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Unitat de Farmacologia, Department de Farmacologia i Química Terapèutica, Facultat de Farmàcia, Universitat de Barcelona, Barcelona, Spain
Received December 24, 2002; accepted June 13, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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(PPAR), affected the expression
of several of these genes. A single-day troglitazone administration (100
mg/kg/day) did not significantly alter plasma free fatty acids or triglyceride
levels. In contrast, a 10-day period of troglitazone treatment significantly
reduced plasma free fatty acids and triglyceride levels by 74% (P
< 0.001) and 56% (P < 0.01), respectively. Cardiac mRNA
expression of acyl-CoA oxidase (ACO) increased (8.3-fold induction) after
1-day troglitazone treatment, whereas after 10 days of treatment ACO mRNA
levels were dramatically reduced (98% reduction, P < 0.02), as
well as those of uncoupling protein 3 (41% reduction, P = 0.05). The
mRNA expression of PPAR
and several PPAR target genes, such as medium
chain acyl-CoA dehydrogenase or fatty acid translocase were not altered after
10 days of troglitazone treatment, whereas muscle-type carnitine
palmitoyltransferase I increased 1.7-fold (P < 0.05). The
reduction in ACO expression in the hearts of 10-day troglitazone-treated mice
was accompanied by an increase in the protein levels of the transcriptional
repressor chicken ovalbumin upstream promoter transcription factor II (COUP-TF
II). Electrophoretic mobility shift assays performed with COUP-TF II antibody
to examine its interaction with a labeled peroxisome proliferator response
element probe showed enhanced binding of COUP-TFII in cardiac nuclear extracts
from troglitazone-treated mice for 10 days but not in the control nuclear
extracts. Overall, the findings presented here show that 10 days of
troglitazone treatment decreased expression of the ACO gene through a
mechanism involving the transcriptional repressor COUP-TF II.
). The heart, in contrast to other tissues such as the brain,
adapts its metabolism to substrate availability. For example, in cardiac
hypertrophy and heart failure, a dramatic reduction in fatty acid oxidation is
detected, because a shift in the source of energy is observed from fatty acids
to glucose (Van Bilsen et al.,
1998). In contrast, diabetes induces a rapid change in substrate
preference of the heart characterized by a decrease in glucose uptake and
utilization, and an increase in fatty acid oxidation
(Stanley et al., 1997). In
these processes, the adjustments of cardiac metabolism to the substrate
availability seem to involve changes in the transcriptional control of genes
implicated in the transport and metabolism of fatty acids and glucose.
Transport and metabolism of these substrates are controlled by a class of
transcription factors called peroxisome proliferator-activated receptors
(PPARs). Three different PPAR subtypes (, 
/
, and )
have been identified to date. PPAR is expressed primarily in tissues
that have a high level of fatty acid catabolism such as liver, brown fat,
kidney, heart, and skeletal muscle
(Braissant et al., 1996;
Desvergne and Wahli, 1999).
PPAR/ is ubiquitously expressed, and PPAR has a restricted
pattern of expression, mainly in white and brown adipose tissues, whereas
other tissues such as skeletal muscle and heart contain limited amounts
(Braissant et al., 1996). PPARs
are activated by ligands, such as naturally occurring fatty acids, which are
activators of all three PPAR subtypes
(Kliewer et al., 1997;
Krey et al., 1997). In
addition to fatty acids, several synthetic compounds bind and activate
specific PPAR subtypes. Thiazolidinediones (troglitazone, ciglitazone, and
rosiglitazone), which bind and selectively activate PPAR
(Forman et al., 1995;
Lehmann et al., 1995), are a
novel class of insulin-sensitizing agents with potent antidiabetic activity in
vivo. To be transcriptionally active, PPARs need to heterodimerize with the
retinoid-X-receptor (RXR). PPAR-RXR heterodimers binds to DNA-specific
sequences called peroxisome proliferator-response elements (PPRE), consisting
of an imperfect direct repeat of the consensus binding site for nuclear
hormone receptors (AGGTCA) separated by one nucleotide (DR-1). This direct
repeat is known to bind potential competitors such as homodimers of other
nuclear receptors, including RXR, chicken ovalbumin upstream promoter
transcription factor II (COUP-TF-II, also called apolipoprotein A1 regulatory
protein) and hepatic nuclear factor-4
(Desvergne and Wahli,
1999).
