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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zomer, A. W. M.
Right arrow Articles by van der Saag, P. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zomer, A. W. M.
Right arrow Articles by van der Saag, P. T.
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?

Journal of Lipid Research, Vol. 41, 1801-1807, November 2000
Copyright © 2000 by Lipid Research, Inc.

Original Article

Pristanic acid and phytanic acid: naturally occurring ligands for the nuclear receptor peroxisome proliferator-activated receptor {alpha}

Anna W. M. Zomera,b, Bart van der Burga, Gerbert A. Jansenc, Ronald J. A. Wandersc,d, Bwee Tien Poll-Theb, and Paul T. van der Saaga
a Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands
b Department of Pediatrics/Metabolic Diseases, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands
c Departments of Pediatrics, Laboratory for Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands
d (Emma Children's Hospital) and Clinical Chemistry, Laboratory for Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands

Correspondence to: Anna W. M. Zomer


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Phytanic acid and pristanic acid are branched-chain fatty acids, present at micromolar concentrations in the plasma of healthy individuals. Here we show that both phytanic acid and pristanic acid activate the peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) in a concentration-dependent manner. Activation is observed via the ligand-binding domain of PPAR{alpha} as well as via a PPAR response element (PPRE). Via the PPRE significant induction is found with both phytanic acid and pristanic acid at concentrations of 3 and 1 µM, respectively. The trans-activation of PPAR{delta} and PPAR{gamma} by these two ligands is negligible. Besides PPAR{alpha}, phytanic acid also trans-activates all three retinoic X receptor subtypes in a concentration-dependent manner. In primary human fibroblasts, deficient in phytanic acid {alpha}-oxidation, trans-activation through PPAR{alpha} by phytanic acid is observed. This clearly demonstrates that phytanic acid itself, and not only its metabolite, pristanic acid, is a true physiological ligand for PPAR{alpha}. Because induction of PPAR{alpha} occurs at ligand concentrations comparable to the levels found for phytanic acid and pristanic acid in human plasma, these fatty acids should be seen as naturally occurring ligands for PPAR{alpha}.

These results demonstrate that both pristanic acid and phytanic acid are naturally occurring ligands for PPAR{alpha}, which are present at physiological concentrations. — Zomer, A. W. M., B. van der Burg, G. A. Jansen, R. J. A. Wanders, B. T. Poll-The, and P. T. van der Saag. Pristanic acid and phytanic acid: naturally occurring ligands for the nuclear receptor peroxisome proliferator-activated receptor {alpha}. J. Lipid Res. 2000. 41: 1801;–1807.

Supplementary key words: natural ligand, peroxisomal disorder, retinoic X receptor, phytol derivatives


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is an isoprenoid-derived 3-methyl fatty acid, which is derived solely from external sources. This branched-chain fatty acid originates from the phytol side chain of chlorophyll ( Fig 1). Microorganisms, which are present in the rumen of ruminants, are thought to release phytol from chlorophyll, after which phytol is converted into phytanic acid (1). Because humans are not capable of phytol release from chlorophyll (2), all phytanic acid enters the human body via the diet. In particular, ruminant fats, fish, and dairy products are rich sources of phytanic acid. Phytanic acid is metabolized into pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) in a process called {alpha}-oxidation. Pristanic acid, like phytanic acid, is present in the lipids from many sources (3). Peroxisomes play an indispensable role in both phytanic acid {alpha}-oxidation and pristanic acid ß-oxidation (4) (5). Although the subcellular localization of the enzymes involved in phytanic acid degradation has not been resolved definitively, phytanoyl-CoA hydroxylase (PhyH), the first enzyme in the phytanic acid {alpha}-oxidation pathway, is strictly peroxisomal in both rats (6) (7) (8) and humans (9). With respect to pristanic acid ß-oxidation, studies have shown that pristanic acid undergoes three cycles of ß-oxidation in the peroxisome, followed by further ß-oxidation in the mitochondrion (10).



View larger version (13K):
[in this window]
[in a new window]
  		Figure 1. Metabolic pathway from phytol to pristanic acid.

Phytanic acid and pristanic acid accumulate in a variety of inherited disorders. First, in patients with a defect in peroxisome biogenesis, both metabolites are elevated in plasma and tissues because of a deficient oxidation of both phytanic acid and pristanic acid. Second, phytanic acid and pristanic acid accumulate in patients with D-bifunctional protein deficiency. In these patients pristanic acid ß-oxidation is deficient (11). Despite the fact that {alpha}-oxidation can proceed normally, phytanic acid accumulates, probably as a result of feedback inhibition of the {alpha}-oxidation pathway by high levels of pristanic acid caused by deficient pristanic acid ß-oxidation. In two other peroxisomal disorders phytanic acid accumulates whereas pristanic acid levels are normal. These two disorders are Refsum disease, in which PhyH is deficient because of mutations in the PHYH gene (12) (13), and rhizomelic chondrodysplasia punctata type 1. In the latter disease, the import of a distinct set of peroxisomal proteins equipped with a peroxisome-targeting signal type 2 (PTS2) is impaired because of mutations in the gene encoding the PTS2 receptor. As a consequence, all PTS2-containing proteins cannot be imported, including PhyH, resulting in a deficient {alpha}-oxidation (4). In the plasma of healthy individuals phytanic acid and pristanic acid are present at micromolar concentrations (<10 and <3 µM, respectively) (14). In patients the plasma concentration of phytanic acid and pristanic acid can rise, depending on the disorder, to 1,300 and 80 µM, respectively (5).

Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors belonging to the family of nuclear hormone receptors. So far, three subtypes have been identified ({alpha}, {delta}/ß, and {gamma}) from humans, rodents, and Xenopus (15). The transcriptional regulation by PPARs is achieved through PPAR-retinoid X receptor (RXR) heterodimers, which bind to PPAR response elements (PPREs) in the promoter of target genes (16) (17). RXRs consist of RXR{alpha}, RXRß, and RXR{gamma} (18), which are activated by 9-cis-retinoic acid. Besides heterodimerization with various members of the nuclear hormone receptor family such as PPAR and retinoic acid receptor, RXRs can also homodimerize and regulate trans-activation of target genes.

In general, PPARs are activated by a variety of natural and synthetic ligands such as fatty acids (e.g., arachidonic acid), eicosanoids [e.g., 15-deoxy-prostaglandin J2 and leukotriene B4 (LTB4)], plasticizers [e.g., di(2-ethylhexyl)phthalate], and hypolipidemic agents (e.g., clofibrate) (19) (20) (21) (22) (23). There are differences in the ability of the different compounds to activate the various receptor subtypes. In line with the fact that the major function of PPAR{alpha} is the regulation of genes involved in lipid homeostasis (24) (25), fatty acids have been identified as PPAR{alpha}-specific ligands. On the basis of this functional specificity we have searched for novel PPAR{alpha}-specific ligands among naturally occurring fatty acids. We report the effect of different phytol-derived branched-chain fatty acids on transcriptional activation by RXRß and PPAR{alpha}. We then investigated the concentration-dependent trans-activation by two of these phytol derivatives, which were found to be ligands. Finally, we investigated the transcriptional activation by phytanic acid of PPAR{alpha} in phytanic acid {alpha}-oxidation-deficient cell lines.


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

Materials
Phytanic acid and pristanic acid were synthesized as described previously (26). Phytol and ß-cyclodextrin were from Sigma (St. Louis, MO). 4,8,12-Trimethyltridecanoic acid, 4,8-dimethylnonanoic acid, and 2,6-dimethylheptanoic acid were a generous gift from G. Dacremont (Ghent, Belgium). SuperFect was from Qiagen (Hilden, Germany). The Luclite luciferase gene reporter assay kit and Topcount liquid scintillation counter were from Packard Instruments (Meriden, CT).

Expression and reporter plasmids
pSG5-RXRß was a gift from P. Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, College de France, Strasbourg, France). GAL4-mRXR{alpha}-LBD, GAL4-mRXRß-LBD, and GAL4-mRXR{gamma}-LBD have been described previously (27). pBK-CMV-Gal4-rPPAR{alpha}-LBD, pBK-CMV-Gal4-rPPAR{delta}-LBD, and pBK-CMV-Gal4-rPPAR{gamma}-LBD were gifts from E. Treuter (Biosciences at Novum, Karolinska Institute, Huddinge, Sweden). 3*PPRE-tk-Luc, pCMX-mPPAR{alpha}, pCMX-mPPAR{delta}, and pCMX-mPPAR{gamma} were gifts from R. M. Evans (Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA).

Cell culture
Monkey COS-1 cells and human HepG2 cells were purchased from the American Type Culture Collection (Rockville, MD). Human primary fibroblasts from a Refsum disease patient, a Zellweger syndrome patient, and a healthy individual (control) were from the Mutant Cell Repository (Laboratory for Genetic Metabolic Diseases, University of Amsterdam, The Netherlands). The diagnoses of Refsum disease and Zellweger syndrome, respectively, were based on clinical symptoms and confirmed by detailed studies of fibroblasts showing an isolated deficiency of PhyH and a complete loss of peroxisomal functions, respectively (14). All cell lines were grown at 37°C in the presence of 7.5% CO2. COS-1 cells were cultured in DF medium (1:1 mixture of Dulbecco's minimal essential medium and Ham's F12, buffered with 44 mM NaHCO3) supplemented with 7.5% fetal calf serum (FCS). HepG2 cells were cultured in Dulbecco's minimal essential medium supplemented with 10% FCS. The primary fibroblasts were cultured in Ham's F12 buffered with 44 mM NaHCO3 and supplemented with 10% FCS.

