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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:2313-2319, October 2005

Biochemical and Molecular Actions of Nutrients

Fatty Acids Induce L-CPT I Gene Expression through a PPAR{alpha}-Independent Mechanism in Rat Hepatoma Cells

Cédric Le May, Michèle Caüzac, Claire Diradourian, Dominique Perdereau, Jean Girard, Anne-Françoise Burnol and Jean-Paul Pégorier1

Institut Cochin, INSERM U567, CNRS UMR 8104, Université PARIS V, Département d’Endocrinologie, 24 rue du Faubourg St Jacques, 75014 Paris

1To whom correspondence should be addressed. E-mail: pegorier{at}cochin.inserm.fr.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Liver carnitine palmitoyl transferase (L-CPT) I is a key regulatory enzyme of long-chain fatty acid (LCFA) oxidation that ensures the first step of LCFA import into the mitochondrial matrix. In rat hepatocytes, we showed previously that L-CPT I gene expression was induced by LCFAs as well as by fibrates. The aim of this study was to determine whether LCFA-induced L-CPT I gene expression was mediated by PPAR{alpha}. For this purpose, we constructed a PPAR{alpha}-dominant negative receptor to inhibit endogenous PPAR{alpha} signaling. Highly conserved hydrophobic and charged residues (Leu459 and Glu462) in helix 12 of the ligand-binding domain were mutated to alanine. These mutations led to a total loss of transcriptional activity due to impaired coactivator recruitment. Furthermore, competition studies confirmed that the mutated PPAR{alpha} receptor abolished the wild-type PPAR{alpha} receptor action and thus acted as a powerful dominant negative receptor. When overexpressed in rat hepatoma cells (H4IIE) using a recombinant adenovirus, the mutated PPAR{alpha} receptor antagonized the clofibrate-induced L-CPT I gene expression, whereas it did not affect LCFA-induced L-CPT I. These results provide the first direct demonstration that LCFAs regulate L-CPT I transcription through a PPAR{alpha}-independent pathway, at least in hepatoma cells.

KEY WORDS: • dominant negative PPAR{alpha} • recombinant adenovirus • carnitine palmitoyl transferase I • hepatoma cell • fatty acid–induced gene expression

Liver carnitine palmitoyl transferase (L-CPT)2 I is a key regulatory enzyme of long-chain fatty acid (LCFA) oxidation that ensures the first step of LCFA import into the mitochondrial matrix (1). L-CPT I gene expression is induced by saturated and unsaturated LCFAs as well as by fibrates (2,3). On the basis of a comparison between the transcriptional effects of these compounds and in vitro ligand binding assays, it was suggested that LCFA could regulate gene expression through the activation of peroxisome proliferator-activated receptors (PPARs). PPARs belong to the nuclear receptor superfamily of ligand-activated transcription factors [reviewed in (4)]. Three isotypes have been identified ({alpha}, {delta}, {gamma}). They ensure different function depending upon their tissue distribution and their specific ligands [reviewed in (5)]. PPAR{alpha} is the main isoform expressed in the liver, and the phenotypic characterization of PPAR{alpha} null mice suggests that this receptor is involved in the control of hepatic fatty acid and glucose metabolism (5). Like other members of the nuclear receptor superfamily, PPAR{alpha} possesses a modular structure composed of functional domains (5). PPAR{alpha} receptors contain a NH2-terminal region that harbors a ligand-independent transactivating domain [activator function (AF)1], and a core DNA binding domain containing 2 highly conserved zinc finger motifs that target the receptor as a heterodimer with the retinoid X receptor (RXR) to specific DNA sequences, the peroxisome proliferator responsive element (PPRE). PPAR{alpha} also presents a hinge region that allows protein flexibility, and a large and highly conserved C-terminal region that encompasses the ligand binding domain (LBD), dimerization interface, and a ligand-dependent transactivating domain (AF2) (5). Structural analysis of PPARs reveals that ligands bind in a pocket of the LBD (6), leading to an active conformation of the receptor through the stabilization of the AF2 domain (7). This conformational change leads to the dissociation of the corepressor complex from the PPAR/RXR heterodimer and the recruitment of the coactivator complex to enable transcriptional activation (7).