Recently, it has been reported that cardiac hypertrophic growth, which
involves a shift in the substrate utilization from fatty acids to glucose, is
associated with deactivation of PPAR
(Barger et al., 2000). These
results suggest that reduced activity of this transcription factor may account
for the down-regulation of enzymes involved in fatty acid oxidation.
Similarly, in a previous study (Cabrero et
al., 2000) we reported that in C2C12 myotubes, cultured in the
presence of glucose but in the absence of fatty acids, troglitazone strongly
repressed the mRNA expression of genes involved in fatty acid oxidation. The
reduction in the expression of these genes correlated with decreased
PPAR expression after thiazolidinedione treatment, suggesting that the
impaired expression of this transcription factor was involved in the effects
of these drugs. It is noteworthy that in the presence of oleic acid in the
culture medium, the effects of troglitazone on PPAR mRNA levels were
reversed. Here, we examine whether troglitazone induces similar changes in the
heart of mice. Drug administration for 1 day strongly increased acyl-CoA
oxidase (ACO) mRNA levels in heart, whereas after 10 days of treatment, when a
fall in plasma values of free fatty acids and triglycerides was detected, a
dramatic reduction in its transcript levels was observed. Interestingly,
troglitazone treatment strongly increased DNA-protein binding activity of
cardiac nuclear proteins to a PPRE probe in gel mobility shift assays after 10
days but not after 1 day of troglitazone treatment. In addition, drug
treatment for 10 days strongly increased COUP-TF II protein levels in heart,
suggesting that increased amounts of this known transcriptional repressor were
responsible for the reduced expression of the ACO gene. In fact, antibody
supershift studies showed enhanced binding of COUP-TF II to the PPRE probe in
the specific complexes from 10-day troglitazone-treated mice. Overall, the
results presented here suggest that COUP-TF II is involved in the reduction of
ACO expression in hearts of troglitazone-treated mice.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Animals and Treatment. Twenty male Swiss mice from Harlan (Barcelona, Spain) were used. They were maintained under standard conditions of illumination (12-h light/dark cycle) and temperature (21 ± 1°C) and fed a standard diet. The mice were randomly distributed into two groups. Each group was administered, respectively, either 0.5% carboxymethyl cellulose (control group) or 100 mg/kg/day of troglitazone (dissolved in 0.5% carboxymethyl cellulose). Each compound was administered per os once a day for 1 or 10 days (1 ml/100 g of body weight). Food and water were given ad libitum. Twenty-four hours after the last administration, mice were killed under pentobarbitone anesthesia to collect blood samples and to isolate hearts. Blood samples, obtained by cardiac puncture, were collected in EDTA tubes and plasma was obtained by centrifugation at 2,200g for 10 min at 4°C. Hearts were rapidly removed, washed in ice-cold 0.9% NaCl, frozen in liquid nitrogen, and stored at –80°C. Animal handling and disposal were performed in accordance with law 5/1995, 21 July, of the Generalitat de Catalunya.
Plasma Determinations. Plasma cholesterol (Roche Diagnostics, Barcelona, Spain), triglycerides (Sigma, Madrid, Spain), nonesterified fatty acids (Wako Pure Chemicals, Neuss, Germany), and glucose (Roche Diagnostics) concentration were determined by colorimetric tests.