Transient transfections
COS-1 and HepG2 cells were transiently transfected in 24-well plates by the calcium phosphate coprecipitation method, using 1.0 µg of reporter construct, 0.25 µg of ß-galactosidase expression vector SV2LacZ, 0.125 µg of receptor construct, and pBluescript SK- to a total of 1.5 µg of DNA per well (28). After 16 h medium was refreshed, and cells were exposed to various additions as indicated. All ligands were solubilized in ß-cyclodextrin (10 mg/ml in 100 mM Tris-HCl, pH 8.0). Cells were harvested 24 h later. Primary fibroblasts were transiently transfected in 12-well plates with SuperFect according to the manufacturer instructions by using 0.7 µg of reporter construct, 0.2 µg of SV2LacZ, 0.1 µg of receptor construct, and 5 µl of SuperFect per well. Medium was refreshed after 3 h and after 16 h, when ligands were added as indicated. Cells were harvested 24 h later. Cell extracts were analyzed for luciferase activity by using the Luclite luciferase gene reporter assay kit and measured in a Topcount liquid scintillation counter. Values were corrected for ß-galactosidase activity as a measure of transfection efficiency (29).


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES

Phytol derivatives differentially stimulate RXR and/or PPAR{alpha}
To examine whether different phytol-derived branched-chain fatty acids can mediate transcriptional effects through PPAR{alpha}/RXR, we used a chimeric receptor system in which the respective ligand-binding domains (LBDs) were fused to the DNA-binding domain of the yeast transcription factor GAL4. By making use of transient transfections, the various branched-chain fatty acids were tested both in COS-1 and HepG2 cells. From the various phytol derivatives tested, only phytanic acid activated GAL4-RXRß ( Fig 2A). No activation of GAL4-RXRß was observed with pristanic acid. Previously, the transcriptional activation of full-length RXR{alpha} by phytanic acid has been described (30) (31). Two of the tested phytol derivatives were found to have a stimulatory effect on GAL4-PPAR{alpha}: Phytanic acid and, with 10-fold higher induction levels, pristanic acid (Fig 2B). The other derivatives, including 4,8,12-trimethyltridecanoic acid, 4,8-dimethylnonanoic acid, and 2,6-dimethylheptanoic acid, which are all intermediates of pristanic acid ß-oxidation, did not show significant stimulation, either of GAL4-RXRß or of GAL4-PPAR{alpha} (Fig 2A and Fig B). The effects of phytanic acid and pristanic acid on the other two PPAR subtypes, determined via GAL4-PPAR{delta} and GAL4-PPAR{gamma}, were negligible ( Fig 3). All three subtypes of RXR were stimulated by phytanic acid in a concentration-dependent manner ( Fig 4). The highest activation was seen with GAL4-RXRß. However, this difference in activation was not specific for phytanic acid because the same difference in activation was seen with 9-cis-retinoic acid (data not shown). Moreover, the difference in activation, observed with the RXR subtypes, might be due to a difference in expression levels of these receptors. Further studies are necessary to elucidate the observed activation differences.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 2. trans-Activation of GAL4-RXRß and GAL4-PPAR{alpha} by phytol derivatives. HepG2 and COS-1 cells were transiently transfected with the reporter construct GAL4-tata-Luc, ß-galactosidase expression vector SV2LacZ, and an expression vector (see Materials and Methods). The various phytol derivatives were added at a final concentration of 50 µM, from 2 mM stock solutions. (A) Expression vector GAL4-RXRß. (B) Expression vector GAL4-PPAR{alpha}. Data are presented as the mean (± SEM) fold induction. COS-1 (open columns); HepG2 (solid columns).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. Activation through the LBD of the PPAR subtypes with phytanic acid and pristanic acid. COS-1 cells were transiently transfected with the reporter construct GAL4-tata-Luc, ß-galactosidase expression vector SV2LacZ, and the expression vector GAL4-PPAR{alpha}, GAL4-PPAR{delta}, or GAL4-PPAR{gamma}. Data are presented as means (± SEM) fold induction. Cells were stimulated with 50 µM ligand. Phytanic acid (solid columns); pristanic acid (shaded columns). As a control cells were also grown in the presence of 300 µM mono(2-ethylhexyl) phthalate (open columns).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Concentration-dependent activation through the LBD of RXR subtypes with phytanic acid. COS-1 cells were transiently transfected with the reporter construct GAL4-tata-Luc, ß-galactosidase expression vector SV2-LacZ, and the expression vector GAL4-RXR{alpha}, GAL4-RXRß, or GAL4-RXR{gamma} by making use of the calcium phosphate coprecipitation method. Phytanic acid concentrations used: 10 µM (solid columns); 30 µM (shaded columns); 100 µM (open columns).

