Molecular and Cellular Biochemistry 259: 115–129, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.

115

Liver fatty acid binding protein expression enhances branched-chain fatty acid metabolism Barbara P. Atshaves, Stephen M. Storey, Huan Huang and Friedhelm Schroeder Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX, USA Received 4 March 2003; accepted 10 September 2003

Abstract Although liver fatty acid binding protein (L-FABP) is known to enhance uptake and esterification of straight-chain fatty acids such as palmitic acid and oleic acid, its effects on oxidation and further metabolism of branched-chain fatty acids such as phytanic acid are not completely understood. The present data demonstrate for the first time that expression of L-FABP enhanced initial rate and average maximal oxidation of [2,3-3H] phytanic acid 3.5- and 1.5-fold, respectively. This enhancement was not due to increased [2,3-3H] phytanic acid uptake, which was only slightly stimulated (20%) in L-FABP expressing cells after 30 min. Similarly, L-FABP also enhanced the average maximal oxidation of [9,10-3H] palmitic acid 2.2-fold after incubation for 30 min. However, the stimulation of L-FABP on palmitic acid oxidation nearly paralleled its 3.3-fold enhancement of uptake. To determine effects of metabolism on fatty acid uptake, a non-metabolizable fluorescent saturated fatty acid, BODIPY-C16, was examined by laser scanning confocal microscopy (LSCM). L-FABP expression enhanced uptake of BODIPY-C16 1.7-fold demonstrating that L-FABP enhanced saturated fatty acid uptake independent of metabolism. Finally, L-FABP expression did not significantly alter [2,3-3H] phytanic acid esterification, but increased [9,10-3H] palmitic acid esterification 4.5-fold, primarily into phospholipids (3.7-fold) and neutral lipids (9-fold). In summary, L-FABP expression enhanced branched-chain phytanic acid oxidation much more than either its uptake or esterification. These data demonstrate a potential role for L-FABP in the peroxisomal oxidation of branched-chain fatty acids in intact cells. (Mol Cell Biochem 259: 115–129, 2004) Key words: L-FABP, fatty acids, phytanic acid, palmitic acid, fatty acid oxidation, fatty acid uptake. Abbreviations: L-FABP – liver fatty acid binding protein; PPAR – peroxisome proliferator-activated receptor; [1]RXR – retinoid X receptor; phytanic acid – 3,7,11,15-tetramethylhexadecoanoic acid; PL – phospholipid; PE – ethanolamine glycerophospholipid; PI – phosphatidylinositol; PS – phosphatidylserine; PC – choline glycerophospholipid; SM – sphingomyelin; PA – phosphatidic acid; TG – triacylglycerides; DG – diacylglycerides; MG – monoacylglycerides; CE – cholesteryl ester; C – cholesterol; FA – fatty acid.

Introduction Although fatty acid binding proteins (FABPs) are present at very high concentration (2–5% of cytosolic protein, 100– 400 µM) in tissues involved in fatty acid metabolism such as liver, intestine, kidney, adipose, and heart, the physiological function(s) of FABPs are only beginning to be resolved.

Liver fatty acid binding protein (L-FABP) is the first known and perhaps most thoroughly examined member of the FABP family (reviewed in [2]). Both subcellular fractionation and immunogold electron microscopy show that in liver hepatocytes, the tissue with the highest level of L-FABP expression, the L-FABP is primarily cytosolic with small amounts detectable in the nucleus [42]. Similarly, overexpression of

Address for offprints: F. Schroeder, Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843-4466, USA (E-mail: [email protected])

116 L-FABP in L cells and embryonic cells (both at levels lower than in liver) resulted in a primarily cytosolic localization of L-FABP, with small amounts present in the nucleus [3, 4]. In keeping with the primarily cytosolic localization of L-FABP, studies with transfected cells and hepatocytes suggest that LFABP enhances multiple aspects of straight-chain fatty acid metabolism including: (i) cellular fatty acid uptake [2, 5–8], (ii) intracellular fatty acid transport/diffusion [2, 8–11], and (iii) modulating microsomal fatty acyl CoA synthase and fatty acyl CoA pool size [12–15]. In summary, while evidence suggests a potential role for L-FABP in straight-chain (e.g. palmitic acid, oleic acid) fatty acid metabolism [2, 16], less is known regarding potential role(s) of L-FABP in oxidation and metabolism of branched-chain fatty acids such as phytanic acid. Phytanic acid, a 16-carbon chain-length fatty acid with 4 methyl branches, is a common dietary constituent derived from meat and dairy products (reviewed in [17]). While normally present in low levels in the serum, phytanic acid and its metabolites reach high, toxic levels in a variety of peroxisomal disorders of branched-chain fatty acid oxidation [18, 19]. Unlike straight-chain fatty acids such as palmitic acid which are β-oxidized primarily in mitochondria, the branchedchain fatty acids (phytanic acid) are first α-oxidized in peroxisomes, followed by several cycles of chain-shortening by β-oxidation, transfer of shortened fatty acids out of peroxisomes, and finally transport to mitochondria for completion of β-oxidation [20]. Evidence supporting involvement of L-FABP in the peroxisomal oxidation of branched-chain fatty acids such as phytanic acid includes the fact that L-FABP binds phytanic acid [21, 22]. Furthermore, phytanic acid is a ligand for nuclear transcription factors such as peroxisome proliferator-activated receptor (PPAR) [1, 23] and retinoid X receptor (RXR) [24] involved in peroxisomal proliferation. Since L-FABP expression increases the distribution of straightchain fatty acids to the nucleus [25], L-FABP may act similarly for the branched-chain fatty acids. In contrast to these potentially stimulatory role(s) of L-FABP in phytanic acid oxidation are studies showing that L-FABP overexpressing cells fed high levels of phytanic acid exhibit inhibition of phytanic acid oxidation [26]. Likewise, studies with SCP-2/ SCP-x gene ablated mice fed high levels of phytol (precursor to phytanic acid) showed inhibition of phytanic acid oxidation [27] concomitant with a 4-fold increase in L-FABP expression and increased levels of enzymes involved in peroxisomal fatty acid oxidation [1, 23, 27]. However, whether the latter finding represents an inability of L-FABP to enhance phytanic acid oxidation, or results from the absence of SCPx (a peroxisomal 3-ketoacyl CoA thiolase specific for oxidation of branched fatty acids) and SCP-2 which binds both phytanic acid [28] and phytanoyl CoA [29] with high affinity (Kds in nM-µM range), is unknown. In summary, the potential role of L-FABP in the peroxisomal oxidation of

branched-chain fatty acids such as phytanic acid remains unclear. The objective of the present investigation was to establish a role for L-FABP in branched chain fatty acid oxidation and metabolism without the conflicting presence of other lipid transport proteins. The influence of phytanic acid uptake on oxidative capacity was also examined.

