their offspring after outbreeding with
C57BL/6J. Genomic DNA from wild type mice when digested with
BamHI and EcoRI and probed with probe 1 yielded a
single 4.3-kb fragment, and in L-PBE+/
mouse
DNA, two bands, 6.1 and 4.3 kb, were visualized (Fig. 1B). In L-PBE
/
mice, there is a single 6.1-kb
band, indicating that both alleles were modified (Fig. 1B).
At 2 weeks of age, the F1 progeny exhibited a predicted frequency of
25% homozygous mutant offspring (L-PBE
/
),
indicating no lethality associated with the null allele.
L-PBE
/
mice exhibited no growth retardation
during the first 4 weeks and displayed no apparent morphological
abnormalities. Both male and female homozygous mice were fertile.
Northern blot analysis using poly(A)+ RNA isolated from
liver showed an absence of L-PBE mRNA in
L-PBE
/
mouse (Fig. 1C). Western
blotting performed with anti-L-PBE antibody revealed the
presence of two bands close to each other in wild type mouse liver
(Fig. 1D). One of these proteins (the lower band) was confirmed to be L-PBE, because it disappeared when the
antibodies were preincubated with purified rat L-PBE
protein. The upper nonspecific band cross-reacting with anti-PBE
antibody is present in mitochondrial and not in peroxisomal fraction
(data not shown). In L-PBE
/
mouse liver, we
also detected this cross-reacting protein (upper band) but
found no authentic L-PBE protein. Immunohistochemically, L-PBE staining appeared highly prominent in the liver of a
wild type mouse treated with a peroxisome proliferator for 2 weeks, whereas no staining was evident in L-PBE
/
mouse liver (Fig. 2). The absence of
L-PBE protein in L-PBE
/
mouse
liver peroxisomes was further confirmed at the ultrastructural level by
protein A-gold immunocytochemical procedure (Fig.
3).

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Fig. 1.
Generation of L-PBE-deficient
mice. A, schematic representation of the mouse
L-PBE gene, targeting vector, and structure of the locus
following gene targeting. Exons 2-5 (E2 to E5)
in the L-PBE gene are shown as closed boxes.
Restriction sites are indicated. Locations of hybridization probes
(probes 1 and 2) used for Southern blot analysis and of primers
(P1 to P4) used for polymerase chain reaction are
shown. B, Southern blot analysis of genomic DNA. Genomic DNA
(5 µg) isolated from tail tips of pups was digested with
BamHI and EcoRI and hybridized with the 5'-probe
(probe 1 shown in A). Lanes:
L-PBE+/+ wild type,
L-PBE+/ heterozygous,
L-PBE / homozygous mice. C,
Northern blot analysis using poly(A+) RNA (5 µg/lane) isolated from liver probed with L-PBE
or -actin cDNA. D, Western blot analysis of liver
homogenates for L-PBE expression in wild type
(+/+) and L-PBE null ( / ) mice.
The antibodies recognize L-PBE (lower band) and
also an unidentified protein x (upper band) in the wild type
mice. In L-PBE null mice only the upper band is
identifiable, but not the band representing L-PBE.
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Fig. 2.
Immunoperoxidase staining for
L-PBE in the liver. Wild type mouse (A) and
L-PBE / mice (B) were fed
ciprofibrate (0.0125% in diet) for 2 weeks, and sections of liver were
processed for immunohistochemical localization of L-PBE
using antibodies against L-PBE. Intense cytoplasmic
granular staining for L-PBE is seen in the liver of wild
type mouse but not in L-PBE / mouse.
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Fig. 3.
Immunogold localization of catalase and
L-PBE in liver peroxisomes. A, catalase
localization in L-PBE / mouse liver. Note
the presence of gold particles (black dots) over peroxisome
(p) matrix representing catalase antigenic sites.
B and C represent liver sections from wild type
mouse and L-PBE / mouse, respectively,
stained for L-PBE. No gold particles are seen over
peroxisomes (p) in L-PBE / mouse
liver.