Transcriptional activation of GAL4-PPAR{alpha} with phytanic acid and pristanic acid
Concentration-dependent activation via the LBD of PPAR{alpha} was seen with phytanic acid ( Fig 5A) and pristanic acid (Fig 5B). The induction with pristanic acid is, depending on the cell line used, 6 to 10 times higher than the induction with phytanic acid, indicating a higher affinity of PPAR{alpha} for pristanic acid than for phytanic acid. With phytanic acid at 10 µM no induction was detectable in either COS-1 or HepG2 cells, while the same concentration of pristanic acid elicited a 30-fold induction in COS-1 cells (Fig 5B). In the case of pristanic acid, the median effective concentration (EC50) value for induction was found to be approximately 40 µM. EC50 values for induction by phytanic acid could not be determined, because the phytanic acid curve had not reached a plateau at 100 µM. Higher phytanic acid concentrations could not be tested as these concentrations were found to be toxic in both cell lines.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 5. Activation via the LBD of PPAR{alpha} with concentration curves of phytanic acid and pristanic acid. COS-1 and HepG2 cells were transiently transfected with the reporter construct GAL4-tata-Luc, ß-galactosidase expression vector SV2-LacZ, and the expression vector GAL4-PPAR{alpha}. (A) Phytanic acid, added as indicated. (B) Pristanic acid, added as indicated. COS-1 (open circles); HepG2 (shaded squares).

Transcriptional activation of full-length PPAR{alpha} with phytanic acid and pristanic acid
So far, the trans-activation by phytanic acid and pristanic acid was studied with GAL4-PPAR{alpha} chimeric constructs on a GAL4 reporter. The next step was to use full-length PPAR{alpha} in combination with RXRß and a PPRE. Because no difference in activation was observed between the full-length RXR subtypes (not shown), RXRß was used in all experiments. The maximal induction found with phytanic acid ( Fig 6A) was in the same range as the induction found for pristanic acid (Fig 6B). This was not only true for PPAR{alpha} alone, but also for the combination of PPAR{alpha} and RXRß. Under these conditions the EC50 value for induction by phytanic acid was approximately 20 µM, while for pristanic acid it was about 3 µM. Because the plateau level was not reached completely with phytanic acid, the EC50 value for phytanic acid is a minimal estimate. The EC50 value for pristanic acid (3 µM) is within the physiological concentration at which this branched-chain fatty acid is present in the plasma of healthy individuals. On the basis of these experiments it is clear that the affinity of the full-length receptor is also significantly higher for pristanic acid than for phytanic acid.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. Concentration-dependent activation of full-length PPAR{alpha} with phytanic acid and pristanic acid. COS-1 cells were transiently transfected with the reporter construct 3*PPRE-tk-Luc, ß-galactosidase expression vector SV2-LacZ, and the indicated expression vector pCMX-PPAR{alpha}, pSG5-RXRß, or both (see Materials and Methods). (A) Phytanic acid, added as indicated. (B) Transfections were done as described in Fig 4A. However, pSG5-RXRß alone was not tested because pristanic acid is not a ligand for RXRß. Pristanic acid was added as indicated. RXRß (shaded triangles); PPAR{alpha} (open squares); PPAR{alpha} plus RXRß (shaded circles).

Synergistic trans-activation with PPAR{alpha} and RXRß
The trans-activation by 100 µM phytanic acid of RXRß was found to be 18-fold (Fig 6), while with the same concentration of pristanic acid no stimulation of RXRß was observed (Fig 2A). The activation of PPAR{alpha} caused by 100 µM phytanic acid or pristanic acid was in both cases approximately 60-fold, while the effect of these ligands on PPAR{alpha} in combination with RXRß was about 130-fold (Fig 6). These results show, both with phytanic acid and pristanic acid, synergistic trans-activation of PPAR{alpha} plus RXRß when compared with the level caused by the summation of RXRß and PPAR{alpha} activities. Because phytanic acid can function as a ligand for RXRß while pristanic acid cannot, these results also suggest that the synergistic process of trans-activation of RXRß with PPAR{alpha} is not RXR-ligand dependent.

Phytanic acid trans-activates PPAR{alpha} in {alpha}-oxidation-deficient cell lines
Because pristanic acid is derived from phytanic acid after oxidative decarboxylation of phytanic acid, trans-activation by phytanic acid of PPAR{alpha} might not be caused by phytanic acid itself, but by pristanic acid, derived from in vivo metabolized phytanic acid. Therefore human primary fibroblasts from patients with either Zellweger syndrome or Refsum disease, both deficient in phytanic acid {alpha}-oxidation, were used to study the trans-activation by phytanic acid of the GAL4-PPAR{alpha} chimeric constructs. In both {alpha}-oxidation deficient cell lines a stimulatory effect of phytanic acid could be observed ( Fig 7) that was comparable to the effect found in primary fibroblasts from healthy individuals. In {alpha}-oxidation deficient cell lines a stimulatory effect of pristanic acid was also observed. These results clearly demonstrate that phytanic acid itself, like pristanic acid, is a ligand for PPAR{alpha}.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 7. trans-Activation of PPAR{alpha} in primary human fibroblasts. The GAL-tk-Luc reporter construct was cotransfected with the GAL4-PPAR{alpha} expression vector and the ß-galactosidase expression vector SV2-LacZ by making use of SuperFect (see Materials and Methods). Cells were incubated for 16 h with a 100 µM concentration of the indicated ligand. Luciferase and ß-galactosidase expression was measured from cell lysates of treated and untreated cells. Data are presented as the mean (± SEM) fold induction. ß-Cyclodextrin (solvent; open columns); phytanic acid (grey columns); pristanic acid (dark columns).