Materials and methods Materials Silica Gel G plates were purchased from Analtech (Newark, DE, USA); Silica Gel 60 plates were obtained from EM Industries, Inc. (Darmstadt, Germany); neutral lipid and fatty acid standards were from Nu-Chek Prep, Inc. (Elysian, MN, USA); and phospholipid standards were purchased from Avanti (Alabasta, AL, USA). [9,10-3H] palmitic acid was acquired from Dupont New England Nuclear (Boston, MA, USA) while [2,3-3H] phytanic acid (3,7,11,15-tetramethylhexadecoanoic acid) was prepared by Moravek Biochemicals, Inc. (Brea, CA, USA). BODIPY-C16 (4, 4-difluoro-5, 7dimethyl-4-bora-3a, 4a-diaza-s-indacene-hexadecanoic acid) was purchased from Molecular Probes Inc. (Eugene, OR, USA). All reagents and solvents used were of the highest grade available and were cell culture tested. L-cell culture Murine L cells were cultured at 37°C and 5% CO2 in Higuchi medium [30] containing 10% fetal bovine serum (Hyclone, Logan, UT, USA). For uptake, oxidation and lipid mass experiments, cells were grown on 35 mm dishes (5 × 105 cells/ dish) overnight, followed by serum starvation 24 h before fatty acid addition. Development of mock-transfected cells (designated as control) and cells transfected with cDNA corresponding to the L-FABP protein were as described earlier [31] where levels of L-FABP in transfected L-cells (0.3% cytosol protein) were in the physiological range of liver, intestine, and kidney (reviewed in [2, 32]. Levels of L-FABP expression were checked in duplicate dishes by western blot analysis before the start of each experiment to ensure consistent levels of L-FABP were present. Mouse L cell fibroblasts were a suitable choice for studying the effect of L-FABP on phytanic acid uptake, oxidation, and metabolism since: (i) levels of L-FABP in untransfected L cell fibroblasts were below the level of detectability and examination of L-FABP overexpression cells allowed analysis of the effect of the LFABP protein on cellular function, and (ii) fibroblasts have been used extensively to study disorders of peroxisomal fatty acid oxidation (reviewed in [20, 33].

117 Uptake of [2,3-3H] phytanic acid and [9,10-3H]-palmitic acid Fatty acid uptake was measured at 37°C in serum deprived cells incubated with increasing concentrations of cold fatty acid (25 nM to 1.8 µM) supplemented with radiolabeled fatty acid (1.5 µCi/nmol [2,3-3H] phytanic acid or 1.5 µCi/nmol [9,10-3H] palmitic acid) at increasing time intervals (1 min through 5 h). Based on these results, subsequent experiments with phytanic acid and palmitic acid were performed at concentrations in the linear range of uptake (50 nM) in the absence of BSA. BSA was not present in the medium or added with the fatty acid in order to avoid confusion of the role of BSA vs. L-FABP in fatty acid uptake. At indicated times the culture medium was removed followed by Folch extraction [34]. The medium was separated into an organic phase containing unesterified and esterified [2,3-3H] phytanic acid or [9,10-3H] palmitic acid and an aqueous layer containing [3H]-labeled water and [3H]-labeled NADH derived from oxidation of [2,3-3H] phytanic acid or [9,103 H] palmitic acid as described [33]. The cell monolayer was washed twice with phosphate buffered saline, lipids were extracted [31], and residual cell protein was sedimented (1500 rpm for 30 min) for quantitation [35]. Because [2,33 H] phytanic acid and [9,10-3H] palmitic acid are metabolizable fatty acids, uptake was corrected for release of watersoluble, radioactive oxidation products (primarily radiolabeled water and NADH) into the medium as described in [33]. [2,3-3H] phytanic acid and [9,10-3H] palmitic acid uptake were expressed as cellular uptake (dpm/µg cell protein). The contents of one 35 mm dish (n = 1) was equivalent to 1– 2 × 106 cells or 0.1–0.3 mg of cell protein. Radioactivity was quantitated in liquid scintillation cocktail (Scinti Verse, Fisher Scientific, Pittsburgh, PA, USA) using a Packard 1600TR counter (Meridian, CT, USA).

Laser scanning confocal microscopy (LSCM) fluorescence images were acquired at room temperature with an MRC1024 laser scanning confocal microscope (Bio-Rad, Hercules, CA, USA) equipped with an Axiovert 135 microscope and x63 Plan-Fluor oil immersion objective, numerical aperture 1.45 (Zeiss, Carl Zeiss Inc., Thornwood, NY, USA). The BODIPY-C16 was excited through a 1% neutral density filter by the 488 nm laser line of a Krypton-Argon laser (Coherent, Sunnyvale, CA, USA) set at 1–3% of the laser power (100 mW). Fluorescence emission of BODIPY-C16 was detected through a 525–565 nm band-pass filter. In order to determine the extent of metabolism of BODIPY C16, cells were washed in PBS, then incubated with BODIPYC16 at 37°C. At selected time points of incubation for (5 or 30 min), uptake of fluorescent fatty acids was terminated by removal of incubation medium and washing 3 times with PBS. Total lipids were extracted in hexane:isopropanol (3:2, v/v) and centrifuged at 1,500 rpm for 10 min. The pelleted protein was dried overnight at room temperature and quantitated by BioRad protein assay (Hercules, CA, USA). An aliquot of the supernatant was dried under N2, dissolved in CHCl3, and spotted on silica gel G thin layer chromatography (TLC) plates pre-activated at 100°C for 1 h. The unesterified fluorescent BODIPY-C16 free fatty acids were separated from potential BODIPY-C16 metabolites by running the TLC plates in a solvent system comprised of petroleum ether: diethyl ether: glacial acetic acid (70:30:1, v/v/v). Relative proportion of BODIPY-C16 fluorescence remaining in unesterified form was determined by fluorescent imaging and quantitating the spots on the TLC plates s with the ChemiImager System (Alpha-Innotech Inc., San Leandro, CA, USA) and analysis with FluorChem v. 2.0 software (Alpha-Innotech Inc., San Leandro, CA, USA).

Oxidation of [2,3-3H] phytanic acid and [9,10-3H]palmitic acid Uptake and metabolism of BODIPY-C16 For laser scanning microscopy (LSCM), cells were seeded in Lab-Tek chambered cover glass slides (Nunc, Naperville, IL, USA or VWR, Sugarland, TX, USA) at 25,000 cells/ chamber (107 cells/ml) and grown for 36–48 h. To determine BODIPY-C16 uptake, cells were washed once with phosphate buffered saline (PBS), then 1 ml PBS containing BODIPYC16 was added from a concentrated stock solution in ethanol. The final solvent concentration was < 0.1% and had no cytotoxic effect on the cells. In order to maintain fluorescence signals in the linear range of uptake, the concentration of BODIPY-C16 was varied as a function of time for 30 min. The optimal concentration of BODIPY-C16 for linear uptake under these conditions was 100 nM.

