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J. Biol. Chem., Vol. 275, Issue 49, 38768-38773, December 8, 2000

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Alterations of Peroxisome Proliferator-activated Receptor  Activity Affect Fatty Acid-controlled Adipose Differentiation^*

Claire Bastie, Serge Luquet, Dorte Holst, Chantal Jehl-Pietri, and Paul A. Grimaldi

From the Institut de Recherche Signalisation, Biologie du Développement et Cancer, INSERM U470, Centre de Biochimie, Faculté des Sciences, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France

Received for publication, July 19, 2000, and in revised form, September 5, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fatty acids have been postulated to regulate adaptation of adipose mass to nutritional changes by controlling expression of genes implicated in lipid metabolism via activation of nuclear receptors. Ectopic expression of the nuclear receptors PPAR or PPAR promotes adipogenesis in fibroblastic cells exposed to thiazolidinediones or long-chain fatty acids. To investigate the role of PPAR in fatty acid regulation of gene expression and adipogenesis in a preadipose cellular context, we studied the effects of overexpressing the native receptor or the dominant-negative PPAR mutant in Ob1771 and 3T3-F442A cells. Overexpression of PPAR enhanced fatty acid induction of the adipose-related genes for fatty acid translocase, adipocyte lipid binding protein, and PPAR and fatty acid effects on terminal differentiation. A transactivation-deficient form of PPAR mutated in the AF2 domain severely reduced these effects. Findings are similar in Ob1771 or 3T3-F442A preadipose cells. These data demonstrate that PPAR plays a central role in fatty acid-controlled differentiation of preadipose cells. Furthermore, they suggest that modulation of PPAR expression or activity could affect adaptive responses of white adipose tissue to nutritional changes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Dietary long-chain fatty acids (LCFA)¹ control adipose tissue mass by regulating both the number and the size, i.e. the lipid accumulation, of adipocytes. This was illustrated in vivo by the findings that high fat diets promote hyperplastic and hypertrophic development of adipose tissue and massive obesity in adult rodents (1-3). Adipogenic effects of LCFA have also been documented in vitro by demonstrating that exposure of preadipose cells to native or non-metabolized fatty acids, such as 2-bromopalmitate (2BrP), increased the number of cells committed to differentiate as well as expression levels of adipose-related genes (4).

Cellular effects of fatty acids and some of their metabolites are related, at least in part, to activation of nuclear receptors called PPARs. Two different PPAR subtypes,  and , are expressed in preadipose and adipose cells. PPAR has been shown to play a central role in the control of gene expression and adipogenesis (5-8). Synthetic and naturally occurring PPAR activators, such as thiazolidinediones or 15-deoxy-^12-14-prostaglandin J2, are potent stimulators of terminal differentiation of cultured preadipose cells (9, 10). We have recently proposed that PPAR acts as an early player in LCFA induction of terminal differentiation by promoting PPAR expression. Fibroblasts ectopically expressing PPAR respond to LCFA by transcriptional activation of genes for fatty acid translocase (FAT/CD36), adipocyte lipid binding protein (ALBP), and PPAR. Although treatment with fatty acids alone was not sufficient to trigger adipogenesis, exposure to a combination of PPAR and PPAR activators, for example 2BrP and thiazolidinedione, or to a pan-PPAR activator, such as prostacyclin, promotes the expression of a typical adipose differentiation program and adipogenesis (11).

These experiments, which documented the role of PPAR in the adipogenic action of LCFA, also illustrated a major difference between fibroblasts and preadipose cells. Fibroblasts expressing PPAR strictly require exposure to strong PPAR activators to trigger terminal differentiation, whereas preadipose cells do not. A similar situation had already been described for PPAR-expressing fibroblasts (5). This discrepancy may reflect the ability of differentiating preadipose cells to synthesize and to accumulate enough endogenous PPAR activator to undergo terminal differentiation. Because many other differences could exist between preadipose cells and PPAR-expressing fibroblasts, it is crucial to examine the role of these transcription factors in a preadipose cellular context to confirm their role in adipocyte differentiation. Therefore, we have investigated the effects of overexpression of PPAR and dominant-negative PPAR mutant on the control by LCFA of gene expression and terminal differentiation in Ob1771 preadipose cells. The dominant-negative PPAR was generated by substitution of a glutamate residue by a proline in the loop preceding the AF-2 domain. This was based on numerous studies (12-16) with various members of the nuclear receptor superfamily showing that the AF-2 domain is important for activity. Receptors mutated in, or near, the AF-2 region, fail to either release corepressors or interact with coactivators and are inactive. Mutations in the C-terminal region of RAR, i.e. close to the AF-2 domain, were also reported to yield dominant-negative mutant receptors (17). The crystal structures of some nuclear receptors, including PPAR (18), strongly support the hypothesis that ligand binding promotes a conformational change resulting in release of corepressors and interaction with coactivators, permitting the transition from an inactive to an active transcriptional complex (reviewed in Ref. 19).

