Received for publication, October 1, 2002, and in revised form, October 18, 2002
Peroxisome proliferator activated-receptor
(PPAR) isoforms,
and
, function as important coregulators of
energy (lipid) homeostasis. PPAR
regulates fatty acid oxidation
primarily in liver and to a lesser extent in adipose tissue, whereas
PPAR
serves as a key regulator of adipocyte differentiation and
lipid storage. Of the two PPAR
isoforms, PPAR
1 and PPAR
2
generated by alternative splicing, PPAR
1 isoform is expressed in
liver and other tissues, whereas PPAR
2 isoform is expressed
exclusively in adipose tissue where it regulates adipogenesis and
lipogenesis. Since the function of PPAR
1 in liver is not clear, we
have, in this study, investigated the biological impact of
overexpression of PPAR
1 in mouse liver. Adenovirus-PPAR
1 injected
into the tail vein induced hepatic steatosis in
PPAR
/
mice. Northern blotting and gene
expression profiling results showed that adipocyte-specific genes and
lipogenesis-related genes are highly induced in
PPAR
/
livers with PPAR
1 overexpression. These
include adipsin, adiponectin, aP2, caveolin-1, fasting-induced adipose
factor, fat-specific gene 27 (FSP27), CD36,
9
desaturase, and malic enzyme among others, implying adipogenic transformation of hepatocytes. Of interest is that hepatic steatosis per se, induced either by feeding a diet deficient in
choline or developing in fasted PPAR
/
mice, failed
to induce the expression of these PPAR
-regulated adipogenesis-related genes in steatotic liver. These results suggest that a high level of PPAR
in mouse liver is sufficient for the induction of adipogenic transformation of hepatocytes with adipose tissue-specific gene expression and lipid accumulation. We conclude that excess PPAR
activity can lead to the development of a novel type of adipogenic hepatic steatosis.
 |
INTRODUCTION |
Peroxisome proliferator-activated receptor
(PPAR
),1 a member of
the nuclear receptor superfamily, is a key regulator of adipogenesis
(1-4). The PPAR subfamily consists of three isotypes, namely PPAR
,
PPAR
, and PPAR
/
, and like all other nuclear receptors, PPARs
possess a highly conserved DNA-binding domain that recognizes peroxisome proliferator response elements (PPREs) in the promoter regions of target genes (5-9). After ligand binding PPARs
heterodimerize with retinoid-X-receptors (RXR) and PPAR-RXR
heterodimers bind to PPRE to initiate the transcriptional regulation of
target genes, in particular those involved in lipid homeostasis
(5-10). The three PPAR isotypes are products of separate genes, and
they exhibit distinct patterns of tissue distribution (7). PPAR
is
expressed at a relatively high concentration in liver, plays a central
role in regulating enzymes involved in the oxidation of fatty acids, and is essential for the pleiotropic responses induced in liver by
structurally diverse chemicals known as peroxisome proliferators (8-10). PPAR
is present in two isoforms, PPAR
1 and PPPAR
2, resulting from alternate promoter usage (1, 8, 9). PPAR
2 contains an
additional 30 amino acids at the N-terminal end relative to PPAR
1.
PPAR
2 expression is limited exclusively to adipose tissue where it
play a key role in adipogenesis (1, 2). On the other hand, PPAR
1 is
expressed at relatively low levels in many tissues including liver, but
the function of this isoform in non-adipose tissue locations is not
well delineated (1, 9, 13). Forced expression of PPAR
2 or PPAR
1
can initiate the differentiation of fibroblasts to adipocytes, and in
the process the fibroblasts express adipocyte-specific genes and
accumulate lipid (2, 14). Although there is some suggestion that
PPAR
ligands may induce adipocyte-specific gene expression in
certain tumor cells, it is uncertain as to whether PPAR
1 or PPAR
2
expression in vivo, in non-fibroblast mesenchymal cells, or
in epithelial tissues can lead to adipocyte-specific gene expression
and adipogenesis (15).
It is well known that CCAAT/enhancer binding family of transcription
factors C/EBP
, -
, and -
have been shown to play an important
role in adipogenesis in that high expression of each member of this
family will direct fibroblasts to differentiate into adipocytes, and
this conversion is mediated through down-stream regulator PPAR
and
PPAR coactivator PBP/TRAP220/DRIP205 (4, 16-19). C/EBPs and PBP are
expressed in liver, but the significance of this expression in terms of
adipogenesis and or lipid accumulation in liver cells remains unclear.
