A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPAR-dependent and-independent pathways

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR ) in vitro . To investigate the effects of these physiological compounds in vivo, wild-type and PPAR -deficient (PPAR / ) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPAR / mice, the already elevated fatty acid levels increased. In addition, PPAR / mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPAR / mice. Investigation of carnitine biosynthesis revealed that PPAR is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPAR -dependent induction of various peroxisomal and mitochondrial -oxidation enzymes. In addition, a PPAR -independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPAR in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders. —Gloerich, J., 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 -dependent and -independent pathways. J. Lipid Res. 2005. 46: 716–726. Supplementary key words peroxisome proliferator-activated receptor • peroxisomes • mitochondria • fatty acid -oxidation • very long-chain fatty acids • branched-chain fatty acids • acylcarnitines • peroxisomal disorders Peroxisome proliferator-activated receptor (PPAR ) is one of the PPARs that form a subfamily of the nuclear hormone receptor superfamily. PPARs are ligand-activated transcription factors: after ligand binding, PPARs heterodimerize with the retinoic X receptor and modulate the expression of target genes by binding to specific peroxisome proliferator response elements in the promotor region of regulated genes (1). Three PPAR isoforms are known: PPAR , PPAR , and PPAR . The three isoforms have different tissue distributions and functions. PPAR is mostly expressed in organs with a high rate of fatty acid catabolism, such as brown adipose tissue, liver, kidney, and heart, and it plays an important role in various aspects of lipid and glucose metabolism (2, 3). PPAR has a broad range of both artificial and natural ligands also called peroxisome proliferators. The artificial ligands of PPAR consist of a variety of compounds, including hypolipidemic drugs (e.g., clofibrate and Wy-14,643), phthalate ester plasticizers, herbicides, and several chlorinated hydrocarbons. A broad array of unsaturated fatty acids, but also long-chain fatty acids and branched-chain fatty acids (e.g., phytanic acid), are natural ligands for PPAR (4–8). Administration of peroxisome proliferators to rodents results in hepatomegaly and an increase in the Abbreviations: AMACR, -methylacyl-CoA racemase; BB, -butyrobetaine; BBD, -butyrobetaine dioxygenase; BCOX, branched-chain acylCoA oxidase; CAT, carnitine acetyltransferase; COT, carnitine octanoyltransferase; CPT2, carnitine palmitoyltransferase 2; CYP4A1, cytochrome P450 hydroxylase 4A1; DBP, d -bifunctional protein; Elovl, long-chain fatty acid elongase; ESI, electrospray ionization; LBP, l -bifunctional protein; LCAD, long-chain acyl-CoA dehydrogenase; MCAD, mediumchain acyl-CoA dehydrogenase; MTP, mitochondrial trifunctional protein; PhyH, phytanoyl-CoA hydroxylase; PMP70, peroxisomal membrane protein 70; PPAR, peroxisome proliferator-activated receptor; SBCHAD, short branched-chain 3-hydroxyacyl-CoA dehydrogenase; SCAD, shortchain acyl-CoA dehydrogenase; SCHAD, short-chain 3-hydroxyacyl-CoA dehydrogenase; SCOX, straight-chain acyl-CoA oxidase; SCPx, sterol carrier protein x; THIO, peroxisomal 3-ketoacyl-CoA thiolase; TMABADH, trimethylaminobutyraldehyde dehydrogenase; TML, trimethyllysine; VLCAD, very long-chain acyl-CoA dehydrogenase; VLCFA, very long-chain fatty acid. 1 To whom correspondence should be addressed. e-mail: s.ferdinandusse@amc.uva.nl Manuscript received 7 September 2004 and in revised form 16 December 2004. Published, JLR Papers in Press, January 16, 2005. DOI 10.1194/jlr.M400337-JLR200 by gest, on O cber 1, 2017 w w w .j.org D ow nladed fom

