J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moya-Camarena, S. Y.
Right arrow Articles by Belury, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moya-Camarena, S. Y.
Right arrow Articles by Belury, M. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Journal of Lipid Research, Vol. 40, 1426-1433, August 1999
Copyright © 1999 by Lipid Research, Inc.

Original Article

Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPAR{alpha}

Silvia Y. Moya-Camarenaa, John P. Vanden Heuvelb, Steven G. Blanchardc, Lisa A. Leesnitzerc, and Martha A. Belurya
a Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907
b Department of Veterinary Science, Center for Molecular Toxicology, Penn State University, University Park, PA 16802
c Department of Molecular Biochemistry, Glaxo Wellcome Research and Development, Research Triangle Park, NC 27709

Correspondence to: Martha A. Belury


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

We have previously shown that a mixture of dietary conjugated derivatives of linoleic acid (conjugated linoleic acid, CLA) induces peroxisome proliferator-responsive enzymes and modulates hepatic lipid metabolism in vivo. The present studies demonstrate that CLA is a high affinity ligand and activator of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and induces accumulation of PPAR-responsive mRNAs in a rat hepatoma cell line. Using a scintillation proximity assay (SPA), CLA isomers were shown to be ligands for human PPAR{alpha} with a rank order of potency of (9Z,11E)>(10E,12Z)>(9E,11E)> furan-CLA (IC50 values from 140 nM to 400 nM). Levels of acyl-CoA oxidase (ACO), liver fatty acid-binding protein (L-FABP), and cytochrome P450IVA1 (CYP4A1) mRNA were induced by CLA in FaO hepatoma cells. Even though linoleate and CLA were incorporated into lipids of hepatoma cells to the same extent, linoleate had little or no effect on ACO, CYP4A1, or L-FABP mRNA. In agreement with its binding potency, (9Z,11E)-CLA was the most efficacious PPAR{alpha} activator in the mouse PPAR{alpha}-GAL4(UAS)5-CAT reporter system.

These data indicate that CLA is a ligand and activator of PPAR{alpha} and its effects on lipid metabolism may be attributed to transcriptional events associated with this nuclear receptor. Also, (9Z,11E)-CLA is one of the most avid fatty acids yet described as a PPAR{alpha} ligand.—Moya-Camarena, S. Y., J. P. Vanden Heuvel, S. G. Blanchard, L. A. Leesnitzer, and M. A. Belury. Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPAR{alpha}. J. Lipid Res. 1999. 40: 1426;–1433.

Supplementary key words: ligand affinity, gene expression, lipid metabolism


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Conjugated linoleic acid (CLA) refers to a mixture of octadecadienoate derivatives which have a conjugated double-bound system (8;–10, 9;–11, 10;–12, or 11;–13 in Z and/or E configurations) (1). CLA is synthesized in vivo (by rumen bacteria) from linoleic acid through oxidative pathways and enzymatic isomerization (2). Recently, it has been reported that CLA may be formed by conversion from dietary trans-fatty acids in humans (3). In foods, CLA is found in ruminant meats and dairy products where its content ranges from 2.5 to 15.0 mg/g lipid (1) (4). CLA has unique properties such as anti-cancer (5) (6) (7) (8), anti-atherogenic (9), and anti-diabetogenic effects in rodent models (10). Several hypotheses to explain the pleiotropic effects of CLA have been proposed but the mechanisms of action of CLA are not fully understood. Previous studies in our laboratory have shown that dietary CLA decreased the incidence of skin tumors in female SENCAR mice but also caused lower body weight and increased hepatic lipid accumulation when compared to mice fed non-CLA containing diets (7) (11). These observations suggested that CLA plays a significant role in the modulation of lipid metabolism that may explain its anti-carcinogenic, anti-atherogenic, and anti-diabetogenic properties.

The ligand-activated transcription factor, peroxisome proliferator-activated receptor (PPAR), modulates lipid metabolism. Specific PPAR responsive elements have been identified in the regulatory regions of genes encoding lipid metabolizing enzymes, such as ACO, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase [bi-(tri) functional enzyme], cytochrome P450 4A family (CYP4A), L-FABP, acyl Co-A synthase, lipoprotein lipase, and many others. Three subtypes of PPARs have been found in amphibians, rodents, fish, and humans. PPAR{alpha} is predominantly expressed in liver, kidney and heart, PPARß (also called {delta}) is expressed in almost every tissue, and PPAR{gamma} is found almost exclusively in adipose tissue and has been linked to adipocyte differentiation (12). Among the synthetic activators of PPAR{alpha}, collectively known as peroxisome proliferators for their ability to increase peroxisomes in rodent liver, are the fibrate class of hypolipidemic drugs (i.e., clofibric acid, bezafibrate, or their analogs). Recently it has been reported that the fatty acid derivative leukotriene B4 (LTB4) and the fibrate analogs [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid] (Wy-14,643) and 2-(4-[2-(3-[2,4-difluorophenyl]-1-heptylureido)ethyl]phenoxy)-2-methylbutyric acid (GW2331) bind to PPAR{alpha} (13) (14). Fatty acids and eicosanoids have been also identified as PPAR ligands (14) (15) (16).