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Characterization of Liver Phenotype in
L-PBE
/
Mice and Comparison with Mice
Deficient in AOX--
The histological architecture of liver as well
as the appearance of hepatocytes in L-PBE
/
mice did not differ significantly from that of wild type animals (Fig.
4A and B). Of
particular interest is that mice deficient in L-PBE
displayed no hepatic fatty metamorphosis, a feature that is striking in
young AOX deficient mice (Fig. 4C). Progressive hepatocellular regeneration occurring in older AOX
/
mice
leads to the emergence of hepatocytes with abundant eosinophilic cytoplasm suggestive of spontaneous peroxisome proliferation (Fig. 4,
C and D). We then surveyed for alterations, if
any, in peroxisome population in liver cells of
L-PBE
/
mice to ascertain if deficiency of
this enzyme also leads to spontaneous peroxisome proliferation such as
that encountered in AOX
/
mice (10). Peroxisomes can be
visualized at the light microscopic level in sections processed
cytochemically for localizing peroxisomal marker enzyme catalase (10).
In L-PBE
/
mouse hepatocytes, peroxisomes
are few, randomly distributed in the cytoplasm, and appear as
diaminobenzidine-positive brown granules (Fig. 4E). The
numerical density of these organelles in
L-PBE
/
mice on normal diet appeared similar
to that observed in wild type mice (Fig. 4F). Thus,
disruption of L-PBE gene fails to induce spontaneous
peroxisome proliferation such as that encountered in liver cells of
mice lacking AOX (Fig. 4, G and H).

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Fig. 4.
Liver morphology and changes in peroxisome
population in L-PBE / and
AOX / mice. A-D, liver sections stained
with hematoxylin and eosin. A,
L-PBE / mouse. B, wild type
litter mate. C, young AOX / mouse with
microvesicular fatty change and few regenerated hepatocytes
(arrows) with abundant eosinophilic cytoplasm, reflective of
spontaneous peroxisome proliferation (see G below).
D, older AOX / mouse liver with regenerated
hepatocytes containing granular eosinophilic cytoplasm and scattered
foamy lipid-laden macrophages (arrows). E-H,
sections of liver that were processed for the cytochemical localization
of peroxisomal catalase (11). L-PBE null mouse liver
(E) and wild type mouse liver (F) show few
peroxisomes in hepatocytes (brown dots, arrows).
G, liver of a young AOX / mouse with numerous
peroxisomes (arrows) in few liver cells such as those
depicted by arrows in C above, whereas
hepatocytes with microvesicular fatty change (Li) show few
or no detectable peroxisomes. H, in older AOX / mice, all
regenerated liver cells (see D above) show extensive
spontaneous peroxisome proliferation.
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Analysis of Fatty Acid-metabolizing Enzymes--
The constitutive
and inducible levels of fatty acid-metabolizing enzymes in livers of
L-PBE
/
and wild type mice were evaluated by
immunoblotting. Constitutive levels of expression of several
peroxisomal (AOX, PTL, D-PBE, carnitine
octanoyltransferase, SCPx, urate oxidase, and CTL) and mitochondrial
(VLCAD, LCAD, medium chain acyl-CoA dehydrogenase, and short chain
acyl-CoA dehydrogenase) enzymes were similar in wild type and
L-PBE
/
mice (Fig.
5). The hepatic levels of AOX, PTL,
D-PBE, carnitine octanoyltransferase, LCAD, and medium
chain acyl-CoA dehydrogenase were increased in wild type and
L-PBE
/
mice fed ciprofibrate for 2 weeks
(Fig. 5). In wild type mice treated with ciprofibrate, hepatic levels
of L-PBE protein increased substantially, and as expected,
this protein was not detected in L-PBE
/
mice
maintained on control or ciprofibrate-containing diet (Fig. 5). These
livers showed the presence of a nonspecific, cross-reacting band
described in Fig. 1D, and this protein was not induced in L-PBE
/
mice treated with a peroxisome
proliferator. Ciprofibrate did not increase the levels of SCPx, CTL,
VLCAD, and short chain acyl-CoA dehydrogenase in wild type and
L-PBE
/
mouse liver.