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES

PPAR{alpha} is activated by a variety of natural and synthetic ligands such as various long-chain fatty acids (linoleic acid and arachidonic acid), eicosanoids (leukotriene B4 and 8(S)-hydroxyeicosatetraenoic acid), and hypolipidemic drugs (clofibric acid and Wy14,643). The structural similarities between the different peroxisome proliferators are not stringent. With the exception of an acidic group observed in all of them, their structures vary widely. Because of the involvement of PPAR{alpha} in lipid homeostasis, we focused our research for two main reasons on the phytol pathway. First, two phytol derivatives, phytanic acid and pristanic acid, are known to accumulate in a variety of inherited disorders and second, various phytol derivatives are fatty acids and thus might be potential ligands for PPAR{alpha}.

The results described in this article show that the naturally occurring fatty acids phytanic acid and pristanic acid can act as ligands for PPAR{alpha}. Previous studies by Seedorf et al. (32) (33) have focused on phytanic acid as a ligand for PPAR{alpha}. These studies were done in mice in which the gene encoding sterol carrier protein 2 was disrupted (33). Interestingly, this gene produces two mRNA transcripts, which correspond to a 58-kDa protein with both thiolase and sterol carrier protein 2 activity and a 13-kDa protein with sterol carrier protein activity only. The 58-kDa thiolase/sterol carrier protein plays a crucial role in the peroxisomal oxidation of 2-methyl fatty acids such as pristanic acid, which explains the deficient oxidation of pristanic acid in these mice (33). Under normal feeding conditions there is no accumulation of pristanic acid and phytanic acid because the standard laboratory diet is low in phytanic acid. Feeding phytol to the mice, however, caused a strong increase in serum levels of phytanic acid and pristanic acid. Furthermore, a variety of PPAR{alpha}-regulated genes was found to be upregulated, which led to the identification of phytanic acid as a natural ligand for PPAR{alpha}. However, as we have shown in this article, pristanic acid is a much more powerful ligand for PPAR{alpha} than phytanic acid, which suggests that the effects observed in the sterol carrier protein-deficient mice on feeding phytol may well be due to pristanic acid rather than phytanic acid. Our findings may also be of importance for other inherited diseases in which the peroxisomal oxidation of pristanic acid and phytanic acid is deficient. In patients with D-bifunctional protein deficiency for instance, oxidation of pristanic acid is blocked at the level of the second and/or third step of ß-oxidation (11) (34) (35). However, in such patients the oxidation of other fatty acids, such as C26:0, is also deficient, leading to their accumulation and possibly disturbances of additional pathways.

A noticeable difference between the two ligands is that phytanic acid is also a ligand for RXR, albeit at supraphysiological concentrations. These supraphysiological concentrations, however, are readily reached in patients with Refsum disease, in whom phytanic acid plasma concentrations can rise to 1,300 µM (5). Aberrant activation of RXRs could, therefore, also contribute to the pathology of Refsum disease. Because RXRs are the natural partners of PPAR{alpha}, accumulation of both PPAR{alpha}/RXR ligands would lead to overactivation of pathways under the control of these nuclear receptors, which could lead to ectopic (over) expression of target genes. However, because RXRs also dimerize with several other different nuclear hormone receptors, other pathways may be stimulated as well.

We show in this article that pristanic acid is a PPAR{alpha} ligand at physiological concentrations. Because of a deficient {alpha}-oxidation of phytanic acid, the plasma concentration of pristanic acid in patients with Refsum disease is lower compared when healthy individuals (5). It could therefore be reasoned that the pristanic acid concentration in patients with Refsum disease becomes too low to be able to trans-activate PPAR{alpha}, which also might contribute to the pathology of this peroxisomal disorder. The identification of pristanic acid as a physiological ligand for PPAR{alpha} gives us new leads, which may well have important implications for our understanding of the pathogenesis of peroxisomal disorders.


