From the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota 55108
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ABSTRACT |
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Fatty acid transport protein (FATP),
a plasma membrane protein implicated in controlling adipocyte
transmembrane fatty acid flux, is up-regulated as a consequence of
adipocyte differentiation and down-regulated by insulin. Based upon the
sequence of the FATP gene upstream region (Hui, T. Y., Frohnert,
B. I., Smith, A. J., Schaffer, J. A., and Bernlohr,
D. A. (1998) J. Biol. Chem. 273, 27420-27429) a
putative peroxisome proliferator-activated receptor response element
(PPRE) is present from Obesity, defined as an excessive accumulation of body fat, has
become an increasingly common health concern in industrialized societies. Excessive adiposity has been linked to the pathogenesis of
many diseases, including type 2 diabetes mellitus, coronary artery
disease, and hypertension. The increased awareness of the detrimental
effects of obesity contributes to the search for a greater
understanding of the molecular mechanisms controlling the accumulation
of adipose tissue and its metabolism.
A central issue in the function of fat tissue is the method by which
adipocytes take up and release fatty acids. This process has been the
source of considerable debate (1-3). Because free fatty acids are
hydrophobic, they freely crossed membranes by passive diffusion.
However, studies in adipocytes, hepatocytes, jejunal enterocytes,
skeletal muscle, and heart myocytes support a saturable,
protein-mediated mechanism for fatty acid transport (4-8). Thus far,
five putative mammalian fatty acid transporters have been identified:
fatty acid-binding protein (plasma membrane) (9), 56-kDa renal fatty
acid-binding protein (10), caveolin (11), fatty acid translocase (12),
and fatty acid transport protein
(FATP)1 (13). FATP is an
integral plasma membrane protein with four to six predicted
membrane-spanning regions with the highest levels found in skeletal
muscle, heart, and fat with lower levels in brain, kidney, lung, and
liver. Although FATP mRNA is present at low levels in 3T3-L1
preadipocytes, it is up-regulated 5-7-fold as a consequence of adipose
conversion (13, 14). This increase is consistent with the increase in
oleic acid uptake shown during preadipocyte differentiation (15).
The differentiation of adipose precursor cells into adipocytes has been
shown to be mediated by three groups of transcription factors:
peroxisome proliferator-activated receptor PPARs constitute a subfamily of the steroid hormone receptor
superfamily. PPAR FATP expression has been shown to be up-regulated in mouse liver by
PPAR Materials--
Troglitazone and Delta Selective C were gifts
from Alan Saltiel (Parke-Davis) and David E. Moller (Merck). WY14643
was purchased from Cayman Chemical. Linoleic acid and
9-cis-retinoic acid were purchased from Biomol. Expression
plasmids prPPAR Plasmid Constructs--
Polymerase chain reaction was used to
introduce NheI sites into the upstream sequence of FATP.
Reporter constructs pNH11 ( Cell Culture and Transfection--
CV-1 cells were cultured in
DMEM with 10% fetal bovine serum (FBS) until the day prior to
transfection experiments, when the cells were plated in phenol red-free
DMEM and 10% charcoal-treated FBS. CV-1 cells were transiently
transfected using the calcium-phosphate method as described previously
(33, 34). Briefly, each well of a 12-well culture plate was transfected
with 1 µg of firefly (Photinus pyralis) luciferase
reporter construct with or without 0.5 µg of expression plasmid for
mouse PPAR
3T3-L1 preadipocytes were grown to confluence and induced to
differentiate to adipocytes as described previously(35). Briefly, preadipocytes were cultured in DMEM and 10% calf serum. Two days after
reaching confluence, cells were differentiated by treatment with 174 nM insulin, 0.5 mM methylisobutylxanthine, and
0.25 µM dexamethasone in DMEM containing 10% FBS. Cells
were treated for an additional 2 days with insulin and maintained in
DMEM with 10% FBS until they are fully mature adipocytes at day 8.