Peroxisome proliferator-activated receptor ␣ (PPAR ␣ ) is one of the PPARs that form a subfamily of the nuclear hormone receptor superfamily.PPARs are ligand-activated transcription factors: after ligand binding, PPARs heterodimerize with the retinoic X receptor ␣ and modulate the expression of target genes by binding to specific peroxisome proliferator response elements in the promotor region of regulated genes (1).Three PPAR isoforms are known: PPAR ␣ , PPAR ␤ , and PPAR ␥ .The three isoforms have different tissue distributions and functions.PPAR ␣ is mostly expressed in organs with a high rate of fatty acid catabolism, such as brown adipose tissue, liver, kidney, and heart, and it plays an important role in various aspects of lipid and glucose metabolism (2,3).
PPAR ␣ has a broad range of both artificial and natural ligands also called peroxisome proliferators.The artificial ligands of PPAR ␣ consist of a variety of compounds, including hypolipidemic drugs (e.g., clofibrate and Wy-14,643), phthalate ester plasticizers, herbicides, and several chlorinated hydrocarbons.A broad array of unsaturated fatty acids, but also long-chain fatty acids and branched-chain fatty acids (e.g., phytanic acid), are natural ligands for PPAR ␣ (4-8).Administration of peroxisome proliferators to rodents results in hepatomegaly and an increase in the number and size of peroxisomes.In addition, it changes the expression of a variety of genes involved in various aspects of lipid metabolism, ranging from fatty acid transport and mitochondrial and peroxisomal fatty acid ␤ -oxidation to microsomal fatty acid -oxidation.Chronic treatment of rodents with peroxisome proliferators results in hepatocellular carcinomas (9).These changes are all mediated by PPAR ␣ , as demonstrated by studies with PPAR ␣ -deficient (PPAR ␣ Ϫ / Ϫ ) mice.Under normal conditions, PPAR ␣ Ϫ / Ϫ mice are indistinguishable from wild-type animals and have normal levels of hepatic peroxisomes.Upon fasting, however, PPAR ␣ Ϫ / Ϫ mice are unable to switch to fatty acid oxidation; they develop a fatty liver and become severely hypoglycemic (10).Similarly, feeding these mice a highfat diet results in massive accumulation of lipids in the liver, attributable to their inability to enhance fatty acid degradation (11).PPAR ␣ Ϫ / Ϫ mice do display an altered constitutive expression of several mitochondrial and peroxisomal enzymes involved in the oxidation of fatty acids (12).However, PPAR ␣ Ϫ / Ϫ mice are nonresponsive to treatment with peroxisome proliferators; hence, they do not show any physiological, toxicological, or carcinogenic responses to peroxisome proliferators (13).
In the studies mentioned above, artificial ligands such as clofibrate and Wy-14,643 were used as PPAR ␣ ligands.Relatively little is known about the effects of natural PPAR ␣ ligands, such as the branched-chain fatty acids phytanic and pristanic acid, which have been shown to activate PPAR ␣ in vitro, in contrast to their precursor phytol (6,14).Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is derived from the chlorophyll component phytol and undergoes ␣ -oxidation in the peroxisome, which leads to shortening of the chain by one carbon atom, yielding pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) and carbon dioxide.Pristanic acid is then further degraded in the peroxisome via ␤ -oxidation (15).In patients affected by different peroxisomal disorders, there is an accumulation of phytanic and/or pristanic acid.Patients suffering from Refsum disease have a deficiency of phytanoyl-CoA hydroxylase (PhyH), the first enzyme of the ␣ -oxidation system, and as a consequence accumulate phytanic acid in tissues and plasma (16).In ␣ -methylacyl-CoA racemase (AMACR) or d -bifunctional protein (DBP) deficiency, two enzymes involved in the peroxisomal ␤ -oxidation of branched-chain fatty acids, there is an accumulation of pristanic acid.Patients suffering from a peroxisome biogenesis disorder accumulate both phytanic and pristanic acid because they are deficient in both peroxisomal ␣and ␤ -oxidation (reviewed in 17).
In this study, we investigated the effects of the accumulation of these branched-chain fatty acids by feeding mice a diet enriched with phytol.Thus, we mimicked the situation in patients suffering from a peroxisomal disorder, because this results in an increase of phytol metabolites in tissues and plasma (4,18).In several mouse models for peroxisomal ␤ -oxidation disorders in which branchedchain fatty acids are increased, an altered expression of various fatty acid-metabolizing enzymes has been reported (19)(20)(21)(22).
We studied the expression of both peroxisomal and mitochondrial proteins involved in the metabolism of fatty acids in wild-type and PPAR ␣ Ϫ / Ϫ mice after phytol feeding.Furthermore, we investigated the effect of a phytol diet on the levels of various metabolites in plasma and liver, including very long-chain, branched-chain, and polyunsaturated fatty acids, acylcarnitines, and carnitine biosynthesis intermediates.