We report that overexpression of PPAR enhances LCFA responsiveness of preadipose cells in terms of maximal response and sensitivity and promotes terminal adipose differentiation. By opposition, expression of a PPAR mutant, that exerts a dominant-negative action, strongly decreases both the short term, i.e. activation of gene expression, and long term, i.e. adipogenesis, responses to LCFA.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmids-- The retroviral constructs containing PPAR cDNA or PPARE411P mutant cDNA were derived from pSG5-FAAR (20) and cloned into the BamHI site of pBizeoneo retroviral vector (Dr. K. Kristiansen, University of Odense, Denmark).

The PPARE411P was obtained by the site-directed mutagenesis method of Viville (21) using the 5'-CATCATCTGGGCGTGTGGGGTGACCAGCTG-3' oligonucleotide, and the construct was verified by sequencing.

Cell Culture-- Cells were grown in Dulbecco's modified Eagle's medium complemented with 8% fetal calf serum, 200 units/ml penicillin, 50 µg/ml streptomycin, 33 µM biotin, 17 nM calcium pantothenate (standard medium). For differentiation, cells were shifted after confluence to a standard medium supplemented with 17 nM insulin and 1 nM triiodothyronine (differentiation medium). Medium was changed every other day. Oil Red O staining was performed as described previously (22).

Stable Cell Lines-- BOSC23 cells were transfected at 50-70% of confluence by lipofection (Fugene 6, Roche Molecular Biochemicals) by 8 µg of pBizeoneo or pBizeoneoPPAR or pBizeoneoPPARE411P expression vectors. After 8 h, cells were re-fed with fresh standard medium and viral supernatants were collected 48 h later. Ob1771 (23) or 3T3F442A (24) cells grown in standard medium were infected with equal titers of recombinant virus for 6 h and then maintained for 48 h in fresh standard medium and then replated with a 1:5 dilution in standard medium containing 0.4 mg/ml Geneticin. Stable populations were obtained after 7-10 days of selection.

Transient Transfection-- HEK-293 cells were grown in standard medium and plated in 24-well plates. At 80% confluence, cells were transfected by Fugene 6 with 0.5 µg/well 3xACOPPRE-Tk luciferase reporter vector, 25 ng/well pCMV-RXR expression vector, 15 ng/well pCMV-Galactosidase vector, and various amounts of pBizeoneoPPAR or pBizeoneoPPARE411P expression vectors. After 6 h, cells were re-fed with fresh standard medium with or without 50 µM 2BrP. A 50 mM stock solution of 2BrP and dilutions were prepared in Me₂SO. Luciferase and galactosidase activities were analyzed 48 h later using the luciferase assay system (Promega France), and the Galacto-Light assay system (Tropix, PerkinElmer, France), respectively. Each transfection was performed in triplicate, and the fluorescence of the samples was measured using a 1450 Micro Beta luminometer (Wallac, Finland).

RNA and Protein Analysis-- Total RNA was prepared and analyzed by Northern blotting as described previously (4). Probes were labeled with [-³²P]dCTP using the random priming kit from Stratagene, and hybridizations were performed at 42 °C in a 50% formamide buffer. Blots were subjected to digital imaging (FujixBAS1000). GAPDH mRNA, which is not affected by adipose differentiation, was monitored as the internal standard.

Total cell extracts were prepared from the various virally infected Ob1771 and 3T3F-442A populations, in a buffer containing 50 mM Tris (pH 7, 4), 250 mM NaCl, 5 mM EDTA, 1 mM vanadate, 0,5 mM phenylmethylsulfonyl fluoride, 0,1% Nonidet P-40. The extracts were separated on 10% polyacrylamide SDS gels and blotted to nitrocellulose membranes. PPAR and PPARE441P mutant proteins were detected using a polyclonal antiserum raised against the A/B domain of mouse PPAR and which recognizes native and mutated PPAR. Immunodetection was performed by chemiluminescence using an ECL reagent from Amersham Pharmacia Biotech (France).

