Effect of branched-chain fatty acid on lipid dynamics in mice lacking liver fatty acid binding protein gene

Barbara P. Atshaves, Avery L. McIntosh, H. Ross Payne, John Mackie, Ann B. Kier, Friedhelm Schroeder

Abstract

Although a role for liver fatty acid protein (L-FABP) in the metabolism of branched-chain fatty acids has been suggested based on data obtained with cultured cells, the physiological significance of this observation remains to be demonstrated. To address this issue, the lipid phenotype and metabolism of phytanic acid, a branched-chain fatty acid, were determined in L-FABP gene-ablated mice fed a diet with and without 1% phytol (a metabolic precursor to phytanic acid). In response to dietary phytol, L-FABP gene ablation exhibited a gender-dependent lipid phenotype. Livers of phytol-fed female L-FABP−/− mice had significantly more fatty lipid droplets than male L-FABP−/− mice, whereas in phytol-fed wild-type L-FABP+/+ mice differences between males and females were not significant. Thus L-FABP gene ablation exacerbated the accumulation of lipid droplets in phytol-fed female, but not male, mice. These results were reflected in the lipid profile, where hepatic levels of triacylglycerides in phytol-fed female L-FABP−/− mice were significantly higher than in male L-FABP−/− mice. Furthermore, livers of phytol-fed female L-FABP−/− mice exhibited more necrosis than their male counterparts, consistent with the accumulation of higher levels of phytol metabolites (phytanic acid, pristanic acid) in liver and serum, in addition to increased hepatic levels of sterol carrier protein (SCP)-x, the only known peroxisomal enzyme specifically required for branched-chain fatty acid oxidation. In summary, L-FABP gene ablation exerted a significant role, especially in female mice, in branched-chain fatty acid metabolism. These effects were only partially compensated by concomitant upregulation of SCP-x in response to L-FABP gene ablation and dietary phytol.

  • gene targeting
  • phytanic acid

although the liver fatty acid binding protein (L-FABP) is the first known and perhaps most thoroughly studied member of the large family of intracellular transport proteins involved in long-chain fatty acid (LCFA) metabolism, its full physiological function is only now beginning to be resolved (reviewed in Ref. 40). Early studies focused on the structure of L-FABP, resolution of its ligand binding site, and determination of ligand specificity (reviewed in Refs. 15, 17, 40, 48, 64, 72). Although L-FABP has high affinity for both straight (reviewed in Refs. 15, 17, 40)- and branched (16, 75)-chain LCFAs, as well as their CoA derivatives, most previous investigations to elucidate L-FABP's physiological function focused on its roles in the uptake and metabolism of straight-chain LCFAs.

Cultured cell and in vitro studies suggest that L-FABP enhances multiple aspects of straight-chain LCFA metabolism, including 1) cellular LCFA uptake (9, 37, 40, 44, 45, 5153, 74), 2) cytoplasmic LCFA and LCFA-CoA binding capacity and pool size (37, 38), 3) intracellular LCFA transport and diffusion (35, 40, 44, 69, 70) and LCFA-CoA transport (54, 55), 4) LCFA targeting to microsomal LCFA-CoA synthase and LCFA-CoA pool size regulation (10, 20, 41, 47), 5) LCFA-CoA targeting for microsomal transacylation to phosphatidic acid (7, 27, 29, 30, 59, 61), and 6) LCFA-CoA targeting to mitochondria for LCFA oxidation (1, 4, 46), but only under conditions of fasting or starvation, where LCFA levels are very high, i.e., the millimolar range (46). Recently, it was also shown that L-FABP enhances LCFA and LCFA-CoA targeting to nuclei of living cells (24, 25). In addition, it was shown that L-FABP directly interacts with peroxisome proliferator-activated receptor (PPAR)α in the nucleus (24, 73) and that L-FABP overexpression enhances the transcriptional activity of PPARα (73). Furthermore, because LCFA-CoAs (less so LCFAs) also interact with hepatocyte nuclear factor (HNF)-4α (23, 49, 50), L-FABP may influence transcriptional regulation of genes involved in both LCFA and glucose metabolism.

In contrast, relatively little is known regarding the physiological role of L-FABP in the metabolism of branched-chain LCFAs, which occurs primarily in peroxisomes (13). Branched-chain LCFAs such as phytanic acid are present at significant levels in the human diet, especially in dairy products (butter, margarine, cheese), where phytanic acid can represent up to 0.5 g/100 g wet weight (62). Although healthy humans can readily metabolize dietary phytanic acid (typically <0.1 g/day; Ref. 66), this is not the case for those with peroxisomal disorders (62). Genetic defects in peroxisomal branched-chain LCFA oxidation result in accumulation of excess branched-chain LCFAs and/or metabolites, which at sufficient levels become toxic (62, 66). Although L-FABP is not a peroxisomal protein, recent studies suggest that L-FABP enhances the peroxisomal oxidation of low levels of branched-chain LCFAs such as phytanic acid in transfected cells overexpressing L-FABP (4), whereas phytanic acid oxidation is inhibited in primary hepatocytes cultured from L-FABP gene-ablated mice (1). However, at high levels of phytanic acid, the overexpression of L-FABP inhibits phytanic acid oxidation and exacerbates phytanic acid toxicity in transfected cells (6). Consistent with this possibility, fourfold upregulation of liver L-FABP in sterol carrier protein (SCP)-2 and/or SCP-x gene-ablated mice correlates with inhibition of peroxisomal phytanic acid oxidation (60). It is unclear, however, whether the latter results from saturation of the ability of L-FABP to enhance phytanic acid oxidation, from the absence of SCP-x [a peroxisomal 3-ketoacyl CoA thiolase required for oxidation of branched fatty acids (FAs)], and/or from the absence of SCP-2, which binds both phytanic acid (63) and phytanoyl CoA (14) with high affinity (Kd in nanomolar to micromolar range).

Finally, it must be noted that L-FABP not only may exert direct effects on delivering branched-chain LCFAs to peroxisomes for oxidation but also may indirectly influence peroxisomal oxidation of branched-chain FAs. Because L-FABP expression increases the distribution of straight-chain LCFAs and LCFA-CoAs to the nucleus (25) and L-FABP binds phytanic acid with high affinity (15, 16, 75), the protein may act similarly to enhance branched-chain LCFA distribution into nuclei for interaction with regulatory proteins such as PPARα (12, 75) and retinoid X receptor (RXR) (33) that influence transcription of genes involved in peroxisomal branched-chain LCFA oxidation.

To address these issues in a physiological context, the present study examined the effect of L-FABP gene ablation on the metabolism of dietary branched-chain phytol (rapidly converted to phytanic acid) and lipid phenotype. Studies were designed to determine whether L-FABP gene ablation alters the mouse phenotype in response to dietary phytol by determining body phenotype (weight gain, fat and lean tissue mass), liver morphology, liver lipid and protein dynamics, and serum lipids. The data show for the first time that L-FABP gene ablation significantly alters the lipid phenotype induced by phytol in a gender-dependent fashion. This was despite concomitant upregulation of SCP-x induced by dietary phytol, especially in female mice. Together these findings were consistent with both L-FABP and SCP-x participating in branched-chain FA metabolism.

MATERIALS AND METHODS

Materials.

Silica Gel G and Silica Gel 60 thin-layer chromatography plates were from Analtech (Newark, DE) and EM Industries (Darmstadt, Germany), respectively. Neutral lipid and FA standards were purchased from Nu-Chek Prep (Elysian, MN), and phospholipid standards were obtained from Avanti (Alabaster, AL). Phytanic acid and phytol were purchased from Sigma (St. Louis, MO). Rabbit polyclonal antisera to recombinant human SCP-2, mouse SCP-x, porcine glutamic-oxaloacetic transaminase (GOT), rat acyl-CoA binding protein (ACBP), and rat L-FABP were prepared as described previously (3). Sheep anti-bovine catalase was purchased from Biodesign (Kennebunk, ME). Rabbit polyclonal anti-caveolin-1 was purchased from BD Transduction Laboratories (Lexington, KY). Rabbit polyclonal anti-fatty acid transport protein (FATP) was a generous gift from Dr. J. Schaffer (Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO). All reagents and solvents used were of the highest grade available and were cell culture tested.

Animal studies.

L-FABP-null (L-FABP−/−) mice were generated by targeted disruption of the L-FABP gene through homologous recombination (37, 38). Wild-type littermates with no disruption (L-FABP+/+) were designated as controls. The genotype of each animal was verified by a PCR screen indicating the presence of the L-FABP or replacement cassette (37, 38). Animal protocols were approved by the Animal Care and Use Committee of Texas A&M University. Mice in the facility were monitored quarterly for infectious diseases and were specific pathogen free, particularly in reference to mouse hepatitis virus. Mice were kept under constant light-dark cycles and had access to food and water ad libitum.