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Fig. 5.
Immunoblot analysis of selected fatty
acid-metabolizing and other enzymes in liver. Liver homogenates
from L-PBE / and
L-PBE+/+ mice fed either control or
ciprofibrate (Cipro)-containing diet were subjected to
SDS-polyacrylamide gel electrophoresis and immunoblotting (3 mice each
group). AOX (5 µg), L-PBE (10 µg; note only the upper
band, representing protein x, is present in
L-PBE / mouse liver), PTL (10 µg);
D-PBE (20 µg); SCPx (10 µg); carnitine
octanoyltransferase (COT) (l0 µg); VLCAD (20 µg), LCAD
(20 µg), medium chain acyl-CoA dehydrogenase (MCAD) (20 µg); short chain acyl-CoA dehydrogenase (SCAD) (10 µg);
urate oxidase (UOX) (2 µg), and CTL (2 µg).
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We also measured total hepatic
-oxidation using palmitic acid in
mice fed either a control or ciprofibrate-containing diet. The basal
level of total fatty acid
-oxidation was similar in wild type and
L-PBE
/
mice. As expected, a 4- to 6-fold
increase in total hepatic
-oxidation activity occurred in wild type
mice treated with ciprofibrate, and the response of
L-PBE
/
mice was essentially similar (data
not presented). In contrast, the ciprofibrate-induced increase in the
activity of cyanide-insensitive (peroxisomal) fatty acid
-oxidation
system in L-PBE
/
mouse liver was ~75%
that observed in wild type mice treated with ciprofibrate (data not presented).
Induction of mRNAs--
Constitutive levels of acyl-CoA
synthetase, AOX, PTL, CYP4A1, CYP4A3, D-PBE, and fatty
acid-binding protein mRNAs were similar in the livers of both wild
type and L-PBE
/
mice, indicating that
unlike in AOX
/
mice, there is no spontaneous
up-regulation of PPAR
-regulated genes in
L-PBE
/
mice (Fig.
6). Following treatment with
ciprofibrate, a 10-to 20-fold increase in liver AOX, PTL, CYP4A1, and
CYP4A3 mRNA levels occurred in both wild type and L-PBE
null mice, and the PTL, CYP4A1, and CYP4A3 increases were similar to
spontaneous increases occurring in AOX
/
mice (Fig. 6).
Fig. 6 also confirms the expected absence of AOX and L-PBE
mRNAs in the livers of AOX
/
and of
L-PBE
/
mice, respectively. The spontaneous
increase in L-PBE mRNA in AOX
/
mouse
liver is comparable in magnitude to that occurring in
ciprofibrate-treated wild type mouse (Fig. 6). We also found ~5-fold
increase in D-PBE mRNA level in the liver of both wild
type and L-PBE
/
mice treated with
ciprofibrate.

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Fig. 6.
Northern blot analysis of total RNA extracted
from liver. Total RNA (20 µg) from
L-PBE / and wild type littermate mice on
control ( ) or ciprofibrate (Cipro, 0.0125%
w/w)-containing diet (+) for 2 weeks was hybridized with
32P-labeled cDNA probes as indicated. Liver RNA from
6-month-old AOX null mice on control diet is used for comparison to
show spontaneous up-regulation of PPAR -controlled genes in
AOX / mouse liver. Acyl-CoA synthetase (ACS),
AOX, L-PBE, PTL, CYP4A1, CYP4A3, D-PBE, and
fatty acid-binding protein (FABP) genes are
transcriptionally activated by PPAR . D-PBE mRNA is
also increased but to a lesser extent than that of L-PBE in
wild type mice fed ciprofibrate. 28 S RNA is used as loading
indicator.