  ACKNOWLEDGMENTS

We thank P. Chambon for providing pSG5-RXRß; E. Treuter for pBK-CMV-Gal4-rPPAR{alpha}-LBD, pBK-CMV-Gal4-rPPAR{delta}-LBD, and pBK-CMV-Gal4-rPPAR{gamma}-LBD; and R. M. Evans for 3*PPRE-tk-Luc, pCMX-mPPAR{alpha}, pCMX-mPPAR{delta}, and pCMX-mPPAR{gamma}. We also thank J. Heinen and F. Vervoordeldonk for graphical assistance.

Manuscript received April 20, 2000; and in revised form July 10, 2000

Abbreviations: EC50, median effective concentration; FCS, fetal calf serum; LBD, ligand-binding domain; PhyH, phytanoyl-CoA hydroxylase; PPAR, peroxisome proliferator-activated receptor; PTS2, peroxisome-targeting signal type 2; PPRE, PPAR response element; RXR, retinoid X receptor


  REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

  1. Patton, S., Benson, A. A. 1966. Phytol metabolism in the bovine. Biochim. Biophys. Acta. 125:22-32[Medline].

  2. Baxter, J. H. 1968. Absorption of chlorophyll phytol in normal man and in patients with Refsum's disease. J. Lipid Res. 9:636-641[Abstract].

  3. Hansen, R. P. 1980. Phytol: its metabolic products and their distribution. A review. N. Z. J. Sci. 23:259-275.

  4. Wanders, R. J. A., Tager, J. M. 1998. Lipid metabolism in peroxisomes in relation to human disease. Mol. Aspects Med. 19:69-154[Medline].

  5. Verhoeven, N. M., Wanders, R. J. A., Poll-The, B. T., Saudubray, J. M., Jakobs, C. 1998. The metabolism of phytanic acid and pristanic acid in man: a review. J. Inher. Metab. Dis. 21:697-728[Medline].

  6. Jansen, G. A., Ofman, R., Denis, S., Ferdinandusse, S., Hogenhout, E. M., Jakobs, C., Wanders, R. J. A. 1999. Phytanoyl-CoA hydroxylase from rat liver. Protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid alpha-oxidation. J. Lipid Res. 40:2244-2254[Abstract/Free Full Text].

  7. Croes, K., Casteels, M., De Hoffmann, E., Mannaerts, G. P., Van Veldhoven, P. P. 1996. alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver. Production of formic acid instead of CO2, cofactor requirements, subcellular localization and formation of a 2-hydroxy-3-methylacyl-CoA intermediate. Eur. J. Biochem. 240:674-683[Medline].

  8. Mihalik, S. J., Rainville, A. M., Watkins, P. A. 1995. Phytanic acid alpha-oxidation in rat liver peroxisomes. Production of alpha-hydroxyphytanoyl-CoA and formate is enhanced by dioxygenase cofactors. Eur. J. Biochem. 232:545-551[Medline].

  9. Jansen, G. A., Mihalik, S. J., Watkins, P. A., Moser, H. W., Jakobs, C., Denis, S., Wanders, R. J. 1996. Phytanoyl-CoA hydroxylase is present in human liver, located in peroxisomes, and deficient in Zellweger syndrome: direct, unequivocal evidence for the new, revised pathway of phytanic acid alpha-oxidation in humans. Biochem. Biophys. Res. Commun. 229:205-210[Medline].

  10. Verhoeven, N. M., Roe, D. S., Kok, R. M., Wanders, R. J., Jakobs, C., Roe, C. R. 1998. Phytanic acid and pristanic acid are oxidized by sequential peroxisomal and mitochondrial reactions in cultured fibroblasts. J. Lipid Res. 39:66-74[Abstract/Free Full Text].

  11. van Grunsven, E. G., van Berkel, E., Mooijer, P. A., Watkins, P. A., Moser, H. W., Suzuki, Y., Jiang, L. L., Hashimoto, T., Hoefler, G., Adamski, J., Wanders, R. J. 1999. Peroxisomal bifunctional protein deficiency revisited: resolution of its true enzymatic and molecular basis. Am. J. Hum. Genet. 64:99-107[Medline].

  12. Jansen, G. A., Ofman, R., Ferdinandusse, S., Ijlst, L., Muijsers, A. O., Skjeldal, O. H., Stokke, O., Jakobs, C., Besley, G. T., Wraith, J. E., Wanders, R. J. 1997. Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene. Nat. Genet. 17:190-193[Medline].

  13. Mihalik, S. J., Morrell, J. C., Kim, D., Sacksteder, K. A., Watkins, P. A., Gould, S. J. 1997. Identification of PAHX, a Refsum disease gene. Nat. Genet. 17:185-189[Medline].

  14. Wanders, R. J., Schutgens, R. B., Barth, P. G., Tager, J. M., van den Bosch, H. 1993. Postnatal diagnosis of peroxisomal disorders: a biochemical approach. Biochimie. 75:269-279[Medline].