Conditions for determining [2,3-3H] phytanic acid and [9,10H] palmitic acid oxidation were the same as used for determining total uptake. At timed intervals, the extent of [9,10-3H] palmitic acid or [2,3-3H] phytanic acid oxidation was determined by measuring the water-soluble tritiated fatty acid oxidation products released into the culture medium as described earlier [33]. Briefly, the medium was removed at timed intervals and extracted by the method of Folch [34]. Radioactivity in the aqueous layer (containing water soluble fatty acid oxidation products) was determined in liquid scintillation cocktail (Scinti Verse, Fisher Scientific, Pittsburgh, PA, USA). Oxidation was expressed as dpm/µg cell protein, with protein determined by the method of Bradford [35]. The percentage of fatty acid taken up that was oxidized was also 3

118 determined in order to compare the extent of oxidation between two fatty acids with different uptake rates. The percent oxidized was measured by dividing the amount of fatty acid that was oxidized by the total uptake. Targeting of [2,3-3H] phytanic acid and [9,10-3H]palmitic acid into specific lipid classes The distribution of radiolabeled palmitic acid and phytanic acid into specific lipid classes was determined under conditions described above. At timed intervals, the culture medium was removed and the cell monolayer was washed twice with PBS. Following freezing of the monolayer on liquid nitrogen, total lipids were extracted with n-hexane-2-propanol 3:2 (v/v). After centrifuging the sample at 1500 rpm for 15 min, sedimented protein residues were separated, allowed to dry, and quantitated [35]. The supernatant was evaporated to dryness, redissolved in chloroform, with lipid classes resolved using Silica gel G thin-layer chromatography (TLC) plates. TLC plates were developed in the following solvent system: petroleum ether-diethyl ether-methanol-acetic acid 90:7:2:0.5 (v/v). The monoacylglyceride, diacylglyceride, and triacylglyceride, along with cholesteryl ester, free fatty acid, and phospholipid fractions were identified by comparison to known standards run on the same TLC plates. Each fraction was scraped, eluted with chloroform, dried under a stream of N2, and radioactivity quantified in liquid scintillation cocktail (Scinti Verse, Fisher Scientific, Pittsburgh, PA, USA) with a Packard 1600TR liquid scintillation counter (Meridian, CT, USA). For determination of individual phospholipid classes, a portion of the total phospholipid sample was applied to Silica Gel 60 TLC plates and resolved into individual phospholipid classes using the following solvent system: chloroform:methanol:water:glacial acetic acid (150:112.5:10.5:6, v/v). Known standards were used to identify individual phospholipid classes. Lipids were stored under an atmosphere of N2 to limit oxidation. All glassware was washed with sulfuric acid-chromate before use. Determination of [2,3-3H] phytanic acid and [9,10-3H]palmitic acid specific activity in lipid classes Mass (nmol/mg cell protein) of individual lipid classes was determined as described earlier [31,36]. Specific activity (dpm/pmol lipid class) was determined by division of (dpm/ mg protein) by lipid mass (pmol/mg protein).

Statistics All values were expressed as the mean ± S.E.M. with n and p indicated in the Results section. Statistical analyses were

performed using Student’s t-test (GraphPad Prism, San Diego, CA, USA). Values with p < 0.05 were considered statistically significant.

Results Effect of methyl-branching on fatty acid uptake in L-cell fibroblasts Although both phytanic acid and palmitic acid are saturated fatty acids with an equivalent 16 carbon chain backbone length, the effect of the 4 methyl branches present in phytanic acid on cellular uptake is not known. The extent of fatty acid uptake was determined under conditions of increasing phytanic acid or palmitic acid (from 25 nM to 0.6 µM) after 5 h incubation in order to establish the concentration dependence in control L-cells (Fig. 1A). The initial rate of phytanic acid uptake was over 34-fold lower as compared to that of palmitic acid (p < 0.006, n = 3–6). In addition, the average maximal uptake of phytanic acid was 2.5-fold lower than that of palmitic acid (p < 0.0001, n = 3–6). The time dependence of phytanic acid and palmitic acid uptake was also examined under conditions where L-cells was incubated with non-saturable levels (50 nM) of phytanic acid or palmitic acid for increasing time intervals (1 min to 5 h, Fig. 1B). Under these experimental conditions, while the average maximal uptake of phytanic acid uptake was 3.9-fold lower than that of palmitic acid (p < 0.001, n = 3–6), the initial rate of uptake was not significantly different. These data indicated that methylbranching significantly affected kinetic parameters related to fatty acid uptake. When total uptake of [2,3-3H] phytanic acid was compared to that of [9,10-3H] palmitic acid in control L-cells under the above optimized conditions (i.e. 50 nM fatty acid, 1–30 min incubation), phytanic acid and palmitic acid uptake were nearly linear (Fig. 2). However, by 5 min incubation, uptake of phytanic acid was over 3-fold slower than palmitic acid uptake. Even after 30 min incubation, uptake of phytanic acid remained slower (2.0-fold) than that of palmitic acid. Thus, the presence of 4 methyl branches in phytanic acid resulted in significantly (p < 0.01) decreased uptake as compared to the equivalent 16-carbon chain-length, non-branched palmitic acid. Effect of L-FABP expression on [2,3-3H] phytanic acid and [9,10-3H] palmitic acid uptake Although the ability of L-FABP to stimulate straight-chain fatty acid uptake [2, 5–8, 25, 37] is well-documented, the effect of methyl-branching on L-FABP mediated fatty acid uptake has not heretofore been determined in intact cells.

119 Therefore, the effect of L-FABP expression on uptake of [2,3H] phytanic acid was examined and compared to that of [9,10-3H] palmitic acid (Fig. 2). The total uptake of [2,3-3H] phytanic acid was initially not significantly affected by L-FABP expression, and then only modestly (20%, p < 0.037, n = 3–6) at later time points (Fig. 2A). In contrast, L-FABP expression increased the total uptake of [9,10-3H] palmitic acid in L-FABP expressing cells 2.5-, 2.7-, and 3.4-fold at 5, 15, and 30 min respectively, as compared to control cells (p < 0.001, n = 3–6, Fig. 2B). It was evident from these findings that L-FABP differentially enhanced the uptake of straight-chain as compared to branched3

Fig. 2. Total uptake of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid in control and transfected L cells overexpressing L-FABP. Total uptake of [2,3- 3H] phytanic acid (A) and [9,10-3H] palmitic acid (B) was determined in control cells (open bars) and L-FABP expressing cells (closed bars) over a 30 min time interval as described in Materials and methods. Values represent means ± S.E.M. *Indicates significance p < 0.04, n = 3–6 as compared to the control cells.

chain fatty acid.