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Blunted Peroxisome Proliferative Response in
L-PBE
/
Mouse Liver--
Rats and mice
treated with peroxisome proliferators such as ciprofibrate and
Wy-14,643 exhibit abundant peroxisome proliferation in hepatocytes with
peroxisomes occupying ~20 to 25% of cytoplasmic volume (6, 8). In
AOX
/
mice we also found profound spontaneous
proliferation of peroxisome, and such changes have been ascribed to
sustained activation of PPAR
by endogenous ligands that require AOX
for metabolism or inactivation (10). We first established that
deficiency of L-PBE neither caused spontaneous peroxisome
proliferation nor led to an increase in protein and mRNA levels of
genes regulated by PPAR
. We then evaluated the impact of
L-PBE gene disruption on peroxisome proliferation induced
by peroxisome proliferators (Fig. 7).
Light microscopic analysis of liver sections processed for cytochemical localization of catalase revealed massive proliferation of peroxisomes in liver cells of wild type mice treated with either ciprofibrate or
Wy-14,643 for 2 weeks (Fig. 7A). In contrast,
peroxisome-proliferative response appeared blunted in
L-PBE
/
mouse liver (Fig. 7B).
These observations were confirmed at the ultrastructural level in that
peroxisome proliferation appeared robust in peroxisome
proliferator-treated wild type mice but subdued in
L-PBE
/
mouse liver cells (not illustrated).
In L-PBE
/
mice treated with peroxisome
proliferators, peroxisomal profiles tended to be smaller than those
seen in wild type liver. Analysis of liver samples by
SDS-polyacrylamide gel electrophoresis showed no increase in the
78-kilodalton L-PBE protein in L-PBE
/
mice treated with a peroxisome proliferator (Fig.
8).

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Fig. 7.
Response of PBE / and wild
type littermate mice to a peroxisome proliferator. Ciprofibrate, a
peroxisome proliferator, was fed in the diet for 2 weeks (0.0125%
w/w). Livers were processed for cytochemical localization of
peroxisomal catalase. L-PBE /
(A) and wild type mice (B) were treated with
ciprofibrate. Peroxisomes appear as black dots in these
black and white photographs; in
L-PBE / mouse liver, ciprofibrate-induced
peroxisome proliferation is somewhat subdued.
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Fig. 8.
SDS-polyacrylamide gel electrophoresis of
liver homogenates. Liver samples from wild type and
L-PBE / mice fed control diet ( ) or a diet
containing Wy-14,643 (+) were subjected to SDS-polyacrylamide gel
electrophoresis, and the gel was stained with Coomassie Brilliant R
Blue. Note the presence of massive amounts of 78-kDa L-PBE
protein in the liver of wild type mouse treated with Wy-14,643
(arrow); it is not present in
L-PBE / mice.
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Liver Cell Proliferation--
Increased liver cell proliferation
has been noted in mice deficient in AOX
/
, attributable
in part to cell loss resulting from extensive steatosis (10). Because
peroxisome proliferators are also known to induce transient liver cell
proliferation (8), we assessed the impact of L-PBE gene
disruption on hepatocyte proliferation induced by the administration of
a peroxisome proliferator in the diet for 3 days. No significant
difference in hepatocyte nuclear labeling was found between wild type
and L-PBE
/
mice treated with a peroxisome
proliferator (data not shown).