Animals
Male wild-type and PPAR ␣ Ϫ / Ϫ mice on a Sv/129 genetic background were used for this study (13).Six week old wild-type and PPAR ␣ Ϫ / Ϫ mice were fed pelleted mouse chow (Hope Farms, Woerden, The Netherlands) containing no (control) or 0.5% (w/w) phytol for 1, 2, 4, or 8 weeks.Each group consisted of three animals.At the end of the experiment, mice were anesthetized using isoflurane, blood was collected by cardiac puncture, and tissues were harvested.The animals were always killed at the same time of day, and animals had free access to water and food until that moment.Tissues were snap-frozen in liquid nitrogen and stored at Ϫ 80 Њ C until further analysis.A small piece of tissue was treated immediately with RNA later RNA stabilization reagent (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions and stored at Ϫ 80 Њ C until RNA isolation.All animal experiments were approved by the University of Amsterdam Animals Experiments Committee.

Metabolite analysis in plasma and liver
Total very long-chain fatty acids (VLCFAs) up to 26 carbon atoms and branched-chain fatty acids in plasma and liver were analyzed using GC-MS (23).Total fatty acids with more than 26 carbon atoms were analyzed using electrospray ionization (ESI)-tandem MS (24).Because some of the necessary standards for precise quantitative analysis of these extremely long-chain fatty acids are not commercially available, only a comparative analysis between the different groups could be performed.For this reason, the amount of each fatty acid present in wild-type mice fed a control diet was set to 1.Total polyunsaturated fatty acids in plasma were measured by GC analysis (25).Free carnitine and acylcarnitines in plasma and liver were analyzed as their propyl esters using ESItandem MS as described previously (26).The carnitine biosynthesis intermediates trimethyllysine (TML) and ␥ -butyrobetaine (BB) in plasma and liver were analyzed using ESI-tandem MS (27).
Phytol levels were determined in freshly prepared liver homogenates in PBS.As an internal standard, 1 nmol of C19-OH dissolved in ethanol was added to samples containing 0.5 mg of liver protein.Subsequently, samples were subjected to alkaline hydrolysis by adding 2 ml of 1 M NaOH in methanol and incubated for 45 min at 110 Њ C.After cooling to room temperature, the pH was decreased by adding 480 l of 37% HCl.Phytol was then extracted with 2 ml of hexane.The organic layer was evaporated to dryness under nitrogen at 40 Њ C. Samples were dissolved in 0.5 ml of heptane and purified on a silica gel column (J.T. Baker, Philipsburg, NJ) using 92:8 heptane-diethyl ether as eluent.Samples were evaporated to dryness under nitrogen at 40 Њ C and derivatized with N -methyl-N -( tert -butyldimethylsilyl)trifluoroacetamide (Pierce, Rockford, IL) and pyridine (50 l each) at 80 Њ C for 30 min.Samples were evaporated to dryness under nitrogen at 40 Њ C, dissolved in 100 l of hexane, and subjected to GC-MS analysis, essentially as described previously (28).The [M-57] ϩ ions of phytol and C19-OH (corresponding to 353. 3 and 341.3, respectively) were detected.The metabolites were quantified using a calibration curve of phytol.

Enzyme activity measurements
All enzyme activity measurements were done in freshly prepared liver homogenates.

Statistical analyses
Data are expressed as means Ϯ SD.Statistical significance was evaluated using an unpaired Student's t -test.The results were considered significant at P Ͻ 0.01.