Although the role of PPAR{alpha} in mediating the fibrate transcriptional effect was clearly established (8), the contribution of PPAR{alpha} in mediating LCFA transcriptional effects remains controversial, at least for the L-CPT I gene. For instance, in fetal rat hepatocytes, linoleate induced the transcription of L-CPT I but not CPT II (the second enzyme involved in the transfer of LCFA into mitochondria) (2), whereas in the same cells, clofibrate induced both L-CPT I and CPT II gene expression (2). Furthermore, LCFA-induced L-CPT I gene transcription was reversed by insulin, but fibrate-induced L-CPT I and CPT II gene expression was not (2). These results suggest that LCFA and clofibrate could regulate gene expression through different mechanisms. To determine whether LCFA-induced L-CPT I gene expression was dependent on PPAR{alpha} activation, we studied the effect of LCFA in cultured hepatocytes from wild-type (WT) and PPAR{alpha} null mice. Unfortunately, we found that LCFA did not induce L-CPT I gene expression in either WT or PPAR{alpha} null mice (3); consequently we could not use these PPAR{alpha} null mice to answer the question. Therefore, the aim of this work was to test the effect of LCFA on L-CPT I gene expression in hepatoma cells overexpressing a dominant negative PPAR{alpha} receptor that inhibits endogenous PPAR{alpha} transcriptional activity.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plasmid constructs

    Mammalian expression vectors. Full-length rat PPAR{alpha} cDNA were cloned by RT-PCR from total rat liver RNA and cloned either into the pSG5 expression vector (Stratagene) or into the pcDNA3myc (Invitrogen). Two highly conserved and functionally amino acids (Leu 459 and Glu 462) located in the AF2 domain (9,10) were mutated to alanine using site-directed mutagenesis (Quickchange, Stratagene). pSVSport-PGC-1 was a gift from Dr. Spiegelman (Harvard Medical School) and pCMX-hSRC1 was a gift from Dr. Evans (Salk Institute for Biological Studies). Full-length WT or L459/E462A PPAR{alpha} double mutant (L*/E* PPAR{alpha}) cDNAs were cloned into pBind (CheckMateTM Mammalian Two-Hybrid System, Promega) to yield GAL4-PPAR{alpha}.

    Reporter constructs. The bifunctional enzyme gene (BFE)-luc was described elsewhere (11). pSG5-luc was included in the CheckMate Mammalian Two-Hybrid System (Promega).

    Electrophoretic mobility-shift assays (EMSAs). Proteins for electrophoretic mobility shift assays (EMSAs) were obtained by in vitro transcription/translation using the TNT T7-coupled reticulocyte lysate system (Promega). EMSAs were performed as described previously (3). The sense oligonucleotide used was: 5'-AGCTGATCCTTCCCGAACGTGACCTTTGTCCTGGTC-CCCTTTTGCTC-3' for acyl-CoA oxidase-PPRE (AOX-PPRE).

    Cell culture and transfection studies. Kidney monkey fibroblastic cells (COS-7, Promochem) were cultured as described previously (12). Cells were transiently transfected using lipofectamine according to the manufacturer’s instructions (Gibco BRL) with the following amounts of constructs depending upon the experimental condition tested (defined in the Results section): 500 ng of the BFE-Luc reporter gene, 100 ng of RSV-ß-Gal control plasmid, 100 ng of a RXR{alpha} expression vector, and either 100 ng of WT or mutated L*/E* PPAR{alpha} expression vector (pSG5). Six hours after transfection, linoleate 18:2 (0.3 mmol/L) complexed to bovine serum albumin (BSA; 0.2%), clofibrate [0.3 mmol/L dissolved in 0.3% dimethyl sulfoxide (DMSO)], or vehicle control (DMSO 0.3% or BSA 0.2%) was added to the cells. Cells were harvested 24 h later in cell lysis buffer (Promega), and luciferase activity was assayed on 20 µL of supernatant using the luciferase assay system (Promega). Protein-protein interaction assays were performed using the mammalian 2-hybrid system kit assay according to the manufacturer’s instructions (CheckMate, Promega).