GPDH Enzymatic and Triglyceride Assays-- Glycerophosphate dehydrogenase (GPDH) activity, which provides the glycerol 3-phosphate required for triglyceride synthesis and is induced during terminal differentiation, was assayed spectrophotometrically as described previously (25). Enzyme activity was expressed in milliunits, i.e. nanomoles of product formed per min/mg of protein. Protein content of samples was determined according to Lowry et al. (26) using bovine serum albumin as standard.

Cellular triglyceride content was measured by using the Sigma Triglycerides 320-UV kit according to the manufacturer's instructions. Triolein was used as standard.

Materials-- Culture media, fetal calf serum, and Geneticin were from Life Technologies, Inc. (France). 2BrP and other chemical products were purchased from Sigma and Aldrich (France). Radioactive materials and nylon membranes were from Amersham Pharmacia Biotech (France). Rosiglitazone was a kind gift from SmithKline Beecham Pharmaceuticals (United Kingdom).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of a Dominant-Negative PPAR Mutant-- The PPARE411P mutant was obtained by replacing Glu-411 by a proline residue. This mutated PPAR was assayed for activation of a PPAR-responsive reporter gene and for dominant-negative activity against either native PPAR or PPAR. HEK-293 cells were transfected with a PPAR-responsive luciferase reporter (27) and an expression vector for the obligate partner RXR. As shown in Fig. 1, treatment of these cells with 2BrP, a non-metabolized long-chain fatty acid (28) and potent activator of PPAR (20) resulted in a very moderate induction of luciferase. Cells transfected with the PPAR expression vector showed a 12-fold 2BrP-dependent activation. In contrast, no activation was observed in cells transfected with the mutated PPAR, indicating that this mutant is inactive.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Characterization of a dominant-negative mutation of PPAR. A, HEK-293 cells were transfected as described under "Experimental Procedures" with luciferase and galactosidase plasmids, 25 ng of RXR expression vector, and the indicated amounts in nanograms of PPAR and PPARE411P expression vectors. Cells were then maintained for 48 h in the absence (open bars) or presence (solid bars) of 50 µM 2BrP prior to determination of luciferase and galactosidase activities. B, HEK-293 cells were treated as in A but transfected with a PPAR expression vector instead PPAR expression vector and exposed (hatched bars) or not (open bars) to 50 µM rosiglitazone for 48 h prior to enzymatic assays. Results are presented by taking as 1 the value obtained for cells maintained in control medium and transfected with PPAR (A) or PPAR (B) vectors and are the mean ± S.D. of three separate experiments.

The dominant-negative activity of PPARE411P was then investigated by transfection of HEK-293 cells with a constant amount of PPAR (Fig. 1A) or PPAR (Fig. 1B) expression vector and increasing amounts of PPARE411P expression vector. These experiments revealed that PPARE411P inhibits, in a dose-dependent manner, the PPAR-mediated transactivation. Luciferase induction was decreased by 50% and 85% when the mutated PPAR was used in a 4- and 20-fold excess, respectively (Fig. 1A). In contrast, PPARE411P did not inhibit rosiglitazone-induced and PPAR-mediated transactivation of the reporter gene (Fig. 1B). Taken together, these observations indicated that the PPARE411P exerts a dominant-negative activity specifically on the PPAR-mediated transactivation.

Isolation of Stable Ob1771 Cell Population Expressing Either Native or Dominant-Negative Mutant PPAR-- Expression of either PPAR or dominant-negative mutant PPARE411P was accomplished in Ob1771 by retroviral infection (11). As shown in Fig. 2A, Ob1771Biz cells expressed at day 1 post-confluence the endogenous PPAR mRNA at the expected size of 3.5 kb. A stronger signal was found at about 5 kb corresponding to the viral transcript in both Ob1771PPAR and Ob1771PPARE411P. Further experiments revealed that the level of endogenous PPAR mRNA increased by 4-fold during the first week after confluence in all cell populations, whereas expression of BizeoneoPPAR and BizeoneoPPARE411P mRNA remained unchanged (not shown). Western blot analysis, performed with an antiserum directed against the PPAR A/B domain and thus cross-reacting with both native and mutated proteins, revealed that at day 1 post-confluence Ob1771PPAR and Ob1771PPARE411P cells contained, respectively, 8- and 6-fold more PPAR protein than did control Ob1771Biz cells (Fig. 2B).