The inability of C/EBP and PBP to induce adipogenesis in normal
hepatocytes may be due to the fact that down-stream regulator PPAR
(PPAR
1 in liver) may be rate-limiting in hepatocytes (19-21). In
this study, we used an adenoviral gene delivery system to overexpress
PPAR
1 in mouse liver to determine whether this would trigger the
expression of adipocyte-specific genes and lipid accumulation
(steatosis) in liver cells. To avoid the confounding effect of PPAR
,
we used PPAR
/
mice (22), and found that
overexpression of PPAR
1 in these livers leads to adipocyte-specific
gene expression and lipid accumulation. We also demonstrate that fatty
liver induced by starvation or that developing after feeding a diet
deficient in choline is not associated with the induction of genes
associated with adipogenesis unlike that accompanying PPAR
1
overexpression in liver. These results strongly suggest that the low
level of PPAR
1 appears to prevent liver cells from becoming
adipocytes despite the prominence of C/EBP
gene expression in these
cells and that overexpression of PPAR
1 leads to adipogenic hepatic steatosis.
 |
MATERIALS AND METHODS |
Mice and Treatment--
Wild type (C57BL/6J) mice and
PPAR
/
mice (22), 3 to 4 months of age and weighing
25-35 g, were used in this study. PPAR
/
mice were
maintained on powdered chow with or without troglitazone (0.1% w/w)
for 5 days prior to adenovirus injection and killed on day 2, 3, 4, 5, or 6 after injections while still on the same diet. For dose response
of Ad/PPAR
1, mice were maintained on powdered chow, injected with
different concentrations of virus, and killed 6 days after injection.
For the induction of fatty liver, PPAR
/
mice were
either fasted for 96 h (23) or fed a choline-deficient diet
(Dyets, Bethlehem, PA) for 15 days (24, 25). All animal procedures used
in this study were reviewed and preapproved by the Institutional Review
Boards for Animal Research of the Northwestern University.
Primers for Endogenous Mouse PPAR
--
To assess the level of
expression of endogenous PPAR
1 under forced expression of
Ad/PPAR
1, the following primers were used to distinguish the ectopic
expression from endogenous. They are endogenous PPAR
1 sense:
5'-cggagggacgcggaagaagag-3'; endogenous PPAR
2:sense:
5'-tgacccagagcatggtgccttc-3'; adenoviral PPAR
1:sense: 5'-cggggatcctctagagtcga-3'. All three PPAR
RT-PCRs were performed using the same antisense primer: 5'-tgtggcatccgcccaaacc-3'. Primers for
the internal control
-actin were sense: 5'-ggttccgatgccctgaggctc-3', antisense: 5'-tgctccaaccaactgctgtcgc-3'. PCR reactions were denatured at 94 °C for 2 min, cycled at 94 °C for 10 s, 60 °C for
30 s, 72 °C for 45 s, 30 cycles for PPAR
and 25 cycles
for
-actin by using the GeneAmp PCR system 9700 (PE Applied Biosystems).
Adenoviral Gene Transfer--
Construction of recombinant
adenovirus containing the mouse PPAR
1 cDNA (Ad/mPPAR
1) was as
follows. Mouse PPAR
1 cDNA (8) was cloned into pShuttle-CMV
expression vector at SalI site (Quantum Biotechnologies,
Inc.). The linearized shuttle vector and AdEasy vector (Quantum
Biotechnologies, Inc.) were then co-transformed into Escherichia
coli strain BJ5183. Positive recombinant plasmid Ad/mPPAR
1 was
selected. The Ad/mPPAR
1 virus was then generated as described
previously (26). Adenoviral construct of Ad/LacZ was the generous gift
of Dr W. El-Deiry (University of Pennsylvania, Philadelphia, PA) and
has been described (27). Mice were intravenously injected (tail vein)
in a volume of 200 µl with 1 ×× 1011 virus particles of
Ad/LacZ or Ad/mPPAR
1 and killed 6 days later. Mice injected with PBS
served as controls in some cases.