CLA has some structural features similar to peroxisome proliferators. Moreover the physiological responses observed in mice (reduced body weights, hepatic lipid accumulation, hypolipidemia) are characteristic of this group of chemicals. These similarities lead us to hypothesize that CLA activates hepatic PPAR{alpha}. Previously, we had shown that SENCAR mice fed increasing levels of CLA, accumulated several PPAR-responsive enzymes including ACO, L-FABP, and CYP4A1 at mRNA and protein levels (17). The present studies were performed to determine the affinity of several CLA isomers for PPAR{alpha}, the extent of PPAR{alpha} activation, and resultant changes in gene expression. These studies are the first to demonstrate that CLA is a ligand and activator of PPAR{alpha}, and as CLA induces PPAR-responsive genes in cultured cells, this dietary fatty acid may regulate lipid metabolism through a PPAR{alpha}-mediated mechanism. Interestingly, the most biologically relevant isomer, (9Z,11E)-CLA, is among the most avid PPAR{alpha} ligands of dietary origin yet described.


  MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES

Materials
[14C]CLA and [14C]linoleic acid were from American Radiolabeled Chemicals (St. Louis, MO). A mixture of CLA isomers [41.2% (9Z, 11E)- and (9E,11Z)-CLA, 44.1% (10E,12Z)-CLA, 1.1% (9Z,11Z)-, 9.4% (10Z,12Z)-, 1.3% (9E,11E)-, and (10E, 12E)-CLA, 0.7% linoleate and 2.2% unidentified compound] was from NuChek Prep, Inc. (Elysian, MN). Pure CLA isomers (9Z,11E)-CLA (97% purity), (9E,11E)-CLA (98% purity), (10E, 12Z)-CLA, and the furan form 8-(5-hexyl-2-furyl)-octanoic acid were from Matreya Inc. (Pleasant Gap, PA). Wy-14,643 was purchased from BIOMOL (Plymouth Meeting, PA) and Chemsyn (Lenexa, KS). Bezafibrate and troglitazone were from Sigma (St. Louis, MO) and Parke-Davis (Ann Arbor, MI), respectively. The expression plasmid pcDNA3-mPPAR{alpha} and psV-GL2-PPRE luciferase reporter plasmid were generously donated by Dr. J. Tugwood (Zeneca Central Toxicology Laboratory, UK). The pSG5-mGAL4-PPAR{alpha} chimera expression construct and (UAS)5-tk-CAT reporter construct were generated as reported previously (18). The psV-ß-galactosidase plasmid was from Promega (Madison, WI). [3H]-GW2331 was prepared as previously described (14).

Ligand binding assays
The binding affinity of several CLA isomers for human PPAR{alpha} was determined by scintillation proximity assay (SPA) using bacterially expressed PPAR{alpha} ligand binding domain as previously described for PPAR{gamma} (19). The (9Z,11E)-, (10E,12Z)-, (9E,11E)-CLA isomers and the putative furan-CLA metabolite were tested in duplicate as inhibitors of [3H]-GW2331 (14) binding to PPAR{alpha}.

Reporter gene assays
Mouse PPAR{alpha}-GAL4/(UAS)5-CAT reporter system. COS-1 cells were co-transfected with GAL4-PPAR{alpha}, (UAS)5-tk-CAT, and pSV-ß-galactosidase (Promega, Madison, WI) as described previously (20). Transfected cells were treated with the test chemicals for 6 h. Chloramphenicol acetyltransferase (CAT) levels in cell lysates were assessed by enzyme-linked immunoabsorbent assay (GIBCO). Data were quantified relative to ß-galactosidase activity and expressed as a percent of the DMSO vehicle treated cells.

Full-length mouse PPAR{alpha}/PPRE-luciferase reporter system. CV-1 cells were co-transfected with pcDNA3-PPAR{alpha}, psV-GL2-PPRE-luciferase and pSV-ß-galactosidase. Cells were transfected using LipofectAMINETM (GIBCO). Transfected cells were treated for 6 h with the test chemicals. Luciferase and ß-galactosidase activities were assayed on cell lysates (Promega, Madison, WI). Data were quantified relative to ß-galactosidase activity and expressed as a percent of DMSO vehicle treated cells.

Total RNA and protein extraction
FaO cells were treated with various doses of (9Z,11E)-CLA isomer, linoleate, Wy-14,643 (100 µM) or 0.1% vehicle DMSO. Total RNA was extracted from FaO hepatoma cells using TRI-Reagent® (Molecular Research Center, Cincinnati, OH). For protein analyses, FaO cells were treated for 24 h with various concentrations of (9Z,11E)-CLA (50 µM, 100 µM and 200 µM), linoleate, Wy-14,643, bezafibrate or troglitazone (100 µM) and 0.1% DMSO as control. Cell lysates were obtained by homogenization with lysis buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS with 100 µg/ml phenylmethylsulfonyl fluoride, 300 µg/ml aprotinin, and 100 µg/ml sodium orthovanadate) and centrifugation (15,000 g for 30 min at 4°C). Total RNA and protein extracts were frozen (-80°C) until analyses were performed.