  15. Sorensen, H. N., Treuter, E., Gustafsson, J. A. 1998. Regulation of peroxisome proliferator-activated receptors. Vitam. Horm. 54:121-166[Medline].

  16. Yu, V. C., Delsert, C., Andersen, B., Holloway, J. M., Devary, O. V., Naar, A. M., Kim, S. Y., Boutin, J. M., Glass, C. K., Rosenfeld, M. G. 1991. RXR beta: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell. 67:1251-1266[Medline].

  17. Kliewer, S. A., Umesono, K., Noonan, D. J., Heyman, R. A., Evans, R. M. 1992. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature. 358:771-774[Medline].

  18. Chambon, P. 1996. A decade of molecular biology of retinoic acid receptors. FASEB J. 10:940-954[Abstract].

  19. Forman, B. M., Chen, J., Evans, R. M. 1996. The peroxisome proliferator-activated receptors: ligands and activators. Ann. N.Y. Acad. Sci. 804:266-275[Abstract].

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

  21. 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 and gamma. Proc. Natl. Acad. Sci. USA. 94:4318-4323[Abstract/Free Full Text].

  22. Lin, Q., Ruuska, S. E., Shaw, N. S., Dong, D., Noy, N. 1999. Ligand selectivity of the peroxisome proliferator-activated receptor alpha. Biochemistry. 38:185-190[Medline].

  23. 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 coactivator-dependent receptor ligand assay. Mol. Endocrinol. 11:779-791[Abstract/Free Full Text].

  24. Lemberger, T., Desvergne, B., Wahli, W. 1996. Peroxisome proliferator-activated receptors: a nuclear receptor signaling pathway in lipid physiology. Annu. Rev. Cell Dev. Biol. 12:335-363[Medline].

  25. 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].

  26. ten Brink, H. J., C. Jakobs, J. L. Baan, and F. Bickelhaupt. 1989. Synthesis of deuterium labelled analogues of pristanic and phytanic acid for use as internal standards in stable isotope dilution analysis. In Synthesis and Applications of Isotopically Labelled Compounds. T. A. Baillie and J. R. Jones, editors. Elsevier, Amsterdam. 717;–722.

  27. Pijnappel, W. W. M., Folkers, G. E., Dejonge, W. J., Verdegem, P. J. E., Delaat, S. W., Lugtenburg, J., Hendriks, H. F. J., Vandersaag, P. T., Durston, A. J. 1998. Metabolism to a response pathway selective retinoid ligand during axial pattern formation. Proc. Natl. Acad. Sci. USA. 95:15424-15429[Abstract/Free Full Text].

  28. Wissink, S., van Heerde, E. C., Schmitz, M. L., Kalkhoven, E., van der Burg, B., Baeuerle, P. A., van der Saag, P. T. 1997. Distinct domains of the RelA NF-kappaB subunit are required for negative cross-talk and direct interaction with the glucocorticoid receptor. J. Biol. Chem. 272:22278-22284[Abstract/Free Full Text].

  29. Pfahl, M., Tzukerman, M., Zhang, X. K., Lehmann, J. M., Hermann, T., Wills, K. N., Graupner, G. 1990. Nuclear retinoic acid receptors: cloning, analysis, and function. Methods Enzymol. 189:256-270[Medline].

  30. Kitareewan, S., Burka, L. T., Tomer, K. B., Parker, C. E., Deterding, L. J., Stevens, R. D., Forman, B. M., Mais, D. E., Heyman, R. A., McMorris, T., Weinberger, C. 1996. Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR. Mol. Biol. Cell. 7:1153-1166[Abstract].

  31. Lemotte, P. K., Keidel, S., Apfel, C. M. 1996. Phytanic acid is a retinoid X receptor ligand. Eur. J. Biochem. 236:328-333[Medline].

  32. Ellinghaus, P., Wolfrum, C., Assmann, G., Spener, F., Seedorf, U. 1999. Phytanic acid activates the peroxisome proliferator-activated receptor alpha (PPAR alpha) in sterol carrier protein 2-/sterol carrier protein x-deficient mice. J. Biol. Chem. 274:2766-2772[Abstract/Free Full Text].

  33. Seedorf, U., Raabe, M., Ellinghaus, P., Kannenberg, F., Fobker, M., Engel, T., Denis, S., Wouters, F., Wirtz, K. W., Wanders, R. J., Maeda, N., Assmann, G. 1998. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-x gene function. Genes Dev. 12:1189-1201[Abstract/Free Full Text].

  34. Suzuki, Y., Jiang, L. L., Souri, M., Miyazawa, S., Fukuda, S., Zhang, Z., Une, M., Shimozawa, N., Kondo, N., Orii, T., Hashimoto, T. 1997. D-3-Hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency: a newly identified peroxisomal disorder. Am. J. Hum. Genet. 61:1153-1162[Medline].