Analysis of phytol and its metabolites phytenic, phytanic, and pristanic acid in plasma and liver
Branched-chain fatty acids were measured in liver and plasma to establish the extent of the accumulation of these phytol metabolites after the phytol-enriched diet ( Table 1 ).In liver from phytol-fed animals, there was a marked increase in hepatic levels of phytenic, phytanic, and pristanic acid.The levels increased with an increasing diet period, but strikingly, the increase was much stronger in wild-type animals than in PPAR ␣ Ϫ / Ϫ mice.These results led us to investigate hepatic phytol levels.Interestingly, we found that there was a greater accumulation of phytol in PPAR ␣ Ϫ / Ϫ mice than in wild-type animals, suggesting a PPAR ␣ -dependent upregulation of the breakdown pathway of phytol to phytanic acid in wild-type animals upon phytol feeding.Remarkably, the plasma levels of phytenic, phytanic, and pristanic acid were increased after the phytol diet but did not increase with a longer duration of the diet.After 1 week of phytol feeding, the maximum plasma levels were reached.Therefore, the results in plasma are presented in Table 2 as a mean of all animals of the same genotype on the same diet (n ϭ 12 per group), regardless of the length of the diet.
Although the hepatic levels of phytol and its metabolites increased with longer periods of the diet, no differences were observed for all other parameters that were investigated regarding the different diet periods.Based on these data, we conclude that already after 1 week of phytol feeding sufficient amounts of phytol metabolites have accumulated to induce the effects described in this study.Therefore, these effects are presented throughout this study as a mean of all animals of the same genotype on the same diet, regardless of the length of the diet.

Plasma analysis of fatty acids
Plasma values of straight-chain and polyunsaturated fatty acids were measured to investigate the effect of a phytolenriched diet on fatty acid metabolism (Table 2).In wildtype mice, the levels of virtually all fatty acids were decreased on the phytol-enriched diet compared with the control diet.In PPAR ␣ Ϫ / Ϫ mice, the levels of most fatty ac-ids were increased, and this effect was even stronger after phytol feeding.In contrast to all other straight-chain fatty acids, the C26:0 levels were increased in both wild-type and PPAR ␣ Ϫ / Ϫ mice as a result of the phytol diet.Therefore, we investigated fatty acids longer than C26:0 using ESI-tandem MS.Interestingly, C28:0 and C30:0 levels (Table 2) and their monounsaturated counterparts (data not shown) were significantly higher after the phytol diet in PPAR ␣ Ϫ / Ϫ mice but not in wild-type mice.
An increase of VLCFAs in PPAR ␣ Ϫ / Ϫ mice on a phytol diet could be attributable to a diminished breakdown or an increased formation of these fatty acids by elongation.This led us to investigate the mRNA expression levels of Elovl2, Elovl3, and Elovl4 using quantitative real-time PCR analysis.We found no significant differences in mRNA levels of Elovl2 (in liver and kidney), Elovl3 (in liver), and Elovl4 (in kidney) between the different experimental groups (data not shown), indicating that the increased lev- The amount of fatty acid present in plasma of wild-type mice on a control diet was set to 1.0.els of C26:0, C28:0, and C30:0 are not caused by increased elongation of long-chain fatty acids.

Acylcarnitines and carnitine biosynthesis intermediates
To further characterize the effects of a phytol diet in wildtype and PPAR␣ Ϫ/Ϫ mice, we investigated the levels of acylcarnitines and free carnitine in plasma and liver (Table 3).In response to the phytol diet, the free carnitine level in liver increased significantly in wild-type animals.This trend was also observed in plasma, but the increase in plasma was not statistically significant.In PPAR␣ Ϫ/Ϫ mice, however, no increase of free carnitine after the phytol diet was observed, and PPAR␣ Ϫ/Ϫ mice on the control diet had markedly reduced levels of free carnitine in plasma (1.9-fold) and liver (1.7-fold) compared with wild-type animals.
Phytol feeding did not induce changes in acetylcarnitine levels in mice from either genotype, but PPAR␣ Ϫ/Ϫ mice had significantly decreased plasma acetylcarnitine levels compared with wild-type mice.Liver acetylcarnitine levels varied considerably, probably because the animals were not fasted before they were killed.Propionylcarnitine levels increased significantly in plasma of both wild-type and PPAR␣ Ϫ/Ϫ mice on the phytol diet.Levels of palmitoylcarnitine were slightly increased in PPAR␣ Ϫ/Ϫ mice compared with wild-type animals, but this was only significant on the control diet.No significant changes were observed for any of the other acylcarnitines that were analyzed.
In search of the cause of the decreased levels of free carnitine and acetylcarnitine in PPAR␣ Ϫ/Ϫ mice and the increase of free carnitine in liver after phytol feeding, we measured the levels of the carnitine biosynthesis intermediates in plasma and liver and the activity of three enzymes involved in carnitine biosynthesis (Table 4).The only two intermediates that were detected in mouse plasma and liver were TML and the direct precursor of carnitine, BB.After phytol feeding, BB levels were decreased in liver from wild-type and PPAR␣ Ϫ/Ϫ mice.Furthermore, TMA-BADH and BBD activities were decreased in PPAR␣ Ϫ/Ϫ mice both on the control and the phytol-enriched diet.