RNA Preparation and Analysis. Total RNA was isolated by using the Ultraspec reagent (Biotecx, Houston, TX). Relative levels of specific mRNAs were assessed by the reverse transcription (RT)-polymerase chain reaction. Complementary DNA was synthesized from RNA samples by mixing 0.5 µg of total RNA, 125 ng of random hexamers as primers in the presence of 50 mM Tris-HCl buffer, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 200 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA), 20 U of RNAsin (Invitrogen), and 0.5 mM each dNTP (Sigma-Aldrich, St. Louis, MO) in a total volume of 20 µl. Samples were incubated at 37°C for 60 min. A 5-µl aliquot of the RT reaction was then used for subsequent PCR amplification with specific primers.
Each 25-µl PCR reaction contained 5 µl of the RT reaction, 1.2 mM
MgCl2, 200 µM dNTPs, 1.25 µCi of [32P]dATP (3,000
Ci/mmol; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK),
1 unit of Taq polymerase (Ecogen, Barcelona, Spain), 0.5 µg of
each primer, and 20 mM Tris-HCl, pH 8.5. To avoid unspecific annealing, cDNA
and Taq polymerase were separated from primers and dNTPs by using a
layer of paraffin (reaction components contact only when paraffin fuses, at
60°C). The sequences of the sense and antisense primers used for
amplification are shown in Table
1. PCR was performed in a thermocycler (MJ Research, Watertown,
MA) equipped with a peltier system and temperature probe. After an initial
denaturation for 1 min at 94°C, PCR was performed for 18 (ANF), 20 (MCAD
and FAT/CD36), 23 (PGC-1), 25 (UCP-3 and PPAR), 26 (RXR and
RXR), 27 (CTE), and 28 (M-CPT-I and ACO) cycles. Each cycle consisted
of denaturation at 92°C for 1 min, primer annealing at 60°C, and
primer extension at 72°C for 1 min and 50 s. A final 5-min extension step
at 72°C was performed. Five microliters of each PCR sample was
electrophoresed on a 1-mm-thick 5% polyacrylamide gel. The gels were dried and
subjected to autoradiography using Kodak X-ray film to show the amplified DNA
products. Amplification of each gene yielded a single band of the expected
size (ANF, 271 bp; PPAR, 645 bp; M-CPT-I, 222 bp; MCAD, 216 bp; UCP-3,
179 bp; ACO, 195 bp; FAT/CD36, 256 bp; CTE, 224 bp; RXR, 202 bp;
RXR, 220 bp; PGC-1, 228 bp; and APRT, 329 bp). Preliminary experiments
were carried out with various amounts of cDNA to determine nonsaturating
conditions of PCR amplification for all the genes studied. Thus, cDNA
amplification was performed in comparative and semiquantitative conditions
(Freeman et al., 1999).
Radioactive bands were quantified by video-densitometric scanning (Vilbert
Lourmat Imaging, Marne-de-Vallee, France). The results for the expression of
specific mRNAs are always presented relative to the expression of the control
gene (aprt).
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Isolation of Nuclear Extracts. Crude nuclear extracts were isolated
using the Dignam method (Dignam et al.,
1983) with the modifications described by Helenius et al.
(1996). Frozen hearts were
weighed, transferred to Corning tubes, and ice-cold hypotonic buffer (1.5 mM
MgCl2, 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT, and 10 mM HEPES, pH 7.9)
was added to each sample. The volume was proportional to the weight of the
tissue, so as to give 15% homogenates. The tissues were left to thaw in an ice
bath and homogenized (2 x 5 s) using a Polytron homogenizer (Kinematica,
Basel, Switzerland). Homogenates were incubated for 10 min on ice and
centrifuged (25,000g, 15 min, 4°C). Pellets were washed once with
the same volume of hypotonic buffer used in the homogenization step and
centrifuged (10,000g,4°C, 15 min). Supernatants were discarded
and pellets were suspended in ice-cold low salt buffer (25% glycerol, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT, 20 mM KCl, and 20 mM
HEPES, pH 7.9) using half of the volume of the hypotonic buffer. Nuclear
proteins were released by adding high salt buffer (25% glycerol, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT, 1.2 M KCl, and 20 mM
HEPES, pH 7.9) drop by drop using half of the volume of the low salt buffer.