Transient transfection of 3T3-L1 preadipocytes was performed by calcium
phosphate precipitation as described above. Each well was transfected
with 1 µg of firefly luciferase reporter construct and 10 ng of
pRL-SV40 control vector. In the first set of transfections, cells were
maintained in DMEM containing 10% calf serum until transfection
mixture was washed off of cells. Cells were then treated with either
linoleic acid or ethanol for 24 h in serum-free DMEM. For the
second set of preadipocyte transfections, cells were maintained in DMEM
with 10% calf serum for the duration of the experiments. After washing
transfection mixture from cells, they were treated with either a PPAR
activator or its vehicle (Me2SO) for 48 h.
3T3-L1 adipocytes grown in 12-well culture plates were transiently
transfected as described previously (32) with 500 ng of firefly
luciferase construct and 20 ng of pRL-SV40 control vector per well.
Briefly, DNA in DMEM was complexed with LipofectAMINE (Life
Technologies, Inc.) at the ratio of 1:8 (w/v) for 45 min before
addition to the cells. After 4 h, an equal volume of 20% fetal
bovine serum in DMEM was added. The transfection mixture was removed
after 20 h. The cells were then refed with DMEM containing 10%
FBS and treated with either PPAR activator or vehicle for 48 h.
All transiently transfected cells were washed two times with
phosphate-buffered saline, lysed, and luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Firefly luciferase
activities were normalized to sea pansy luciferase activities to adjust
for transfection efficiency. Activities were also normalized to the promoterless reporter construct, pGL3-Basic.
In Vitro Transcription/Translation--
cDNAs for rPPAR Gel Electromobility Shift Assays--
Nuclear extracts were
prepared from day 9 3T3-L1 adipocytes essentially as described
previously (36). To study the binding of nuclear hormone receptors to
the putative PPRE, a double-stranded oligonucleotide, PPREwt, spanning
nucleotides Fatty Acid Uptake--
3T3-L1 adipocytes between days 7 and 9 were treated for 4 days with either 20 µM
troglitazone and 1 µM 9-cis-retinoic acid or
their carrier, Me2SO. The adipocytes were then assayed for uptake of oleate essentially as described previously (37). Briefly, cells were first preincubated for 2-4 h in serum-free DMEM and then
for 10-30 min at 25 °C in Krebs-Ringer phosphate solution with 2 mM glucose. Oleate uptake was measured at 25 °C by
incubating cells in a 100 µM, 1:1 oleate:BSA mixture,
which contained trace [3H]oleate (approximately 12,000 dpm/nmol). At various time points, uptake was terminated by aspirating
the oleate:BSA mixture from the cells and washing three times with
ice-cold phosphate-buffered saline containing 0.1% albumin and 200 µM phloretin. Cell lysate was then quantitated for
radioactivity using a Beckman 3801 liquid scintillation counter.
The Mouse Fatty Acid Transport Protein Gene Contains a
Functional PPRE--
Previous studies of FATP regulation in mice
indicated that transcription was activated in both liver and white
adipose tissue by treatment of mice with activators of PPAR
In order to determine whether the putative PPRE identified in the FATP
upstream sequence was able to mediate transcriptional activation,
portions of the FATP 5'-flanking sequence were tested for their ability
to mediate PPAR-activated transcription of a reporter gene. Four
luciferase reporter constructs were made, each containing portions of
the FATP 5'-flanking region linked to a promoterless firefly luciferase
gene. Two of these plasmids, pNH11 and pNH13, contained the putative
PPRE sequence, while the other two, pNH13
In the first set of experiments, two of these constructs, pNH13 and
pNH13
To further characterize the responsiveness of this PPRE to the various
PPAR subtypes, all four reporter constructs were transfected into CV-1
cells and assayed for luciferase activity in the presence and absence
of various PPARs and their activators. The activators used were
troglitazone, a thiazolidinedione (PPAR PPRE Involved in Differentiation-dependent Regulation
of FATP--
In order to determine the role of the FATP PPRE in the
process of its gene regulation during the process of adipose
differentiation, the luciferase reporter constructs were introduced
into both 3T3-L1 preadipocytes as well as mature adipocytes. These
experiments relied upon endogenous PPARs to mediate transcriptional
activation. Cells were also treated with activators of the various PPAR
subtypes to determine whether transcription could be further increased. In preadipocytes, which contain low levels of PPAR Synergistic Activation by PPAR PPARs and RXR PPAR The critical role of PPARs in the regulation of lipid metabolism
has become increasingly apparent. Many genes whose products take part
in some aspect of fatty acid catabolism, synthesis, or trafficking have
been shown to contain functional PPREs (Table I). FATP, which has been
argued to play a role fatty acid uptake, is a likely candidate for
regulation by this group of nuclear hormone receptors. Indeed, two
previous studies have shown that treatment of various cell types with
PPAR activators leads to an increase in FATP mRNA levels (30, 31).
