Phytanic acid is ligand and transcriptional activator of murine liver fatty acid binding protein.
(1/98)
Branched-chain phytanic acid is metabolized in liver peroxisomes. Sterol carrier protein 2/sterol carrier protein x (SCP2/SCPx) knockout mice, which develop a phenotype with a deficiency in phytanic acid degradation, accumulate dramatically high concentrations of this fatty acid in serum (Seedorf at al. 1998. Genes Dev. 12: 1189-1201) and liver. Concomitantly, a 6.9-fold induction of liver fatty acid binding protein (L-FABP) expression is observed in comparison to wild-type animals fed standard chow, possibly mediated by the peroxisome proliferator-activated receptor alpha (PPARalpha). Cytosolic transport of phytanic acid to either peroxisomal membranes or to the nucleus for activation of PPARalpha may be mediated by L-FABP, which gives rise to the question whether phytanic acid is a transactivator of this protein. Here we show first that phytanic acid binds to recombinant L-FABP with high affinity. Then the increase of the in vivo phytanic acid concentration by phytol feeding to mice results in a 4-fold induction of L-FABP expression in liver, which is in the order of that attained with bezafibrate, a known peroxisome proliferator. Finally to test in vitro whether this induction is conferred by phytanic acid, we cotransfected HepG2 cells with an expression plasmid for murine PPARalpha and a CAT-reporter gene with 176 bp of the murine L-FABP promoter, containing the peroxisome proliferator responsive element (PPRE). After incubation with phytanic acid, we observed a 3.2-fold induction of CAT expression. These findings, both in vivo and in vitro, demonstrate that phytanic acid is a transcriptional activator of L-FABP expression and that this effect is mediated via PPARalpha. (+info)
Refsum disease diagnostic marker phytanic acid alters the physical state of membrane proteins of liver mitochondria.
(2/98)
Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), a branched chain fatty acid accumulating in Refsum disease to high levels throughout the body, induces uncoupling of rat liver mitochondria similar to non-branched fatty acids (e.g. palmitic acid), but the contribution of the ADP/ATP carrier or the aspartate/glutamate carrier in phytanic acid-induced uncoupling is of minor importance. Possible deleterious effects of phytanic acid on membrane-linked energy coupling processes were studied by ESR spectroscopy using rat liver mitochondria and a membrane preparation labeled with the lipid-specific spin probe 5-doxylstearic acid (5-DSA) or the protein-specific spin probe MAL-TEMPO (4-maleimido-2,2,6, 6-tetramethyl-piperidine-1-oxyl). The effects of phytanic acid on phospholipid molecular dynamics and on the physical state of membrane proteins were quantified by estimation of the order parameter or the ratio of the amplitudes of the weakly to strongly immobilized MAL-TEMPO binding sites (W/S ratio), respectively. It was found, that phytanic acid (1) increased the mobility of phospholipid molecules (indicated by a decrease in the order parameter) and (2) altered the conformational state and/or the segmental mobility of membrane proteins (indicated by a drastic decrease in the W/S ratio). Unsaturated fatty acids with multiple cis-double bonds (e.g. linolenic or arachidonic acid), but not non-branched FFA (ranging from chain length C10:0 to C18:0), also decrease the W/S ratio. It is hypothesized that the interaction of phytanic acid with transmembrane proteins might stimulate the proton permeability through the mitochondrial inner membrane according to a mechanism, different to a protein-supported fatty acid cycling. (+info)
Phytanic acid alpha-oxidation: identification of 2-hydroxyphytanoyl-CoA lyase in rat liver and its localisation in peroxisomes.
(3/98)
Phytanic acid is broken down by alpha-oxidation in three steps finally yielding pristanic acid. The first step occurs in peroxisomes and is catalysed by phytanoyl-CoA hydroxylase. We have studied the second step in the alpha-oxidation pathway, which involves conversion of 2-hydroxyphytanoyl-CoA to pristanal catalysed by 2-hydroxyphytanoyl-CoA lyase. To this end, we have developed a stable isotope dilution gas chromatography-mass spectrometry assay allowing activity measurements in rat liver homogenates. Cell fractionation studies demonstrate that in rat liver 2-hydroxyphytanoyl-CoA lyase is localised in peroxisomes. This finding may have important implications for inherited diseases in man characterised by impaired phytanic acid alpha-oxidation. (+info)
Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids.
(4/98)
The formability, current-voltage characteristics and stability of the planar lipid bilayer membranes from the synthetic phytanyl-chained glycolipids, 1, 3-di-O-phytanyl-2-O-(beta-glycosyl)glycerols (Glc(Phyt)(2), Mal(N)(Phyt)(2)) were studied. The single bilayer membranes were successfully formed from the glycolipid bearing a maltotriosyl group (Mal(3)(Phyt)(2)) by the folding method among the synthetic glycolipids examined. The membrane conductance of Mal(3)(Phyt)(2) bilayers in 100 mM KCl solution was significantly lower than that of natural phospholipid, soybean phospholipids (SBPL) bilayers, and comparable to that of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers. From the permeation measurements of lipophilic ions through Mal(3)(Phyt)(2) and DPhPC bilayers, it could be presumed that the carbonyl groups in glycerol backbone of the lipid molecule are not necessarily required for the total dipole potential barrier against cations in Mal(3)(Phyt)(2) bilayer. The stability of Mal(3)(Phyt)(2) bilayers against long-term standing and external electric field change was rather high, compared with SBPL bilayers. Furthermore, a preliminary experiment over the functional incorporation of membrane proteins was demonstrated employing the channel proteins derived from octopus retina microvilli vesicles. The channel proteins were functionally incorporated into Mal(3)(Phyt)(2) bilayers in the presence of a negatively charged glycolipid. From these observations, synthetic phytanyl-chained glycolipid bilayers are promising materials for reconstitution and transport studies of membrane proteins. (+info)
Phytanoyl-CoA hydroxylase from rat liver. Protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid alpha-oxidation.