Morphology--
Tissue fixed in 10% neutral buffered formalin
was embedded in paraffin by using standard procedures. Sections (4-µm
thick) were cut and stained with hematoxylin and eosin. For visualizing
-galactosidase activity, sections of liver were incubated in PBS
containing 5 mM potassium ferricyanate, 2 mM
MgCl2, and 1/20 volume of 20 mg/ml
5-bromo-4-chloro-3-indolyl
-D-galactoside in
dimethylformamide at 37 °C (26, 27). Immunohistochemical localization of PPAR
was performed as described previously using polyclonal anti-PPAR
antibodies (Santa Cruz, CA). Frozen sections of
formalin-fixed liver (5-µm thick) were stained with Oil Red O and
counterstained with Giemsa. Histological analysis and image processing
were carried out using Leica DMRE microscope equipped with Spot digital
camera as described (24, 26-30).
Northern and Immunoblot Procedures--
Total RNA isolated from
liver using Trizol reagent (Invitrogen) was glyoxylated,
electrophoresed on 0.8% agarose gel, and then transferred to nylon
membrane. These nylon membranes were then hybridized at 42 °C in
50% formamide hybridization solution using 32P-labeled
cDNA probes. Equal loading was verified by the intensity of
methylene blue-stained 18 S and 28 S RNA or by hybridizing the filters
for glyceraldehyde-3-phosphate dehydrogenase. For immunoblotting, liver
extracts were subjected to 7.5% or 10% SDS-PAGE, transferred to
nitrocellulose membranes, and immunoblotted as previously described
(26, 28, 29). The aP2 antibody was a generous gift from Dr. G. S. Hotamisligil (Harvard School of Public Health, Boston, MA).
Microarray Approach--
Total RNA was isolated from the livers
of PPAR
/
mice injected with Ad/LacZ or
Ad/mPPAR
1 and killed 6 days after injection (26). Reverse
transcription, second-strand synthesis, and probe labeling were all
performed using 10 µg of total liver RNA as a template for cDNA
synthesis. Biotin-labeled cRNA was produced using the above cDNA as
a template, purified, fragmented, and hybridized to U74Av2 arrays
(Affymetrix, Santa Clara, CA). After hybridization, bound cRNA was
fluorescently labeled using R-phycoerythrin-streptavidin (Molecular
Probes), and the fluorescence was intensified by the antibody-amplification method. Data were collected and analyzed using
the Affymetrix Microarray suite 4.01 software.
 |
RESULTS |
Fatty Liver Resulting from PPAR
1 Overexpression--
We have
investigated the morphological changes in the liver of
PPAR
/
mice injected with 1 × 1011
adenoviral-PPAR
1 particles intravenously. PPAR
1 overexpression in
the liver of PPAR
/
mice caused extensive lipid
accumulation in hepatocytes located in the periportal and midzonal
regions of liver lobules at 6 days (Fig.
1). The accumulation of fat in liver was
grossly evident between 3 and 6 days after Ad/PPAR
1 injection. The
degree and zonality of lipid accumulation in liver in mice
overexpressing PPAR
1 appeared essentially similar whether or not
they were on troglitazone, the PPAR
1 ligand (Fig. 1, B
and C). Mice given troglitazone and injected with either PBS
or with Ad/LacZ failed to reveal lipid accumulation in hepatocytes
(Fig. 1, A and D). Oil red O staining of liver
sections obtained from PPAR
/
mice infected with
Ad/mPPAR
1 confirmed hepatic lipid accumulation (Fig. 1E),
whereas no appreciable lipid accumulation was evident in the liver of
mice treated with Ad/LacZ (Fig. 1F). Immunohistochemical analysis revealed PPAR
nuclear staining in ~70% of hepatocytes with microvesicular steatosis between 4 and 6 days following
Ad/mPPAR
1 injection into the PPAR
/
mice (Fig.
1G). No detectable immunostaining for PPAR
was noted in
the livers of Ad/LacZ-injected mice (Fig. 1H). As expected, ~60-70% of hepatocytes stained positively for
-galactosidase after Ad/LacZ injection (Fig. 1, I). Wild type
(PPAR
+/+) mice dosed with Ad/mPPAR
1 also exhibited
hepatic lipid accumulation but not as severe as that seen in
PPAR
/
livers with PPAR
1 overexpression (not
illustrated), suggesting that the presence of PPAR
in the liver
facilitates the up-regulation of fatty acid oxidation systems and
reduces lipid accumulation (11, 23).

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Fig. 1.