In this paper we have identified a sequence in the 5' region of the
murine FATP gene, which is very similar (16/17) to the consensus
sequence for previously identified PPREs (see Table I).
By deletion analysis and mutation of this putative PPRE, we have
demonstrated that this PPRE is necessary for the PPAR-mediated up-regulation of FATP expression. Transfection into 3T3-L1
preadipocytes showed that FATP transcription can indeed be activated by
the naturally occurring compound, linoleic acid. This fatty acid has been shown previously to be able to activate transcription via both the
In addition to showing the functionality of the FATP PPRE, our
experiments demonstrate a differential activation of gene expression by
the various PPAR subtypes. Although both PPAR We and others have reported the up-regulation of FATP during adipose
conversion (13, 14). We hypothesized that this
differentiation-dependent regulation of FATP is mediated by
PPAR Several previous studies have shown a synergistic effect of the RXR The ability of the various PPAR subtypes to bind to the FATP PPRE
in vitro was examined by electromobility shift assay. As has
been shown in other systems, neither the PPARs nor RXR In order to correlate FATP regulation by PPARs with its putative
function, oleate uptake was measured in cells that were treated with
troglitazone and 9-cis-retinoic acid. Uptake was
significantly increased in activator-treated cells when compared with
control-treated cells. This leads to an interesting model for positive
feedback regulation of FATP. Increased FATP expression has been shown
to result in increased fatty acid uptake (13). Fatty acids, in turn,
are activators of PPAR We have reported previously the regulation of FATP by insulin via an
insulin-responsive element (PLE3) in the upstream region of the FATP
gene (458 to
474. To determine whether the FATP
PPRE was functional, and responded to lipid activators, transient
transfection of FATP-luciferase reporter constructs into CV-1 and
3T3-L1 cells was carried out. In CV-1 cells, FATP-luciferase activity
was up-regulated 4- and 5.5-fold, respectively, by PPAR
and PPAR
in the presence of their respective activators in a
PPRE-dependent mechanism. PPAR
, however, was unable to
mediate transcriptional activation under any condition. In 3T3-L1
cells, the PPRE conferred a small but significant increase in
expression in preadipocytes, as well as a more robust up-regulation of
FATP expression in adipocytes. Furthermore, the PPRE conferred the
ability for luciferase expression to be up-regulated by activators of
both PPAR
and retinoid X receptor
(RXR
) in a synergistic
manner. PPAR
and PPAR
activators did not up-regulate FATP
expression in 3T3-L1 adipocytes, however, suggesting that these two
subtypes do not play a significant role in
differentiation-dependent activation in fat cells.
Electromobility shift assays showed that all three PPAR subtypes were
able to bind specifically to the PPRE as heterodimers with RXR
.