Quantitative reverse transcriptase-PCR (RT-PCR)
Quantitative RT-PCR using recombinant RNA as an internal standard was performed as reported previously (17) (21) to determine ß-actin, ACO, C Y P4A1, or L-FABP mRNA levels.

Protein analysis
Protein samples (20 µg) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Sheep anti-rat CYP4A1 or rabbit anti-rat albumin primary antibodies were used, followed by anti-sheep Ig-biotinylated antibody and streptavidin-horseradish peroxidase conjugate (Amersham Life Sciences, Arlington Heights, IL) or anti-rabbit Ig-horseradish peroxidase-linked secondary antibody (Bio-Rad Corp., Richmond, CA). Immunoreactive bands were detected using enhanced chemiluminescence (Amersham Life Sciences, Arlington Heights, IL) and the films were quantified using pdiTM scanning densitometry.

Incorporation of [14C]CLA and [14C]linoleate into FaO hepatoma cells
Cells were incubated with 0.1 µCi/ml in 0.1% bovine serum albumin-containing media of [14C]CLA or [14C]linoleate for 1, 3, 6, 9, 12, or 24 h. Percentage of incorporation into cells was determined by scintillation counting.

Total lipid extraction, neutral and phospholipids fractionation, and fatty acid analyses
FaO cells were incubated with serum-free media for 12 h and treated with vehicle (0.1% DMSO), linoleate or (9Z,11E)-CLA (100 µM) in serum-free media supplemented with 0.01% bovine serum albumin for 12 h. Total lipids were extracted from FaO cell homogenates and analyzed as reported previously (11).


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES

CLA is a ligand for human PPAR{alpha}. Scintillation proximity assay (SPA) is a novel assay system useful for quantitation of receptor;–ligand equilibria. Major advantages of the method are simplicity and speed as separation of receptor-bound from free ligand is not necessary. We have recently reported an SPA for screening potential ligands for PPAR{gamma} with the results obtained with SPA showing an excellent correlation with the traditional gel filtration system (19). In addition, it has been previously shown that the fibrate analogue GW2331 is a high affinity ligand for both PPAR{alpha} and PPAR{gamma} (14). We tested whether individual CLA isomers were able to inhibit specific binding of [3H]-GW2331 to PPAR{alpha} by competitive SPA. Figure 1 represents a typical inhibition curve obtained for (9Z,11E)-CLA isomer (IC50 = 140 ± 90 nM) demonstrating that this isomer is a potent PPAR{alpha} ligand. The other CLA isomers tested were also potent ligands for PPAR{alpha}. IC50 values for CLA isomers determined by inhibition of [3H]-GW2331 binding were similar and ranged from 140 to 420 nM ( Table 1). Interestingly, CLA also binds to PPAR{gamma} but with lower affinity (IC50s = 5;–7 µM) when compared to PPAR{alpha} (M. A. Belury, unpublished data).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. CLA binds to PPAR{alpha}. Competitive binding assays were performed by scintillation proximity assay (SPA) for human PPAR{alpha} ligand binding domain and 10 nM [3H]-GW2331 in the presence of increasing concentrations (up to 100 µM) of non-radioactive (9Z, 11E)-CLA as competitor.


 
View this table:
[in this window]
[in a new window]
  		Table 1. IC50 values of PPAR{alpha} in the competitive binding scintillation proximity assay

CLA activates mouse PPAR{alpha}. After determining that CLA is a high affinity ligand for PPAR{alpha}, we determined the extent that CLA is able to activate PPAR{alpha}. The GAL4-mPPAR{alpha};–ligand binding domain (LBD) chimera reporter assay is very responsive to peroxisome proliferators (20) and as an artificial receptor is being used, has little interference from endogenous expression of PPAR subtypes. The isomeric mixture of CLA resulted in a dose-dependent activation of PPAR{alpha} (5-fold) ( Figure 2A). Activation of mPPAR{alpha} by individual isomers of CLA was also examined and compared to that of Wy-14,643 and perfluorodecanoic acid as positive controls (Figure 2B). As expected, Wy-14,643 and perfluorodecanoic acid activated mPPAR{alpha} with a maximal induction of 50- and 4-fold, respectively, above vehicle-treated cells. The (9Z,llE)-CLA isomer was a very efficacious activator of mPPAR{alpha} in the GAL4 system resulting in a 26-fold increase in activity (Figure 2B). In regard to other mPPAR{alpha} activators, treatment with the (9Z,11Z)- and furan-CLA isomers also resulted in substantial increases in reporter expression.