Experiments were performed with male and female mice 2 mo of age and weighing 25–35 g. One week before the start of experiments, mice were switched to a modified AIN-76A phytol-free rodent diet (5% calories from fat; no. D11243, Research Diets, New Brunswick, NJ). After 1 wk, one-half of the mice remained on the phytol-free diet, and the rest were switched to a modified AIN-76A rodent diet supplemented with 1.0% phytol (5% calories from fat; diet no. D01020601, Research Diets). Mouse body weights and food intake were monitored every other day. At the end of the study (day 11), animals were fasted overnight and anesthetized (ketamine 100 mg/kg, zylaxine 10 mg/kg), and blood was collected by cardiac puncture, followed by cervical dislocation. Fat tissue mass (FTM) and lean tissue mass (LTM) were determined by dual-energy X-ray absorptiometry (DEXA) with a Lunar PIXImus densitometer (Lunar, Madison, WI). Livers were examined grossly and weighed. Liver slices were excised near the porta hepatis for light microscopy and histological examination. Remaining portions of liver were snap-frozen on dry ice and stored at −80°C for lipid and Western blot analyses.

Histopathology.

Liver slices were fixed in 10% neutral-buffered formalin for 24 h and stored in 70% alcohol until processing and embedding in paraffin. Liver sections (5 μm thick) were stained with hematoxylin and eosin for histological examination under a light microscope. Severity of lesions was graded as 0, normal; 1, minimal change; 2, mild change; 3, moderate change; and 4, severe change.

Morphometric measurements of lipid volume.

Small samples of liver (1- to 2-mm3 segments) were fixed by immersion in glutaraldehyde and formaldehyde at room temperature. Fat droplets in the liver tissues were stained by a histological procedure described previously (19), with slight modifications. Briefly, liver tissue was incubated for 8 h in 1% osmium tetroxide-2.5% potassium dichromate, dehydrated in a graded ethanol series, and embedded in Spurr's epoxy resin. Semithin sections, 0.75 μm thick, were mounted on glass slides, coverslipped, and examined without counterstaining. These liver sections with darkly stained lipid droplets were imaged with a ×40 light microscope objective and recorded with a charge-coupled device (CCD) camera. Four representative images were randomly selected from each liver, for a total image area of 0.6 mm2 per treatment group per mouse. The relative volume density of lipid droplets in the image field was analyzed with the program ImageJ [developed at the National Institutes of Health (NIH) and available on the Internet at http://rsb.info.nih.gov/ij/].

Western blotting.

Protein expression levels in liver samples were determined by Western blot analysis as described previously (2). Proteins were quantified by densitometric analysis after image acquisition with a single-chip CCD video camera and a computer workstation (IS-500 system; Alpha Innotech, San Leandro, CA). Image files were analyzed (mean 8-bit gray scale density) with NIH Image (available by anonymous FTP).

Lipid analyses.

Lipids were extracted from liver homogenates and serum with n-hexane-2-propanol 3:2 (vol/vol) and divided into portions for mass and FA composition determination as described previously (1). One-half of each extracted lipid sample was resolved into individual lipid classes with Silica Gel G thin-layer chromatography plates developed in petroleum ether-diethyl ether-methanol-acetic acid 90:7:2:0.5 (1, 21, 26). Total cholesterol (Chol), free FA (FFA), triacylglyceride (TG), cholesteryl ester (CE), and phospholipid (PL) content were determined by the method of Marzo et al. (39). Protein concentration was determined by the method of Bradford (8) from the dried protein extract residue digested overnight in 0.2 M KOH.

Transesterification.

Acid-catalyzed transesterification was performed on the remaining half of the extracted lipid fraction from each sample to convert the lipid acyl chains to fatty acid methyl esters (FAMEs). FAMEs were extracted into n-hexane and separated by gas-liquid chromatography (GLC) on a GLC-14A (Shimadzu, Kyoto, Japan) equipped with a RTX-2330 capillary column (0.32-mm internal diameter × 30-m length; Restek, Bellefonte, PA). The injector and detector temperatures were set at 260°C with the temperature program of 100°C for 1 min, 10°C/min to 140°C, 2°C/min to 220°C, hold 1 min, and then ramp 20°C/min to 260°C. A Waters SAT/IN analytical-to-digital interface was used to collect peak area data that were converted to peak area with Millenium32 3.2 software. With the exception of phytol metabolites, individual peaks were identified by comparison to known FAME standards (Nu-Chek Prep) and referenced against a set concentration of 15:0 added before analysis. Phytol metabolites (phytanic acid and pristanic acid) were confirmed by gas chromatography (GC)-mass spectroscopy on a Thermo-Finnegan Trace DSQ single-quadrapole Mass Spect with trace GC in chemical ionization (CI) mode.

Serum analyses.

Collected blood samples were allowed to sit overnight at 4°C, followed by centrifugation at 14,000 rpm for 20 min at 4°C, and the serum fraction was removed. Serum was analyzed for lipids as described above for liver lipids.

Statistics.

Each feeding group consisted of five to seven animals. All values are expressed as means ± SE. Statistical analysis was performed by ANOVA combined with the Newman-Keuls multiple-comparisons test (GraphPad Prism, San Diego, CA). Values with P < 0.05 were considered statistically significant.

RESULTS

Effect of L-FABP gene ablation on response of body weight to dietary phytol.

The effects of L-FABP gene ablation in response to a 1% phytol diet were determined in 2-mo-old male and female mice. Food consumption and body weight were measured every 2 days. The dietary study was terminated after 11 days because thereafter the phytol-rich diet elicited severe weight loss, especially in female mice, despite similar food consumption. When comparing wild-type vs. knockout mice on the control diet, there was no significant difference in body weights at the end of the 11-day dietary study (Fig. 1A). Likewise, L-FABP gene ablation did not significantly alter the percent weight gain, but L-FABP+/+ female mice lost more weight than their male counterparts (Fig. 1B). In contrast, a substantial (15–20%) weight loss was observed in both male and female phytol-fed wild-type L-FABP+/+ and L-FABP−/− mice (Fig. 1). Whereas the wild-type L-FABP+/+ female, but not male, mice on the 1% phytol diet exhibited significantly (P < 0.001) lower body weight, both male (P < 0.02) and female (P < 0.05) L-FABP−/− mice exhibited lower final body weight on the 1% phytol diet (Fig. 1A). However, although the body weight of female L-FABP+/+ and −/− mice on the 1% phytol diet was significantly lower than that of the male mice on the same diet, there was no difference in body weight between female L-FABP+/+ and −/− mice. In summary, the 1% phytol diet elicited significant weight loss in both male and female mice, an effect observed to equal extent in L-FABP +/+ and L-FABP−/− mice. Differences in body weight or weight gain were not due to altered food consumption, because all mouse groups consumed similar quantities of food over the course of the feeding study. L-FABP gene ablation did not exacerbate the phytol-induced weight loss.

Fig. 1.

Body weight and % change in weight. Body weight (A) and % change in body weight (B) were measured in wild type L-FABP+/+ and L-FABP−/− male and female mice on control and 1% phytol diets. Values represent means ± SE (n = 5–7). *Significance (P < 0.05) vs. male wild-type L-FABP+/+ mice on the control diet; **significance (P < 0.001) vs. female wild-type L-FABP+/+ mice on the control diet; @significance (P < 0.02) vs. male L-FABP−/− mice on the control diet; +significance (P < 0.05) vs. female L-FABP−/− mice on the control diet; ^significance (P < 0.001) vs. male wild-type L-FABP+/+ mice on the 1% phytol diet; #significance (P < 0.007) vs. male L-FABP−/− mice on the 1% phytol diet; %significance (P < 0.02) vs. female wild-type L-FABP+/+ mice on the 1% phytol diet.

Effect of L-FABP gene ablation and phytol diet on body phenotype.

To determine the effect of L-FABP gene ablation and 1% phytol diet on body composition, the relative proportions of FTM and LTM were determined at the beginning and end of the dietary study by DEXA. DEXA scans of wild-type L-FABP+/+ and L-FABP−/− mice fed a control diet for 11 days showed that wild-type L-FABP+/+ and L-FABP−/− female (Fig. 2, A vs. C) as well as male (not shown) mice were basically similar. However, after 11 days on the control diet both the male L-FABP+/+ and −/− mice gained weight (Fig. 1B), the relative percent distribution of FTM (Fig. 2E) and LTM (Fig. 2F) was not significantly altered. In contrast, over the same time period the relative proportion of FTM in both female L-FABP+/+ and L-FABP−/− mice was significantly increased (P < 0.006) compared with LTM.