The expression of peroxisomal ␤-oxidation enzymes
To determine the effect of the phytol diet on the expression of peroxisomal fatty acid-metabolizing enzymes and the role of PPAR␣ therein, immunoblot analyses were performed followed by densitometric analysis of the immunoblots (Fig. 1, Table 5).In PPAR␣ Ϫ/Ϫ mice, the constitutive expression of several peroxisomal ␤-oxidation enzymes was lower, which is in agreement with previously reported data (12), but this decrease was only significant for LBP and THIO.Phytol feeding of wild-type mice resulted in a significant increase in the expression of SCOX, LBP, DBP, THIO, SCPx, catalase, COT, and PhyH, with levels of induction ranging from 4.5-fold for SCOX to 1.6-fold for COT.In contrast, phytol feeding of PPAR␣ Ϫ/Ϫ mice did not result in an induction of LBP, DBP, and SCPx, indicating that the effect of the phytol-enriched diet on the expression of these enzymes is PPAR␣ dependent.SCOX, THIO, and COT were induced by phytol treatment in PPAR␣ Ϫ/Ϫ mice.However, this induction was less than in wild-type mice, indicating that there is both a PPAR␣-dependent and -independent effect on their expression.Interestingly, catalase and PhyH were induced to the same extent by the phytol diet in both wild-type and PPAR␣ Ϫ/Ϫ mice.As a marker for peroxisome proliferation, the expression of the peroxisomal membrane protein PMP70 was studied, but no changes in the amount of protein were observed between the different experimental groups.In addition to the immunoblot analyses, enzyme activity measurements were performed for SCOX, BCOX, DBP, SCPx, and catalase (Fig. 2).Basal DBP activity was decreased in PPAR␣ Ϫ/Ϫ mice compared with wild-type animals.Furthermore, in wild-type mice, the activity of SCOX, DBP (both the enoyl-CoA hydratase and the 3-hydroxyacyl-CoA dehydrogenase activity), SCPx, and catalase increased on the phytol diet.In PPAR␣ Ϫ/Ϫ mice, catalase and SCOX activities increased as a result of the phytol treatment, although the increase in SCOX activity was lower than that observed in wild-type animals.In contrast, BCOX activity decreased in PPAR␣ Ϫ/Ϫ mice after the phytol diet.

The expression of mitochondrial ␤-oxidation enzymes
Besides the peroxisomal fatty acid-metabolizing enzymes, PPAR␣ also regulates the expression of several enzymes involved in the mitochondrial ␤-oxidation system.To investigate the influence of the branched-chain fatty acid metabolites of phytol on the expression of mitochondrial fatty acid-metabolizing enzymes, immunoblot analysis was performed using specific antibodies against several of these enzymes (Fig. 1, Table 6).The constitutive levels of SCAD and LCAD were significantly decreased in PPAR␣ Ϫ/Ϫ mice.For SCHAD, an increased constitutive expression in PPAR␣ Ϫ/Ϫ mice was reported previously ( 12), but we could not confirm this.No differences in the constitutive expression of SCHAD, MCAD, and SBCHAD were found.The phytol diet did not induce significant changes in protein expression for the investigated mitochondrial ␤-oxidation enzymes in mice of either genotype.A significant increase was found for the expression of CYP4A1, a microsomal enzyme involved in -oxidation of fatty acids, in phytol-fed wild-type mice.This increase in expression of CYP4A1 was not found in PPAR␣ Ϫ/Ϫ mice on a phytol diet, indicating that the induction of CYP4A1 is PPAR␣ dependent.The amount of protein present in liver from wild-type mice on a control diet was set to 1.0.Values represent means Ϯ SD. n ϭ number of animals.
a P Ͻ 0.01, phytol-enriched diet versus control diet in mice of the same genotype.b P Ͻ 0.01, PPAR␣ Ϫ/Ϫ versus wild-type mice on the same diet.
To investigate the effect of the phytol diet on the activity of the enzymes involved in mitochondrial fatty acid metabolism, enzyme activity measurements were performed for SCAD, MCAD, VLCAD, LCAD, CPT2, and CAT (Fig. 3).The basal activity levels of VLCAD, LCAD, and CPT2 were significantly decreased in PPAR␣ Ϫ/Ϫ mice.On the phytol diet, a significant increase in enzyme activity was measured for SCAD, LCAD, CPT2, and CAT in wild-type animals.In PPAR␣ Ϫ/Ϫ mice, no induction of enzyme activity was observed for these enzymes after phytol feeding, except for a slight but significant increase in CAT activity.However, this induction of CAT in PPAR␣ Ϫ/Ϫ mice was less than in wild-type mice, indicating that there is both a PPAR␣-dependent and -independent effect of the phytol diet on CAT activity.The absence of induction of SCAD, LCAD, and CPT2 activity in PPAR␣ Ϫ/Ϫ mice indicates that these enzymes are solely regulated via PPAR␣, at least with the branched-chain fatty acid metabolites of phytol as ligands.