View larger version (75K):
[in this window]
[in a new window]
  		Figure 2. Activation of mPPAR{alpha} by CLA and its different geometric isomers. A: Transfected cells were treated for 6 h with increasing concentrations of CLA mixture (0 µM, 50 µM, 100 µM, or 250 µM). CAT/ß-galactosidase ratios are expressed relative to vehicle treated cells (0.1% DMSO, n = 3). B: COS-1 cells were transfected with GAL4-mPPAR{alpha} along with a CAT-reporter and ß-galactosidase vectors. Transfected cells were treated for 6 h with the following compounds (100 µM): CLA mixture, (9Z,11E)-CLA, (9E,11E)-CLA, (9Z,11Z)-CLA, 8-(5-hexyl-2-furyl)-octanoic acid (furan-CLA), linoleate, perfluorodecanoic acid (PFDA), or Wy-14,643. CAT/ß-galactosidase ratios are expressed relative to vehicle treated cells (0.1% DMSO, n = 3). C: CV-1 cells were co-transfected with pcDNA3-PPAR{alpha}, psV-GL2-PPRE-luciferase and pSV-ß-galactosidase vectors. Transfected cells were treated for 6 h with increasing concentrations of CLA mixture. Luciferase/ß-galactosidase ratios are expressed relative to vehicle treated cells (0.1% DMSO). Asterisks denote values that are significantly different from DMSO treated cells (P < 0.05, n = 3). D: Transfected cells were treated for 6 h with the following compounds (100 µM): CLA mixture, (9Z,11E)-CLA, (9E,11E)-CLA, (10E,12Z)-CLA, 8-(5-hexyl-2-furyl)-octanoic acid (furan-CLA), linoleate, or Wy-14,643. Luciferase/ß-galactosidase ratios are expressed relative to vehicle treated cells (0.1% DMSO). Different letters denote significant differences (P < 0.05, n = 3).

The full-length mouse PPAR{alpha}/PPRE;–luciferase reporter assay is generally less responsive than the GAL4 system described above but is closer to the natural cell environment. This system may suffer from interference by endogenously expressed PPAR. Despite its limitations, the luciferase reporter gene system co-transfected with the full-length mPPAR{alpha}, was responsive to the activation of mPPAR{alpha} by CLA and its individual isomers (Figure 2C). Wy-14,643 activated PPAR{alpha} with a maximal induction of 7.5-fold over DMSO-treated cells (Figure 2D). The CLA mixture also activated PPAR{alpha} to the same extent as the furan-CLA metabolite (2.7- and 2.9-fold induction, respectively). The individual CLA isomers (10E,12Z), (9E,11E), and (9Z,11E) were activators of PPAR{alpha} (4- to 4.5-fold induction) with no apparent differences in efficacy.

CLA induces mRNA and protein of PPAR-responsive enzymes. The FaO cell line is a well-differentiated subclone derived from the rat hepatoma H4IIEC3 line (22) which expresses both PPAR{alpha} and PPARß but very low PPAR{gamma}1 (23). To determine the effect of CLA on PPAR-responsive gene expression, we used RT-PCR to quantify mRNA accumulation of ACO, L-FABP, and CYP4A1 ( Figure 3). Messenger RNA levels of the housekeeping gene ß-actin were found to be similar among treatments and time points (data not shown). FaO cells were incubated with a (9Z,11E)-CLA, linoleate, or Wy-14,643 (100 µM) in DMEM supplemented with fetal bovine serum (Figure 3A) for 6 and 24 h. ACO mRNA levels showed a slight increase at 6 h for all treatments but were not significantly different from control treated cells (P > 0.01). As expected, FaO cells treated with the prototypical peroxisome proliferator, Wy-14,643, for 24 h showed a higher induction (7-fold, P < 0.01) of ACO mRNA compared to vehicle- treated cells. The accumulation of ACO mRNA was significantly increased (P < 0.01) when FaO cells were treated with 200 µM CLA for 24 h in charcoal-stripped fetal bovine serum-containing media compared to vehicle-treated cells (data not shown).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Effect of CLA on (A) ACO (B) L-FABP, and (C) CYP4A1 mRNA accumulation in FaO hepatoma cells. Cells were incubated for 6 and 24 h with DMSO ({square}), 100 µM CLA .

), 100 µM linoleate .

), or 100 µM Wy-14,643 .

). Data are expressed relative to vehicle treated cells (0.1% DMSO). Total RNA was isolated and RT-PCR was performed as described in Materials and Methods. Asterisks designate values that are significantly different among treatments (* and **P < 0.05, ***P < 0.0001, n = 6;–9).

Levels of L-FABP mRNA were more responsive to CLA and Wy-14,643 (100 µM) than ACO mRNA after 24 h (Figure 3B), although there was no change after 6 h of treatment with CLA or linoleate. CLA induced L-FABP mRNA (P < 0.05) 11-fold after 24 h treatment in FaO cells compared to cells treated with vehicle, whereas Wy-14,643 induced the levels of L-FABP mRNA approximately 940-fold (P < 0.0001, Figure 3B). L-FABP mRNA levels for linoleate treated cells were slightly higher than control cultures (P < 0.05).