Fig. 2.

Distribution of fat and lean tissue mass. Lunar PIXImus dual-energy X-ray absorptiometry (DEXA) scans were performed on female wild-type L-FABP+/+ (A and B) and L-FABP−/− (C and D) mice fed control (A and C) and 1% phytol (B and D) diets. Percent changes in fat tissue mass (FTM; E) and lean tissue mass (LTM; F) were determined as described in materials and methods. Values represent means ± SE (n = 5–7). *Significance (P < 0.006) vs. mice wild-type L-FABP+/+ male on the control diet; **significance (P < 0.01) vs. female wild-type L-FABP+/+ mice on the control diet; @significance (P < 0.001) vs. male L-FABP−/− mice on the control diet; +significance (P < 0.001) vs. female L-FABP−/− mice on the control diet; ^significance (P < 0.005) vs. male wild-type L-FABP+/+ mice on the 1% phytol diet; #significance (P < 0.001) vs. male L-FABP−/− mice on the 1% phytol diet; %significance (P < 0.05) vs. female wild-type L-FABP+/+ mice on the 1% phytol diet. WT indicates wild-type L-FABP+/+ mice. KO indicates knockout L-FABP−/− mice.

Although similar images were obtained with 1% phytol-fed male L-FABP+/+ vs. L-FABP−/− mice (not shown), DEXA scans of female wild-type L-FABP+/+ (Fig. 2B) and L-FABP−/− (Fig. 2D) mice fed the 1% phytol diet showed that the females were noticeably thinner than their control-fed counterparts (Fig. 2, A and C, respectively). When the relative contributions of FTM and LTM in these mice were determined, it was observed that feeding the male L-FABP+/+ mice a 1% phytol diet elicited a significant (P < 0.006) decrease in both FTM (Fig. 2E) and LTM (Fig. 2F). This effect was even more pronounced in the female mice.

Although L-FABP gene ablation did not alter the loss in FTM in male mice on a 1% phytol diet, female L-FABP−/− mice were less affected by the 1% phytol diet compared with their wild-type L-FABP+/+ counterparts (Fig. 2E). Similarly, the female L-FABP−/− mice exhibited slightly less LTM loss on the 1% phytol diet compared with their wild-type L-FABP+/+ counterparts (P < 0.05, Fig. 2F). Thus increased FTM contributed more to weight gain in female wild-type L-FABP+/+ and −/− mice, whereas increased LTM contributed more to weight gain in male mice.

The 1% phytol diet elicited significantly reduced FTM and LTM in both male and female L-FABP+/+ mice. Although L-FABP gene ablation did not prevent the decrease in FTM and LTM in male mice fed 1% phytol, L-FABP−/− female mice fed 1% phytol exhibited smaller losses in FTM and LTM than their wild-type L-FABP+/+ female counterparts fed the same 1% phytol diet. Thus L-FABP gene ablation did not exacerbate the effects of phytol on reducing FTM and LTM. In fact, L-FABP gene ablation somewhat protected the female, but not male, mice from loss in FTM and LTM induced by 1% phytol feeding. These results demonstrated that L-FABP gene ablation modestly modified the sexually dimorphic response to phytol.

Effect of L-FABP gene ablation and dietary phytol on liver mass and peroxisomal proteins.

Because L-FABP is expressed at the highest level in liver, the effect of dietary phytol and L-FABP gene ablation on liver mass and protein was determined. Although a 1% phytol diet significantly (P < 0.001) increased liver weight of both male and female wild-type L-FABP+/+ mice, L-FABP gene ablation did not further exacerbate or significantly alter the phytol-induced increase in liver weight (Fig. 3A). Similar results were obtained when liver weight was expressed per 100 g of body weight (Fig. 3B). In contrast, when liver weight was expressed as milligrams of protein per gram of liver weight, neither L-FABP gene ablation nor 1% phytol diet significantly altered this parameter (Fig. 3C). Together, these data indicate that after 11 days on a 1% phytol diet, liver mass was significantly increased in both male and female mice. In addition, the increased liver mass and liver mass/100 g body weight and unaltered protein/liver weight induced by the 1% diet suggested increased hepatocyte mass rather than accumulation of water, lipid, or glycogen within the liver. Overall, L-FABP gene ablation did not markedly alter this pattern.

Fig. 3.

Liver weight and protein content in wild type L-FABP+/+ and L-FABP−/− mice fed a defined diet with or without 1% phytol. Liver weight (A), liver/body weight (B), and protein/liver weight (C) were determined in male and female wild-type L-FABP+/+ and L-FABP−/− mice fed a control or phytol diet. Values are means ± SE (n = 5–7). *Significance (P < 0.001) vs. male wild-type L-FABP+/+ mice on the control diet; **significance (P < 0.001) vs. female wild-type L-FABP+/+ mice on the control diet; @significance (P < 0.001) vs. male L-FABP−/− mice on the control diet; +significance (P < 0.001) vs. female L-FABP−/− mice on the control diet; ^significance (P < 0.001) vs. male wild-type L-FABP+/+ mice on the 1% phytol diet.

Because phytanic acid is a PPARα agonist, the expression levels of several proteins regulated by PPARα were examined by Western blotting (Fig. 4). L-FABP gene ablation and the phytol diet significantly altered the expression of several cytoplasmic proteins involved in FA uptake (L-FABP) and FA-fatty acyl CoA transport (L-FABP, ACBP). These results were consistent with the facts that L-FABP is a known PPARα target gene (11) and that, although ACBP is a PPARγ target gene, some data suggest that it may also be a nonclassic PPARα target gene under certain conditions (22). Results from Western blotting indicated that levels of L-FABP in the liver were higher in both male and female phytol-fed wild-type L-FABP+/+ mice (Fig. 4A). This observation was confirmed by quantitative analysis of multiple Western blots, which showed a 1.8-fold increase (P < 0.05) in L-FABP in livers of male and female wild-type L-FABP+/+ mice (Fig. 5A). As expected, L-FABP was absent in the livers of L-FABP−/− mice (Fig. 4A).

Fig. 4.

Western blot analysis of liver proteins in male and female wild-type L-FABP+/+ and L-FABP−/− mice fed a defined diet with or without 1% phytol. Western blots of liver cell homogenates (2 μg) from wild-type L-FABP+/+ and L-FABP−/− mice fed a control (C) or phytol (P) diet were probed with the following affinity-purified antibodies as described in materials and methods: anti-L-FABP (A), anti-sterol carrier protein (SCP)-2 (B), anti-SCP-x (C), anti-catalase (D), anti-fatty acid transport protein (FATP) (E), anti-caveolin-1 (F), anti-glutamic-oxaloacetic transaminase (GOT) (G), and anti-acyl-CoA binding protein (ACBP) (H).

Fig. 5.

Hepatic protein expression levels in male and female wild-type L-FABP+/+ and L-FABP−/− mice fed a defined diet with or without 1% phytol. Expression levels of L-FABP (A), SCP-2 (B), SCP-x (C), ACBP (D), FATP (E), caveolin-1 (F), GOT (G), and catalase (H) were determined as described in materials and methods. Values are means ± SE (n = 5–7). *Significance (P < 0.05) vs. male wild-type L-FABP+/+ mice on the control diet; **significance (P < 0.02) vs. female wild-type L-FABP+/+ mice on the control diet; @significance (P < 0.05) vs. male L-FABP−/− mice on the control diet; +significance (P < 0.02) vs. female L-FABP−/− mice on the control diet; ^significance (P < 0.01) vs. male wild-type L-FABP+/+ mice on the 1% phytol diet; #significance (P < 0.01) vs. male L-FABP−/− mice on the 1% phytol diet; %significance (P < 0.001) vs. female wild-type L-FABP+/+ mice on the 1% phytol diet.

With ACBP, levels in control-fed female wild-type L-FABP+/+ mice were 2.5-fold lower (P < 0.05) than in their male counterparts (Fig. 5D). A 1% phytol diet significantly decreased the level of ACBP by 1.6-fold in male wild-type L-FABP+/+ mice (P < 0.01), with little effect on female wild-type L-FABP+/+ mice. Levels of ACBP were 2.7-fold lower in male L-FABP−/− mice compared with wild-type L-FABP+/+ mice. In contrast, a slight increase in ACBP expression was observed in female L-FABP−/− mice compared with female L-FABP +/+ mice. A 1% phytol diet significantly increased levels of ACBP 3.0-fold in male and 1.5-fold in female L-FABP−/− mice (P < 0.05; Fig. 5D). Overall, these results were consistent with phytol-induced PPARα activation of L-FABP in wild-type mice and ACBP expression in L-FABP gene-ablated animals.