DISCUSSION
Many studies have been performed on the effect of artificial PPAR␣ ligands on fatty acid metabolism and the expression of the enzymes involved.Much less is known about the effect of natural ligands such as the branched-  The amount of protein present in liver from wild-type mice on a control diet was set to 1.0.Values represent means Ϯ SD. n ϭ number of animals.
a P Ͻ 0.01, PPAR␣ Ϫ/Ϫ versus wild-type mice on the same diet.b P Ͻ 0.01, phytol-enriched diet versus control diet in mice of the same genotype.
chain fatty acids phytanic and pristanic acid.These fatty acids accumulate in several peroxisomal disorders, such as adult Refsum disease, AMACR deficiency, and peroxisome biogenesis disorders.In several mouse models for peroxisomal disorders in which these fatty acids accumulate, an altered pattern of gene expression is reported.For example, AMACR Ϫ/Ϫ mice have a normal phenotype on a control diet, but phytol feeding results in liver injury, peroxisome proliferation, and changes in the expression of fatty acid-metabolizing enzymes (22).These changes are likely mediated via PPAR␣.Furthermore, the expression of the H 2 O 2 -producing SCOX is strongly increased in SCPx Ϫ/Ϫ and DBP Ϫ/Ϫ mice, which could lead to an overproduction of reactive oxygen species and therefore oxidative stress.This indeed has been shown to occur in patients with a deficiency of DBP (45).Because of the accumulation of natural ligands of PPAR␣ in patients with an inherited peroxisomal disorder, it is important to have full insight into the effect of these compounds.Therefore, we set out to study the effect of phytol metabolites on fatty acid metabolism and the role of PPAR␣ therein.
Analysis of phytol and its branched-chain fatty acid metabolites revealed increased levels in plasma and liver of mice after the phytol-enriched diet.The increased levels of phytol and its metabolites in liver suggest that once phytol is taken up by the body it is transported to the liver, where it is broken down.Interestingly, we found that the level of hepatic phytol was higher in PPAR␣ Ϫ/Ϫ mice, whereas the levels of the phytol metabolites were lower compared with wild-type animals, suggesting that the breakdown pathway of phytol to phytanic acid is under the control of PPAR␣.Further research is under way to investigate this.The observed effect of the phytol diet on all of the parameters studied was maximal after 1 week of diet, and increasing hepatic concentrations of branched-chain fatty acids and longer exposure to increased levels of these fatty acids did not influence the extent of the observed effects.
In this study, we found that the protein level and/or activity of all of the peroxisomal ␤-oxidation enzymes increased in wild-type mice after phytol feeding, except for the activity of BCOX.Interestingly, the expression of BCOX is increased in DBP Ϫ/Ϫ mice (19), but this apparently is not attributable to the accumulation of branched-chain fatty acids in this mouse model.COT and CAT are also induced by phytol treatment in wild-type animals.Both of these enzymes are involved in the (peroxisomal) conversion of shortened acyl-CoAs to carnitine esters, so that the latter can move to the mitochondrion for further breakdown (42).Surprisingly, a response to the phytol diet was also observed in PPAR␣ Ϫ/Ϫ mice for SCOX, THIO, COT, and CAT.However, the increase in expression in PPAR␣ Ϫ/Ϫ mice was not as high as in wild-type animals; therefore, the enhanced expression of these genes in wild-type mice is probably regulated by PPAR␣ as well as via another pathway that is still present in PPAR␣ Ϫ/Ϫ mice.A PPAR␣independent induction on the phytol diet was also observed for catalase and PhyH, the first enzyme of the peroxisomal ␣-oxidation pathway.Most likely, one of the phytol metabolites not only activates PPAR␣ but also another, yet unknown, transcription factor, resulting in upregulation of a subset of peroxisomal enzymes.It is unlikely that phytol-mediated peroxisome proliferation, which has been described previously (46), is involved in the increased protein levels/enzyme activity we observed, because the expression of the peroxisomal membrane protein PMP70 was not increased.This is supported by the observation that peroxisome proliferation can be achieved independently from induction of PPAR␣-regulated fatty acid ␤-oxidation genes, suggesting a separate regulation of these processes (47).
The effect of the phytol diet on the expression of the enzymes involved in mitochondrial ␤-oxidation was less pronounced compared with that on the peroxisomal enzymes.CPT2, LCAD, and SCAD showed a slight PPAR␣-regulated induction on the phytol diet.This was not observed for MCAD, VLCAD, and MTP, which have been reported to be regulated via PPAR␣ (12).