(5/98)
Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid alpha-oxidation pathway. Recently, several studies have shown that in humans, phytanic acid alpha-oxidation is localized in peroxisomes. In rat, however, the alpha-oxidation pathway has been reported to be mitochondrial. In order to clarify this differential subcellular distribution, we have studied the rat PhyH protein. We have purified PhyH from rat liver to apparent homogeneity as judged by SDS-PAGE. Sequence analysis of two PhyH peptide fragments allowed cloning of the rat PHYH cDNA encoding a 38. 6 kDa protein. The deduced amino acid sequence revealed strong homology to human PhyH including the presence of a peroxisome targeting signal type 2 (PTS2). Heterologous expression of rat PHYH in Saccharomyces cerevisiae yielded a 38.6 kDa protein whereas the PhyH purified from rat liver had a molecular mass of 35 kDa. This indicates that PhyH is probably processed in rat by proteolytic removal of a leader sequence containing the PTS2. This type of processing has been reported in several other peroxisomal proteins that contain a PTS2. Subcellular localization studies using equilibrium density centrifugation showed that PhyH is indeed a peroxisomal protein in rat. The finding that PhyH is peroxisomal in both rat and humans provides strong evidence against the concept of a differential subcellular localization of phytanic acid alpha-oxidation in rat and human. (+info)
Sterol carrier protein-2.
(6/98)
The compartmentalization of cholesterol metabolism implies target-specific cholesterol trafficking between the endoplasmic reticulum, plasma membrane, lysosomes, mitochondria and peroxisomes. One hypothesis has been that sterol carrier protein-2 (SCP2, also known as the non-specific lipid transfer protein) acts in cholesterol transport through the cytoplasm. Recent studies employing gene targeting in mice showed, however, that mice lacking SCP2 and the related putative sterol carrier known as SCPx, develop a defect in peroxisomal beta-oxidation. In addition, diminished peroxisomal alpha-oxidation of phytanic acid (3,7,11, 15-tetramethylhexadecanoic acid) in these null mice was attributed to the absence of SCP2 which has a number of properties supporting a function as carrier for fatty acyl-CoAs rather than for sterols. (+info)
Phytanoyl-CoA hydroxylase activity is induced by phytanic acid.
(7/98)
Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid present in various dietary products such as milk, cheese and fish. In patients with Refsum disease, accumulation of phytanic acid occurs due to a deficiency of phytanoyl-CoA hydroxylase, a peroxisomal enzyme containing a peroxisomal targeting signal 2. Recently, phytanoyl-CoA hydroxylase cDNA has been isolated and functional mutations have been identified. As it has been shown that phytanic acid activates the nuclear hormone receptors peroxisome proliferator-activated receptor (PPAR)alpha and all three retinoid X receptors (RXRs), the intracellular concentration of this fatty acid should be tightly regulated. When various cell lines were grown in the presence of phytanic acid, the activity of phytanoyl-CoA hydroxylase increased up to four times, depending on the particular cell type. In one cell line, HepG2, no induction of phytanoyl-CoA hydroxylase activity was observed. After addition of phytanic acid to COS-1 cells, an increase in phytanoyl-CoA hydroxylase activity was observed within 2 h, indicating a quick cell response. No stimulation of phytanoyl-CoA hydroxylase was observed when COS-1 cells were grown in the presence of clofibric acid, 9-cis-retinoic acid or both ligands together. This indicates that the activation of phytanoyl-CoA hydroxylase is not regulated via PPARalpha or RXR. However, stimulation of PPARalpha and all RXRs by clofibric acid and 9-cis-retinoic acid was observed in transient transfection assays. These results suggest that the induction of phytanoyl-CoA hydroxylase by phytanic acid does not proceed via one of the nuclear hormone receptors, RXR or PPARalpha. (+info)
Pristanic acid and phytanic acid: naturally occurring ligands for the nuclear receptor peroxisome proliferator-activated receptor alpha.
(8/98)
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 (PPARalpha) in a concentration-dependent manner. Activation is observed via the ligand-binding domain of PPARalpha 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 microM, respectively. The trans-activation of PPARdelta and PPARgamma by these two ligands is negligible. Besides PPARalpha, 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 PPARalpha 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 PPARalpha. Because induction of PPARalpha 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 PPARalpha. These results demonstrate that both pristanic acid and phytanic acid are naturally occurring ligands for PPARalpha, which are present at physiological concentrations. (+info)