Nuclear extracts from 3T3-L1 adipocytes also showed a specific
gel-shift complex with the FATP PPRE. To correlate the expression of
FATP to its physiological function, treatment of 3T3-L1 adipocytes with
PPAR
and RXR
activators resulted in an increased uptake of
oleate. Moreover, linoleic acid, a physiological ligand, up-regulated FATP expression 2-fold in a PPRE-dependent manner. These
results demonstrate that the FATP gene possesses a functional PPRE and is up-regulated by activators of PPAR
and PPAR
, thereby linking the activity of the protein to the expression of its gene. Moreover, these results have implications for the mechanism by which certain PPAR
activators such as the antidiabetic thiazolidinedione drugs affect adipose lipid metabolism.
INTRODUCTION
Top
Abstract
Introduction
References
(PPAR
1 and 2) the
CCAATT enhancer-binding proteins (C/EBP), and the sterol-response element binding proteins (SREBP or ADD1) (16). The importance of PPARs
for the development and maintenance of the adipocyte phenotype can be
more directly shown by the existence of peroxisome proliferator
response elements (PPREs) in the promoters of several genes whose
protein products are critical for lipid metabolism and the development
of the adipocyte phenotype such as lipoprotein lipase,
phosphoenolpyruvate carboxykinase, acyl-CoA synthetase, malic enzyme,
and adipocyte lipid-binding protein (ALBP or aP2) (16-21).
is predominantly expressed in liver, heart, kidney, and adipose tissue, whereas PPAR
(also known as NUC1 or
FAAR) shows a similar expression, with the exception of low levels in
liver (22-25). Two isoforms of mouse PPAR
have been cloned,
1
(22, 24, 26) and
2 (19), which are transcribed from the same gene
and alternatively spliced (27, 28). PPAR
1 is expressed in liver,
heart, and kidney, similar to PPAR
, whereas PPAR
2 is primarily
expressed in adipose tissue (29). The two PPAR
isoforms do not
appear to differ significantly in ligand binding affinities, ability to
bind DNA response elements, or ability to activate transcription. The
critical difference identified, thus far, is the distribution of expression.
activators and in white adipose tissues by activators of
PPAR
(30, 31). This evidence, together with the
differentiation-dependent regulation of FATP in 3T3-L1
adipocytes, led us to investigate the upstream region of the recently
cloned FATP gene (32) for a possible PPRE and to examine the role of
the various PPAR subtypes in FATP expression. In this report we detail
the regulation of the FATP gene, the identification of a functional
PPRE, and its up-regulation by PPAR
and PPAR
agonists.
EXPERIMENTAL PROCEDURES
, pSG5-FAAR (PPAR
), pBS-PPAR
2, and pRS-hRXR
were provided by Drs. Donald Jump, Paul A. Grimaldi, Bruce M. Spiegelman, David J. Mangelsdorf, and Ronald M. Evans, respectively.
971/+84), pNH13 (
556/+84), and pNH15
(
160/+84) were constructed by ligating varying lengths of FATP
upstream sequence into the NheI and HindIII sites
of the pGL3-Basic luciferase expression vector (Promega). Construct
pNH13
P was generated by single-stranded mutagenesis of construct
pNH13 using the Muta-Gene in vitro mutagenesis kit (Bio-Rad,
Hercules, CA) and the oligonucleotide:
5'-CTGGAACATCTCCTGAGTACTTCCTCCTCTCCC-3'. Sequence fidelity and
the introduced mutation were verified by sequencing. The PPAR
2
expression construct, pAH215, was made by cutting pBS-PPAR
2 with
KpnI and XbaI and ligating into pCDNA3.1 (Promega). The expression construct for RXR
, pAH232, was subcloned from pRS-hRXR
into the EcoRI site of pCDNA3.1.
2, rat PPAR
, or mouse PPAR
. 20 ng of pRL-SV40
(Promega), a vector containing a sea pansy (Renilla
reniformis) luciferase gene driven by an SV40 promoter, was
cotransfected into each well as a transfection control. After approximately 20 h, the calcium phosphate/DNA precipitate was removed by washing three times with phosphate-buffered saline. Cells
were then refed and treated for 48 h with either the appropriate PPAR activator or its vehicle, dimethyl sulfoxide
(Me2SO).