Uptake and metabolism of BODIPY-C16

Fig. 1. Extent of total fatty acid uptake of [2,3-3H] phytanic acid and [9,103 H] palmitic acid at different fatty acid concentrations and incubation times. (A) Total uptake of [2,3-3H] phytanic acid (closed circles) and [9,10-3H] palmitic acid (open circles) was determined in control cells at different fatty acid concentrations (25 nM through 0.6 µM) after 5 h incubations as described in Materials and methods. (B) Total uptake of [2,3-3H] phytanic acid (closed circles) and [9,10-3H] palmitic acid (open circles) was determined in control cells at increasing time intervals (1 min to 5 h) at 50 nM concentration of fatty acid. Values represent means ± S.E.M. (n = 3–6).

To differentiate the effects of L-FABP expression on fatty acid uptake from those on metabolism in real-time, in living cells, it was essential to study the effect of L-FABP on fluorescent saturated fatty acids that remained in unesterified form over the time period of uptake. Therefore, studies with BODIPYC16, a fluorescent fatty acid with a C16 back-bone chain length, were performed. Image analysis on L cells incubated with BODIPY-C16 (100 nM) for 30 min demonstrated the cytosolic distribution of the fluorescent fatty acid (Fig. 3A). Total uptake of BODIPY-C16 was determined by analyzing increased fluorescence intensity over time in both control and

120 in transfected L cells overexpressing L-FABP (Fig. 3B). Expression of L-FABP enhanced total uptake of BODIPY-C16 at all time points studied. In order to determine if the fluorescent fatty acid BODIPYC16 was metabolized during the time interval of the uptake experiments, L-cell fibroblasts were incubated for up to 30 min with BODIPY-C16, then lipid extracted, and resolved on TLC plates using the following system: petroleum ether: diethyl ether: glacial acetic acid (70:30:1, v/v/v) where the unesterified BODIPY-C16 was clearly separated from esterified BODIPYC16. Under these conditions, analysis revealed that > 98% of the BODIPY-C16 remained unmetabolized after 30 min (Fig. 3B, inset). In summary, these results show that L-FABP expression markedly enhanced the uptake of both non-metabolizable fluorescent as well as metabolizable saturated fatty acids.

Effect of methyl-branching on fatty acid oxidation in L-cell fibroblasts Basal oxidation of phytanic acid and palmitic acid in L-cells was determined at increasing concentrations (from 25 nM to 1.8 µM) of phytanic acid and palmitic acid (Fig. 4A). While maximal oxidation for both phytanic acid and palmitic acid was reached at levels > 100 nM, at this concentration maximal oxidation of phytanic acid was 8.6-fold less than that of palmitic acid (p < 0.001, n = 3–6, Fig. 4A). Next, in order to establish the optimal time of L-cell incubation for determination of fatty acid oxidation under non-saturating conditions, cells were incubated for increasing time intervals at 50 nM phytanic acid or palmitic acid (Fig. 4B). Under these conditions, both phytanic acid and palmitic acid oxidation levels increased linearly under 30 min. From these results, it was determined that subsequent oxidation measurements would be performed with incubations of 50 nM fatty acid from 1– 30 min, at levels below saturation. While the above data showed much lower total oxidation of [2,3-3H] phytanic acid as compared to total oxidation of [9,10-3H] palmitic acid, differences in phytanic acid and palmitic acid uptake must also be considered to compare oxidation values. In order to account for the much slower uptake of phytanic acid vs. palmitic acid, the % [2,3-3H] phytanic acid taken up and oxidized at each time point was calculated (Table 1). Within the first minute of uptake, the % of [2,33 H] phytanic acid taken up and oxidized was 30.2 ± 5.2%. With increasing time of incubation, this decreased nearly 4fold to 8.2 ± 1.5% and 7.3 ± 0.5% at 5 and 30 min, respectively. In contrast, within the first minute of [9,10-3H] palmitic acid uptake, 92.6 ± 3.9% was oxidized in control cells. With increasing time the % of [9,10-3H] palmitic acid taken up that was oxidized decreased to 59.9 ± 2.7% and 47.0 ± 3.1% at 5 and 30 min, respectively (Table 1). It was apparent that, over

Fig. 3. Distribution, uptake, and metabolism of BODIPY-C16 in L cells. Laser scanning confocal microscopy (LSCM) was used to study the distribution (A), uptake (B), and metabolism (inset) of BODIPYC-16 in control cells (open bars) and L-FABP expressing cells (closed bars) as described in Materials and methods. Fluorescence intensity values were expressed in arbitrary units, as measured during the time course. Values represent means ± S.E.M. *Indicates significance p < 0.01, n = 20–30 cells, as compared to the control cells.

the same time periods examined, the % of [2,3-3H] phytanic acid taken up and oxidized was 3.1-, 7.3-, and 6.4-fold less than that of [9,10-3H] palmitic acid (Table 1). In summary, at all time points the total oxidation of [2,33 H] phytanic acid, as well as the % of [2,3-3H] phytanic acid taken up that was oxidized, was many-fold lower than that of [9,10-3H] palmitic acid. Further, both [2,3-3H] phytanic acid and [9,10-3H] palmitic acid exhibited decreased percent oxidation at longer time points, consistent with increased intracellular metabolism leading to esterification. Effect of L-FABP expression on oxidation of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid in L-cell fibroblasts In order to determine the role L-FABP plays in the oxidation of branched- vs. straight-chain fatty acids, the total oxidation

121 Table 1. Percent oxidation of [2, 3-3H] phytanic acid and [9,10-3H] palmitic in control and transfected L cells overexpressing L-FABP

Time (min)

% Oxidation of phytanic and palmitic acid Control L-FABP

Phytanic acid 1 5 30

30.2 ± 5.2@ 8.2 ± 1.5@ 7.3 ± 0.5@

83.3 ± 15.8* 23.6 ± 5.6*@ 9.4 ± 0.6*@

Palmitic acid 1 5 30

92.6 ± 3.9 59.9 ± 2.7 47.0 ± 3.1

91.8 ± 4.6 68.3 ± 2.5 29.4 ± 4.1*

Total oxidation of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid was determined as described in Materials and methods. Values reflect the mean ± S.E.M. *Indicates significance p < 0.04, n = 3–6 as compared to control cells labeled with either palmitic or phytanic acid. @Indicates significance p < 0.05, n = 3–6 as compared to the respective cell line labeled with palmitic acid.