Samples were incubated on ice for 30 min. During incubation, the tubes were
smoothly mixed frequently. Samples were centrifuged (25,000g, 30 min,
4°C), and supernatants were collected in Microfuge tubes and stored in
aliquots at –80°C. The protein concentration of the nuclear extracts
was then measured.
Electrophoretic Mobility Shift Assays (EMSAs). EMSAs were performed
using double-stranded oligonucleotides (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) for the consensus binding sites of PPRE
(5'-CAAAACTAGGTCAAAGGTCA-3'), mutant PPRE
(5'-CAAAACTAGCACAAAGCACA-3'), and Oct-1
(5'-TGTCGAATGCAAATCACTAGAA-3'). Oligonucleotides were labeled in
the following reaction: 1 µl of oligonucleotide (20 ng/µl), 2 µl of
5x kinase buffer,5UofT4 polynucleotide kinase, and 3
µlof[-32P]ATP (3,000 Ci/mmol at 10 mCi/ml) incubated at
37°C for 1 h. The reaction was stopped by adding 90 µl of TE buffer (10
mM Tris-HCl, pH 7.4, and 1 mM EDTA). To separate the labeled probe from the
unbound ATP the reaction mixture was eluted in a Nick column (Pharmacia, Sant
Cugat, Spain) according to the manufacturer's instructions. Eight micrograms
of crude nuclear proteins was incubated for 15 min on ice in binding buffer
[10 mM Tris-HCl, pH 8.0, 25 mM KCl, 0.5 mM DTT, 0.1 mM EDTA, pH 8.0, 5%
glycerol, 5 mg/ml bovine serum albumin, 100 µg/ml tRNA, and 50 µg/ml
poly(dI-dC)], in a final volume of 15 µl. Labeled probe (approximately
50,000 cpm) was added and the reaction was incubated for 20 min at room
temperature. Where indicated, specific competitor oligonucleotide was added
before the addition of labeled probe and incubated for 10 min on ice. For
supershift assays with the PPRE probe, antibodies were added after incubation
with labeled probe for a further 30 min at room temperature. Protein-DNA
complexes were resolved by electrophoresis at 4°C on a 5% acrylamide gel
and subjected to autoradiography. Antibodies against COUP-TF II and PPARs were
from Santa Cruz Biotechnology, Inc.
Western Blot Analysis. Crude nuclear extracts (40 µg) from hearts
were subjected to 10% SDS-polyacrylamide gel electrophoresis. Proteins were
then transferred to Immobilon polyvinylidene diflouride transfer membranes
(Millipore, Bedford, MA), and immunological detection was performed using a
goat polyclonal antibody raised against COUP-TF II (dilution 1:1,000).
Detection was achieved using the enhanced chemiluminescence detection system
(Amersham Biosciences UK, Ltd.). Blots were also incubated with a rabbit
antibody against -tubulin (dilution 1:5,000) (Roche Diagnostics,
Mannheim, Germany), used as a control of equal abundance of nuclear extracts
in the samples. Size of detected proteins was estimated using protein
molecular mass standards (Invitrogen).
Statistical Analyses. Results are usually expressed as means ± S.D. of four or five mice. Significant differences were established by Student's t test using the computer program Instat (GraphPad Software Inc., San Diego, CA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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),
confirming the efficacy of the troglitazone treatment. Treatment with
troglitazone did not modify either body weight or the ratio of heart weight to
body weight, compared with the control group. However, when we determined the
expression of the hypertrophic growth marker gene, ANF, we observed a 6.5-fold
induction (P < 0.05) in the heart of 10-day troglitazone-treated
mice, indicating the presence of cardiac hypertrophy
(Fig. 1B).