Adenovirus-PPAR 1 expression and
hepatic steatosis in
PPAR /
mouse. PPAR / mice were treated with
troglitazone for 5 days and then injected with PBS (A),
Ad/PPAR 1 (B), or Ad/LacZ (D) and then
maintained on troglitazone for another 6 days. (C) The
PPAR / mouse not treated with troglitazone was
injected with Ad/PPAR 1. Livers harvested 6 days after tail vein
injection of Ad/PPAR 1, with or without the ligand, show severe
periportal steatosis (B, C). Oil Red O stain
demonstrates fatty change in the Ad/PPAR 1-injected mouse that was
not on troglitazone (E) in contrast to the mouse given
Ad/LacZ (F). Liver of the PPAR / mouse
injected with Ad/PPAR 1 (G) or with Ad/LacZ (H,
I) was immunostained for PPAR localization 6 days after
injection (G, H). I, -galactosidase staining
of 6-day Ad/LacZ PPAR / liver.
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|
PPAR
1-induced Adipogenic Gene Expression in
PPAR
/
Mouse Liver--
PPAR
exists as two
isoforms, PPAR
1 and PPAR
2, generated by alternate promoter usage
(1, 9). The expression of PPAR
2 is restricted mainly to adipocytes
and is the key regulator of adipogenesis, although forced expression of
PPAR
1 isoform can also induce adipogenesis in fibroblasts (2, 14).
Since we noted a striking degree of lipid accumulation in hepatocytes
following PPAR
1 overexpression, it appeared necessary to investigate
the adipogenic action of overexpressed PPAR
1 in liver (Fig.
2). Northern analysis of RNA isolated
from PPAR
/
mouse liver revealed dramatic induction
mRNAs for fat differentiation markers aP2, adipsin and adiponectin
(adipoQ/acrp30) (see Refs. 31, 32) in PPAR
1 overexpressing livers
but not after Ad/LacZ infection (Fig. 2, A and B). Since
these mice are PPAR
/
, it is reasonable to conclude
that PPAR
plays no role in the observed induction of these
adipogenic genes. The mRNA of the adipsin gene reached peak level
at 3 days postinjection, whereas aP2 mRNA level was maximally
expressed at 6 days after Ad/PPAR
1 injection (Fig. 2A).
The induction of adiponectin appeared somewhat delayed when compared
with aP2 and adipsin. Glucose-6-phophatase (Glc-6-P) mRNA
level in liver increased 2 days after Ad/PPAR
1 injection (Fig.
2A). We also determined the hepatic mRNA levels of
several genes 6 days after Ad/PPAR
1 injection to assess the adipogenic and lipogenic profile. Noticeable increases in CD36, glucokinase, malic enzyme, low density lipoprotein receptor, microsomal triglyceride transfer protein, and
9d mRNA levels were detected at 6 days after Ad/PPAR
injection (Fig. 2B). We did not
observe perceptible increases in the levels of C/EBP
, SREBP,
glucokinase, phospho(enol)pyruvate carboxykinase, and Glut-2 mRNAs
in liver (Fig. 2B). We also assessed the mRNA levels of
PPAR
-regulated peroxisomal fatty acid
-oxidation system genes and
found modest increases in straight chain fatty acyl-CoA oxidase, L-PBE,
and peroxisomal 3-ketoacyl-CoA thiolase mRNAs (Fig. 2A),
suggesting that PPAR
1 at very high levels can transcriptionally
activate PPAR
target gene expression and regulate fatty acid
-oxidation in liver in the absence of PPAR
.

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Fig. 2.
Inducibility of PPAR
and PPAR target gene expression in
PPAR /
mouse liver. A, Northern blot showing the expression
levels of PPAR , aP2, adipsin, adiponectin, Glc-6-P, straight chain
fatty acyl-CoA oxidase, L-PBE, and peroxisomal 3-ketoacyl-CoA thiolase
genes after viral injection. PPAR / mice maintained
on troglitazone were infected with 1 × 1011
Ad/PPAR 1 viral particles and killed at 2, 3, 4, 5, and 6 days after
infection. PPAR / mice injected with Ad/LacZ were
killed 6 days after the injection. This blot includes samples from 2, 3, and 6-day post-treatment groups (2 animals). RNAs, 28 S and 18 S,
are shown as a measure of loading control. B, Northern
blot of C/EBP , SREBP1, glucokinase, phosphoenolpyruvate
carboxykinase, Glut-2, low density lipoprotein receptor, aP2,
microsomal triglyceride transfer protein, CD36, 9d and other genes
after viral injection. PPAR / mice (three in each
group) maintained on normal diet were infected with 1 × 1011 Ad/PPAR 1 or Ad/LacZ viral particles and killed 6 days after infection. Total RNAs from liver samples were hybridized
with aforementioned probes. Glyceraldehyde-3-phosphate dehydrogenase is
used for loading control. C, immunoblot of PPAR ,
aP2, and L-PBE genes. PPAR / mice (two in each group)
maintained on troglitazone were infected with 1 × 1011 Ad/PPAR 1 viral particles and killed 2, 3, 4, 5, and
6 days after infection. PPAR / mice injected with
Ad/LacZ were killed 6 days after injection. Liver samples were
immunoblotted for PPAR , aP2, L-PBE, and catalase. The nonresponsive
gene catalase was shown as loading control.