Fig. 4. Extent of fatty acid oxidation of [2,3-3H] phytanic acid and [9,103 H] palmitic acid at different fatty acid concentrations and incubation times. (A) Oxidation of [2,3-3H] phytanic acid (closed circles) and [9,10-3H] palmitic acid (open circles) was determined in control cells at different fatty acid concentrations (25 nM through 1.8 µM) after 5 h incubations as described in Materials and methods. (B) Oxidation of [2,3-3H] phytanic acid (closed circles) and [9,10-3H] palmitic acid (open circles) was determined in control cells at increasing time intervals (1 min to 5 h) at 50 nM concentration of fatty acid. Values represent means ± S.E.M. (n = 3–6).

of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid was examined in L-FABP expressing cells and compared to that in control cells (Fig. 5). L-FABP expression increased the initial rate of [2,3-3H] phytanic acid total oxidation 3.5-fold as well as increased the maximum of [2,3-3H] phytanic acid total oxidation 1.5-fold (p < 0.02, n = 3–6, Fig. 5). In contrast, while L-FABP expression did not increase the initial rate of [9,10-3H] palmitic acid total oxidation, the maximal oxidation was increased 2.2-fold (p < 0.004, n = 3–6, Fig. 5, inset). It should be noted, however, that when expressed as % of [2,3-3H] phytanic acid taken up that was oxidized, LFABP expression increased [2,3-3H] phytanic acid oxidation as much as 2.8-fold from 30.2 ± 5.2% to 83.3 ± 15.8%

at 1 min, 2.9-fold at 5 min, and 1.3-fold at 30 min (p < 0.04, n = 3–6) (Table 1). In contrast, L-FABP expression did not significantly affect [9,10-3H] palmitic acid β-oxidation until 30 min at which point L-FABP expression actually decreased the percentage of [9,10-3H] palmitic acid taken up that was oxidized by 37%, p < 0.027, n = 3–6 (Table 1), suggesting increased metabolism. In summary, L-FABP expression enhanced the total oxidation of both [2,3-3H] phytanic acid and [9,10-3H] palmitic acid. However, when the differential effect of L-FABP expression on the uptake of the two fatty acids was taken into account, L-FABP stimulated [2,3-3H] phytanic acid oxidation independent of alterations in [2,3-3H] phytanic acid uptake. In contrast, L-FABP enhanced both [9,10-3H] palmitic acid oxidation and uptake in parallel, such that the % of [9,10-3H] palmitic acid taken up that was oxidized did not show similar enhancement.

Distribution of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid to the free, unesterified fatty acid pool in control and L-FABP expressing cells The above studies indicated that methyl-branching significantly reduced the uptake and oxidation of a saturated fatty acid (i.e. phytanic acid), suggesting that methyl-branching may also differentially modulate the distribution of branchedchain vs. straight-chain fatty acids into the free, unesterified fatty acid pool. Therefore, control cells were incubated with [2,3-3H] phytanic acid or [9,10-3H] palmitic acid as above, lipids were extracted, resolved by lipid classes, and radioactivity in the free unesterified fatty acid fraction was determined (Fig. 6). The free fatty acid pool was small compared

122

Fig. 5. Total oxidation of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid in control and transfected L cells overexpressing L-FABP. Total oxidation of [2,3-3H] phytanic acid (A) and [9,10-3H] palmitic acid (inset) was determined in control cells (closed circles) and L-FABP expressing cells (open circles) over a 30 min time interval as described in Materials and methods. Values represent means ± S.E.M. (n = 3–6). *Indicates significance p < 0.007 as compared to the control cells.

to other lipids (approximately 3–6% of total lipid mass). The [2,3-3H] phytanic acid taken up entered the free fatty acid pool within 5 min and thereafter was no longer detectable in the free fatty acid pool (Fig. 6A). In contrast, while [9,10-3H] palmitic acid taken up was similarly detectable in the free fatty acid pool by 5 min, highest levels were observed by 15 min of uptake, and thereafter declined to nondetectable levels (Fig. 6B). Furthermore, highest levels of [9,10-3H] palmitic acid (Fig. 6B) were 2.9-fold greater than for [2,3-3H] phytanic acid (Fig. 6A) in the free fatty acid pool. Thus while the free fatty acid pool was small compared to other lipids, the [2,3-3H] phytanic acid was retained in this pool for a shorter time and to a lesser extent than was [9,10-3H] palmitic acid. To determine the effect of L-FABP expression on distribution of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid to the free unesterified fatty acid pool, simultaneous measurements were also made in L-FABP expressing L-cells. LFABP expression did not significantly alter the accumulation of [2,3-3H] phytanic acid in the free unesterified fatty acid pool (Fig. 6A). In contrast, although incorporation of [9,103 H] palmitic acid into the free fatty acids was again maximal at 15 min in L-FABP expressing cells, the maximal level of [9,10-3H] palmitic acid accumulating in the free fatty acid fraction was reduced 2.7-fold in L-FABP overexpressing cells (Fig. 6B), suggesting increased targeting to other lipids was occurring.

Fig. 6. Targeting of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid to total free fatty acids in control and transfected L cells overexpressing LFABP. Targeting of [2,3-3H] phytanic acid (A) and [9,10-3H] palmitic acid (B) to the free fatty acid pool was determined in control cells (open bars) and L-FABP expressing cells (closed bars) as described in Materials and methods. ND was defined as ‘not detected’. Values represent means ± S.E.M. (n = 3–6). *Indicates significance p < 0.015 as compared to control cells incubated with the respective fatty acid.

Effect of L-FABP expression on extent of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid targeting to total esterified lipids In addition to being oxidized, both [2,3-3H] phytanic acid and [9,10-3H] palmitic acid can be esterified to more complex lipids such as phospholipids (PL), and neutral lipids (NL) such as diacylglycerides (DG), triacylglycerides (TG), and cholesteryl esters (CE). The extent of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid targeting to total esterified lipids was determined at 30 min (Fig. 7), the time point at which both free fatty acid pool size (Fig. 6) and percent oxidization of fatty acids (Table 1) were lowest. In control L-cells, [2,3-

123 incorporation into total lipid over time. In order to clearly show how the two catabolic and anabolic processes (oxidation and esterification) were nearly inversely correlated, the extent of total lipid targeting vs. percent oxidation in control cells over the 30 min time course was determined (Fig. 8). The % oxidation of both phytanic acid and palmitic acid was initially high, dropping off after 30 min incubation. In contrast, the extent of phytanic acid and palmitic acid esterification was minimal initially, yet increased at longer incubations. The decreased percent oxidization and increased lipid targeting was reflected in the changing free fatty acid pools (Fig. 6). Expression of L-FABP did not significantly alter [2,3-3H] phytanic acid targeting to total esterified lipids (Fig. 7A). This was in marked contrast to [9,10-3H] palmitic acid whose targeting to total esterified lipids increased 4.5-fold (p ≤ 0.0005,

Fig. 7. Targeting of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid to esterified lipids in control and transfected L cells overexpressing L-FABP. The extent of targeting of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid to total esterified lipids (A), total phospholipids (B), and total neutral lipids (C) were determined in control cells (open bars) and L-FABP expressing cells (closed bars) over a 30 min time interval as described in Materials and methods. Values represent means ± S.E.M. (n = 3–6). *Indicates significance p < 0.05 as compared to cells incubated with [2,3-3H] palmitic acid. @ Indicates significance p < 0.007 as compared to control cells incubated with the respective fatty acid. Total neutral lipids included diacylglycerols, monoacylglycerols, triacylglycerols, and cholesteryl esters.