L-FABP gene ablation and phytol feeding significantly altered expression of several peroxisomal proteins. SCP-2 (Fig. 4B) is enriched in peroxisomes, but significant levels are also localized outside the peroxisome (reviewed in Ref. 18). In contrast, SCP-x (Fig. 4C) is exclusively peroxisomal and is the only 3-ketoacyl-CoA thiolase known to function in oxidation of branched-chain FAs such as phytanic acid (reviewed in Ref. 18). SCP-2, SCP-x, and catalase are PPARα target genes upregulated by PPARα ligands (2, 71). Levels of SCP-2 were increased by the 1% phytol diet in both male (P < 0.05) and female (P < 0.02) wild-type L-FABP+/+ mice (Fig. 5B). Although L-FABP gene ablation significantly increased the level of SCP-2 in control-fed mice, L-FABP gene ablation blunted the response to the 1% phytol diet (Fig. 5B). With regard to the SCP-x, the level of this peroxisomal enzyme was nearly 10-fold lower in female than in male wild-type mice fed the control diet (Fig. 5C). L-FABP gene ablation decreased the level of SCP-x slightly in liver from control-fed male mice but significantly increased the level of SCP-x fivefold (P < 0.02) compared with control fed L-FABP+/+ female mice (Fig. 5C). Although basal levels of SCP-x were nearly 10-fold lower in females vs. males on control diets, a 1% phytol diet increased the expression of SCP-x protein in wild-type L-FABP+/+ female mice to levels indistinguishable from control and phytol-fed wild-type L-FABP+/+ and L-FABP−/− male mice. Thus the 1% phytol diet increased the level of SCP-x in female, but not male, wild-type L-FABP mice. L-FABP gene ablation modified this response such that phytol-fed male and female L-FABP−/− mice both exhibited increased levels of SCP-x, 1.7-fold (P < 0.05) and 3.9-fold (P < 0.001), respectively (Fig. 5C). Thus 1% phytol-fed female L-FABP−/− mice exhibited the highest level of SCP-x, 1.6-fold (P < 0.01) higher than that of phytol-fed male L-FABP−/− mice. In both male and female wild-type L-FABP+/+ and L-FABP−/− mice, the phytol-induced increases in L-FABP, SCP-2, and SCP-x levels correlated with similarly increased levels of catalase, a peroxisomal enzyme marker.

Despite the facts that FATP expression is regulated by PPARα activators such as fibrates (36) and that there is evidence that certain aminotransferase genes such as GOT are regulated through PPARα (65), L-FABP gene ablation and phytol diet had relatively little effect on plasma membrane FA transport/translocase proteins (FATP, GOT, and caveolin-1) involved in FA uptake into the cell. Neither the phytol diet nor L-FABP gene ablation significantly altered the levels of FATP (Figs. 4E and 5E), caveolin-1 (Figs. 4F and 5F), or GOT (Fig. 4G and 5G). Together the above data indicate that a 1% phytol diet increased liver mass, especially in female mice. The 1% phytol diet induced expression of PPARα-regulated proteins typically found in peroxisomes (SCP-2, SCP-x, catalase) and proteins responsible for the peroxisomal oxidation (SCP-x) and transport (SCP-2, L-FABP) of branched-chain FAs in both male and female wild-type L-FABP+/+ mice. The finding that 1% phytol did not induce, but rather inhibited, formation of ACBP in L-FABP+/+ mice was not consistent with ACBP being a classic PPARα target gene. Neither 1% phytol nor L-FABP gene ablation markedly altered the levels of any of the plasma membrane FA transport/translocase proteins. Although L-FABP gene ablation alone did not alter liver mass, L-FABP gene ablation selectively modulated the induction of SCP-2 (abolished the response to phytol) and increased the level of SCP-x as well as catalase in phytol-fed male and even more so in female L-FABP−/− mice. Thus although the highest liver mass observed for the 1% phytol-fed female L-FABP−/− mice correlated with the lowest level of cytoplasmic FA transport protein, these samples also exhibited the highest level of SCP-x, the only known 3-ketoacyl-CoA thiolase enzyme involved in branched-chain FA oxidation in peroxisomes.

Effect of L-FABP gene ablation and dietary phytol on liver histopathology.

L-FABP (1, 4), SCP-2 (6), and SCP-x (6) enhance the oxidation of branched-chain FAs in cultured cells. Because the loss of L-FABP leads to concomitant upregulation of SCP-2 in control-fed and upregulation of SCP-x in phytol-fed L-FABP−/− mice, especially females, it was difficult to predict a priori the response of liver morphology and lipids to dietary phytol in the L-FABP−/− mice.

On microscopic examination, livers of control-fed female (Fig. 6B) and male (data not shown) L-FABP−/− mice appeared similar to those of control-fed, wild-type L-FABP+/+ littermates (Fig. 6A), with no significant difference in lesion scores of fatty vacuolation (steatosis) of hepatocytes and no evidence of necrosis or in inflammation (Fig. 6, E and F). In contrast, phytol feeding was associated with necrosis and loss of hepatocytes, inflammation, and an increase in fatty vacuolation of hepatocytes in male and female L-FABP+/+ and −/− mice compared with control-fed mice (P < 0.001, Fig. 6, C–F). Within phytol-fed animals, female L-FABP−/− mice exhibited slightly more necrosis than male L-FABP−/− mice. The distribution of necrosis, inflammation, and fatty vacuolation was predominantly midzonal (Fig. 6, C and D). The inflammation consisted of mainly macrophages, with occasional multinucleated giant cells and mineralization in more severely affected livers. There was often zonal hypertrophy of intact hepatocytes and an increase in mitotic figures. In addition, minimal to mild hyperplasia of oval cells and/or biliary epithelium was seen in some phytol-fed male and female L-FABP+/+ and L-FABP−/− animals.

Fig. 6.

Histopathology of liver tissue from wild-type L-FABP+/+ and L-FABP−/− mice fed a defined diet with or without 1% phytol. A–D: representative histological sections from female mice stained with hematoxylin and eosin. Examples of portal triads (PT) and central veins (CV) are marked. Liver sections from wild-type L-FABP+/+ (A) and L-FABP−/− (B) mice fed a control diet are essentially normal. Liver sections from phytol-fed L-FABP+/+ (C) and L-FABP−/− (D) mice exhibit a similar degree of necrosis and loss of midzonal hepatocytes (arrows), accompanied by inflammation and mild to moderate fatty vacuolation. Note also hypertrophy of hepatocytes around portal triads and central veins (C and D) and mitotic figures in periportal hepatocytes (D). The extent of fatty vacuolation (E) and necrosis (F) was graded as described in materials and methods. Necrosis is present only in phytol-fed mice (C and D). Values are means ± SE (n = 5–7). *Significance (P < 0.001) vs. male wild-type L-FABP+/+ mice on the control diet; **significance (P < 0.001) vs. female wild-type L-FABP+/+ mice on the control diet; @significance (P < 0.001) vs. male L-FABP−/− mice on the control diet; +significance (P < 0.001) vs. female L-FABP−/− mice on the control diet; #significance (P < 0.02) compared with male L-FABP−/− mice on the 1% phytol diet. Scale bars, 75 μm.

In summary, control-fed L-FABP gene-ablated mice appeared histologically normal compared with their control-fed wild-type L-FABP+/+ counterparts. A 1% phytol diet induced the appearance of fatty vacuolation and necrosis in all mice. However, necrosis appeared more prominent in the phytol-fed female mice. These data would suggest that the concomitant upregulation of SCP-x did not fully compensate for the loss of L-FABP in the L-FABP−/− mice.

Effect of L-FABP gene ablation and dietary phytol on liver lipid accumulation.

Although lipid droplets were randomly distributed in both male and female control-fed wild-type L-FABP+/+ mice, visual inspection suggested that a higher number of lipid droplets were in female (Fig. 7E) than male (Fig. 7A) mice. When the proportion of lipid droplet volume density was quantitatively analyzed as described in materials and methods, livers of control-fed female wild-type L-FABP+/+ mice were 3.5-fold greater (P < 0.02) than in the corresponding male mice fed the control diet (Fig. 7I). In wild-type L-FABP+/+ mice fed a 1% phytol diet, lipid droplets were not randomly distributed but rather exhibited a patchy distribution (Fig. 7, B and F). The 1% phytol appeared to increase the appearance of lipid droplets in wild-type L-FABP+/+ male (Fig. 7B) and less so in female mice (Fig. 7F). Quantitative analysis indicated that 1% phytol nearly doubled the lipid droplet volume density (P < 0.02) in male wild-type L-FABP+/+ mice without significantly altering that in female wild-type L-FABP+/+ mice fed 1% phytol (Fig. 7I).