View larger version (80K): [in this window] [in a new window]   Fig. 2.   Expression of native or mutated PPAR in Ob1771 cells infected with the various retroviral expression vectors. Ob1771Biz (lanes 1), Ob1771PPAR (lanes 2), and Ob1771PPARE411P (lanes 3) were maintained in standard medium until day 1 post-confluence. A, 20 µg of total RNA from each cell population was analyzed by Northern blot for PPAR and GAPDH mRNAs. B, Western blot analysis of PPAR expression in the various cell populations at day 1 post-confluence was performed as described under "Experimental Procedures."

Effects of PPAR and PPARE411P Expression on Fatty Acid Responsiveness-- To investigate the effects of PPAR and the dominant-negative PPAR mutant on responsiveness to LCFA-induced transcription, Ob1771Biz, Ob1771PPAR, and Ob1771PPARE411P cell populations were grown to confluence and then exposed for 2 days to increasing concentrations of 2BrP. As described previously for parental Ob1771 cells (29), control Ob1771Biz cells showed a fatty acid dose-dependent induction of the fatty acid transporter FAT and ALBP mRNA (Fig. 3). Induction of these genes was markedly enhanced in Ob1771 overexpressing PPAR. These cells were also more sensitive to 2BrP than control cells (EC₅₀ of about 10 µM in Ob1771PPAR versus greater than 30 µM in Ob1771Biz).

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Activation of FAT and ALBP mRNA expression by 2BrP in the various Ob1771 cell populations. Ob1771Biz (), Ob1771PPAR (), and Ob1771PPARE411P () cells were grown to confluence in standard medium and then exposed for 48 h to increasing concentrations of 2BrP. GAPDH mRNA was monitored as internal standard. Northern blot analyzes for FAT (upper panel), ALBP (middle panel), and GAPDH (lower panel) mRNAs were performed with 20 µg total RNA from the indicated cells maintained in standard medium (lane 1) or in medium containing 10 µM (lane 2), 30 µM (lane 3), or 100 µM (lane 4) 2BrP. Results presented in B and C are representative of three independent experiments.

In contrast, Ob1771 expressing the dominant-negative PPAR displayed a reduced induction of FAT and ALBP mRNA even at higher fatty acid concentrations. Interestingly, the mutated protein did not completely abolish the transcriptional response to 2BrP.

Taken together, the data indicate that a change in the PPAR activity, i.e. overexpression or inhibition, modulates the action of LCFA on gene expression in preadipose cells.

Effects of PPAR and PPARE411P Expression on Fatty Acid Regulation of Terminal Differentiation of Ob1771 Cells-- To promote adipogenesis, cells were maintained after confluence in differentiation medium and treated for the first 5 days with increasing concentrations of 2BrP. Adipogenesis was estimated by Oil Red O staining (Fig. 4) and by determination of cellular triglyceride amounts (Fig. 5) at day 14 post-confluence and measurements of GPDH activity at days 9 and 14 (Fig. 6).

View larger version (72K): [in this window] [in a new window]   Fig. 4.   Effect of 2BrP on adipogenesis in the various Ob1771 cell populations. Cells were maintained after confluence in differentiation medium and exposed from days 0 to 5 to increasing concentrations of 2BrP. Cells were fixed and stained with Oil Red O at day 14 post-confluence.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effects of PPAR and dominant-negative PPAR on triglyceride accumulation in Ob1771 cells. Ob1771Biz (), Ob1771PPAR (), and Ob1771PPARE411P () cells were maintained as in Fig. 4, and triglyceride mass was determined as described under "Experimental Procedures" at day 14 post-confluence. Results are the mean ± S.D. from three separate experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effects of PPAR and dominant-negative PPAR on GPDH expression in Ob1771 cells. Ob1771Biz (), Ob1771PPAR (), and Ob1771PPARE411P () cells were maintained as in Fig. 4 and GPDH specific activity was determined as described under "Experimental Procedures" at days 9 and 14 post-confluence. Results are the mean ± S.D. from three separate experiments.