Rat hepatoma cells (H4IIE, Promchem) were cultured in HAM F-12 medium supplemented with 2.4 mmol/L L-glutamine, antibiotics (105 units/L penicillin, 100 mg/L streptomycin) and 10% fetal calf serum. All experiments were performed on subconfluent cells at the same stage of differentiation; 24 h before the beginning of treatment, cells were cultured in serum-free medium. The effects of linoleate (0.3 mmol/L) complexed to defatted BSA (0.2%) and clofibrate (0.3 mmol/L) on gene transcription were tested after 8 h of culture as previously described (3). In preliminary experiments, using dose-response curves, we determined the concentration of linoleate or clofibrate necessary to induce maximal L-CPT I gene expression (data not shown). Moreover, before this, we verified that DMSO and BSA did not affect L-CPT I gene expression (data not shown).

    Adenovirus construction and expression. Recombinant type 5 adenoviruses expressing green fluorescent protein (GFP) alone (AdGFP) or GFP and full-length mutated L*/E* PPAR{alpha} (Ad L*/E*PPAR{alpha}) were generated using the Ad Easy Vector System kit (13). Briefly, the full-length mutated L*/E* PPAR {alpha} was subcloned into the shuttle vector pAd Track-CMV. The resultant plasmid was linearized by the restriction endonuclease Pme I and cotransformed with the supercoiled adenoviral vector pAd-Easy 1 into Escherichia coli strain BJ 5183. Recombinants were selected by kanamycine resistance and screened by restriction endonuclease digestion. Then, the recombinant adenoviral construct was cleaved with Pac I and transfected into the packaging cell line 293. Recombinant adenoviruses and viral amplification were performed in collaboration with the Vector Core of the University Hospital of Nantes (France).

COS 7 cells were transfected with various DNA constructs using the lipofectamine method (see above). After the media were changed, cells were incubated for 2 h at 37°C with 10 plaque-forming units/cell (pfu/cell) of AdGFP or Ad L*/E*PPAR{alpha}. The transcriptional effect of different effectors was tested after 24 h of culture.

H4IIE cells grown in 60-mm Petri dishes were incubated for 120 min at 37°C in HAM F-12 medium deprived of fetal calf serum in the presence of 10 pfu/cell of AdGFP or Ad L*/E*PPAR{alpha}. Then the medium was replaced and the transcriptional effects of linoleate and clofibrate were tested after 8 h of culture. Comparable viral infection efficiency was verified by fluorescence microscopy.

    Western blotting. Cells were solubilized at 4°C in lysis buffer (Tris-HCl 20 mmol/L pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA, 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 1% Triton X-100, 0.1% BSA, 1 mg/L pepstatin A, 2 mg/L leupeptin, 5 mg/L aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L orthovanadate). Lysates were cleared by centrifugation at 15,000 x g for 15 min at 4°C. Equal amounts of proteins were subjected to SDS-PAGE analysis, and immunodetected with the indicated antibodies. The immunoreactive bands were revealed using the ECL detection kit (Amersham Pharmacia Biotech).