Although there was a clear effect of dietary phytol on the expression of fatty acid-metabolizing enzymes, the highest induction level was 4.5-fold for SCOX.The reported induction of SCOX after treatment of mice with Wy-14,643 is much stronger (6.6-fold) (12).Thus, our results support the in vitro data that phytanic and pristanic acid are natural ligands for PPAR␣ (4), but we found that they are not as potent as artificial PPAR␣ activators such as clofibrate or Wy-14,643.Besides the PPAR␣-regulated effect, we also found PPAR␣-independent regulation of the expression of peroxisomal enzymes involved in fatty acid metabolism, which has not been reported for the artificial PPAR␣ ligand Wy-14,643 (12).Further research is needed to resolve which underlying mechanism is involved in this regulation.
Analysis of straight-chain fatty acids revealed a phytol diet-induced decrease of total plasma fatty acid levels in wild-type mice.In PPAR␣ Ϫ/Ϫ mice on a phytol diet, the levels of the extremely long-chain fatty acids (with more than 26 carbon atoms) increased significantly.This was not attributable to enhanced elongation by the elongation enzymes; therefore, we suggest that these changes, like the changes in the other saturated and polyunsaturated fatty acids we have found, are attributable to differences in overall ␤-oxidation capacity (i.e., fatty acid breakdown).In PPAR␣ Ϫ/Ϫ mice, the constitutive expression of many fatty acid-metabolizing enzymes is lower than in wild-type animals, resulting in a lower ␤-oxidation capacity and therefore higher levels of fatty acids in plasma.In wildtype animals, such an accumulation of monounsaturated and polyunsaturated fatty acids leads to an upregulation of the ␤-oxidation enzymes (8), but this mechanism is abolished in PPAR␣ Ϫ/Ϫ mice.Decreased levels of acetylcarnitine in plasma of the PPAR␣ Ϫ/Ϫ mice likely reflect a lower production of acetyl-CoA units by fatty acid ␤-oxidation, which supports this hypothesis.However, no increase in acetylcarnitine was observed in wild-type mice after the phytol diet, despite the higher ␤-oxidation capacity under this condition.The increased plasma levels of propionylcarnitine after phytol feeding in both wild-type and PPAR␣ Ϫ/Ϫ mice could represent an increased production of propionyl-CoA during the ␤-oxidation of the phytol metabolite pristanic acid, because one pristanic acid molecule yields three propionyl-CoA units besides the acetyl-CoA units normally produced during ␤-oxidation of fatty acids.
Our results suggest that PPAR␣ is involved in the changes in free carnitine and acetylcarnitine in liver and plasma.The PPAR␣ Ϫ/Ϫ mice are carnitine and acetylcarnitine deficient in both liver and plasma and do not respond to the phytol diet, in contrast to wild-type animals, which display enhanced carnitine levels with this treatment.Previous studies have shown that under fasting conditions there is an increase in hepatic carnitine levels in wild-type mice that is not observed in PPAR␣ Ϫ/Ϫ mice (10).This is supported by the observation that in SCOX Ϫ/Ϫ mice, which have sustained PPAR␣ activation, hepatic carnitine levels are higher than in wild-type mice (10,48).The decreased carnitine levels in PPAR␣ Ϫ/Ϫ mice could be attributable to decreased biosynthesis, decreased renal reabsorption of carnitine, or decreased hepatic uptake of carnitine from the circulation.The activities of the carnitine biosynthesis enzymes TMABADH and BBD were indeed lower in PPAR␣ Ϫ/Ϫ mice, but this did not result in changes in the levels of the carnitine biosynthesis intermediates.Besides decreased production of hepatic carnitine as a result of lower enzymatic activity of the last two enzymes of carnitine biosynthesis, the lower levels of free carnitine and acetylcarnitine in PPAR␣ Ϫ/Ϫ mice could also be the result of a lower flux through the carnitine biosynthesis pathway.This could be caused by decreased input of TML from protein degradation, which is known to be the rate-limiting step of the carnitine biosynthetic pathway (49).In support of this, clofibrate indeed is known to induce protein breakdown in skeletal muscle (50).Further research is needed to fully resolve the role of PPAR␣ in carnitine metabolism.
In summary, our results show that increased levels of the phytol metabolites phytenic, phytanic, and pristanic acid result in the activation of PPAR␣ but possibly also of another yet unknown transcription factor that induces several enzymes involved in peroxisomal fatty acid metabolism.This activation by phytol metabolites resulted in the increased expression of several peroxisomal and mitochondrial ␤-oxidation enzymes, leading to changes in fatty acid metabolism.These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.