Fig. 7.

Morphometric analysis of lipid droplets in liver sections from wild-type L-FABP+/+ and L-FABP−/− mice fed a defined diet with or without 1% phytol. Representative hepatocytes from male (A–D) and female (E–H) wild-type L-FABP+/+ (A, B, E, F) and L-FABP−/− (C, D, G, H) mice on control (A, C, E, G) and phytol (B, D, F, H) diets were stained with osmium tetroxide and potassium dichromate to quantitate lipid droplets as described in materials and methods. Values are means ± SE (n = 5–7). I: values for lipid droplet percent volume density were calculated on the basis of the entire image field of liver tissue without correction for nonparenchymal cells or section thickness. Values represent the means ± SE within the population of droplets from 4 image fields for each of 2 mice per group. *Significance (P < 0.02) vs. male wild-type L-FABP+/+ mice on the control diet; **significance (P < 0.03) vs. female wild-type L-FABP+/+ mice on the control diet; +significance (P < 0.01) vs. female L-FABP−/− mice on the control diet; #significance (P < 0.008) vs. male L-FABP−/− mice on the 1% phytol diet. Bar, 25 μm.

With the L-FABP−/− mice, the pattern of lipid droplet distribution (randomly distributed in control fed vs. patchy in 1% phytol fed) was also observed (Fig. 7, C and G vs. D and H). However, the quantity of lipid droplets and response to 1% phytol diet were markedly altered by L-FABP gene ablation. In control-fed male L-FABP gene-ablated mice, the number of lipid droplets appeared slightly decreased (Fig. 7C) compared with that in control-fed male wild-type mice (Fig. 7A), an observation that did not achieve statistical significance on quantitative analysis (Fig. 7I). L-FABP gene ablation appeared to qualitatively decrease the appearance of lipid droplets in control-fed female L-FABP−/− mice (Fig. 7G) compared with control-fed wild-type L-FABP+/+ mice (Fig. 7E). Quantitative analysis confirmed this observation by showing nearly 50% (P < 0.03) reduced liver lipid volume density in control-fed female L-FABP−/− vs. L-FABP+/+ mice (Fig. 7I). L-FABP gene ablation did not significantly increase the appearance of lipid droplets in male L-FABP−/− mice fed 1% phytol (Fig. 7D) vs. male wild-type L-FABP+/+ mice (Fig. 7B), an effect confirmed by quantitative analysis of lipid droplet volume density (Fig. 7I). However, L-FABP gene ablation significantly increased the appearance of lipid droplets in 1% phytol-fed female L-FABP−/− mice (Fig. 7H) vs. 1% control-fed female L-FABP+/+ mice (Fig. 7F). Quantitative analysis showed that L-FABP gene ablation increased the lipid droplet volume density in 1% phytol-fed female L-FABP−/− mice compared with 1% phytol-fed female wild-type L-FABP+/+ mice (Fig. 7I). Thus the more lipid-specific staining method (osmium tetroxide and potassium dichromate) indicated that a 1% phytol diet substantially increased lipid droplet volume in a gender- and L-FABP-dependent manner. In wild-type L-FABP+/+ mice, the 1% phytol diet increased lipid droplet volume in males. In contrast, in L-FABP gene-ablated mice the 1% phytol diet increased lipid droplet volume in female L-FABP−/− mice, but much less so (not significantly) in male L-FABP−/− mice. L-FABP−/− female mice on a 1% phytol diet exhibited the highest levels of lipid droplet volume. Thus L-FABP gene ablation exacerbated the accumulation of lipid droplets in phytol-fed female, but not male, mice.

Effect of L-FABP gene ablation and dietary phytol on liver lipid accumulation: lipid class distribution.

The effect of L-FABP gene ablation and dietary phytol on 1) total lipid (TL), 2) the content and relative distribution of lipid found primarily in lipid droplets, i.e., CE and TG, and 3) lipids that are primarily membrane constituents, i.e., Chol and PL, was determined as described in materials and methods. Although the TL content of livers from control-fed female wild-type L-FABP+/+ mice tended to be higher than that of the corresponding males, this difference did not achieve statistical significance. Feeding a 1% phytol diet for 11 days increased the TL level in livers from male wild-type L-FABP+/+ mice by 1.9-fold and less so in female mice by 1.3-fold (P < 0.01; Fig. 8A). L-FABP gene ablation differentially affected the TL content of control-fed mice by decreasing TL content 33% (P < 0.01) in male mice without significantly changing that in female mice (Fig. 8A). The phytol-induced accumulation of TL in wild-type mice was exacerbated by L-FABP gene ablation such that TL content was increased 2.8- and 1.6-fold, respectively, in male and female L-FABP−/− mice on the 1% phytol diet (P < 0.05, Fig. 8A).

Fig. 8.

Lipid content in liver of wild-type L-FABP+/+ and L-FABP−/− mice fed a defined diet with or without 1% phytol. Total lipids were extracted from liver homogenates of male and female wild-type L-FABP+/+ and L-FABP−/− mice, followed by resolution into individual lipid fractions as described in materials and methods. Values are means ± SE (n = 5–7). *Significance (P < 0.02) vs. male wild-type L-FABP+/+ mice on the control diet; **significance (P < 0.01) vs. female wild-type L-FABP+/+ mice on the control diet; @significance (P < 0.05) vs. male L-FABP−/− mice on the control diet; +significance (P < 0.05) vs. female L-FABP−/− mice on the control diet; #significance (P < 0.05) vs. male L-FABP−/− mice on the 1% phytol diet; %significance (P < 0.05) vs. female wild-type L-FABP+/+ mice on the 1% phytol diet. A: total lipid. B: cholesterol. C: triglyceride. D: esterified fatty acid. E: phospholipid. F: C-to-P ratio. G: cholesteryl ester. H: unesterified fatty acid.

With regard to the lipids comprising lipid droplets (i.e., CE and TG), L-FABP gene ablation and phytanic acid differentially affected the levels of these lipids. Control-fed male and female wild-type L-FABP+/+ mice did not differ in the level of liver CE (Fig. 8G). L-FABP gene ablation tended to increase the level of CE in female, but not male, control-fed L-FABP−/− mice, but this increase did not achieve statistical significance (Fig. 8G). The 1% phytol diet significantly increased the level of CE in male, but not female, wild-type L-FABP+/+ mice (Fig. 8G). However, L-FABP gene ablation did not further affect this finding (Fig. 8G). In contrast, the level of TG in control-fed female wild-type L-FABP+/+ mice was 1.8-fold higher than that in the corresponding male wild-type L-FABP+/+ mice (Fig. 8C). L-FABP gene ablation significantly (P < 0.01) reduced the level of TG by 3.6-fold in control-fed male, but not female, mice. The 1% phytol diet increased the level of TG in male, but not female, wild-type L-FABP+/+ mice. L-FABP gene ablation exacerbated these 1% phytol-induced increases such that TG levels increased 7.7- and 1.9-fold in male and female mice, respectively (Fig. 8C). Thus both increased CE and TG contributed to elevation in lipid droplet volume in response to a 1% phytol diet, especially in male mice.

Effects of L-FABP gene ablation and 1% phytol diet on lipids found primarily in membranes (e.g., Chol, PL) were complex. In control-fed wild-type L-FABP+/+ mice, Chol levels did not differ between males and females (Fig. 8B), whereas PL levels were lower in female than male mice (Fig. 8E). Consequently, the Chol-to-PL ratio was significantly higher (P < 0.01) in liver lipids of female vs. male control-fed wild-type L-FABP+/+ mice (Fig. 8F). A 1% phytol diet increased the level of Chol (Fig. 8B) but did not statistically significantly alter PL (Fig. 8F) or Chol-to-PL molar ratio (Fig. 8F) in wild-type L-FABP+/+ mice. L-FABP gene ablation did not significantly alter Chol levels in male or female mice (Fig. 8B) but decreased PL content in males (Fig. 8E) and thereby increased the Chol-to-PL ratio (Fig. 8F) in male but not female control-fed mice. A 1% phytol diet significantly increased Chol content in both male and female L-FABP−/− mice (Fig. 8B). However, because the 1% phytol diet also increased the PL content in L-FABP gene-ablated mice, together these effects contributed to an unaltered Chol-to-PL ratio.