    Extraction and analysis of mRNA expression by Northern blot and real-time quantitative RT-PCR. Total RNA was extracted from adenovirus-infected cells according to Chomczynski and Sacchi (14). Northern blot analyses were performed as described previously (3). The L-CPT I probe was an EcoR1-EcoR1 insert from subclone p61a (15). Real-time PCR was performed with the LightCycler instrument (Roche Molecular Biochemicals) using SYBR Green I as described previously (16). The following oligonucleotide primers (Invitrogen) were used: cyclophilin gene (GenBank M19533); 5'-ATGGCACTGGTGGCAAGTCC-3' and 5'-TTGCCATTCCTGGACCCAAA-3'; L-CPTI gene (GenBank LO7736) 5'-TCTTGCAGTCGACTCACCTT-3' and 5'-TCCACAGGAC-ACATA-GTCAGG-3'. SYBR Green I fluorescence emission was determined after each cycle. The relative amounts of all mRNAs were quantified by using the second derivative maximum method of the LightCycler software. Amplification of specific transcripts was confirmed by profiles of melting curves generated at the end of each run. PCR specificity and product length were further checked by agarose gel electrophoresis and ethidium bromide staining.

Statistics

Results are expressed as means ± SEM. Differences among control cells (transfected with empty vectors) and cells transfected with either WT PPAR{alpha} or mutated L*/E* PPAR{alpha} receptor were analyzed by 2-way ANOVA. When the F-test was significant, a posteriori comparisons between means were performed using Fisher’s least significant difference test. Calculations were carried out using the Stat View program. Differences were considered significant at P < 0.05.


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Characterization of mutated L*/E* PPAR{alpha} receptor transcriptional activity. Transcriptional activity of the WT or mutated L*/E* PPAR{alpha} receptor was measured on a luciferase reporter gene containing the PPRE of the peroxisomal BFE. The empty expression vector did not induce the reporter gene even in the presence of clofibrate or when RXR{alpha} was expressed (Fig. 1A). This result confirmed the absence of endogenous PPAR{alpha} receptor expression in COS 7 cells. Cotransfection of the WT PPAR{alpha} expression vector increased the luciferase reporter gene, an effect that was markedly enhanced by the addition of clofibrate alone or in association with the RXR{alpha} expression vector. Similar results were obtained when cells were cultured in the presence of linoleate instead of clofibrate (Fig. 1B). By contrast, under each condition tested, the mutated L*/E* PPAR{alpha} receptor showed a total loss of its transcriptional activity. This was not due to a reduce level of expression as demonstrated by Western blot analysis (Fig. 1C).



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  		FIGURE 1 Effect of clofibrate (A) and linoleate (B) on mutated PPAR{alpha} transcriptional activity in COS-7 cells. Transcriptional activity of WT and mutated L*/E* PPAR{alpha} was determined in COS-7 cells transfected with the BFE-Luc reporter gene. In some experiments, cells were cotransfected with a RXR{alpha} expression vector as indicated. (C) Representative Western blot of WT and mutated L*/E* PPAR{alpha} receptor expression. Myc-tagged PPAR{alpha} proteins were immunodetected using an anti-myc monoclonal antibody. ß-Actin was used as a control of protein loaded. Values are means ± SEM, n = 3. Means without a common letter differ P < 0.05: b,c, compared with an empty vector (pSG5) for the same condition of culture; d,e, compared with vehicle; f, compared with clofibrate in the absence of RXR.

 
    Mutated L*/E* PPAR{alpha} receptor retains DNA binding activity. In gel shift experiments, the WT or mutated L*/E* PPAR{alpha}/RXR{alpha} complexes bound similarly to the specific PPRE oligonucleotides of AOX (Fig. 2). Formation of these complexes was inhibited by an increasing amount of unlabeled AOX-PPRE probe confirming the specificity of binding (Fig. 2). These data demonstrated that the absence of transcriptional activity could not be attributed to a defect in DNA binding and/or to an alteration in heterodimerization with RXR{alpha}.