,
mPPAR
2, mPPAR
, and hRXR
were transcribed and translated
in vitro from the plasmids prPPAR
, pAH215, pSG5-FAAR, and
pAH232, respectively. The TNT Coupled Reticulocyte Lysate System
(Promega) was used according to the manufacturer's instructions. The
following expression plasmids were used prPPAR
, pSG5-FAAR (PPAR
),
pAH215, and pAH232. Translation products were verified by
SDS-polyacrylamide gel electrophoresis.
482 to
453 of the FATP upstream sequence was
32P-labeled with polynucleotide kinase (Promega). A 15-µl
reaction containing and 0.5 ng of PPRE probe and 9 µg of nuclear
extract or 0.5-1 µl of in vitro translation reaction was
incubated for 20 min at 25 °C and 15 min at 4 °C in a buffer
containing 20 mM HEPES (pH 8), 60 mM KCl, 1 mM dithiothreitol, 10% glycerol, and 2 µg poly(dI-dC).
The DNA-protein complexes were resolved from the free probe by
electrophoresis at 4 °C on a 4% polyacrylamide gel in 0.5 × TBE buffer (1 × TBE = 9 mM Tris, 90 mM boric
acid, 20 mM EDTA) (pH 8). Double-stranded oligonucleotides
composed of the following sequences were used for competition analysis: PPREwt, 5'-GATCTAGAGGAGGAAGTGGGGCAAAGGGCACAGGA-3';
PPREmut, 5'-GATCTAGAGGAGGAAGTGGGGCtAtcGGCACAGGA-3. PPRE sequence is underlined. Mutated bases are shown in lowercase letters.
RESULTS
or
PPAR
, respectively (30, 31). Furthermore, FATP expression was
observed to be up-regulated during adipose differentiation, a process
known to be mediated in part by PPAR
(14). The upstream sequence of the recently cloned FATP gene (32) was therefore examined for a
possible PPRE. A putative PPRE was identified, which is similar to the
consensus sequence proposed by Palmer et al. (38) (Fig. 1, Table
I).
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Fig. 1.
Upstream sequence of the FATP gene. The
putative PPRE is double underlined.
Comparison of identified PPRE sequences
P and pNH15 did not (Fig.
2).
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Fig. 2.
Construct map of the luciferase reporter
constructs used in transfection assays. Constructs contain various
portions of FATP upstream sequence, as indicated. The putative PPRE is
denoted by a shaded box.
P, as well as the promoterless pGL3-Basic control construct,
were transfected into 3T3-L1 preadipocytes. Following transfection, the
cells were maintained for 24 h in serum-free media and treated
with 100 µM linoleic acid or its carrier, ethanol. This
experiment was performed in the absence of serum, since serum albumin
binds fatty acids, leaving low levels of available fatty acid
activator. Linoleic acid treatment activated transcription of the
PPRE-containing construct approximately 2-fold over control-treated cells (Fig. 3), but did not affect
transcription of the PPRE-deletion construct. Similar results were seen
in transfection of 3T3-L1 adipocytes (data not shown). These
experiments indicated that the putative PPRE identified in the
FATP upstream sequence was indeed functional.
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Fig. 3.
Activation of FATP expression in 3T3-L1
preadipocytes by linoleic acid. 3T3-L1 preadipocytes were
transfected with the reporter constructs pNH13 and pNH13 P, as well
as the control construct, pGL3-Basic. The cells were then treated for
24 h in serum-free medium with either vehicle (EtOH) or 100 µM linoleic acid. Asterisks indicate
statistical difference from activity of the control-treated construct
(** = p < 0.005).
activator); WY14643, a
fibrate drug (PPAR
activator); and the PPAR
activator, Delta
Selective C. As shown in Fig.
4A, cells transfected with PPAR
and treated with troglitazone demonstrated a 5.5-fold increase in transcription of the PPRE-containing reporter constructs. PPAR
alone was able to activate transcription approximately 3-fold. PPAR
was also able to activate transcription in the PPRE-containing constructs, albeit to a lesser extent; transcription was increased 4- and 2-fold in the presence and absence of activator, respectively (Fig.