3

H] phytanic acid incorporation into total esterified lipids was 2.3-fold higher than [9,10-3H] palmitic acid (Fig. 7A, open bars). This was consistent with less total [2,3-3H] phytanic acid than [9,10-3H] palmitic acid being oxidized (Fig. 4, Table 1). Further, at initial time points, as seen from Table 1, the percentage of oxidation was greatest but decreased with time as the fatty acid was esterified resulting in increased

Fig. 8. Percentage of fatty acid oxidation and total lipid targeting in L cells. The extent of total oxidation (open bars) and total lipid targeting (closed bars) of [2,3-3H] phytanic acid (A) and [9,10-3H] palmitic acid (B) in L cells was determined as described in Materials and methods. Values represent means ± S.E.M. (n = 3–6). *Indicates significance p < 0.005 as compared to the percentage of fatty acid oxidation in L cells incubated with the respective fatty acid.

124 n = 3–6) in L-FABP expressing cells (Fig. 7A). Consequently, the total esterified [2,3-3H] phytanic acid was 1.7-fold less than that of total esterified [9,10-3H] palmitic acid in L-FABP expressing cells. Thus, L-FABP differentially enhanced accumulation of [9,10-3H] palmitic acid, but not [2,3-3H] phytanic acid, in total esterified lipids.

Differential distribution of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid into individual esterified lipids: phospholipids and neutral lipids The targeting of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid into specific esterified lipid classes of control L-cells differed markedly. The [2,3-3H] phytanic acid was distributed nearly equally between these fractions. In contrast, the [9,103 H] palmitic acid was preferentially (i.e. 5.5-fold) esterified into phospholipids as compared to neutral lipids (Figs 7B and 7C). Furthermore, while both fatty acids were esterified to similar extent into phospholipids (22.9 ± 1.4 vs. 18.6 ± 1.4), 8.4-fold more [2,3-3H] phytanic acid was esterified into neutral lipids. Thus, phytanic acid accumulated much more in the neutral lipids as compared to palmitic acid. When the incorporation of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid into phospholipids was expressed in terms of phospholipid mass (dpm/pmol phospholipid), the specific activity increased with increasing time as expected. However, turnover of [2,3-3H] phytanic acid was 1.8-fold slower than [9,10-3H] palmitic acid in control cells (3.5 ± 0.22 vs. 6.3 ± 0.48). Finally, while the ratio of the % [2,3-3H] phytanic acid in the neutral/phospholipids did not decrease significantly, the ratio of the % [9,10-3H] palmitic acid in the neutral/phospholipids decreased more than 5-fold with increasing time in control cells. These results suggested that while [2,33 H] phytanic acid was evenly targeted between phospholipid and neutral lipid fractions in control cells, the distribution of [9,10-3H] palmitic acid in the phospholipid fraction increased over time at the expense of the neutral lipid fraction.

Effect of L-FABP expression on the distribution of [9,10-3H] palmitic acid and [2,3-3H] phytanic acid into phospholipids While it is well documented that L-FABP stimulates incorporation of straight-chain fatty acids (oleic acid, arachidonic acid, palmitic acid) into phospholipids in transfected cells and/or in vitro [38, 39], the effect of methyl-branching on LFABP-mediated incorporation of fatty acids into phospholipids remains unresolved. L-FABP expression increased the incorporation of the branched-chain [2,3-3H] phytanic acid into phospholipids modestly 1.4-fold (Fig. 7B), without altering the specific activity of [2,3-3H] phytanic acid in the phospholipid fraction (data not shown). In contrast, L-FABP

expression was more effective in targeting of the straightchain [9,10-3H] palmitic acid to phospholipids which was increased up to 3.7-fold (p ≤ 0.001, n = 3–6) (Fig. 7B). Furthermore, L-FABP increased the specific activity of [9,10-3H] palmitic acid (dpm/pmol phospholipid) by 2.5-fold after 30 min (data not shown). Since L-FABP enhanced incorporation of straight-chain fatty acids into specific phospholipid classes in vitro [40], individual phospholipid fractions were resolved in order to determine whether [2,3-3H] phytanic acid was also selectively esterified to specific phospholipid classes. Within the phospholipid fraction the [2,3-3H] phytanic acid was 3.6-fold more distributed to anionic (i.e. net negatively charged) phospholipids (p ≤ 0.003, n = 3–6, data not shown). L-FABP expression did not selectively increase the specific activity of [2,3-3H] phytanic acid in either the total phospholipids or any specific phospholipid class (Fig. 9A). Instead, L-FABP expression selectively decreased the specific activity of [2,3-3H] phytanic acid in phosphatidylinositol (PI) and phosphatidylcholine (PC) 2- and 7-fold, respectively (Fig. 9A). In contrast, the pattern of straight-chain [9,10-3H] palmitic acid distribution among phospholipid classes differed markedly from that of the branched-chain [2,3-3H] phytanic acid. The [9,10-3H] palmitic acid was essentially equally distributed between zwitterionic (i.e. net neutral charged) and anionic phospholipid classes in both control and L-FABP expressing cells (data not shown). However, L-FABP expression significantly increased the specific activity (dpm/pmol phospholipid) of [9,10-3H]-palmitic acid into total phospholipids by up to 2.5-fold (data not shown), the increase was primarily in phosphatidic acid (PA), phosphatidylethanolamine (PE) and sphingomyelin (SM) which increased 4.4-, 1.6-, and 2.0-fold, respectively, at the expense of phosphatidylcholine which decreased 3.5-fold (Fig. 9B). In summary, the methyl-branched [2,3-3H] phytanic acid was incorporated significantly less into esterified lipids than the straight-chain [9,10-3H] palmitic acid. Although L-FABP expression enhanced the specific activity of [9,10-3H] palmitic acid, but not [2,3-3H] phytanic acid, in phospholipids, the specific activities of [9,10-3H] palmitic acid and [2,3-3H] phytanic acid were both altered in individual phospholipid classes with the largest changes observed in phosphatidic acid, phosphatidylcholine, and phosphatidylinositol.