As shown in Figure 3C, CYP4A1 mRNA was elevated after 6 h of treatment with CLA and Wy-14,643 (34-fold and 68-fold, respectively). After 24 h the levels of CYP4A1 mRNA were similar for CLA and Wy-14,634-treated cells (9-fold, P < 0.05). Linoleate (a CLA parent compound and ligand for PPAR{alpha}) did not have any effect on CYP4A1 mRNA accumulation at any time point studied.

To determine whether the changes in CYP4A1 mRNA by CLA were associated with enhanced protein expression, Western blot analysis was performed. The dramatic effect of CLA on CYP4A1 protein levels paralleled mRNA levels. CYP4A1 protein levels were increased by (9Z,11E)-CLA in a dose-dependent manner ( Figure 4A). As expected, Wy-14,643 (a PPAR{alpha} selective activator) and bezafibrate (a non-subtype selective PPAR activator) induced the accumulation of this protein (Figure 4B). Troglitazone (a PPAR{gamma} selective activator) did not cause any change in CYP4A1 protein corroborating the low expression of PPAR{gamma} in FaO cells. Linoleate was able to increase CYP4A1 protein to a lesser extent than CLA (2.5- versus 5-fold). Albumin levels were not affected by CLA (P > 0.05).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 4. Effect of CLA on C Y P4A1 protein accumulation in FaO hepatoma cells. A: Inset, FaO cells were treated for 24 h with increasing concentrations of (9Z,11E)-CLA: Lanes 1;–3, DMSO; 4;–6, 50 µM; 7;–9, 100 µM; and 10;–12, 200 µM. A: Densitometric analysis for C Y P4A1 bands. Data (mean ± SD) are presented after normalization with albumin and relative to DMSO-treated cells. Asterisks denote significant differences among treatments (n = 3, P < 0.05). B: Inset, FaO cells were treated for 24 h with: lanes 13;–15, 100 µM linoleate; 16;–18, 100 µM Wy-14,643; 19;–21, 100 µM bezafibrate; and 22;–23, 100 µM troglitazone. B: Densitometric analysis for C Y P4A1 bands. Data (mean ± SD) are presented after normalization with albumin and relative to DMSO-treated cells. Asterisks denote significant differences among treatments (n = 3, P < 0.05).

CLA and linoleate are incorporated into FaO hepatoma cells to the same extent. Because of the striking difference between CLA and the parent compound linoleate on affecting PPAR-responsive gene expression, we compared their incorporation into FaO cells and into neutral and phospholipid fractions. The maximal uptake of [14C]CLA and [14C]linoleate occurred by 9 h ( Figure 5) without significant change after 12 and 24 h (P > 0.01). Incorporation of [14C]CLA or [14C]linoleate in FaO cells was similar at all time points determined. As shown in Table 2, linoleate and (9Z,11E)-CLA were incorporated at the same extent into the neutral and phospholipid fractions. The percentage of incorporation of equimolar mixture of CLA isomers [(9Z,11E)-, (10E,12Z)-, and (9E,11E)-] were also similar in the neutral and phospholipid fractions. These data indicate that the increased efficacy of CLA compared to linoleic at inducing gene expression is due to differences in affinity for PPAR{alpha} (IC50s = 140 nm versus 1.1 µM, present study and ref. (24)) and not due to pharmacokinetic differences.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. Incorporation of [14C]CLA and [14C]linoleate into FaO hepatoma cells. Cells were incubated with 0.1 µCi/ml 0.1% BSA-containing media of [14C]CLA ({circ}) or [14C]linoleate (•) for 1, 3, 6, 9, 12, or 24 h. After the time period indicated, percentage of incorporation into cells was determined by scintillation counting.


 
View this table:
[in this window]
[in a new window]
  		Table 2. Incorporation of CLA isomers into rat hepatoma FaO cells


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES

One of the potential mechanisms of CLA modulation of cancer, atherosclerosis, and diabetes may be through its potent effect on lipid transport and metabolism in vivo (9) (10) (25). In SENCAR mice, we found that CLA supported decreased body weights, increased hepatic lipid accumulation (11), and increased accumulation of lipid metabolizing enzymes (ACO, L-FABP and CYP4A1) (17). These proteins are regulated, in part, by PPAR{alpha}, the pivotal transcription factor for lipid metabolism in the liver (15, 26, (27)).

Searching for the involvement of CLA in modulating lipid metabolism, we found that several of the CLA isomers studied bound to PPAR{alpha} with submicromolar IC50s (140 nM to 400 nM). Although one cannot directly compare the IC50s obtained in different studies due to different methodologies and/or receptor constructs utilized, CLA isomers appear to be among the most avid fatty acid ligands of PPAR{alpha} reported to date (c.f., the eicosanoid derivative leukotriene B4, 8(S)-hydroxyeicosatetraenoic, and the prostaglandin I2 analog carbaprostacyclin which bind PPAR{alpha} with IC50s of 90 nM, 100 nM, and 500 nM, respectively) (14) (15). In particular, the (9Z,11E)- is the CLA isomer with the highest affinity (IC50 = 140 nM) in the present study. Interestingly, linoleate (CLA parent compound) has lower affinity for PPAR{alpha} (IC50 = 1.1 ± 0.14 µM; 24) when compared to CLA isomers under the same conditions of analysis.