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  		FIGURE 2 Electrophoretic mobility-shift analysis of WT and mutated PPAR{alpha} to AOX-PPRE. In vitro translated WT or mutated PPAR{alpha}/RXR{alpha} heterodimers were incubated with 32P-labeled oligonucleotides containing the PPRE sequence of AOX-PPRE and analyzed by EMSA. Competition assays were achieved in the presence of a 10- to 100-fold molar excess of unlabeled AOX-PPRE oligonucleotide. The results are representative of 4 separate experiments.

 
    Coactivator recruitment by the mutated PPAR{alpha} receptor is altered. The WT or mutated L*/E* PPAR{alpha}-PGC-1 interactions [PPAR{gamma} coactivator 1, a well-characterized coactivator (17,18)], were determined using a mammalian cell 1-hybrid system. In the absence of PPAR{alpha}, GAL4 DNA binding domain (DBD) did not induce the reporter gene even in the presence of clofibrate or linoleate or when PGC-1 was overexpressed (Fig. 3A, B). These data clearly demonstrated that there was no nonspecific interaction between GAL4 DBD and PGC1. Then, we fused in frame of GAL4 DBD, the WT PPAR{alpha} receptor. The addition of PPAR{alpha} ligands (clofibrate or linoleate) increased the luciferase activity, suggesting a specific interaction between PPAR{alpha} and endogenous coactivators (Fig. 3A, B). Overexpression of PGC1 markedly enhanced the luciferase activity in the absence or presence of PPAR{alpha} ligands (Fig. 3A, B). By contrast, GAL4-mutated L*/E* PPAR{alpha} did not induce luciferase activity in response to either PPAR ligands or to PGC-1 overexpression (Fig. 3A, B). Similar results were obtained with other coactivators such as steroid receptor coactivator-1 and cAMP-response element-binding protein-binding protein (data not shown). These results demonstrated that the loss of ligand-independent or -dependent transcriptional activity of the mutated L*/E* PPAR{alpha} receptor was due to impaired coactivator recruitment.



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  		FIGURE 3 Effect of clofibrate (A) and linoleate (B) on WT and mutated PPAR{alpha} -PGC-1 interactions in COS-7 cells. COS7 cells were transfected with a luciferase reporter gene containing 5 GAL4 Upstream Activating Sequence (UAS) in the absence or presence of a vector expressing PGC-1. WT or mutated L*/E* PPAR{alpha} receptor was fused to the GAL4 DBD. Bars represent mean relative luciferase units normalized to the value (i.e., 1) obtained with GAL4 DBD cotransfected with the expression vector backbone in the presence of vehicle. Values are means ± SEM, n = 4. Means without a common letter differ, P < 0.05: b,c,d, compared with GAL4 DBD alone; e,f, compared with vehicle; g,h, compared with GAL4 WT PPAR{alpha} in the absence of PGC-1.

 
    Effect of mutated L*/E* PPAR{alpha} receptor on WT receptor transcriptional activity. The putative dominant negative effect of mutated L*/E* PPAR{alpha} on the WT receptor was determined in COS-7 cells by increasing the ratio of mutated:WT PPAR{alpha} expression vectors. This induced a concentration-dependent decrease in luciferase activity (Fig. 4). To rule out the possibility that the decrease in luciferase activity could be due to a reduction in WT PPAR{alpha} receptor expression, similar experiments were performed by replacing the mutated L*/E* PPAR{alpha} expression vector with an empty vector (Fig. 4). Under these conditions, there was no effect on luciferase activity until the amount of WT PPAR expression vector was decreased by 90% (Fig. 4), indicating that the mutated L*/E* PPAR{alpha} receptor was a potent inhibitor of the WT PPAR{alpha} transcriptional activity.



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  		FIGURE 4 Effect of mutated L*/E* PPAR{alpha} receptor on WT PPAR{alpha} transcriptional activity in COS-7 cells. COS 7 cells were transfected with BFE-Luc reporter gene, pSG5 WT PPAR{alpha} and either empty pSG5 or pSG5-mutated L*/E* PPAR{alpha}. Values are means ± SEM, n = 4. Means without a common letter differ, P < 0.05: b,c,d, compared with PPAR{alpha}WT alone; e,f, compared mutated L*/E* PPAR{alpha} with pSG5empty vector.