 |
DISCUSSION |
The classical peroxisome proliferator-inducible
L-hydroxy-specific peroxisomal
-oxidation system that
acts on long and very long straight chain acyl-CoAs, long chain
dicarboxylyl-CoAs, and the CoA esters of prostaglandins consists of
AOX, L-PBE, and PTL (2, 3). The second noninducible
D-3-hydroxy-specific peroxisomal
-oxidation system that
acts on branch chain acyl-CoAs also contains three enzymes namely,
branched chain acyl-CoA oxidase, D-PBE, and SCPx (3, 4,
15). Straight chain acyl-CoAs are almost exclusively metabolized by
classical AOX and not by D-hydroxy-specific branched chain
acyl-CoA oxidase, although the enzymes of
D-hydroxy-specific
-oxidation pathway reveal minor
catalytic activity toward straight chain acyl-CoAs (3, 4, 15). This
assumption is supported by the observations in mice deficient in
classical AOX as they exhibit sustained up-regulation of
PPAR
-induced genes along with spontaneous peroxisome proliferation
in liver parenchymal cells, indicating that this
L-hydroxy-specific AOX is crucial for metabolically degrading biological ligands of PPAR
(10, 16). Because this classical
-oxidation system degrades long and very long straight chain fatty acids, long chain dicarboxylic acids, and prostaglandins or
their respective CoAs, it is reasonable to conclude that these and
possibly other yet to be identified substrates of
L-hydroxy-specific AOX act as natural ligands of PPAR
(10). These AOX-deficient mice also highlight the fact that substrates
of this classical AOX that function as PPAR
ligands are not degraded
by D-hydroxy-specific branched chain acyl-CoA oxidase. Here
we have described the generation and initial characterization of a
mouse homozygous for a targeted disruption of L-PBE, the
enzyme immediately distal to AOX in the classical
L-hydroxy-specific
-oxidation pathway.
L-PBE
/
mice, in combination with
AOX
/
(10), PPAR
/
(17),
X-ALD
/
(X-linked adrenoleukodystrophy gene (18)), SCPx
/
(19), LCAD
/
(20) and other mouse
models should prove valuable in exploring the functional role and/or
redundancy of metabolic pathways in lipid metabolism. These mutant mice
will also be valuable in investigating the role of the enzymes of lipid
metabolism in regulating the function of PPAR
and possibly other
receptors (10, 20).
There are several lines of evidence that support the view that the mice
we generated in this study contain a null mutation in the
L-PBE gene. The gene targeting construct we used was
designed to eliminate the entire exon 4, and the selectable gene
cassette used to disrupt the L-PBE gene introduced nonsense
mutation in all reading frames. Polymerase chain reaction as well as
Southern blot analysis revealed that ES cells contained the correct
targeted event, and Northern blot analysis revealed that liver from
L-PBE
/
mice fed either a control diet or
peroxisome proliferator-containing diet did not contain a detectable
message corresponding to the L-PBE probe. The
L-PBE
/
mice displayed no authentic
L-PBE protein on Western blot analysis; the
L-PBE antibody we used was raised against rat
L-PBE, and in mouse liver, it recognized a nonspecific
protein band, designated protein x, the identity of which, however,
remains to be determined (Fig. 1D). By immunogold technique,
this protein x was detected in mitochondria and not in peroxisomes of
L-PBE
/
mice, further confirming the
L-PBE null mutation. It is also worth noting that this
protein x was not induced in the livers of wild type and
L-PBE
/
mice treated with peroxisome
proliferators. Peroxisome of L-PBE
/
mice did not reveal
any staining for L-PBE by immunogold procedure (Fig.
3C), and when these animals were treated with a peroxisome proliferator, the liver parenchymal cells failed to show any
immunohistochemically detectable L-PBE protein (Fig. 2).
These observations unequivocally establish the disruption of
L-PBE gene, and the absence of L-PBE mRNA
and protein, therefore, establish the functional inactivation of this gene.
The striking results observed in these
L-PBE
/
mice were the absence of hepatic
steatosis and of spontaneous peroxisome proliferation such as that
found in the livers of young AOX
/
mice (10). These
observations provide strong evidence that the classical AOX of the
L-hydroxy-specific
-oxidation system is responsible for
the metabolism of all putative ligands of PPAR
and that
L-PBE enzyme immediately downstream of AOX is not essential for the successful completion of the L-hydroxy-specific
-oxidation. It would appear that once the long and very long
straight chain acyl-CoAs, long chain dicarboxylyl-CoAs, and the CoA
esters of prostaglandins are converted into their respective enoyl-CoAs by the classical AOX, these can be metabolized by D-PBE of
the D-hydroxy-specific peroxisomal
-oxidation system
(Fig. 9) or even by the mitochondrial
-oxidations system. In essence, the AOX null and L-PBE
null mouse models we generated convincingly prove that classical AOX is
pivotal for the metabolic degradation of PPAR
ligands and not the
branched chain acyl-CoA oxidase of the second system. Further studies,
however, are needed to rule out the possibility that straight chain
enoyl-CoAs generated by classical AOX, if left unmetabolized, serve as
PPAR
ligands (10, 21). This issue can be explored by generating mice
deficient in both L-PBE and D-PBE and
characterizing their liver phenotype.