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Troglitazone Differently Modifies ACO mRNA Levels in Heart after 1 or 10
Days of Treatment. We first examined the effects of troglitazone for 1 day
on the mRNA levels of ACO and MCAD genes. ACO catalyzes the rate-limiting step
of peroxisomal -oxidation of fatty acids and its transcription is
controlled by PPAR (Desvergne and
Wahli, 1999). MCAD is also a PPAR-target gene that
catalyzes a rate-limiting step in the mitochondrial -oxidation of
medium-chain fatty acyl-thioesters
(Desvergne and Wahli, 1999). In
the heart of 1-day troglitazone-treated mice the mRNA levels of ACO seemed
increased (8.3-fold induction, P < 0.05) in relation to control
mice (Fig. 2A). In contrast,
MCAD transcript levels were not modified by a single administration of
troglitazone. We also studied the effects of this drug on CTE, which catalyzes
the hydrolysis of acyl-CoAs to free fatty acids and CoA, and so it is an
important mediator in cellular processes regulated by intracellular levels of
nonesterified fatty acids and acyl-CoAs. In the heart of 1-day
troglitazone-treated mice the mRNA levels of CTE were not modified. These
findings indicate that, although PPAR is expressed at low levels in
heart compared with adipose tissue
(Braissant et al., 1996,
Vidal-Puig et al., 1997), in
vivo administration of troglitazone increases ACO mRNA levels, confirming
previous results showing that troglitazone induced transcriptional activity of
neonatal cardiac myocytes transfected with a (ACO-PPRE)3-TK-Luc
reporter plasmid (Takano et al.,
2000). In contrast, MCAD and CTE showed a different behavior after
troglitazone treatment, suggesting that the activity of PPAR may depend
on the nature of the PPRE, as suggested previously
(Marcus et al., 1993). When we
examined the expression of ACO in the heart of 10-day troglitazone-treated
mice, a dramatic reduction was observed (98% reduction, P < 0.02)
(Fig. 2B) compared with control
mice. In contrast, troglitazone treatment did not affect the mRNA expression
of either MCAD or CTE. Similarly, the expression of the fatty acid translocase
(FAT/CD36), a membrane-associated protein that facilitates the uptake of
long-chain fatty acids into cells and is up-regulated after PPAR
activation (Desvergne and Wahli,
1999), was not modified by 10-day troglitazone treatment
(Fig. 2B). Muscle-type
carnitine palmitoyl-transferase (M-CPT-I), a PPAR-target gene
(McGarry and Brown, 1997;
Mascaró et al., 1998)
that catalyzes the entry of long-chain fatty acids into the mitochondrial
matrix (McGarry and Brown,
1997), was significantly increased (1.7-fold induction, P
< 0.05) after troglitazone treatment for 10 days in relation to control
animals. Interestingly, the expression of uncoupling protein 3 (UCP-3), a
mitochondrial carrier localized in the inner mitochondrial membrane, which has
been implicated in fatty acid utilization and it is a PPAR target-gene
(Ricquier and Bouillad, 2000),
was reduced (41%, P = 0.05) after troglitazone treatment
(Fig. 2B). Given the important
role of PPAR in the control of cardiac lipid metabolism
(Djouadi et al., 1999) and the
previous results showing down-regulation of this transcription factor after
troglitazone treatment in C2C12 myotubes
(Cabrero et al., 2000), we
finally studied whether the effects of troglitazone treatment on ACO
expression were mediated by reduced expression of this transcription factor.
The mRNA amounts of this PPAR subtype were not modified after 10 days of
troglitazone treatment (Fig.
2B), suggesting that changes in PPAR expression were not
responsible for the effects of troglitazone. Similarly, no changes were
observed in PPAR transcript levels in heart after troglitazone
treatment for either 1 or 10 days (data not shown).
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Effects of Troglitazone Treatment for 10 Days on RXR and PGC-1 mRNA
Levels in Heart. To study whether reduced availability of the PPAR
heterodimeric partner RXR was responsible for the reduced transcriptional
activity of the ACO gene in 10 days troglitazone-treated mice, we determined
the transcript levels of RXR and RXR. Troglitazone treatment for
10 days did not modify the mRNA expression of these transcription factors in
heart compared with control mice (Fig.