As described previously for the original Ob1771 cell line (4), 2BrP treatment during the preadipose state enhanced terminal differentiation in Ob1771Biz cells in a dose-dependent manner, as shown by the tremendous increase of triglyceride accumulation (Figs. 4 and 5). The adipogenic effect of the fatty acid was also illustrated by the induction of GPDH activity during the course of differentiation (day 9) or at the end of the process (day 14).

Compared with control cells, Ob1771PPAR cells displayed an increased ability to differentiate and enhanced fatty acid sensitivity. This is illustrated by greater increases of both triglyceride accumulation (Figs. 4 and 5) and GPDH activity (Fig. 6) occurring at lower 2BrP concentrations. Interestingly, as shown by the Oil Red O staining, adipose differentiation was nearly complete when the cells were treated by 25 µM fatty acid, which suggests that PPAR overexpression dramatically promoted the commitment of preadipose cells to terminal differentiation.

The decrease of PPAR activity in Ob1771, expressing the dominant-negative form of this nuclear receptor, resulted in a significant reduction of adipose differentiation. Indeed, Ob1771PPARE411P cells did not accumulate lipids when maintained in low 2BrP concentrations. At high concentrations of 2BrP, these cells displayed moderate adipogenesis (Fig. 4) and contained less triglyceride than control cells (Fig. 5). This is confirmed by the significantly lower GPDH activities measured in Ob1771PPARE411P than in control cells under all conditions assayed (Fig. 6).

Effects of PPAR and PPARE411P on Expression of PPAR-- Because PPAR plays a crucial role in terminal differentiation, we investigated its expression pattern in the three cell populations treated with 25 µM 2BrP during the first 5 days after confluence. As described for the original Ob1771 line (20), in Ob1771Biz cells, PPAR mRNA emerged after confluence and gradually increased until terminal differentiation (Fig. 7). In Ob1771PPAR, PPAR mRNA was already detected at confluence and accumulated thereafter to reach a maximal value at day 6. Noteworthy, at the end of adipose differentiation, i.e. day 14, PPAR mRNA amounts were nearly similar in the two cell populations. In contrast, expression of the dominant-negative PPAR resulted in a marked down-regulation of PPAR mRNA. Expression levels remained relatively low and were, at day 14 post-confluence, 6-fold lower than those measured in control cells.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effects of PPAR and dominant-negative PPAR on the time-course of PPAR mRNA expression in Ob1771 cells. Ob1771Biz (), Ob1771PPAR (), and Ob1771PPARE411P () cells were maintained after confluence in differentiation medium and treated from days 0 to 5 with 25 µM 2BrP. RNA was prepared at the indicated days and analyzed by Northern blotting. GAPDH mRNA was used as internal standard. Results are the mean ± S.D. from three separate experiments.