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|
Immunoblotting confirmed the induction of white adipose tissue marker
protein aP2 in PPAR
1-overexpressing liver beginning at day 3 (Fig.
2C). This protein was not detected in normal liver or
Ad/LacZ-injected mouse livers (Fig.
3C). Increase in PPAR
1 and
in L-PBE protein levels were also seen in Ad/PPAR
1-expressing livers
(Fig. 2C). As expected, no change in the amount of catalase protein, the peroxisomal marker enzyme, was discerned.

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Fig. 3.
Expression of aP2 in
PPAR -induced adipogenic hepatic steatosis but
not in other forms of fatty liver. A, immunoblot
analysis of aP2 expression in PPAR /
PPAR +/+ wild type mouse liver. Mice were injected with
PBS, Ad/LacZ, or Ad/PPAR 1 through tail vein with or without
treatment with troglitazone and killed 6 days later. Liver samples from
each group were immunoblotted for PPAR and aP2. The non-responsive
gene catalase is used as loading control. B, expression of
PPAR and aP2 genes in fatty livers. Steatosis that developed in
PPAR / mouse liver after infection with Ad/PPAR 1
for 6 days (lanes 1 and 2, following fasting for 4 days
(lanes 3 and 4), or fed a choline-deficient diet for 15 days
(lanes 5 and 6). Representative liver samples were
immunoblotted (two mice in each group) for PPAR , aP2
(PPAR -responsive gene), and catalase (nonresponsive gene).
C, Northern blot analysis of RNA obtained from fatty livers
that developed in PPAR / mice after infection with
Ad/PPAR 1 (6 days), starvation for 4 days, or after 2 weeks of
feeding a choline-deficient diet. Expression of adipogenesis-associated
genes is confined to PPAR -induced adipogenic hepatic steatosis and
not with other forms of fatty liver.
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aP2 Protein Gene Expression in Liver is
PPAR
-dependent--
We observed that the gene
expression of aP2 in liver was in a PPAR
dose-dependent
manner (Fig. 2C). We also established that the induction of
aP2 expression in the liver of PPAR
/
and wild type
(PPAR
+/+) mice is dependent upon PPAR
1 overexpression
whether or not these mice were on troglitazone, the synthetic PPAR
ligand (Fig. 3A). Since PPAR
1 overexpression induced the
adipogenic aP2 protein as well as hepatic steatosis in
PPAR
/
mice, it appeared necessary to study the
relationship if any of PPAR
1 and aP2 gene expression with hepatic
steatosis and to distinguish hepatic adipogenesis (hepatic adiposis)
from the typical non-alcoholic hepatic steatosis (30). We induced fatty
liver in PPAR
/
mice (Fig.
4) by either fasting these mice for
96 h (23) or feeding them a diet deficient in choline for 15 days
(24). Immunoblotting failed to reveal PPAR
and aP2 protein in fatty
livers induced by starvation or by a choline-deficient diet, but these
two proteins appeared prominent in the fatty liver induced by PPAR
1
overexpression (Fig. 3B). These observations clearly
demonstrate that hepatic fatty change itself is not enough to induce
either PPAR
or its target gene aP2, adipsin, and adiponectin and
that PPAR
1 overexpression leads to a novel type of adipogenic
hepatic steatosis designated hepatic adiposis to distinguish it from
the common hepatic steatosis.

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Fig. 4.
Histological evidence of hepatic
steatosis in
PPAR /
mice. A, fatty liver following 4 days of starvation.
B, fatty liver after 2 weeks of feeding a diet deficient in
choline. C, hepatic steatosis in Ad/PPAR 1-injected mouse.
Insets show high magnification of boxed areas.