In agreement with the binding data, we found that several CLA isomers activated PPAR{alpha} with the (9Z,11E)- isomer being the most efficacious mPPAR{alpha}-GAL4 activator, whereas linoleate activated the chimeric construct weakly. Activation of PPAR{alpha} by long chain fatty acids has been demonstrated previously, with polyunsaturated being slightly better activators than saturated fatty acids (28) (29). The difference in binding affinity to PPAR{alpha} and cellular activity (140 nM vs. 50 µM) is not surprising based on the number of potential metabolic pathways and other factors related to the disposition of CLA in cells, such as incorporation into membrane phospholipids, binding to cytosolic proteins (i.e., L-FABP), and catabolism via ß-oxidation (28) (30). In particular, the incorporation of CLA into the FaO cells in the present study was 30%.

According to our binding and transcriptional activation data, it is not surprising that CLA and linoleate have different abilities to induce L-FABP and CYP4A1 mRNA (Figure 3B and Figure 3C), and these differences are not attributed to preferential incorporation into liver cells or lipid fractions (Table 2). Different potencies of various fatty acids (at 320 µM) on the induction of L-FABP mRNA and protein have been reported previously in FaO cells (31). The potency of linoleate to induce L-FABP mRNA in the present study was comparable to that reported in the Meunier-Durmort et al. study (31) (3.1-fold vs. 3.8-fold, respectively). Interestingly, the linoleate conjugated isomer (9Z,11E-CLA) studied here was a more potent inducer (11-fold) than its parent compound; moreover, the CLA potency obtained was comparable to the PPAR{alpha} activator ETYA (31). Although the differences in potency in the Meunier-Durmort et al. report (31) were attributed to the length of the fatty acid chain when octanoic (no effect), linoleic (3.8-fold), arachidonic (5.0-fold) acids, and the PPAR activator ETYA (14-fold) were compared, our results suggest that the spatial and positional double bond system present in CLA may favor a conformational change important for the binding and activation of PPAR{alpha}.

Based on the current knowledge of peroxisome proliferator-mediated gene expression and our findings, we can speculate that peroxisome proliferators and CLA share a mechanism to explain their hypolipidemic and hypocholesterolemic effects. At the extracellular level, the hypolipidemic effect induced by peroxisome proliferators, may be due to an increased hydrolysis by lipoprotein lipase of triglycerides derived from chylomicrons and very low density lipoprotein particles (32). Evidence suggests that the plasma triglyceride clearance processes are regulated by peroxisome proliferators and, in some steps, directly by hepatic PPAR{alpha} as lipoprotein lipase expression is induced by PPAR activators (32). Inside the cell, free fatty acids (FFAs) are transported by FABP. This binding protein is also up-regulated through a PPAR-mediated mechanism (23) (33) with one functional PPAR response element (PPRE) regulating its expression (34). FFAs regulate other lipid-metabolizing enzymes of the peroxisome, microsome, or cytosol via activation of PPAR{alpha}. In peroxisomes, the rate-limiting enzyme for ß-oxidation ACO and the enzyme responsible for the second and third step in ß-oxidation enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase have functional PPREs (35) (36). Other lipid metabolizing enzymes regulated via PPAR are also found in mitochondria (acyl-CoA synthase, medium chain acyl-CoA dehydrogenase, ß-hydroxy-ß-methylglutaryl-CoA synthase, reviewed in 32), in microsomes (CYP4A, {omega}-hydroxylase, (37)) and in cytosol (phosphoenol pyruvate carboxy kinase, reviewed in 32).

These are the first findings to demonstrate that (9Z,11E)-CLA is a high affinity ligand and activator for PPAR{alpha}. Taken together, these data suggest that CLA regulates lipid metabolism through binding and activation of PPAR{alpha}. Therefore, CLA may be beneficial for those metabolic disorders related to impaired lipid metabolism, such as non-insulin-dependent diabetes mellitus, atherosclerosis, obesity, and cancer.


  ACKNOWLEDGMENTS

We thank A. Kempa-Steczko for gas chromatography lipid analyses, E. Snyder and G. Anchiraico for technical assistance, Dr. J. Tugwood (Zeneca Central Toxicology Laboratory, UK) for providing pCDNA3-mPPAR{alpha}, psV-GL2-PPRE-luciferase, and Dr. T. Consler (Glaxo Wellcome) for the PPAR{alpha} ligand binding domain. We also thank Dr. S. A. Kliewer (Glaxo Wellcome) and Dr. T. M. Willson (Glaxo Wellcome) for helpful discussion of the data and manuscript. This work was supported by AICR#96B071. S. Y. Moya-Camarena was supported by AICR and CONACyT (Mexico).