4B). Finally, the PPAR
subtype was unable to positively regulate transcription of any of the reporter constructs, regardless of
activator treatment (Fig. 4C). Deletion of the PPRE rendered the promoter unresponsive to any PPAR or agonist combination.
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Fig. 4.
The putative PPRE confers selective
responsiveness to PPAR-mediated activation. Reporter constructs
were cotransfected with or without an expression vector for PPAR (A), PPAR
(B), or PPAR
(C) and
treated for 48 h with activator or vehicle (Me2SO
(DMSO)). Activators were 20 µM Troglitazone,
10 µM WY14643, or 40 nM Delta Selective C,
respectively. Normalized luciferase activities are shown as mean ± S.E. (n = 4) and are expressed as -fold induction
relative to the activity in the absence of expression vectors and
activators. Asterisks indicate statistical difference from
activity of the reporter construct alone (* = p < 0.05, ** = p < 0.005).
as well as PPAR
, the PPRE containing construct, pNH13, was expressed at about
1.6-fold the level of the PPRE-deletion construct, pNH13
P, when both
were treated with Me2SO. Treatment with troglitazone had a
small, but not statistically significant, effect on pNH13 expression.
In adipocytes, the PPRE-containing construct was expressed at levels
5-fold higher than the PPRE-deletion construct (Fig. 5). Furthermore, troglitazone treatment
resulted in a further 3-fold increase in expression. Neither WY14643
nor Delta Selective C caused any significant change in luciferase
expression in either preadipocytes or adipocytes.
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Fig. 5.
Activation of FATP expression by endogenous
PPARs in preadipocytes and adipocytes. 3T3-L1 preadipocytes and
adipocytes were transfected with the reporter constructs pNH13 and
pNH13 P as well as the control construct, pGL3-Basic. The cells were
then treated with either vehicle or PPAR activators: 20 µM troglitazone, 10 µM WY14643, or 40 nM Delta Selective C. Luciferase activities were normalized
to pGL3-Basic and are shown as mean ± S.E. (n = 4). Asterisks indicate statistical difference between pNH13
and pNH13
P expression (* = p < 0.05, ** = p < 0.005).
and RXR
Activators--
Issemann et al. (39) showed that the RXR
ligand, 9-cis-retinoic acid enhances PPAR action. To
determine whether RXR
activation affected
PPAR
-dependent transactivation, 3T3-L1 adipocytes were treated with either troglitazone, 9-cis-retinoic acid, or
both. Retinoic acid did not significantly affect transcription by
itself; however, when added to cells in conjunction with troglitazone, it was able to produce an almost 2-fold increase in activity above that
produced by troglitazone alone (Fig. 6).
This result demonstrates the synergistic activation of the FATP gene in
response to activation of both PPAR
and RXR
.
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Fig. 6.
Synergistic activation of FATP expression in
3T3-L1 adipocytes by PPAR and RXR
activators. 3T3-L1 preadipocytes and adipocytes were
transfected with the reporter constructs pNH13 and pNH13
P as well as
the control construct, pGL3-Basic. These cells were then treated with
the PPAR
activator, troglitazone (20 µM), and the
RXR
activator, 9-cis-retinoic acid (1 µM).
Luciferase activities were normalized to pGL3-Basic and are shown as
mean ± S.E. (n = 4). Asterisks
indicate statistical difference from control-treated reporter construct
(** = p < 0.005).