Note the presence of steatohepatitis in choline-deficient liver.
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Gene Expression Profiling--
To investigate whether PPAR
1
overexpression in liver indeed leads to adipogenic transdifferentiation
of hepatocytes, we performed global transcriptional profiling using RNA
isolated from liver 6 days after injection of Ad/PPAR
1 into
PPAR
/
mice. Biotin-labeled RNA probes from either
Ad/PPAR
liver or Ad/LacZ liver were hybridized to Affymatrix
microarray chips containing 12,000 genes. When a 4-fold change is used
as the cut off for either up- or down-regulation, we found that 184 genes were up-regulated and 87 were down-regulated in
Ad/PPAR
1-overexpressing livers. Table
I lists transcripts displaying a 6-fold
or greater increase in liver as a result of overexpression of PPAR
1.
A majority of these genes participate in adipogenic differentiation or
lipid metabolism indicating that the observed lipid accumulation in hepatocytes reflects transformation/transdifferentiation of hepatocytes toward adipocytes. Adipocyte marker genes adipsin (33), aP2 (34, 35),
adiponectin (36, 37), and caveolin-1 (38, 39) were increased to
~137-, 66-, 48-, and 28-fold, respectively. E-FABP (40), FSP27 (41),
CYP4A10 and CYP4A14 (25), hormone-sensitive lipase (42), monoglyceride
lipase (43), and others involved in lipid metabolism were induced
>10-fold in PPAR
1-overexpressing livers. Three apoptosis-associated
genes, namely cell death-inducing DNA fragmentation factor (CIDE) (44),
cyclophilin C (45), and nur77 (46, 47) were increased to ~101-, 33-, and 15-fold, respectively, suggesting that induction of the adipogenic
differentiation program in liver augments hepatocyte apoptosis.
Northern analysis confirmed marked increases in the levels of
cavelolin-1, CIDE-A, and nur77 mRNAs in liver beginning about 3 days after Ad/PPAR
1 injection (Fig.
5). Previously, we found up-regulation of
CIDE in the liver following treatment with Wy-14,643, a PPAR
ligand (43). However, since CIDE was not up-regulated in mice lacking straight
chain fatty acyl-CoA oxidase, which exhibit spontaneous PPAR
activation, we concluded that induction of CIDE may not be dependent
upon PPAR
(43). Further studies are needed to ascertain the
mechanisms and implications of CIDE, cyclophilin C, and nur77
up-regulation following overexpression of PPAR
1 in liver. Among the
genes up-regulated between 5- and 10-fold are farnesyl diphosphate
synthetase (~9-fold), diacylglycerol acyltransferase ~8-fold),
pyruvate carboxylase (~7-fold), long chain fatty acyl elongase
(~7-fold), CD36 (~7-fold), adiponutrin-like protein (~7-fold),
acetyl-CoA acyltransferase (~6-fold), dicarboxylate transporter
(~6-fold), and S3-12 gene (~6-fold), among others (Table I). A
majority of these genes participate in lipid metabolism.

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Fig. 5.
Northern blot analysis for caveolin-1,
CIDE-A, and nur77. Total RNA isolated from liver of
PPAR / mice at 2, 3, 4, 5, and 6 days after
Ad/mPPAR 1 injection or 6 days after Ad/LacZ injection. Note marked
increases in caveolin-1 and CIDE-A mRNAs at 5 and 6 days after
Ad/mPPAR 1 injection. Increases in nur77 mRNA are also
evident.
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Table II lists the genes that were
down-regulated 6-fold or higher in the liver with PPAR
1
overexpression. These include genes encoding for interferon-induced
protein with tetratricopeptide repeats 1 and 2, interferon-induced
15-kDa protein, interferon-
-induced 47-kDa protein, and
asialoglycoprotein receptor among others.
Endogenous PPAR
Expression--
We have ascertained that forced
expression of PPAR
1 does not up-regulate endogenous PPAR
(Fig.
6). PPAR
2 sense primer was designed to
include the N-terminal region of PPAR
2 to distinguish it from
endogenous PPAR
1. For ectopic PPAR
1 the sense primer was from the
cytomegalovirus promoter. The RT-PCR data show no increase in
endogenous PPAR
1 or PPAR
2 mRNA in the liver 6 days after
Ad/PPAR
1 injection.

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Fig. 6.