Manuscript received December 30, 1998; and in revised form March 15, 1999.

Abbreviations: CLA, conjugated linoleic acid; PPAR{alpha}, peroxisome proliferator-activated receptor alpha; ACO, acyl-CoA oxidase; L-FABP, liver fatty acid-binding protein; CYP4A1, cytochrome P450 IVA1; PPRE, PPAR-responsive element; DMSO, dimethyl sulfoxide; SPA, scintillation proximity assay


  REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

  1. Lavillonnière, F., Martin, J. C., Bougnoux, P., Sébédio, J. L. 1998. Analysis of conjugated linoleic acid isomers and content in French cheeses. J. Am. Oil Chem. Soc. 75:343-352.

  2. Chin, S. F., Storkson, J. M., Liu, W., Albright, K. J., Pariza, M. W. 1994. Conjugated linoleic acid (9,11- and 10,12-octadecanoid acid) is produced in conventional but not germ-free rats fed linoleic acid. J. Nutr. 124:694-701.

  3. Salminen, I., Mutanen, M., Jauhiainen, M., Aro, A. 1998. Dietary trans fatty acids increase conjugated linoleic acid levels in human serum. J. Nutr. Biochem. 9:93-98.

  4. Lin, H., Boyslston, H., Chang, M. J., Leudecke, L. O., Shultz, T. D. 1995. Survey of the conjugated linoleic acid content of dairy products. J. Dairy Sci. 78:2358-2365[Abstract].

  5. Ha, Y. L., Grimm, N. K., Pariza, M. W. 1987. Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid. Carcinogenesis. 8:1881-1887[Abstract/Free Full Text].

  6. Ip, C., Singh, M., Thompson, H. J., Scimeca, J. A. 1994. Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat. Cancer Res. 54:1212-1215[Abstract/Free Full Text].

  7. Belury, M.A., Bird, C., Nickel, K. P., Wu, B. 1996. Inhibition of mouse skin tumor promotion by dietary conjugated linoleate. Nutr. Cancer. 26:149-157[Medline].

  8. Ha, Y. L., Storkson, J., Pariza, M. W. 1990. Inhibition of bezo(a)pyrene-induced mouse forestomach neoplasia by conjugated derivatives of linoleic acid. Cancer Res. 50:1097-1101[Abstract/Free Full Text].

  9. Nicolosi, R. J., Laitinen, L. 1996. Dietary conjugated linoleic acid reduces aortic fatty streak formation greater than linoleic acid in hypercholesterolemic hamsters. FASEB J. 10:A477.

  10. Houseknecht, K. L., Vanden Heuvel, J. P., Moya-Camarena, S. Y., Portocarrero, C. P., Peck, L. W., Nickel, K. P., Belury, M. A. 1998. Dietary conjugated linoleic acid normalizes impaired glucose tolerance in the Zucker diabetic fatty fa/fa rat. Biochem. Biophys. Res. Commun. 244:678-682[Medline].

  11. Belury, M. A., Kempa-Steczko, A. 1997. Conjugated linoleic acid modulates hepatic lipid composition in mice. Lipids. 32:199-204[Medline].

  12. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A., Spiegelman, B. M. 1994. mPPAR{gamma}2: tissue specific regulator of an adipocyte enhancer. Genes Dev. 8:1224-1234[Abstract/Free Full Text].

  13. Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F., Wahli, W. 1996. The PPAR{alpha}-leukotriene B4 pathway to inflammation control. Nature. 384:39-43[Medline].

  14. Kliewer, S. A., Sundseth, S. S., Jones, S. A., Brown, P. J., Wisely, G. B., Koble, C. S., Devchand, P., Wahli, W., Willson, T. M., Lenhard, J. M., Lehmann, J. M. 1997. Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors {alpha} an {gamma}. Proc. Natl. Acad. Sci. USA. 94:4318-4323[Abstract/Free Full Text].

  15. Forman, B. M., Chen, J., Evans, R. M. 1997. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors {alpha} and {delta}. Proc. Natl. Acad. Sci. USA. 94:4312-4317[Abstract/Free Full Text].

  16. Krey, G., Braissant, O., L'Horset, F., Kalkhoven, E., Perroud, M., Parker, M. G., Wahli, W. 1997. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by co-activator-dependent receptor ligand assay. Mol. Endocrinol. 11:779-791[Abstract/Free Full Text].

  17. Belury, M. A., Moya-Camarena, S. Y., Liu, K-L., Vanden Heuvel, J. P. 1997. Dietary conjugated linoleic acid induces peroxisome-specific enzyme accumulation and ornithine decarboxylase activity in mouse liver. J. Nutr. Biochem. 8:579-584.

  18. Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Wilson, T. M., Kliewer, S. A. 1995. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor {gamma}. J. Biol. Chem. 270:12953-12956[Abstract/Free Full Text].

  19. Nichols, J. S., Parks, D. J., Consler, T. G., Blanchard, S. G. 1998. Development of a scintillation proximity assay for peroxisome proliferator-activated receptor gamma ligand binding domain. Anal. Biochem. 257:112-119[Medline].