  35. van Grunsven, E. G., van Berkel, E., Ijlst, L., Vreken, P., de Klerk, J. B., Adamski, J., Lemonde, H., Clayton, P. T., Cuebas, D. A., Wanders, R. J. A. 1998. Peroxisomal D-hydroxyacyl-CoA dehydrogenase deficiency: resolution of the enzyme defect and its molecular basis in bifunctional protein deficiency. Proc. Natl. Acad. Sci. USA. 95:2128-2133[Abstract/Free Full Text].


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 has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. A. K. Westin, M. C. Hunt, and S. E. H. Alexson
Peroxisomes Contain a Specific Phytanoyl-CoA/Pristanoyl-CoA Thioesterase Acting as a Novel Auxiliary Enzyme in {alpha}- and beta-Oxidation of Methyl-branched Fatty Acids in Mouse
J. Biol. Chem., September 14, 2007; 282(37): 26707 - 26716.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. Ashibe, T. Hirai, K. Higashi, K. Sekimizu, and K. Motojima
Dual Subcellular Localization in the Endoplasmic Reticulum and Peroxisomes and a Vital Role in Protecting against Oxidative Stress of Fatty Aldehyde Dehydrogenase Are Achieved by Alternative Splicing
J. Biol. Chem., July 13, 2007; 282(28): 20763 - 20773.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
J. Gloerich, D. M. van den Brink, J. P. N. Ruiter, N. van Vlies, F. M. Vaz, R. J. A. Wanders, and S. Ferdinandusse
Metabolism of phytol to phytanic acid in the mouse, and the role of PPAR{alpha} in its regulation
J. Lipid Res., January 1, 2007; 48(1): 77 - 85.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
L. Michalik, J. Auwerx, J. P. Berger, V. K. Chatterjee, C. K. Glass, F. J. Gonzalez, P. A. Grimaldi, T. Kadowaki, M. A. Lazar, S. O'Rahilly, et al.
International Union of Pharmacology. LXI. Peroxisome Proliferator-Activated Receptors
Pharmacol. Rev., December 1, 2006; 58(4): 726 - 741.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
P. Germain, P. Chambon, G. Eichele, R. M. Evans, M. A. Lazar, M. Leid, A. R. De Lera, R. Lotan, D. J. Mangelsdorf, and H. Gronemeyer
International Union of Pharmacology. LXIII. Retinoid X Receptors
Pharmacol. Rev., December 1, 2006; 58(4): 760 - 772.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
T. Hashimoto, N. Shimizu, T. Kimura, Y. Takahashi, and T. Ide
Polyunsaturated Fats Attenuate the Dietary Phytol-Induced Increase in Hepatic Fatty Acid Oxidation in Mice
J. Nutr., April 1, 2006; 136(4): 882 - 886.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
J. Gloerich, N. van Vlies, G. A. Jansen, S. Denis, J. P. N. Ruiter, M. A. van Werkhoven, M. Duran, F. M. Vaz, R. J. A. Wanders, and S. Ferdinandusse
A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPAR{alpha}-dependent and -independent pathways
J. Lipid Res., April 1, 2005; 46(4): 716 - 726.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
K. Savolainen, T. J. Kotti, W. Schmitz, T. I. Savolainen, R. T. Sormunen, M. Ilves, S. J. Vainio, E. Conzelmann, and J. K. Hiltunen
A mouse model for {alpha}-methylacyl-CoA racemase deficiency: adjustment of bile acid synthesis and intolerance to dietary methyl-branched lipids
Hum. Mol. Genet., May 1, 2004; 13(9): 955 - 965.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
J. A. Mobley, I. Leav, P. Zielie, C. Wotkowitz, J. Evans, Y.-W. Lam, B. S. L'Esperance, Z. Jiang, and S.-M. Ho
Branched Fatty Acids in Dairy and Beef Products Markedly Enhance {alpha}-Methylacyl-CoA Racemase Expression in Prostate Cancer Cells in Vitro
Cancer Epidemiol. Biomarkers Prev., August 1, 2003; 12(8): 775 - 783.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Idel, P. Ellinghaus, C. Wolfrum, J.-R. Nofer, J. Gloerich, G. Assmann, F. Spener, and U. Seedorf
Branched Chain Fatty Acids Induce Nitric Oxide-dependent Apoptosis in Vascular Smooth Muscle Cells
J. Biol. Chem., December 13, 2002; 277(51): 49319 - 49325.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
J. M. Little, L. Williams, J. Xu, and A. Radominska-Pandya
Glucuronidation of the Dietary Fatty Acids, Phytanic Acid and Docosahexaenoic Acid, by Human UDP-Glucuronosyltransferases
Drug Metab. Dispos., May 1, 2002; 30(5): 531 - 533.
[Abstract] [Full Text] [PDF]

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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zomer, A. W. M.
Right arrow Articles by van der Saag, P. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zomer, A. W. M.
Right arrow Articles by van der Saag, P. T.
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