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|
Fig. 9.
Schematic representation of peroxisome
proliferator inducible, L-hydroxy-specific, classical
peroxisomal -oxidation system and the
noninducible D-hydroxy-specific system. In mice
deficient in L-PBE, the straight chain enoyl-CoAs are most
likely diverted to the noninducible branched chain -oxidation
pathway for metabolism by D-PBE. In contrast, in
AOX / mice, the straight chain fatty acyl-CoAs and
possibly other PPAR ligands are not metabolized because of the
absence of AOX, and these substrates are not diverted to a noninducible
system for degradation by branched chain acyl-CoA oxidase, thus causing
sustained activation of PPAR .
|
|
Of further interest is that L-PBE
/
mice,
when challenged with a peroxisome proliferator, exhibited somewhat
blunted hepatic peroxisome proliferative response, although there were
no significant differences in other immediate or early pleiotropic
responses attributable to PPAR
activation, with the single notable,
but expected exception that L-PBE mRNA and protein are
undetectable in L-PBE
/
mouse livers. Thus,
the blunting of peroxisome proliferative response is attributed to the
absence of L-PBE protein in L-PBE null animals.
It is important to note that L-PBE, a 78-kilodalton protein, is massively induced in the livers of wild type mice exposed
to peroxisome proliferators (22). This protein, once called peroxisome
proliferator-activated polypeptide (22), becomes the most abundant
protein in the livers of peroxisome proliferator-treated rats and mice,
contributing substantially to the morphological phenomenon of
peroxisome proliferation. The L-PBE null mutation abolishes
the inducibility of L-PBE in response to peroxisome proliferators, and this most likely accounts for the blunting of
peroxisome proliferative response, because all other PPAR
responsive
genes in L-PBE
/
mice are up-regulated.
Therefore, it is conceivable that changes in the levels of expression
of L-PBE gene in nonrodent species in response to
peroxisome proliferators may influence the morphological phenomenon of
peroxisome proliferation, whereas other effects of these PPAR
ligands in nonrodent species may be similar to those seen in rats and
mice. It would be of great interest to investigate whether
L-PBE
/
mice develop liver tumors following
chronic exposure to peroxisome proliferators, despite the subdued
nature of peroxisome proliferative response, because all other
PPAR
-regulated genes are up-regulated.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM 23750 (to J. K. R.), Veterans Affairs merit review grants (to A. V. Y. and M. S. R.), and Joseph L. Mayberry, Sr. Endowment Fund.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Pathology, Northwestern University Medical School, 303 East Chicago
Ave., Chicago, IL 60611-3008. Tel.: 312-503-7948; Fax: 312-503-8249; E-mail: jkreddy{at}nwu.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
AOX, straight chain
fatty acyl-CoA oxidase or palmitoyl-CoA oxidase;
PPAR, peroxisome
proliferator-activated receptor;
L-PBE, enoyl-CoA
hydratase/L-3-hydroxyacyl-CoA dehydrogenase
(L-bifunctional enzyme);
D-PBE, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl
CoA-dehydrogenase (D-bifunctional enzyme);
PTL, peroxisomal
3-ketoacyl-CoA thiolase;
CYP4A1 and CYP4A3, encode microsomal
cytochrome P450 fatty acid
-hydroxylases;
SCPx, sterol carrier
protein x or 3-ketoacyl-CoA thiolase/sterol carrier protein 2;
LCAD, long-chain acyl-CoA dehydrogenase;
VLCAD, very LCAD;
CTL, catalase;
ES, embryonic stem cells;
kb, kilobase(s).
 |
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