Expression of the Dominant-Negative PPAR Severely Decreased 3T3-F442A Cell Differentiation-- The effects of dominant-negative PPAR were also investigated in the 3T3-F442A preadipose cell line, which was established from mouse embryo (24). Cell populations were obtained after infection with retroviral pBizeoneo vector with or without the dominant-negative PPAR coding sequence. Fig. 8A shows that 442Abiz cells expressed a significant level of PPAR at confluence, whereas PPAR protein was 8-fold more abundant in confluent 442APPARE441P cells. Exposure for 4 days after confluence to 10 µM 2BrP was associated with low adipogenesis in 442APPARE411P cells, indicating that the dominant-negative PPAR mutant strongly repressed the process. This is evidenced by the marked reduction of Oil Red O staining in 442APPARE411P population. Although treatment of these cells with 2BrP significantly increased lipid accumulation, levels still remained lower than those observed in 442Abiz cells maintained in control medium without 2BrP (Fig. 8B). The time course of PPAR gene expression was investigated in 442Abiz and 442APPARE411P cells exposed to 2BrP. In 442Abiz, PPAR mRNA emerged at day 2 post-confluence and then accumulated rapidly to reach a maximal level at day 5. On the other hand, the induction of PPAR mRNA was seriously delayed and reduced in 442APPARE411P cells, i.e. emerging only at day 5 and remaining lower than in control cells at all times of determination (Fig. 8C). Consistent with the Oil Red O staining, 442APPARE411P cells expressed lower amounts of ALBP, FAT, and GPDH mRNAs than control cells when maintained in the absence or presence of 2BrP treatment (Fig. 8D). These findings demonstrate that PPAR is important for LCFA regulation of 3T3-F442A differentiation.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Effects of dominant-negative PPAR on terminal differentiation of 3T3F-442A cells. A, Western blot of PPAR expression in 1 day post-confluent 442Abiz and 442APPARE411P cells was performed as described under "Experimental Procedures." B, 442Abiz and 442APPARE411P cells were maintained for 6 days after confluence in differentiation medium and exposed or not to 10 µM 2BrP and then stained with Oil Red O. C, 442Abiz () and 442APPARE411P () cells were maintained from confluence in differentiation medium containing 10 µM 2BrP. RNA was prepared at the indicated days and analyzed by Northern blotting. GAPDH mRNA was used as internal standard. Results are the mean ± S.D. from three separate experiments. D, 442Abiz (gray and black bars) and 442APPARE411P (white and hatched bars) cells were maintained from confluence in differentiation medium with (black and hatched bars) or without (gray and white bars) 10 µM 2BrP. RNA was prepared at day 7 and analyzed by Northern blotting. Results are presented by taking as 100 the maximal signal obtained for each probe and are the mean ± S.D. from three separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study is a first examination of the role of PPAR in mediating LCFA regulation of gene expression and adipogenesis in preadipose cells. Two complementary approaches have been used in the study, i.e. overexpression of the native nuclear receptor and expression of a dominant-negative PPAR mutant.

As would be predicted based on studies with other nuclear receptors (17), the substitution of a glutamate residue by a proline at position 411 in PPAR generates a protein without transcriptional activity and which exerts an inhibitory effect on the endogenous PPAR. With other nuclear receptors, such mutations yield proteins that bind the ligand and the DNA-responsive element but that constitutively remain in a repressed form (19). Consequently, the inhibitory action of the constitutively repressed PPARE411P mutant may reflect its competition with endogenous native PPAR for binding to the PPRE. However, regardless of the underlying mechanism behind the dominant-negative activity of PPARE411P, it is worth noting that the mutant did not exert a complete inhibition on the native receptor even when used in high excess (Fig. 1A) and did not affect the transcriptional activity of PPAR (Fig. 1B). The lack of effect on PPAR probably relates to the higher affinity of the  isoform for the PPRE as compared with that of PPAR. Such a difference in binding affinities between the two isoforms for several natural PPRE has been previously documented by electrophoretic mobility shift assay experiments (30).

This work clearly demonstrates that a change in PPAR level or activity strongly alters the response of Ob1771 cells to LCFA and that this nuclear receptor acts early in the adipose differentiation time course. Preadipose Ob1771PPAR cells, in which the PPAR level was increased by retroviral infection, display a magnified response to fatty acids as shown by higher induction of FAT and ALBP gene expression at low fatty acid concentrations (Fig. 3).

The adipogenic action of fatty acids in promoting terminal differentiation also occurs at lower concentration in Ob1771PPAR as shown by Oil Red O staining (Fig. 4), by GPDH activity (Fig. 5), and by triglyceride accumulation (Fig. 6). Furthermore, the almost complete terminal differentiation, shown by the homogenous Oil Red O staining observed in Ob1771PPAR exposed for only the first 5 days to 2BrP (Fig. 4), strongly supports a crucial role of LCFA activated-PPAR during early confluent phase in promoting the commitment of preadipocytes to adipogenesis. In addition to enhancing the effects of added fatty acids, the increase in PPAR expression also exerts a potent action on terminal adipose differentiation of cells maintained in control medium. Ob1771PPAR cells accumulate significantly more lipids (Fig. 5) and express more GPDH activity (Fig. 6) than do control cells. The considerable increase of terminal differentiation observed in the absence of added 2BrP could be explained by increased sensitivity to the effects of LCFA from the serum or from endogenous origin or to other naturally occurring activators such as prostacyclin, a potent PPAR activator (7) synthesized by early confluent Ob1771 cells (31).