TABLE 1 .
Hepatic levels of phytol and its branched-chain fatty acid metabolites from wild-type and PPAR ␣ Ϫ / Ϫ mice fed a control diet or a 0.5% phytol diet for 1, 2, 4, or 8 weeks PPAR␣ Ϫ/Ϫ , peroxisome proliferator-activated receptor ␣-deficient.Concentrations of phytol and branchedchain fatty acids are expressed in nmol/mg protein.Values represent means Ϯ SD. n ϭ number of animals.
a P Ͻ 0.01, phytol-enriched diet versus control diet in mice of the same genotype.b P Ͻ 0.01, PPAR␣ Ϫ/Ϫ versus wild-type mice on the same diet.

TABLE 2 .
Plasma values of very long-chain, long-chain, branched-chain, and polyunsaturated fatty acids from wild-type and PPAR␣ Ϫ/Ϫ mice fed a control diet or a 0.5% phytol diet b Concentrations of fatty acids are expressed in mol/l.Values represent means Ϯ SD. n ϭ number of animals.aP Ͻ 0.01, phytol-enriched diet versus control diet in mice of the same genotype.bP Ͻ 0.01, PPAR␣ Ϫ/Ϫ versus wild-type mice on the same diet.c

TABLE 3 .
Values of acylcarnitines measured in plasma and liver of wild-type and PPAR␣ Ϫ/Ϫ mice fed a control diet or a 0.5% phytol diet Concentrations of acylcarnitines in plasma are expressed in mol/l and those in liver are expressed in nmol/g wet weight.Values represent means Ϯ SD. n ϭ number of animals.P Ͻ 0.01, phytol-enriched diet versus control diet in mice of the same genotype.
a P Ͻ 0.01, PPAR␣ Ϫ/Ϫ versus wild-type mice on the same diet.b

TABLE 4 .
Concentration of the carnitine biosynthesis intermediates measured in plasma and liver, and carnitine biosynthesis enzyme activities in liver, of wild-type and PPAR␣ Ϫ/Ϫ mice fed a control diet or a 0.5% phytol diet bConcentrations of carnitine biosynthesis intermediates in plasma are expressed in mol/l and those in liver are expressed in nmol/g wet weight.Activities of carnitine biosynthesis enzymes are expressed in pmol/mg/min.Values represent means Ϯ SD. n ϭ number of animals.aP Ͻ 0.01, phytol-enriched diet versus control diet in mice of the same genotype.b P Ͻ 0.01, PPAR␣ Ϫ/Ϫ versus wild-type mice on the same diet.

TABLE 5 .
Immunoblot quantification of peroxisomal proteins in liver of wild-type and PPAR␣ Ϫ/Ϫ mice fed a control diet or a 0.5% phytol diet

TABLE 6 .
Immunoblot quantification of mitochondrial ␤-oxidation enzymes and cytochrome P450 hydroxylase 4A1 in liver of wild-type and PPAR␣ Ϫ/Ϫ mice fed a control diet or a 0.5% phytol diet