3). In addition, we studied the mRNA expression of PGC-1, which
directly interacts with PPAR, and it has been postulated as a regulator
of mitochondrial -oxidation (Vega et
al., 2000). PGC-1 mRNA was not altered in the heart of 10-day
troglitazone-treated mice. All these findings suggest that RXR and PGC-1 are
unlikely to be involved in the changes observed after troglitazone treatment
for 10 days.
|
DNA Binding Activity of Cardiac Nuclear Proteins to a PPRE Probe Was Induced in 10-Day but Not in 1-Day Troglitazone-Treated Mice. EMSAs were performed to examine the interaction of PPARs with its cis-regulatory element using a 32P-labeled PPRE probe and cardiac nuclear extracts from control and troglitazone-treated mice for 1 or 10 days. The PPRE probe formed four complexes with cardiac nuclear proteins (Fig. 4A, complexes I to IV). Competition studies performed with a molar excess of unlabeled probe revealed that the four complexes represented specific PPRE-protein interactions. These results suggest that several endogenous cardiac nuclear proteins bind to PPRE. In nuclear extracts from hearts of 1-day troglitazone-treated mice no significant changes were observed in DNA binding activity to the PPRE probe compared with control animals (Fig. 4B). In contrast, despite the lack of induction in the transcriptional rate of PPAR-target genes after troglitazone treatment for 10 days, this drug increased the binding of cardiac proteins to the PPRE cis-element (Fig. 4C), resulting in an increase of specific complexes, mainly of complex II. No changes were observed in the DNA binding of cardiac proteins from control and troglitazone-treated mice to an Oct-1 probe, indicating that the increase observed for the PPRE probe was specific (Fig. 4D).
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Supershift studies performed using antibodies against PPAR,
PPAR/, and PPAR (Fig.
4, E–G) demonstrated that the incubation mixtures contained
the three different PPAR subtypes.
Troglitazone Treatment Increases COUP-TF II Protein Levels in Heart.
The fall in the transcriptional rate of the ACO gene together with the
increased interaction of the PPRE probe with cardiac proteins in
troglitazone-treated mice suggests that drug treatment induces the expression
of a transcriptional repressor in the heart. Given that the DR-1-type present
in the PPRE is capable of interacting in vitro with multiple nuclear
receptors, including homodimers of the transcriptional repressor COUP-TF
(Desvergne and Wahli, 1999), we
determined whether expression of COUP-TF II parallels the increased
interaction of the PPRE probe in troglitazone-treated mice. Protein levels of
COUP-TF II were assessed by the Western blot technique. Nuclear levels of
COUP-TF II were only slightly increased after 1 day of troglitazone treatment
compared with control animals (Fig.
5A). However, after 10 days of troglitazone treatment a strong
induction in COUP-TF II protein levels in heart was observed compared with
control mice samples (Fig. 5B).
These findings show that through the time course of troglitazone treatment,
there is a good correlation between the fall in plasma lipids and the
induction in COUP-TF II protein levels.
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Increased Binding of the Transcriptional Repressor COUP-TF II to a PPRE Probe in Cardiac Nuclear Extracts of 10-Day Troglitazone-Treated Mice. In EMSAs performed with a PPRE oligonucleotide and cardiac nuclear extracts from control and treated mice for 10 days, a reduction in the intensity of complex II was observed in the presence of COUP-TF II antibody, indicating enhanced levels of COUP-TF bound to the PPRE probe in cardiac nuclear extracts from troglitazone-treated mice (Fig. 6). This finding supports a role for this transcriptional repressor in the changes observed after troglitazone treatment for 10 days.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). We
have recently reported that thiazolidinediones down-regulate the expression of
genes involved in fatty acid oxidation in C2C12 myotubes cultured in the
presence of glucose but in the absence of exogenous fatty acids
(Cabrero et al., 2000). These
changes correlated well with impaired expression of PPAR and were
reverted by addition of fatty acids to the culture medium, suggesting that
different fuel substrate utilization modifies the regulation of the fatty acid
oxidation system. In the normoglycemic mice used in this work troglitazone
treatment for 10 days, but not for 1 day, significantly reduced plasma free
fatty acids and triglycerides, whereas glucose levels were not altered. Thus,
10 days of troglitazone treatment induced a change in plasma substrate
availability, resulting in a high ratio of glucose to fatty acids. Under these
conditions, troglitazone caused a dramatic reduction in ACO mRNA levels,
without changes in the expression of PPAR. The reduction in ACO
expression after 10 days of troglitazone treatment was accompanied by
increased protein levels of COUP-TF II, suggesting that this
PPAR-transcriptional repressor was involved.