The role of PPAR as a nuclear mediator of LCFA effects on gene transcription and adipose differentiation is confirmed by the demonstration that expressing the dominant-negative mutant in Ob1771 cells considerably attenuates 2BrP-induced transcriptions of FAT and ALBP genes in preadipose cells (Fig. 3) and 2BrP enhancement of adipogenesis (Fig. 4). However, the cells remain able to respond to the fatty acid derivative, as shown by exposure to high concentrations of 2BrP. Morphological (Fig. 4) and biochemical investigations (Figs. 5 and 6) indicate that expression of the dominant-negative mutant, by partially inhibiting action of the endogenous PPAR, dramatically impairs terminal differentiation of Ob1771 cells. Ob1771PPARE411P cells do not undergo terminal differentiation when maintained in medium containing low concentrations of activator but display adipogenesis when high concentrations of 2BrP are used. Residual responses during short term or long term exposure to high concentrations of LCFA are not surprising, because transactivation experiments revealed that the dominant-negative PPAR mutant, even when used at a high excess, does not completely suppress fatty acid activation of the native nuclear receptor (Fig. 1). The amount of the mutated protein expressed in Ob1771PPARE411P at day 1 after confluence was estimated to be approximately 6-fold higher than the endogenous PPAR (Fig. 2B). Thus, in Ob1771PPARE411P cells exposed to high concentrations of 2BrP, the amount of activated native PPAR may be high enough to effectively compete with the mutated repressed receptor for binding to the PPRE and to allow transcription of LCFA-responsive genes. It is possible that, at high concentrations of LCFA, there is accumulation of ligand-activated native PPAR, which favors the binding of the active transcription factor to the PPRE of LCFA-responsive gene promoters.

The respective positive or negative actions of either native or dominant-negative PPAR on terminal differentiation are a likely consequence of the alterations of PPAR expression. Although overexpression of the native nuclear receptor did not significantly change the maximal expression of PPAR, it resulted in an earlier induction of its expression when compared with control cells. Expression of the dominant-negative mutant led to an impairment of PPAR expression during the differentiation phase (Fig. 7). Thus, the pattern of PPAR expression in the three different Ob1771-derived cell lines used in this study supports the interpretation that LCFA-activated PPAR controls PPAR gene expression and that PPAR is crucial for the induction of genes related to terminal differentiation.

These findings were applicable to both Ob1771 and 3T3F442A cells and indicated that they are not dependent on nor specific to a particular cell line. For example, despite the fact that terminal differentiation of 3T3-F442A cells is less dependent on fatty acid supply as shown for 442Abiz cells (Fig. 8, A and C), expression of the dominant-negative PPAR in 3T3-F442A cells severely reduces adipogenesis in a way similar to Ob1771 cells.

In summary, the observations reported in this study, clearly establish the role for PPAR as nuclear mediator of LCFA-mediated transcriptional and adipogenic actions in a preadipose cell context. The findings suggest that changes in the amount or activity of this nuclear receptor in preadipose cells may have important functional consequences with respect to the response of adipose mass to nutritional changes. Possibly, up-regulation of the receptor would result in hypersensitivity to LCFA effects in increasing adipose tissue mass, whereas down-regulation may confer resistance to LCFA. PPAR is also expressed in various tissues, including heart, skeletal muscle, and intestine, and it would be of interest to characterize what tissue-specific effects up- or down-regulation of this nuclear receptor would have in the whole animal. The dominant-negative PPAR mutant described in this study can be used to selectively inhibit LCFA-induced transcriptional activation in particular tissues. The construction of transgenic animals expressing the PPARE411P mutant in a tissue-specific manner would provide valuable information on the role of PPAR in various tissues.

	ACKNOWLEDGEMENTS

We thank Nada A. Abumrad (Stony Brook, NY) and Ellen Van Obberghen-Schilling (Nice, France) for critical comments and review of the manuscript and Delphine Brignon for expert technical assistance.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33-492-07-64-34; Fax: 33-492-07-64-02; E-mail: grimaldi@taloa.unice.fr.

Published, JBC Papers in Press, September 15, 2000, DOI 10.1074/jbc.M006450200

	ABBREVIATIONS

The abbreviations used are: LCFA, long-chain fatty acid; ALBP, adipocyte lipid binding protein; 2BrP, 2-bromopalmitate; FAT, fatty acid transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPDH, glycerophosphate dehydrogenase; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR responsive element; RXR, retinoid X receptor; kb, kilobase(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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