Extent of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid targeting to total and individual neutral lipids in control and L-FABP expressing cells In contrast to the straight-chain [9,10-3H] palmitic acid, LFABP expression had little effect on incorporation of [2,33 H] phytanic acid into neutral lipids. L-FABP expression did not enhance incorporation of [2,3-3H] phytanic acid into

125 of [2,3-3H] phytanic acid to neutral lipids (as compared to [9,10-3H] palmitic acid (Fig. 7C) was a result of increased incorporation into di- and tri-acylglycerides (Fig. 10). Within the individual neutral lipid classes, L-FABP expression had little effect on [2,3-3H] phytanic acid incorporation into DG and TG, but decreased [2,3-3H] phytanic acid incorporation into CE about 2-fold (Fig. 10A). In contrast, L-FABP expression enhanced the targeting of [9,10-3H] palmitic acid to diacylglycerols, triacylglycerols, and cholesteryl esters 12-, 4-, and 2-fold, respectively (Fig. 10B). These data indicate that, in control cells, the higher targeting of [2,3-3H] phytanic acid to neutral lipids (as compared to [9,10-3H] palmitic acid (Fig. 7C) was a result of higher incorporation into di- and tri-acylglycerides (Fig. 10). While L-FABP expression did not increase incorporation of [2,33 H] phytanic acid into individual neutral lipid classes (Fig. 10A), L-FABP expression increased [9,10-3H] palmitic acid incorporation into all neutral lipid classes (Fig. 10B).

Fig. 9. Targeting of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid to individual phospholipids in control and transfected L cells overexpressing L-FABP. The extent of targeting of [2,3-3H] phytanic acid (A) and [9,103 H] palmitic acid (B) to individual phospholipids was determined as specific activity (dpm/pmole) in control cells (open bars) and L-FABP expressing cells (closed bars) after 30 min incubations as described in Materials and methods. Phospholipids were defined as total phospholipid, PL; phosphatidic acid, PA; ethanolamine glycerophospholipid, PE; phosphatidylinositol, PI; phosphatidylserine, PS; choline glycerophospholipid, PC; and sphingomyelin, SM. Values represent means ± S.E.M. (n = 3–6). *Indicates significance p < 0.01 as compared to control cells incubated with the respective fatty acid.

neutral lipids (Fig. 7C) or alter the specific activity of [2,3H] phytanic acid in the neutral lipid fraction at the 30 min time point (data not shown). In contrast, L-FABP expression increased the targeting of the straight-chain [9,10-3H] palmitic acid to neutral lipids 9-fold (pmol/mg protein, P ≤ 0.0001, n = 3–6) (Fig. 7C) and increased its specific activity 7.9-fold (dpm/pmol neutral lipid, p ≤ 0.0001, n = 3–6) at 30 min. The neutral lipids were fractionated into individual classes including diacylglycerols (DG), monoacylglycerols (MG), triacylglycerols (TG), and cholesteryl esters (CE). Since the monoacylglycerol fraction was a minor component representing less than 0.1% of the total it was included with the diacylglycerol values. In control cells, the increased targeting 3

Fig. 10. Targeting of [2,3-3H] phytanic acid and [9,10-3H] palmitic acid to individual neutral lipids in control and transfected L cells overexpressing L-FABP. The extent of targeting of [2,3-3H] phytanic acid (A) and [9,103 H] palmitic acid (B) to individual neutral lipids was determined in control cells (open bars) and L-FABP expressing cells (closed bars) after 30 min incubations as described in Materials and methods. Neutral lipids were defined as diacylglycerols/monoacylglycerols, DG; triacylglycerols, TG; and cholesteryl esters, CE. Values represent means ± S.E.M. (n = 3–6). *Indicates significance p < 0.05 as compared to control cells incubated with the respective fatty acid.

126

Discussion Because the toxic effects of high levels of branched-chain fatty acids in animals have been associated with diminished peroxisomal oxidation, considerable attention has been focused on genetic diseases wherein peroxisome assembly is defective or individual enzymes of peroxisomal fatty acid oxidation are mutated (reviewed in [17, 41–43]). These studies have contributed significantly to our understanding of the oxidation pathway of branched-chain fatty acids such phytanic acid. Metabolism of branched-chain fatty acids differs significantly from that of straight-chain fatty acids in that branched chain fatty acids undergo an initial α-oxidation in peroxisomes followed by chain shortening β-oxidation, and then transfer to mitochondria for completion of β-oxidation. In contrast, straight chain fatty acids such as palmitic acid are both oxidized and chain shortened primarily in mitochondria by β-oxidation (8-fold more than in peroxisomes). Nevertheless, despite these advances in resolving the pathway of branched-chain fatty acid oxidation, little is known regarding factors that mediate the uptake and transport/targeting of branched-chain fatty acids for oxidation in peroxisomes and/ or transport of chain-shortened phytanic acid metabolites to mitochondria for completion of oxidation. While substantial in vitro and/or correlative data suggest potential role(s) of LFABP in enhancing many aspects (uptake, mitochondrial oxidation, esterification) of straight-chain fatty acid metabolism, with the exception of an earlier study performed at high levels of phytanic acid [26], to our knowledge there are no published reports directly demonstrating the effect of L-FABP expression on branched-chain peroxisomal oxidation in intact cells. The present work provides several new insights into the oxidation and metabolism of branched-chain fatty acids. L-FABP significantly enhanced the initial rate and average maximal oxidation of [2,3-3H] phytanic acid 3.5- and 1.5fold, respectively. Furthermore, this effect was not due to L-FABP dramatically altering uptake since L-FABP had little effect on phytanic acid uptake at 5 min incubation and increased phytanic acid uptake only slightly (20%) at longer time points. In contrast, while L-FABP binds both palmitic acid and phytanic acid with similar affinity [21, 40], L-FABP expression dramatically enhanced the average maximal oxidation of [9,10-3H] palmitic acid 2.2-fold after incubation for 30 min, consistent with rat hepatocyte data wherein L-FABP levels were altered by hypophysectomy [37]. Furthermore, while palmitic acid was oxidized much more rapidly than phytanic acid, L-FABP’s enhancement of palmitic acid oxidation was driven by its uptake whereas phytanic acid oxidation was not. The present work shows for the first time that peroxisomal phytanic acid oxidation was enhanced by LFABP expression, relatively independent of an effect on phytanic acid uptake. Since L-FABP is primarily a cytosolic protein [44] that binds phytanic acid [22], this suggested that