According to previous studies (Tsai and
Tsai, 1997), the mechanism by which COUP-TF antagonizes PPAR
signaling involves competitive occupation of the DR-1 present in the PPRE. We
observe increased binding of COUP-TF to the PPRE cis-element,
suggesting a role for this transcription factor in the effects caused by
troglitazone. However, different effects were observed after troglitazone
treatment in ACO and MCAD genes. The expression of the former was nearly
abolished by troglitazone, whereas the second was not modified by the
treatment. The data here presented do not allow us to know the reasons for the
lack of effect of troglitazone on MCAD compared with ACO expression. However,
differences in the nature of their PPRE could be involved. In fact, it has
been reported that the PPREs containing two divergent half sites in the DR-1,
such as that present in PPRE-MCAD, are less able to bind the transcriptional
repressor COUP-TF II compared with the ACO-PPRE
(Palmer et al., 1995).
Likewise, the expression of MCAD is strongly reduced in the heart of
PPAR knockout mice, whereas ACO expression was not affected
(Watanabe et al., 2000),
suggesting that MCAD expression strongly depends on PPAR expression.
Therefore, the PPRE present in the MCAD gene may be more specific for
PPAR-RXR than the PPRE present in the ACO gene. In cardiac
hypertrophy induced by pulmonary artery banding the reduction in MCAD is
accompanied by an increase in the expression of COUP-TF II and a strong
reduction in the expression of PPAR
(Sack et al., 1997). In our
study, we have observed an induction in the expression of COUP-TF II, whereas
the expression of PPAR at the mRNA level was unchanged. Therefore,
given the strong dependence of MCAD on PPAR levels, the presence of
normal levels of PPAR in hearts of troglitazone-treated mice may be
responsible for the unchanged MCAD expression.
Alternative mechanisms not directly involving PPAR may also participate in
the regulation of ACO gene expression after troglitazone treatment. Thus, it
is likely that the enhanced sensitivity to insulin caused by troglitazone may
contribute to the reduction in ACO expression without affecting other
PPAR-target genes. This is supported by the fact that peroxisomal fatty acid
oxidation is inhibited at a much lower insulin concentration that is
mitochondrial oxidation (Hamel et al.,
2001).
In contrast to ACO and MCAD, M-CPT-I expression was increased by 10-day
troglitazone treatment. Although the up-regulation of this gene may be
mediated through PPAR activation by troglitazone, the lack of effect of
troglitazone on the well known PPAR target gene FAT/CD36 make this
possibility unlikely. On the other hand, it is well known that increased
glucose entry to skeletal myocytes and white adipocytes, increases the
concentration of malonyl-CoA, a known inhibitor of CPT-I
(McGarry et al., 1989).
Because troglitazone increases glucose utilization it is likely that
upregulation of M-CPT-I was a compensatory mechanism for its inhibition caused
by malonyl-CoA, similar to the increased expression of M-CPT-I in heart after
treatment with the CPT-I inhibitor etomoxir
(Brandt et al., 1998). In
addition, it has been recently reported that troglitazone treatment results in
a down-regulation in heart of the mRNA of malonyl-CoA decarboxylase, the gene
that catalyzes the degradation of malonyl-CoA
(Young et al., 2001).