Bind as Heterodimers to the FATP PPRE--
In
order to determine whether PPARs bind to the PPRE as heterodimers with
RXR
, gel mobility shift assays were performed with a double-stranded
oligonucleotide containing the FATP PPRE (Fig. 7A). The double-stranded
probe, PPREwt, was end-labeled with 32P and incubated with
in vitro translated protein as well as 3T3-L1 nuclear
extract. As shown in Fig. 7B, neither PPARs nor RXR
alone could bind to the PPRE; however, all three PPAR subtypes were able to
bind as heterodimers with RXR
to the probe. Furthermore, nuclear
proteins from 3T3-L1 adipocytes were able to form an in vitro complex with the PPRE (Fig. 7C). In order to test
the specificity of the protein-DNA interactions, an excess of unlabeled
oligonucleotide (PPREwt) was added to the reactions. The unlabeled
oligonucleotide was able to compete for binding of all three
PPAR-RXR
-DNA complexes, as well as the nuclear protein-DNA complex.
The introduction of 3-base pair substitutions (Fig. 7A)
produced an oligonucleotide, PPREmut, which was no longer able to
significantly compete for protein binding.
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Fig. 7.
PPARs and RXR bind
as heterodimers to the FATP PPRE. A, sequences of
oligonucleotides used in gel-shift studies. PPRE sequence is
underlined; mutated bases are in lowercase.
B, the double-stranded probe, PPREwt, was end-labeled with
32P and incubated with in vitro translated PPARs
and RXR
. The competitors PPREwt and PPREmut were used in 20- and
100-fold molar excess. Protein-DNA complexes were analyzed by
electrophoretic mobility shift assay. C, labeled PPREwt
probe was incubated with 3T3-L1 adipocyte nuclear extracts with or
without competitor. Competitors PPREwt, PPREmut, and NS (a
nonspecific competitor oligonucleotide) were added in 20-, 50-, and
200-fold molar excess.
and RXR
Activators Cause an Increase in Oleate
Uptake--
Finally, in order to correlate PPAR-mediated
transcriptional activation with putative in vitro protein
function, fatty acid uptake was analyzed in 3T3-L1 adipocytes. 3T3-L1
adipocytes were treated for 4 days with either troglitazone and
retinoic acid or their carrier Me2SO. Cells were then
incubated with [3H]oleate:BSA mixture (1:1), washed,
lysed at various time points, and assayed for radioactivity. As shown
in Fig. 8, treatment with the activators
of PPAR
and RXR
resulted in a significant increase in oleate
uptake. This result is consistent with the up-regulation of FATP
transcription by troglitazone and 9-cis-retinoic acid, as
shown by the previously described transfection studies, and correlates
well with the increase in FATP mRNA expression upon treatment of
3T3-L1 adipocytes with the PPAR
agonist BRL49653, as shown by Martin
et al. (30).
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Fig. 8.
Oleate uptake by 3T3-L1 adipocytes treated
with troglitazone and retinoic acid. 3T3-L1 adipocytes were
treated for 4 days with either 20 µM troglitazone and 1 µM 9-cis-retinoic acid or their carrier
Me2SO (DMSO). Cells were then incubated with 1:1
oleate:BSA mixture in Krebs-Ringer phosphate buffer which contained
trace [3H]oleate. Cells were washed and lysed at various
time points and cell lysate was assayed for radioactivity.
Asterisks indicate statistical difference between uptake of
treated and nontreated cells (* = p < 0.05).
DISCUSSION
and
PPAR subtypes (40-42). Further transfection experiments into CV-1 cells demonstrated that FATP transcription was activated by
both PPAR
and PPAR
. Both subtypes activate transcription upon
treatment with synthetic activator; however, the receptors are also
able to activate transcription in the absence of exogenous activator.
This could be explained either by the presence of an endogenous
activator, such as a fatty acid or its metabolite, or by a
ligand-independent activity of these subtypes (43).
and PPAR
are able
to activate transcription, our studies show that PPAR
mediates a
greater response both in the presence and absence of a synthetic activator. PPAR
, in contrast, did not significantly activate transcription, either alone or upon treatment with activator. This
difference has been demonstrated previously in other systems (44) and
has been hypothesized to reflect the differing roles of the PPAR
subtypes in the regulation of fatty acid metabolism. PPAR
has been
best characterized in the liver, where it up-regulates many genes
involved in the catabolism of fatty acids. PPAR
is chiefly active in
the adipose tissue, where it contributes to lipid accumulation and the
development of the adipose phenotype. In contrast to the other two
subtypes, the role of PPAR
in whole-body fatty acid metabolism has
not been well defined.
and is dependent on the presence of the FATP PPRE. This was
investigated in a further series of transfections, which compared
transcriptional activity in both 3T3-L1 preadipocytes and adipocytes,
in both the presence and absence of synthetic activators. The PPRE
conferred a 1.6-fold activation of FATP transcription in preadipocytes.