L-FABP enhances phytanic acid diffusion/transport through the cytosol to peroxisomes for oxidation. This prediction was based on the observation that L-FABP enhances diffusion/ transport of straight-chain fatty acids within the cell [2, 8–10]. Furthermore, while L-FABP expression only weakly affected branched chain fatty acid uptake, uptake of straight-chain fatty acids containing no branched chains was enhanced 2-4 fold in (i) transfected L-cells overexpressing L-FABP [5, 6, 45], and in (ii) normal hepatocytes vs. hepatocytes of hypophysectomized rats, with the latter having lower L-FABP levels [37]. The preferential L-FABP enhancement of straight-chain fatty acid uptake was not due to an inability of L-FABP to interact with phytanic acid since L-FABP binds both phytanic acid [22] and straight-chain unbranched fatty acids [21, 40]. Isothermal titration calorimetry [21], radiolabeled ligand (reviewed in [40]), and fluorescence competition [46] fatty acid binding assays showed that L-FABP has two fatty acid binding sites. Furthermore, the L-FABP high affinity binding site exhibits binding constants (Kd) for branched (phytanic acid) and straight (palmitic acid) chain fatty acids of 0.015 µM for phytanic acid and a range of 60 nM- 62 µM for palmitic acid (depending on the assay), respectively. The higher affinity of branched chain fatty acids such as phytanic acid for L-FABP was further confirmed using displacement studies where phytanic acid was shown to be 3-fold more effective than palmitic acid in displacing cis-paranaric acid [22]. Furthermore, while L-FABP has high affinity for phytanic acid [21] it does not bind fatty acids ≤ C-12 chain-length [21, 40]. Thus, L-FABP apparently enhances phytanic acid oxidation by forming a complex with phytanic acid to enhance its transport to peroxisomes rather than by enhancing uptake or altering transport of the chain-shortened phytanic acid metabolites (i.e. C-7 and C-9 chain-length) from peroxisomes to mitochondria for completion of β-oxidation. Finally, it should be noted that phytanic acid, rather than its CoA derivative, is utilized by L-FABP for uptake into peroxisomes-a process that involves conversion to phytanoyl CoA by a peroxisomal membrane acyl CoA synthetase [47]. Since L-FABP is known to enhance the intracellular movement/diffusion of straightchain fatty acids [2, 8, 9] and binds with high affinity to phytanic acid, it is likely that L-FABP also enhances intracellular movement of phytanic acid to peroxisomes where it is converted to phytanoyl CoA and translocated into the peroxisomal matrix for oxidation. L-FABP differentially targeted branched- vs. straight-chain fatty acid to esterified lipids. In the basal state, much more phytanic acid than palmitic acid was esterified. Furthermore, esterified phytanic acid was nearly equally distributed between neutral and phospholipids while esterified palmitic acid was targeted primarily into phospholipids. However, despite L-FABP’s affinity for branched-chain fatty acids (and their CoA derivatives), L-FABP did not stimulate phytanic acid esterification into total lipids. This effect was in contrast to

127 L-FABP expression resulting in enhanced straight-chain fatty acid esterification into more complex lipids both in vitro [38, 39, 48–50] and in transfected cells [5, 6, 51]. Thus, despite L-FABP’s high affinity for branched- as well as straight-chain fatty acids, expression of L-FABP had relatively little effect on levels of total lipid esterification of phytanic acid as compared to that of straight-chain fatty acids. However, it is of interest to note that within the esterified lipids both phytanic acid and palmitic acid were highly localized to phosphatidic acid and diacylglycerides where specific activities were very high. This was to be expected since phosphatidic acid is the precursor of phospholipids as well as neutral glycerides (e.g. mono-, di-, and tri-acylglycerides) synthesized de novo. While L-FABP is known to enhance microsomal incorporation of straight-chain fatty acyl CoAs into phosphatidic acid in vitro [39, 48], the fact that L-FABP did not increase the specific activity of phytanic acid in these lipids indicated that L-FABP did not stimulate branched-chain fatty acid accumulation in highly dynamic esterified pools. This was consistent with the much lower activity of acyl CoA synthetases with phytanic acid as compared to palmitic acid [47]. In contrast, L-FABP expression greatly enhanced (4.5-fold) palmitic acid esterification, consistent with earlier in vitro experiments [39, 48, 50] where L-FABP was shown to enhance microsomal incorporation of straight chain fatty acyl CoAs into phosphatidic acid, the precursor of both triglycerides and phospholipids [38, 39, 48]. Likewise, L-FABP was also shown to stimulate microsomal incorporation of straight chain fatty acyl CoAs into cholesteryl esters in vitro [49, 50]. The observation that the L-FABP expression stimulated the oxidation of phytanic acid while not greatly affecting its esterification may help explain recent observations of phytanic acid toxicity in SCP-x/SCP-2 gene ablated mice where phytanic acid was present at higher levels and the expression of L-FABP was upregulated 4-fold [27]. Since the promoter region of the L-FABP gene contains a PPARα response element and phytanic acid acts as a potent PPARα agonist ligand that dramatically upregulates transcription of the L-FABP gene in mice [1, 23, 27], the present data suggest that L-FABP expression may be upregulated in the SCP-x/SCP-2 gene ablated mice as a compensatory effect to enhance phytanic acid oxidation. This would be especially important since these mice lack SCP-x, a peroxisomal thiolase specifically involved in oxidation of branched chain fatty acids (i.e. phytanic acid). Taken together, these data suggest that even though L-FABP was upregulated in the SCP-x/SCP-2 null mice, the lack of the SCP-x peroxisomal thiolase resulted in decreased phytanic acid oxidation which in turn elicited phytanic acid accumulation and cytotoxicity [27]. In summary, the data presented herein for the first time directly demonstrated that expression of L-FABP enhanced the oxidation, uptake, and intracellular esterification of branchedchain (phytanic acid) under conditions where cells were sup-

plemented with low levels of phytanic acid. This finding was in contrast to earlier observations where feeding high levels of phytanic acid to L-FABP expressing cells apparently saturates the oxidation pathway and inhibits phytanic acid oxidation [26]. These data were consistent with a potential role for L-FABP not only in enhancing the oxidation of straightchain fatty acids but also that of branched-chain fatty acids. Furthermore, taken together with data in the literature, the results presented herein suggested that when peroxisomal oxidative mechanism(s) are compromised, concomitant upregulation of L-FABP may actually enhance branched-chain fatty acid toxicity due its stimulation of uptake and targeting of branched-chain fatty acid to peroxisomal oxidation rather than esterification. The data presented herein for the first time directly examined the effects of L-FABP expression on oxidation as well as uptake and esterification of saturated branched chain fatty acids. Finally, L-FABP expression enhanced the uptake of the nonmetabolizable fluorescent fatty acid BODIPY-C16 1.7-fold. This increase was similar to that observed with the metabolizable fatty acid palmitic acid but less than with phytanic acid, a result indicating that the use of nonmetabolizable fatty acids provides a useful tool for discriminating L-FABP effects on saturated fatty acid uptake vs. intracellular metabolism in living cells.

Acknowledgements This work was supported in part by the USPHS, National Institutes of Health grant DK41402.

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