 
    Effect of adenoviral expression of mutated L*/E* PPAR{alpha} receptor on WT PPAR{alpha}-dependent transcription. Before overexpressing mutated L*/E* PPAR{alpha} receptor into hepatoma cells, we first verified that mutated L*/E* PPAR{alpha} receptor adenovirus-infected COS cells strongly expressed the mutated L*/E* PPAR{alpha} receptor (Fig. 5A). These results confirmed that adenoviral overexpression of the mutated L*/E* PPAR{alpha} receptor blocked the WT PPAR{alpha} ligand-dependent transactivation, whereas this transactivation was not modified in cells infected with control adenovirus expressing GFP alone (AdGFP) (Fig. 5B). These data clearly demonstrated that the mutated L*/E* PPAR{alpha} receptor is a powerful dominant negative receptor that can be used as a tool to antagonize the endogenous PPAR{alpha} signaling.



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  		FIGURE 5 Adenoviral overexpression of mutated PPAR{alpha} (A) and effect of clofibrate (B) on WT transcriptional activity in COS-7 cells. Panel A: Representative Northern and Western blots of mutated PPAR{alpha} expression in COS 7 cells infected with 1–50 pfu/cell of Ad-L*/E* PPAR{alpha}. Panel B: COS-7 cells were transfected with BFE-Luc, and pSG5 RXR{alpha} + pSG5 PPAR{alpha}. Then, cells were infected with no virus or with 10 pfu/cell of Ad-GFP or Ad-L*/E* PPAR{alpha}. Results were expressed as a percentage, with luciferase activity as the reference value (100%) obtained in the absence of adenoviral infection and in the presence of vehicle. Values are means ± SEM, n = 3. Means without a common letter differ, P < 0.05.

 
    Effect of linoleate on L-CPT I gene expression in hepatoma cells overexpressing mutated PPAR{alpha} receptor. In preliminary experiments, H4IIE hepatoma cells were infected with various AdGFP or Ad L*/E*PPAR{alpha} adenovirus titers. We determined that 10 pfu/cell induced 90% infection of H4IIE cells (data not shown). When cells were infected with the AdGFP, L-CPT I gene expression in response to clofibrate or linoleate was similar to that observed in noninfected hepatoma cells (3). After adenofection with mutated L*/E* PPAR{alpha} receptor adenovirus, the induction of L-CPT I gene expression by clofibrate was markedly reduced (81 ± 7%, n = 4, P < 0.05) compared with cells infected with Ad GFP (Fig. 6). This result confirmed that the mutated L*/E* PPAR{alpha} receptor was able to antagonize endogenous PPAR{alpha} signaling in hepatoma cells. By contrast, under the same conditions, linoleate-induced L-CPT I gene expression was not altered (Fig. 6). This provided the first direct evidence that in hepatoma cells, LCFA regulated L-CPT I gene expression through a PPAR{alpha}-independent pathway.