At this point in the differentiation process, PPAR
is present in low
levels relative to fully differentiated adipocytes. Exogenous
activators of the three PPAR subtypes did not significantly increase
transcription above that of untreated cells. In adipocytes, the PPRE
conferred a 5-fold increase in FATP expression, and this activation was further stimulated by the PPAR
activator, troglitazone. Neither the
activator of PPAR
nor PPAR
was able to increase FATP expression over untreated cells. This can be explained by the lack of significant PPAR
expression in adipocytes. PPAR
, while present in adipocytes, has been demonstrated in the previously described studies to be unable
to activate FATP expression.
activators, 9-cis-retinoic acid on PPAR-activated expression (18, 19, 21, 45-48). This convergence of the PPAR and RXR signaling
pathways was also demonstrated in transfection studies of FATP reporter
constructs in 3T3-L1 adipocytes. Although the RXR
activator alone
was unable to increase expression of FATP in 3T3-L1 adipocytes, it was
able to enhance the troglitazone-mediated activation of expression.
were able to
bind to the PPRE as homodimers (20, 21, 45-50); however, all three
PPAR subtypes were able to bind as heterodimers. These protein-DNA
complexes were sequence-specific, as shown by competition analysis, and
were dependent upon the presence of an intact PPRE. Mutation of 3 base
pairs of the PPRE abolished the formation of protein complexes on this
element. It is interesting to note that PPAR
, while unable to
activate transcription, was able to form a heterodimer complex with the
PPRE. This indicates that binding of the receptor heterodimers to an
element is not equivalent with transcriptional activity.
and PPAR
, which are able up-regulate the
expression of FATP.
1353 to
1347). The down-regulation of FATP by insulin, an
anabolic hormone, seems counterintuitive, since fatty acid uptake would
be expected to rise in response to insulin stimulation. It is important
to note, however, that regulation of FATP at the transcriptional level
is unlikely to be the result of the transient postprandial insulin
peak, but rather a more chronic hyperinsulinemia, such as in type 2 diabetes mellitus. Furthermore, the majority of type 2 diabetics are
obese, a condition associated with a down-regulation of PPAR
expression in adipose tissue (51). The combination of these two factors
may contribute to the elevation in serum free fatty acid levels
observed in type 2 diabetics. This leads to a possible mechanism for
the antidiabetic effects of the drug, troglitazone; by reversing the
effects of hyperinsulinemia and obesity on FATP regulation,
troglitazone may enable adipose tissue improve fatty acid uptake.
Further studies on the function of FATP and its regulation in the
diabetic state may lead to insight into both normal and deranged fatty
acid metabolism.
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ACKNOWLEDGEMENTS |
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We thank the members of the Bernlohr laboratory for their comments in preparing this manuscript. In particular we would like to thank Dr. Ann Hertzel for her insightful assistance with transfection analysis and interpretation of the results.
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FOOTNOTES |
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* This work supported by National Institutes of Health Grant DK 49807.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.
Present address: Dept. of Biology, San Diego State University, San
Diego, CA 92182.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, MN 55108. Tel.: 612-624-2712; Fax: 612-625-5780; E-mail: david-b{at}biosci.cbs.umn.edu.
The abbreviations used are: FATP, fatty acid transport protein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PPRE, peroxisome proliferator response element; PPAR, peroxisome proliferator-activated receptor; Me2SO, dimethyl sulfoxide; BSA, bovine serum albumin; RXR, retinoid X receptor.
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REFERENCES |
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