RT-PCR to determine the endogenous and
ectopic PPAR mRNA level in liver. RNA
from liver of mice 6 days after injecting Ad/LacZ or Ad/PPAR 1 was
analyzed for endogenous PPAR 1 (Endo-PPAR 1), and
endogenous PPAR 2 (Endo-PPAR 2) and for ectopic PPAR 1
(Viral PPAR ). -Actin served as internal control. A
slight increase in endogenous PPAR in Ad/LacZ livers is attributed
to subtle inflammatory response.
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|
 |
DISCUSSION |
The nuclear receptor PPAR
is essential for adipogenesis and
lipid storage (1-4). PPAR
is present in two isoforms, PPAR
1and PPAR
2, generated by alternate promoter usage and splicing (9). PPAR
2 isoform, which is expressed exclusively in adipocytes, plays a
pivotal role in adipocyte differentiation and adipocyte-specific gene
expression, but there is some controversy as to whether PPAR
1 isoform can initiate and sustain adipogenic gene expression (2, 14,
48). Tontonoz et al. (2) reported that both PPAR
1and PPAR
2 could stimulate adipogenesis when introduced into fibroblasts, whereas Ren et al. (48), using engineered zinc finger
repressors to inhibit the expression of PPAR
isoforms, have
concluded that PPAR
1 overexpression exerted no adipogenic effect.
Recently, Mueller et al. (14) using PPAR
null fibroblasts
demonstrated convincingly that both PPAR
1and PPAR
2 isoforms have
intrinsic ability to induce robust adipogenesis. While most of the work on PPAR
-induced adipogenesis involved the use of fibroblast cell lines, the key question as to whether PPAR
1 isoform, which is the
only isoform expressed in liver and many other tissues, can stimulate
fat cell differentiation or transformation of hepatocytes into
adipocytes. In this study, we have shown that overexpression of
PPAR
1 in mouse liver induced adipocyte-specific gene expression as
well as microvesicular steatosis in this organ. Furthermore, we have
demonstrated that hepatic steatosis resulting from PPAR
1 overexpression is associated with adipogenic gene expression, whereas
hepatic steatosis induced by starvation or developing after feeding a
choline-deficient diet failed to stimulate adipogenic gene expression.
Accordingly, we propose that PPAR
overexpression leads to the
development of a novel form of hepatic steatosis, which is designated
adipogenic hepatic steatosis or simply "hepatic adiposis" to
distinguish this entity from the common forms of hepatic steatosis.
Our results showed the expression of adipocyte-specific genes in liver
upon overexpression of PPAR
1 whether or not these mice were treated
with troglitazone. This may be due to the availability of putative
endogenous ligands in liver generated as part of the normal lipid
metabolism (49, 50). Because PPAR
1 is endogenously expressed at a
low level in mouse liver it is unlikely that this receptor exerts any
adipogenic effect under normal physiological conditions. Furthermore,
the relative abundance of PPAR
in normal liver serves as a key
regulator of fatty acid catabolism thereby minimizing the need for
adipogenesis in liver to store lipids (11, 30). In that sense
functionally active PPAR
and fatty acid oxidation systems keep the
PPAR
1 in check (30). Our results clearly establish that the
overexpression of PPAR
1 isoform in liver in PPAR
null background
triggers the expression of adipogenesis-related genes and fatty change
in liver. This process essentially represents conversion of hepatocytes
into cells with active adipogenic gene expression profile, and in that
regard this represents transformation or transdifferentiation of
hepatocyte into adipocytes, i.e. the conversion of
fat-burning hepatocytes into fat storage cells (31, 32, 51). It is
important to note that the expression of adipocyte-specific genes and
microvesicular steatosis observed in liver is not associated with
overexpression of the endogenous PPAR
gene. RT-PCR analysis showed
no induction of endogenous PPAR
1 or PPAR
2 isoforms (Fig. 6). Thus
all the changes observed in gene expression patterns and
morphological changes are the result of the exogenous
expression of adenovirally driven PPAR
1 overexpression. These
results suggest that maintenance of low levels of PPAR
in liver is
crucial for preventing hepatocytes from encountering an adipogenesis
fate. The maintenance of marginal PPAR
1 gene expression in liver may be achieved by unknown mechanisms designed to prevent positive feedback
between C/EBP
and PPAR
because the downstream adipogenic events
could manifest successfully in the presence of abundant PPAR
protein
(4).