Therefore, it is likely that the decreased malonyl-CoA decarboxylase
expression may also contribute to the up-regulation of M-CPT-I, as a mechanism
to compensate inhibition of this enzyme by malonyl-CoA. Finally, the reduction
in plasma free fatty acids levels after troglitazone treatment may account for
the fall in UCP-3 transcripts, as reported previously
(Ricquier and Bouillad,
2000).
In the present study, troglitazone treatment for 10 days caused cardiac
hypertrophy, in agreement with previous studies showing that all
thiazolidinediones have been associated with cardiac hypertrophy in animal
studies, at doses far exceeding those recommended for therapeutic use
(Ghazzi et al., 1997). In
addition, it has been reported that although troglitazone does not initiate
hypertrophy, it can sensitize cardiomyocytes to growth effects of serum
(Bell and McDermott, 2000),
indicating that the actions of this drug may promote changes favoring
increased growth of these cells under some circumstances. In contrast, recent
studies have shown that thiazolidinediones inhibit cardiac hypertrophy
(Yamamoto et al., 2001,
Asakawa et al., 2002). These
differences could be attributed to the different concentrations of
thiazolidinediones used. Thus, it is well known that at low concentrations
PPAR activators antagonize the activity of NF-
B
(Desvergne and Wahli, 1999),
whereas in our study we found increased NF-B activation (A. Cabrero, M.
Vázquez-Carrera, unpublished data) in hearts after 10 days of
troglitazone treatment. Because NF-B activation is required for the
hypertrophic growth of cardiomyocytes
(Purcell et al., 2001), the
different effects of thiazolidinediones on cardiac hypertrophy may depend on
the concentration used.
It is still a matter of controversy whether changes in intracellular
substrate and metabolite levels in cardiomyocytes are the consequence or the
reason for the shift of cardiac metabolism from fatty acids to glucose
observed in cardiac hypertrophy (Van
Bilsen et al., 1998). In fact, an increase in the activities of
several glycolytic enzymes has been reported before cardiac hypertrophy
(Taegtmeyer and Overturf,
1998), suggesting that increased glucose metabolism may induce the
metabolic changes observed in cardiac hypertrophy. Because troglitazone
improves insulin responsiveness in skeletal muscle by facilitating glucose
transport activity, which thereby leads to increased rates of muscle oxidation
(Petersern et al., 2000), it
is likely that these changes initiate a shift toward a fetal metabolic pattern
in the heart, which mainly relies on glucose utilization. This explanation
seems not to be in accordance with the absence of a reduction in plasma
glucose levels after 10 days of troglitazone treatment. However, it has been
recently postulated that normoglycemic animals are protected from
troglitazone-induced hypoglycemia by a mechanism that maintains plasma glucose
levels through elevation of both gluconeogenesis and glycogenolysis
(Dea et al., 2000). Therefore,
increased glucose utilization by muscle after troglitazone treatment occurs
without changes in plasma glucose levels in normal animals.
In summary, we report that troglitazone treatment results in the adjustment
of the expression of genes implicated in peroxisomal fatty acid
-oxidation through a mechanism involving the transcriptional repressor
COU-TF II.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor;
RXR, retinoid-X-receptor; PPRE, peroxisome proliferator response element;
COUP-TF II, chicken ovalbumin upstream promoter transcription factor II; ACO,
acyl-CoA oxidase; RT, reverse transcription; PCR, polymerase chain reaction;
ANF, atrial natriuretic factor; M-CPT-I, muscle-type carnitine
palmitoyl-transferase; MCAD, medium chain acyl-CoA dehydrogenase; UCP-3,
uncoupling protein 3; FAT/CD36, fatty acid translocase; CTE, cytosolic
acyl-CoA thioesterase; PGC-1, peroxisome proliferator-activated
receptor- coactivator 1; bp, base pair; PMSF, phenylmethylsulfonyl
fluoride; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay;
NF-B, nuclear factor-B.
Address correspondence to: Dr. Manuel Vázquez-Carrera, Unitat de Farmacologia, Facultat de Farmàcia, Diagonal 643, E-08028 Barcelona, Spain. E-mail: mvaz{at}farmacia.far.ub.es
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