In summary, a 1% phytol diet elicited major increases in TL and in lipids typically found in lipid droplets (CE, TG, PL) in a gender-dependent fashion. The phytol-induced increase in lipid droplet volume (Fig. 7I) correlated with increased levels of CE, TG, and Chol in male wild-type L-FABP+/+ mice. L-FABP gene ablation altered this gender-dependent pattern of phytol-induced lipid accumulation, such that not only did 1% phytol-fed male L-FABP−/− mice accumulate elevated lipid classes typically enriched in lipid droplets but also phytol-fed female L-FABP−/− mice accumulated significantly higher levels of lipid droplet-enriched lipids (TG) as well as Chol and PL. Together, these lipid analysis data confirmed the overall pattern observed by morphometric analysis of lipid droplet volume density.

Effect of L-FABP gene ablation and dietary phytol on liver FAs: total unesterified vs. total esterified FA.

To determine whether L-FABP gene ablation and/or phytol altered the proportion of esterified vs. unesterified FA, total esterified and total unesterified FA content were determined as described in materials and methods. In mice fed the control diet, levels of esterified FA were greater in female wild-type L-FABP+/+ mice than male wild-type L-FABP+/+ mice (P < 0.02; Fig. 8D). L-FABP gene ablation decreased liver total esterified FA content in male mice 1.9-fold (P < 0.02), whereas levels in female mice were unaltered. The 1% phytol diet increased the level of esterified lipid by 1.7-fold (P < 0.02) in male wild-type L-FABP+/+ mice, with little effect on female wild-type L-FABP+/+ mice. L-FABP gene ablation exacerbated this effect of the phytol diet and increased total esterified FA 4-fold (P < 0.002) in livers of male L-FABP−/− mice and 1.8-fold in livers of female L-FABP−/− mice.

There was little difference in the level of unesterified FAs in male vs. female control-fed mice (Fig. 8H). L-FABP gene ablation decreased the level of unesterified FAs in livers of male, but not female, mice modestly by 29%. A 1% phytol diet increased liver unesterified FAs 2.5- and 2.2-fold in male and female wild-type L-FABP+/+ mice, respectively. L-FABP gene ablation did not further exacerbate or alter this effect of 1% phytol diet on increasing liver unesterified FAs. In summary, the male mice appeared to tolerate the 1% phytol diet better than female mice, as evidenced by less accumulation of esterified lipid in liver, regardless of the presence or absence of L-FABP.

Accumulation of select FAs in liver lipids of L-FABP gene-ablated mice.

The fatty acyl chain distribution of liver lipids and accumulation of phytol metabolites were determined in livers from wild-type male L-FABP+/+ and L-FABP−/− mice (Table 1). The most prevalent FAs in lipids of control-fed male wild-type L-FABP+/+ mice were 16:0 > 18:1–9 and 18:2–9,12 > 20:4 > 18:0 > 16:1 and 22:6, followed by trace amounts of other FAs, especially polyunsaturated FAs. The overall ratio of saturated to unsaturated FAs in control-fed wild-type male L-FABP+/+ mice was 0.7 ± 0.1 (Table 1). The pattern in control-fed female wild-type L-FABP+/+ mice was basically similar except that levels of 18:1–9 were greater than those of 16:0 and 18:2–9,12. There was very little difference between control-fed male and female wild-type L-FABP +/+ mice in terms of the quantity of unsaturated (Fig. 9A), saturated (Fig. 9B), polyunsaturated (Fig. 9C), or monounsaturated (Fig. 9D) FAs in the liver lipids. As a result, the overall ratio of saturated to unsaturated FAs in control-fed wild-type female L-FABP+/+ mice was 0.5 ± 0.1, only slightly lower than in their male counterparts (Table 1).

Fig. 9.

Liver and serum levels of fatty acids in wild-type L-FABP+/+ and L-FABP−/− mice fed a defined diet with or without 1% phytol. Unsaturated (A and E), saturated (B and F), polyunsaturated (PUFA; C and G), and monounsaturated (MUFA; D and H) fatty acids in liver (A–D) and serum (E–H) were quantitated as described in materials and methods. Values are means ± SE (n = 5–7). *Significance (P < 0.04) vs. male wild-type L-FABP+/+ mice on the control diet; **significance (P < 0.01) vs. female wild-type L-FABP+/+ mice on the control diet; @significance (P < 0.001) vs. male L-FABP−/− mice on the control diet; +significance (P < 0.006) vs. female L-FABP−/− mice on the control diet; ^significance (P < 0.05) vs. male wild-type L-FABP+/+ mice on the 1% phytol diet; #significance (P < 0.03) vs. L-FABP−/− male mice on the 1% phytol diet.

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Table 1.

Liver lipid FA mass and acyl chain distribution in wild-type and L-FABP knockout mice on control and 1% phytol diets

L-FABP gene ablation significantly altered the quantities of the individual FAs in control-fed male mice, especially 18:0, 18:1–9, and 18:2–9,12 (Table 1). These and other smaller differences resulted in L-FABP gene ablation significantly (P < 0.04) decreasing the quantities of unsaturated (Fig. 9A), saturated (Fig. 9B), polyunsaturated (Fig. 9C), and monounsaturated (Fig. 9D) FAs in the lipids of control-fed male mice. However, when these data were expressed together as the overall ratio of saturated to unsaturated FAs, this ratio in control-fed male L-FABP−/− mice was 1.2 ± 0.2, significantly higher than in their male wild-type L-FABP+/+ counterparts (P < 0.05, Table 1).

In control-fed female L-FABP−/− mice there was much less dramatic change in any one FA except 18:2–9,12 (Table 1). There was no significant difference between control-fed female wild-type L-FABP+/+ and L-FABP−/− mice in terms of the quantity of unsaturated (Fig. 9A), saturated (Fig. 9B), polyunsaturated (Fig. 9C), and monounsaturated (Fig. 9D) FAs in the liver lipids. As a result, the overall ratio of saturated to unsaturated FAs in control-fed female L-FABP−/− mice was 0.50 ± 0.04, not significantly different from their female wild-type counterparts (0.5 ± 0.1; Table 1).

The 1% phytol diet significantly altered the pattern of FA distribution in liver lipids of male wild-type L-FABP+/+ mice, increasing the level of 18:1–9, 18:2–9,12, 22:6, and other FAs present in trace amounts (Table 1). As a result of these and other smaller changes in FA composition, the quantity of unsaturated (Fig. 9A), saturated (Fig. 9B), polyunsaturated (Fig. 9C), and monounsaturated (Fig. 9D) FAs was markedly increased in male wild-type L-FABP+/+ liver lipids. Overall, these changes increased the overall ratio of saturated to unsaturated FAs in phytol-fed male L-FABP+/+ mice to 0.8 ± 0.1, slightly higher than their male control-fed counterparts (Table 1).

In 1% phytol-fed female L-FABP+/+ mice there was an even greater change in FA composition, especially in 18:1–9, 18:2–9,12, 16:0, 16:1, 22:4, and 22:6 (Table 1), such that the quantity of unsaturated (Fig. 9A), saturated (Fig. 9B), polyunsaturated (Fig. 9C), and monounsaturated (Fig. 9D) FAs all increased in the liver lipids. As a result, the overall ratio of saturated to unsaturated FAs in phytol-fed female L-FABP+/+ mice was increased to 0.9 ± 0.1, 1.8-fold higher than in their female control-fed counterparts (P < 0.02; Table 1). The 1% phytol diet affected female L-FABP−/− mice similarly so that the overall ratio of saturated to unsaturated FAs in phytol-fed female L-FABP−/− mice was increased to 0.9 ± 0.1, 1.8-fold higher than in their female control-fed counterparts (P < 0.02; Table 1).

Accumulation of phytol metabolites in liver lipids of L-FABP gene-ablated mice.

The effect of L-FABP gene ablation and 1% phytol diet on the appearance of phytol metabolites (phytanic acid, pristanic acid) in liver lipids was examined as described in materials and methods. In control-fed male and female wild-type L-FABP+/+ mice, phytanic acid and pristanic acid are normally present at very low levels and represent <0.2% of total FAs therein (Table 1). The 1% phytol diet increased the level of phytanic acid in livers of wild-type male and female L-FABP+/+ mice by 110-fold and 240-fold, respectively (P < 0.001; Table 1). Concomitantly, the 1% phytol diet also markedly increased the liver level of pristanic acid.

When comparing female vs. male wild-type L-FABP+/+ mice fed a 1% phytol diet, levels of phytanic acid and pristanic acid were 2.2-fold (P < 0.001) and 2.1-fold (P < 0.001) higher, respectively, in female mice. L-FABP gene ablation further exacerbated the difference between male and female mice in response to 1% phytol diets, such that the content of phytanic acid + pristanic acid was 2.3-fold higher than in the corresponding male L-FABP−/− mice.