We performed Northern analysis and global transcriptional profiling to
define the pattern of genes expressed in liver as a result of PPAR
1
overexpression. Our studies indicate that overexpression of PPAR
1
isoform in liver results in the up-regulation of many genes known to be
up-regulated during adipocyte differentiation of 3T3-L1 fibroblasts
(31, 32, 52-54). Adipogenesis genes up-regulated in liver include
PPAR
target genes adipsin, aP2, adiponectin, caveolin, fat-specific
protein 27, and others (Table I). Caveolae and caveolin-1 protein
expression are most abundant in adipocytes (39, 55). The overexpression
of caveolin-1 mRNA in liver expressing PPAR
is further
indication of adipogenic transformation of hepatocytes in that
caveolin-1 appears to participate in facilitating the conversion of
triglycerides in lipoprotein form to triglycerides in lipid droplet
storage from (39). Caveolin-1 appears functionally necessary to
maintain lipid droplet integrity, and the absence of this protein in
caveolin-1-deficient mice leads to abnormalities in adipocyte function
(39). Increase in caveolin-1 gene expression in PPAR
1-overexpressing
livers is further indication of attaining an adipocyte phenotype. In
addition, numerous adipocyte-enriched genes involved in lipogenesis and
lipid metabolism, in particular E-FABP (keratinocyte lipid binding
protein) (40), cyp4a10, cyp4A14 (25), fasting-induced adipose factor
(angiopoietin-like 4) (56, 57), CD36 (58), glycerophosphate
dehydrogenase (52), and hormone-sensitive lipase (42), were markedly
up-regulated in liver following PPAR
1 overexpression. Thus, our
in vivo observations clearly establish the adipogenic
conversion of liver when PPAR
1 is overexpressed in this organ.
In this study, we noted marked up-regulation of three genes that have
recently been shown to participate in apoptosis. These include CIDE-A
(44), cyclophilin C (45), and nur77 (46, 47). CIDEs belong to a novel
family of recently identified cell death-inducing proteins that induce
apoptosis when overexpressed (59). CIDE is not normally expressed in
liver, but increased levels of CIDE mRNA have been noted in livers
of mice that were treated with Wy-14,643, a PPAR
ligand (43).
However, the CIDE expression was not dependent upon PPAR
(43). The
functional implications of up-regulation of three apoptotic genes in
liver with adipogenic hepatic steatosis suggests that the acquisition of adipogenic phenotype places hepatocytes at greater risk for apoptosis. Additional studies are needed to ascertain if the CIDE, cyclophilin C, and nur77 genes are PPAR
targets for regulation. Studies are also needed to ascertain if DNA synthesis is a prerequisite for the adipogenic conversion of hepatocytes.
It is of interest that this new form of hepatic adiposis or adipogenic
hepatic steatosis resulting from PPAR
1 overexpression may have
potential clinical implications. Individuals with relatively low
hepatic levels of PPAR
and hyperactive PPAR
1 resulting from single nucleotide polymorphism or by some other mechanism could develop
adipogenic hepatic steatosis similar to that described in this report.
It is also worth noting that ob/ob mice exhibit increased levels of
PPAR
mRNA in their livers (60). Increased expression levels of
aP2 and CD36 mRNA were seen in these livers when these mice were
given PPAR
ligand troglitazone (61). Thus, it is important to
consider PPAR
1 overexpression or hyperactivity as a possible
molecular mechanisms responsible for a special type of non-alcoholic
hepatic steatosis, designated as hepatic adiposis or adipogenic hepatic steatosis.
Finally, we have identified a number of novel genes as modestly
up-regulated in PPAR
1-overexpressing liver, which remain to be
characterized (Table I). This includes a gene we designated as
promethin (AY167031), which is induced in liver by PPAR
overexpression. Additional studies are needed to further characterize
these and other genes to gain appreciation of the role of PPAR
in
liver in relation to glucose homeostasis, energy utilization, and
insulin resistance (61).
We thank Dr. Pradip Raychaudhuri of the
University of Illinois School of Medicine for the nur77 cDNA.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M210062200
The abbreviations used are:
PPAR, peroxisome
proliferator-activated receptor;
PPRE, peroxisome proliferator response element(s);
L-PBE, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA
dehydrogenase bifunctional protein;
Glc-6-P, glucose-6-phosphatase;
C/EBP
, CCAAT enhancer-binding protein
;
SREBP1, sterol regulatory
element-binding protein 1;
aP2, adipose fatty acid-binding protein;
RT, reverse transcriptase.
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