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  		FIGURE 6 Effect of a mutated L*/E* PPAR{alpha} on clofibrate- or linoleate-induced L-CPT I gene expression in rat hepatoma cells. Twenty-four hours after infection with 10 pfu/cell of AdGFP or Ad L*/E*PPAR{alpha}, hepatoma cells were cultured in the absence (control) or presence of 0.3 mmol/L clofibrate or 0.3 mmol/L linoleate for 8 h. L-CPT I mRNA levels were standardized with the cyclophilin housekeeping gene. Values are means ± SEM, n = 4. Means without a common letter differ, P < 0.05.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The phenotypic analysis of PPAR{alpha} null mice, which has provided interesting clues in the regulation of gene expression by LCFA, also demonstrated that PPAR is not the sole transcription factor involved in this regulatory process. For instance, genes encoding fatty acid synthase, spot 14, or L-pyruvate kinase were still inhibited by PUFA in the liver of PPAR{alpha} null mice (19,20) because the regulation of these genes by LCFA depends upon the recruitment of other transcription factors such as sterol regulatory element binding protein-1c or liver X receptor (LXR) [reviewed in (21,22)]. For the L-CPT I gene, the situation is quite different because, unlike rat liver, this gene was not induced by LCFA in either PPAR{alpha} null or WT mice (3), thus preventing any conclusions about the role of PPAR{alpha} in the regulation of the L-CPT I by LCFA. This observation led us to construct a dominant negative PPAR{alpha} receptor to inhibit endogenous PPAR{alpha} action. The crystal structure of PPAR LBD revealed that several amino acids were crucial for stabilizing the PPAR receptor in an active conformation because they were necessary for the establishment of a combination of hydrogen bonds and hydrophobic interactions with the ligand (7). The resulting stabilization of the AF-2 domain ensured the recruitment of a coactivator complex (7). Previous studies performed on other members of the nuclear receptor superfamily (9,10) showed that mutations of highly conserved amino acids located in the middle of the AF-2 domain altered the recruitment of coactivators, leading to the loss of transactivating functions without modifications of ligand binding capacity. Mutations of Leu459 and Glu462 in the AF-2 domain of PPAR{alpha} led to the same functional consequences, confirming that ligand binding capacity and transactivating function could be dissociated. Indeed, a recent report provided similar results (23).

Functional analysis demonstrated that the L*/E* PPAR{alpha} receptor was able to inhibit the transcriptional activity of endogenous PPAR{alpha} as demonstrated recently by another group (23). This could occur by at least 2 distinct and/or complementary mechanisms. First, because mutated L*/E* PPAR{alpha} retained the ability for dimerization, its overexpression led to an increase in the formation of the mutated PPAR{alpha}/RXR{alpha} heterodimer, thus decreasing the proportion of the functional PPAR{alpha}/RXR{alpha} heterodimer. Indeed, previous studies reported that the level and activity of other nuclear receptor signaling pathways using RXR{alpha} as a partner in heterodimerization modulated PPAR{alpha} action by controlling the availability of a limited amount of RXR protein (24–27). Second, because the mutated L*/E* PPAR{alpha} bound to PPRE with the same efficiency as did the WT receptor, this suggested that inhibition of transactivating activity of mutated L*/E* PPAR{alpha} was also due to a competition with the WT receptor for PPRE occupancy in PPAR{alpha} target genes. Such antagonism was demonstrated previously for hepatocyte nuclear factor 4-{alpha} (HNF4{alpha}) or chicken ovalbumin upstream-promotor transcription factor homodimers that compete with PPAR{alpha} signaling by displacing PPAR/RXR from its binding sites (28–31).