  20. Kliewer, S. A., Forman, B. M., Blumberg, B., Ong, E. S., Borgmeyer, U., Mangelsdorf, D. J., Umesono, K., Evans, R. M. 1994. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc. Natl. Acad. Sci. USA. 91:7355-7359[Abstract/Free Full Text].

  21. Vanden Heuvel, J. P., Clark, G. C., Kohn, M. C., Tritscher, A. M., Greenlee, W. F., Lucier, G. W., Bell, D. A. 1994. Dioxin-responsive genes: examination of dose-response relationships using quantitative reverse transcriptase-polymerase chain reaction. Cancer Res. 54:62-68[Abstract/Free Full Text].

  22. Bayly, A. C., French, N. J., Dive, C., Roberts, R. A. 1993. Non-genotoxic hepatocarcinogens in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis. J. Cell Sci. 104:307-315[Abstract].

  23. Poirier, H., Braissant, O., Noit, I., Wahli, W., Besnard, P. 1997. 9-cis-Retinoic acid enhances fatty acid-induced expression of the liver-fatty acid binding protein gene. FEBS Lett. 412:480-484[Medline].

  24. Xu, H. E., Lambert, M. H., Montana, V. G., Parks, D. J., Blanchard, S. G., Brown, P. J., Sternbach, D. D., Lehmann, J. M., Wisely, G. B., Willson, T. M., Kliewer, S. A., Milburn, M. V. 1999. Molecular recognition of fatty acids by peroxisome proliferator-activated receptors. Mol. Cell. In press.

  25. Belury, M. A., Vanden Heuvel, J. P. 1997. Protection against cancer and heart disease by CLA: potential mechanism of action. Nutr. Dis. Update. 1:58-63.

  26. Keller, H., Dreyer, C., Medin, J., Mahfoudi, A., Ozato, K., Wahli, W. 1993. Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers. Proc. Natl. Acad. Sci. USA. 90:2160-2164[Abstract/Free Full Text].

  27. Lee, S. S-T., Pineau, T., Drago, J., Lee, E. J., Owens, J., Kroetz, D. L., Fernandez-Salguero, P. M., Westphal, H., Gonzalez, F. 1995. Targeted disruption of the {alpha} isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol. Cell Biol. 15:3012-3022[Abstract].

  28. Bocos, C., Gottlicher, M., Gearing, K., Banner, C., Enmark, E., Teboul, M., Crickmore, A., Gustafsson, J. A. 1995. Fatty acid activation of peroxisome proliferator activated receptor (PPAR). J. Steroid Biochem. Mol. Biol. 53:467-473[Medline].

  29. Gottlicher, M., Widmark, E., Li, Q., Gustafsson, J. A. 1992. Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor. Proc. Natl. Acad. Sci. USA. 89:4653-4657[Abstract/Free Full Text].

  30. Issemann, I., Prince, R. A., Tugwood, J. D., Green, S. 1993. The peroxisome proliferator-activated receptor:retinoid X receptor heterodimer is activated by fatty acids and fibrate hypolipidaemic drugs. J. Mol. Endocrinol. 11:37-47[Abstract/Free Full Text].

  31. Meunier-Durmort, C., Poirier, H., Niot, I., Forest, C., Besnard, P. 1996. Up-regulation of the expression of the gene for liver fatty acid-binding protein by long chain fatty acids. Biochem. J. 319:483-487.

  32. Schoonjans, K., Staels, B., Auwerx, J. 1996. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J. Lipid Res. 37:907-925[Abstract].

  33. Sterchele, P. F., Vanden Heuvel, J. P., Davis, J. W., II, Shargo, E., Knudsen, J., Peterson, E. 1994. Induction of hepatic acyl-CoA binding protein and liver fatty acid binding protein by perfluorodecanoic acid in rats. Lack of correlation with hepatic long chain acyl-CoA levels. Biochem. Pharmacol. 48:955-966[Medline].

  34. Issemann, I., Prince, R., Tugwood, J., Green, S. 1992. A role for fatty acids and liver fatty acid binding protein in peroxisome proliferation? Biochem. Soc. Trans. 20:823-827.

  35. Dreyer, C., Krey, G., Keller, H., Givel, F., Helftenbein, G., Wahli, W. 1992. Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors. Cell. 68:879-887[Medline].

  36. Tugwood, J. D., Issemann, I., Anderson, R. G., Bundell, K. R., McPheat, W. L., Green, S. 1992. The mouse peroxisome proliferator-activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl CoA oxidase gene. EMBO J. 11:433-439[Medline].

  37. Johnson, E. F., Palmer, C. A. N., Griffin, K. J., Hsu, M-H. 1996. Role of peroxisome proliferator-activated receptor in cytochrome P450 A gene regulation. FASEB J. 10:1241-1248[Abstract].


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moya-Camarena, S. Y.
Right arrow Articles by Belury, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moya-Camarena, S. Y.
Right arrow Articles by Belury, M. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today