Effect of L-FABP gene ablation and dietary phytol on serum lipids.

To determine whether the effects of L-FABP gene ablation and 1% phytol observed in liver were also present in serum lipids, serum was extracted and analyzed as described in materials and methods. The serum TL content from control-fed female wild-type L-FABP+/+ mice did not differ from that of their male counterparts (Fig. 10A). However, the male and female serum lipids from control-fed wild-type L-FABP+/+ mice differed significantly in terms of specific lipid classes: CE (Fig. 10G) and TG (Fig. 10C) were lower in female mice, whereas Chol (Fig. 10B) and PL (Fig. 10E) were not significantly different. The overall effect was that serum total esterified FAs (Fig. 10D) and unesterified FAs (Fig. 10H) were not significantly different in male vs. female control-fed wild-type L-FABP+/+ mice.

Fig. 10.

Lipid content in serum of wild-type L-FABP+/+ and L-FABP−/− mice fed a defined diet with or without 1% phytol. Serum lipids were extracted from liver homogenates of male and female wild-type L-FABP+/+ and L-FABP−/− mice, followed by resolution into individual lipid fractions as described in materials and methods. Values are means ± SE (n = 5–7). A: total lipid. B: cholesterol. C: triglyceride. D: esterified fatty acid. E: phospholipid. F: C-to-P ratio. G: cholesteryl ester. H: unesterified fatty acid. *Significance (P < 0.03) vs. male wild-type L-FABP+/+ mice on the control diet; **significance (P < 0.04) vs. female wild-type L-FABP+/+ mice on the control diet; @significance (P < 0.01) vs. male L-FABP−/− mice on the control diet; +significance (P < 0.02) vs. female L-FABP−/− mice on the control diet; ^significance (P < 0.04) vs. male wild-type L-FABP+/+ mice on the 1% phytol diet; #significance (P < 0.04) compared with male L-FABP−/− mice on the 1% phytol diet.

In response to the 1% phytol diet, serum TL of wild-type male L-FABP+/+ mice was decreased by 32% (P < 0.03) compared with control-fed wild-type L-FABP+/+ mice, whereas serum total TL was unaltered in female mice (Fig. 10A). These decreases in the male mice were primarily due to 60% lowered CE (Fig. 10G) and a trend to lowered TG (Fig. 10C). As a result, both serum total esterified FAs (Fig. 10D) and unesterified FAs (Fig. 10H) trended to lower levels in male with little difference observed in female phytol-fed wild-type mice.

L-FABP gene ablation decreased serum TL 25% (P < 0.03) in control-fed L-FABP−/− male mice and tended to decrease (albeit not significantly) serum unesterified FA levels (Fig. 10H) while increasing those in control-fed female L-FABP−/− mice 50% (Fig. 10A) and 42% (Fig. 10H), respectively. Because of selective alterations in specific lipid classes (Fig. 10), levels of total serum esterified FAs in male phytol-fed L-FABP−/− mice were similar in control-fed male L-FABP−/− mice and phytol-fed male L-FABP+/+ mice (Fig. 10D).

With female control-fed L-FABP−/− mice, the loss of L-FABP significantly increased serum TL 1.5-fold (Fig. 10A), primarily because of a 2.5-fold increase in CE (Fig. 10G), a 2.7-fold increase in TG (Fig. 10C), and a 1.4-fold increase in unesterified FAs (Fig. 10H). In the phytol-fed female L-FABP−/− mice, the serum total esterified FAs (Fig. 10D) and serum unesterified FAs (Fig. 10H) were lowered 12% and 37%, respectively. In summary, in control-fed female mice L-FABP gene ablation elicited a serum hyperlipidemic effect (higher TL, CE, TG, unesterified and esterified FAs), whereas in control-fed male mice L-FABP gene ablation elicited a hypolipidemic effect (lowered TL, CE, and TG). In wild-type L-FABP+/+ mice, a 1% phytol diet lowered TL, TG, and CE in male but not female mice. In contrast, in L-FABP gene-ablated mice, the 1% phytol diet raised serum lipids in male mice but lowered those in female mice. Thus L-FABP gene ablation significantly and differentially modified the response of serum lipids in male and female mice fed 1% phytol.

Accumulation of select FAs in serum lipids of L-FABP gene-ablated mice.

Although the qualitative pattern of different types of FA in serum lipids (Table 2) was similar to that in liver (Table 1), there were marked quantitative differences. In contrast to the liver FAs (Table 1), the most common FAs in serum lipids of control-fed male wild-type L-FABP+/+ mice were 16:0, 18:2–9,12, 20:4 > 18:1–9 > 22:6, followed by trace amounts of other FAs, especially polyunsaturated FAs (Table 2). The ratio of saturated to unsaturated FAs in serum of control-fed wild-type male L-FABP+/+ mice was 0.5 ± 0.1 (Table 2), lower than that in liver of control-fed wild-type male L-FABP+/+ mice (0.7 ± 0.1; Table 1). The FA pattern in control-fed female wild-type L-FABP+/+ mice was basically similar except that the levels of 16:0, 18:2–9,12, and 20:4 were lower than in males (Table 2). The serum of control-fed female wild-type L-FABP+/+ mice contained significantly lower levels of unsaturated (Fig. 9E), polyunsaturated (Fig. 9G), and monounsaturated (Fig. 9H) FAs than those in male mice. As a result, the overall ratio of serum saturated to unsaturated FAs in control-fed female wild-type L-FABP+/+ mice was 0.6 ± 0.1, somewhat higher than in their male counterparts (Table 2). L-FABP gene ablation significantly altered the quantities of the individual FAs in control-fed male mice, especially 18:2–9 (Table 2). However, these and other smaller differences (Table 2) did not result in L-FABP gene ablation altering the overall proportions of unsaturated (Fig. 9E), saturated (Fig. 9F), polyunsaturated (Fig. 9G), and monounsaturated (Fig. 9H) FAs in the serum lipids of control-fed male mice.

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Table 2.

Serum lipid FA mass and acyl chain distribution in wild-type and L-FABP knockout mice on control and 1% phytol diets

In response to the 1% phytol diet the male wild-type L-FABP mice exhibited a significantly decreased level of unsaturated FAs (18:1–9, 18:2–9,12, and 20:4) (Table 2). This was reflected in significantly lower unsaturated (Fig. 9E), polyunsaturated (Fig. 9G), and monounsaturated (Fig. 9H) FAs. As a result of these and other changes in serum lipid FA composition, the 1% phytol diet increased the ratio of saturated to unsaturated FAs 2.2-fold in serum lipid FAs from male wild-type L-FABP+/+ mice (P < 0.001; Table 2). L-FABP gene-ablated male mice fed 1% phytol exhibited a similar pattern of changes.

With regard to female wild-type L-FABP+/+ mice, the 1% phytol diet exacerbated many of these alterations in serum lipid FA composition except that the relative proportion of saturated FAs was increased (Fig. 9F) and the ratio of saturated to unsaturated FAs was increased to even higher levels, 1.2 ± 0.2 (Table 2). Female L-FABP gene-ablated mice fed a 1% phytol diet had a pattern of FA alterations similar to that of the female wild-type mice fed 1% phytol (Fig. 10, E–H; Table 2) and exhibited a saturated-to-unsaturated FA ratio of 1.2 ± 0.1 (Table 2). Thus the 1% phytol diet increased the ratio of saturated to unsaturated FA in serum lipid total FAs in both male and female L-FABP+/+ mice. In addition, L-FABP gene ablation did not increase the ratio of serum saturated to unsaturated total FAs in control- or phytol-fed male or female mice.

Accumulation of phytol metabolites in serum lipids of L-FABP gene-ablated mice.

As with liver, serum levels of phytol metabolites (phytanic acid, pristanic acid) were very low in both male and female control-fed wild-type L-FABP mice (0.3% and 0.4%, respectively; Table 2). In male wild-type L-FABP+/+ mice fed phytol, phytanic acid and pristanic acid accounted for ∼19% and 2% of total serum FAs (Table 2), basically similar to the percent distribution of these phytol metabolites in liver of 1% phytol-fed male mice, i.e., 16% and 3%, respectively (Table 1). Although L-FABP gene ablation did not further significantly increase the mass of phytanic acid metabolites in 1% phytol-fed male L-FABP−/− mice, the percentages of phytanic acid and pristanic acid were increased to 23% and 3% of total serum FAs (Table 2). In female wild-type L-FABP+/+ mice, the 1% phytol diet increased the mass of phytol metabolites even more than in their male counterparts (Table 2). As a result, phytanic and pristanic acids represented 30% and 3% of serum total FAs, 1.6-fold more than that in the male wild-type L-FABP+/+ mice fed 1% phytol (Table 2). L-FABP gene ablation did not further increase the mass or percent composition of phytol metabolites in the female L-FABP−/− mice fed 1% phytol (Table 2).