When overexpressed in hepatoma cells, mutated L*/E*PPAR{alpha} receptor blocked the fibrate-mediated L-CPT I gene expression, whereas it had no effect on LCFA-induced L-CPT I gene transcription. PPAR{alpha}-independent regulation of gene transcription by fatty acids was demonstrated only in terms of downregulation of gene expression (19,20), whereas the present work represents the first demonstration that LCFA could also enhance gene expression through a PPAR{alpha}-independent pathway, at least in rat hepatoma cells. The mechanism and transcription factors that relayed the transcriptional effects of LCFA on L-CPT I gene expression remained actually unresolved. Several transcription factors were involved in the regulation of gene transcription by LCFA [reviewed in (22,32,33)]. For instance, it was shown recently that LCFA bound to LXR and to RXR and activated their respective target genes (34,35). However, because both of these receptors dimerized with RXR, its sequestration by mutated L*/E* PPAR{alpha} would also reduce these signaling pathways. Furthermore, PPAR{alpha} heterodimerized with LXR as efficiently as with RXR (36), suggesting that overexpression of mutated L*/E* PPAR{alpha} could lead to the formation of an inactive PPAR/LXR heterodimer. Finally, LXR binds exclusively unsaturated fatty acids (34), whereas L-CPT-I gene expression was induced by both saturated and unsaturated fatty acids (2). Thus, it seemed unlikely that these nuclear receptors could be involved in the regulation of L-CPT I gene expression by LCFA. HNF4{alpha} represents another transcription factor involved in the control of gene transcription by fatty acids (37). Because HNF4{alpha} bound with high affinity to long-chain fatty acyl-CoA but not FFA (37), it seems unlikely that this receptor would be involved in LCFA-induced L-CPT I gene expression because native fatty acids rather than CoA esters were the metabolite signals responsible for the transcriptional effects of LCFA (3). Moreover, we showed that the region responsible for the stimulatory effect of LCFA was located in the first intron of the L-CPT I gene (3). Indeed, computer analysis of this intronic sequence reinforced these conclusions because no consensus DR1 (which binds PPAR{alpha}, ß, and {gamma}; HNF4 or RXR) or DR4 (which binds LXR) sequences were present in the first intron of the L-CPT I gene (3). The precise location of the sequence responsible for the transcriptional effect of LCFA is still under investigation, but the former observations suggest that unidentified transcription factors are involved in the regulation of the L-CPT I gene expression by LCFA.

Interestingly, it was shown recently that liver fatty acid binding protein (L-FABP) transported fatty acids to the nucleus and delivered them to a nuclear receptor through a direct interaction (38). Indeed, a pull-down assay and immunocoprecipitation clearly demonstrated that L-FABP interacts directly with PPAR{alpha} (39). However, PPAR{alpha} is not the only nuclear protein able to interact with L-FABP. Far-Western analysis confirmed the presence of several FABP-interacting proteins in rat liver nuclear extracts including a very prominent 33-kDa protein (38). The identity of these FABP-interacting proteins is still undetermined but this suggests that the list of potential transcription factors involved in fatty acid–mediated gene expression is probably not exhausted.

In conclusion, our work provides evidence that mutated PPAR{alpha} is a potent inhibitor of the transcriptional activity of the endogenous receptor and thus represents a useful and tool other than the mouse models with which to study LCFA-induced gene transcription.


   ACKNOWLEDGMENTS
 
We thank the Vector Core of the University Hospital of Nantes supported by the Association Française contre les Myopathies (AFM) for providing the adenovirus vectors. We also thank Drs. B. Spiegelman and R. Evans for providing us with the pSVSport-PGC-1 and pCMXhSRC1 expression vectors.


   FOOTNOTES
 
2 Abbreviations used: Ad-GFP, adenovirus expressing green fluorescent protein; Ad-L*/E*PPAR{alpha}, adenovirus expressing mutated PPAR{alpha}; AF, activator function; AOX, acyl-CoA oxidase; BFE, bifunctional enzyme gene; BSA, bovine serum albumin; DBD, DNA binding domain; DMSO, dimethyl sulfoxide; EMSA, electrophoretic mobility shift assays; GFP, green fluorescent protein; HNF4, hepatocyte nuclear factor 4; LBD, ligand binding domain; LCFA, long-chain fatty acid; L-CPT, liver carnitine palmitoyl transferase; L*/E*PPAR{alpha}, L459/E462A PPAR{alpha} double mutant; L-FABP, liver fatty acid binding protein; LXR, liver X receptors; pfu, plaque-forming units; PGC-1, PPAR{gamma} coactivator 1; PPAR, peroxisome proliferator activated receptor; PPRE, PPAR response element; RXR, retinoic X receptor; WT, wild-type. Back

Manuscript received 27 April 2005. Initial review completed 23 May 2005. Revision accepted 13 July 2005.


   LITERATURE CITED
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. McGarry J. D., Brown N. F. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur. J. Biochem. 1997;244:1-14.[Medline]

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