In summary, levels of phytol metabolites were gender dependent, i.e., higher in female than male mice. L-FABP gene ablation increased the proportion of phytol metabolites slightly in male, but not female, mice fed 1% phytol. These patterns of phytol metabolites qualitatively reflected those observed in liver, except for the observation that L-FABP gene ablation significantly increased the mass and percent distribution of phytol metabolites in liver from female L-FABP−/− mice fed 1% phytol.

DISCUSSION

Although most studies of branched-chain LCFA metabolism have focused on peroxisomal enzymes necessary for the oxidation of branched-chain FAs (reviewed in Refs. 6668), little is known regarding the role of extraperoxisomal factors such as FA binding proteins in FA metabolism. Recent data suggest that intracellular FA binding proteins may facilitate the process of transfer of branched-chain FAs such as phytanic acid to the peroxisome. Both in vitro studies (1416, 63, 75) and recent investigations with cultured cells (1, 46) indicate that in the liver at least two families of intracellular lipid binding/transfer proteins may be involved: L-FABP and the SCP-2/SCP-x family. L-FABP is a nonenzymatic, primarily cytoplasmic FA binding protein, occurring at high levels (2–3% of cytosolic protein, 200–400 μM) in the liver (reviewed in Ref. 40). A significant body of evidence obtained with cultured cells and in vitro studies shows that L-FABP functions in LCFA uptake (reviewed in Ref. 40), intracellular diffusion (reviewed in Refs. 34, 35, 40, 44, 57, 69), and intracellular targeting to multiple organelles such as endoplasmic reticulum (reviewed in Refs. 27, 29, 40, 59) and nuclei (24, 25). The SCP-2/SCP-x family represents the second intracellular lipid binding protein family that has high affinity for branched-chain FAs (16, 60). Through alternate transcription sites the SCP-2/SCP-x gene encodes for two functionally and spatially distinct proteins: 1) pro-SCP-2, which is rapidly cleaved to SCP-2 such that it is distributed in highest concentration in peroxisomes and less so in extraperoxisomal sites (reviewed in Refs. 18, 31, 58), and 2) SCP-x, a 3-ketoacyl-CoA thiolase found exclusively in peroxisomes and the only known LCFA oxidative enzyme utilizing branched-chain substrates. The data presented herein with L-FABP gene-ablated mice fed 1% phytol provide several new insights into the physiological role of the L-FABP in branched-chain (phytanic acid) LCFA metabolism, as well as functional interrelationships between L-FABP and SCP-x in facilitating branched-chain FA metabolism.

First, although neither 1% dietary phytol nor L-FABP gene ablation altered food consumption, L-FABP gene ablation altered the whole body phenotype in a gender-dependent manner. In wild-type L-FABP+/+ mice, the 1% phytol diet elicited significant loss in body weight, FTM, and LTM in males and even more so in female littermates. Consistent with these findings of higher sensitivity of female L-FABP+/+ mice to 1% dietary phytol, an earlier study performed at a lower dose (i.e., 0.5% phytol) showed that female, but not male, inbred C57BL/6 mice lost body weight, both as FTM and LTM (2). Together these data suggest that the whole body phenotype of female wild-type L-FABP+/+ mice was more sensitive to dietary phytol than that of their male counterparts. Male L-FABP+/+ littermates exhibited qualitatively similar whole body phenotype only at significantly higher levels of dietary phytol. L-FABP gene ablation significantly exacerbated this gender-dependent effect of 1% dietary phytol on body phenotype such that not only female but also male body weights were decreased by the 1% phytol diet. In fact, when expressed as percent weight loss the phytol-fed male L-FABP−/− mice lost nearly 15% body weight, whereas the control-fed male L-FABP−/− mice gained 15% body weight. This range of change was similar to that for 1% phytol-fed female L-FABP−/− mice, which lost nearly 20% body weight, whereas the control-fed female L-FABP−/− mice gained 2% body weight. Whether these weight losses were due to loss in FTM or LTM was resolved by whole body DEXA. L-FABP gene ablation increased the %FTM in 1% phytol-fed female mice compared with control-fed female littermates, whereas that in the male littermates was not further influenced. In contrast, L-FABP gene ablation significantly ameliorated the 1% phytol-induced loss in %LTM in male but not female L-FABP−/− mice.

Second, 1% dietary phytol significantly altered liver morphology and phenotype. Dietary 1% phytol increased liver mass, fatty vacuolation, necrosis, and expression of PPARα-regulated proteins such as L-FABP and peroxisomal proteins such as catalase and SCP-2 in both male and female mice. Because lower dietary phytol (i.e., 0.5%) increased liver mass and elicited significant lesions only in female, but not male, inbred C57BL/6 mice (2), these data suggested that the livers of female L-FABP+/+ mice were more sensitive to dietary phytol, although at sufficiently high dietary phytol qualitatively similar changes were observed in livers of male phytol-fed mice. These findings were rationalized by the fact that livers of female control-fed mice had nearly 10-fold lower levels of SCP-x (the only known 3-ketoacyl-CoA thiolase enzyme involved in branched-chain FA peroxisomal oxidation) than male littermates. Lower levels of SCP-x in female animals have also been reported in inbred strains of C57BL/6 mice (1), FVB mice (1), BALB/c mice (56), and rats (42, 43). The data showing that 1% phytol increased liver mass and upregulated the expression of PPARα-regulated proteins were consistent with several lines of evidence suggesting that L-FABP may directly and indirectly influence PPARα transcriptional activity. For example, phytanic acid is a peroxisome proliferator (2, 12, 75). L-FABP enhances the distribution of bound ligand to the nucleus (24, 25, 32), and small quantities of L-FABP have been detected in the nucleus associated with PPARα (24, 73). Because L-FABP transcription is also regulated by PPARα, L-FABP may regulate its own transcription by transport of bound ligand (75). Because L-FABP binds phytanic acid with high affinity (16, 75) and phytanic acid enhances PPARα transcriptional activity (12, 75), L-FABP may also enhance targeting of phytanic acid to the nucleus and deliver bound phytanic acid to PPARα for enhancing transcriptional activity of L-FABP as well as peroxisomal proteins involved in phytanic acid oxidation.

Third, L-FABP gene ablation significantly altered the liver morphology and phenotype in response to 1% phytol. The above-described gender-dependent induction of SCP-x expression by 1% phytol diet in wild-type L-FABP+/+ mice was highly exacerbated on L-FABP gene ablation, such that livers of male and much more so of female L-FABP−/− mice fed 1% phytol expressed highly elevated levels of SCP-x. This effect of L-FABP gene ablation in 1% phytol-fed mice was specific because there was no concomitant upregulation of SCP-2 or plasma membrane LCFA translocase or transport proteins in gene-ablated mice. These data suggest that both L-FABP and SCP-x are important for efficient peroxisomal oxidation of branched-chain FAs. In support of this possibility, L-FABP expression is upregulated fourfold in phytol-fed SCP-2/SCP-x gene-ablated mice (60). Furthermore, L-FABP overexpression in transfected cells enhances oxidation of low levels of phytanic acid (4). The significance of L-FABP gene ablation potentiating the upregulation of SCP-x in response to 1% dietary phytol provides new insights into the interrelationships between these proteins to facilitate peroxisomal phytanic acid oxidation basically. On the basis of the fact that L-FABP not only binds phytanic acid (16, 75) and is known to enhance the intracellular transport of straight-chain FAs (reviewed in Refs. 34, 35, 40, 44, 57, 69), it may be postulated that L-FABP also enhances intracellular transport of phytanic acid. If so, then L-FABP gene ablation would be expected to reduce phytanic acid transport to the peroxisome, and thereby phytanic acid oxidation, and phytanic acid should accumulate in liver and serum. Consistent with this prediction, livers and serum of 1% phytol-fed L-FABP−/− mice accumulated the highest levels of phytanic acid (25–31% of total FAs) and pristanic acid (2–4%). This was despite the fact that SCP-x, a branched-chain FA oxidative peroxisomal enzyme, was concomitantly upregulated to highest levels in 1% phytol-fed female and less so in male L-FABP−/− mice.

In summary, the results presented herein are consistent with L-FABP gene ablation exerting a significant physiological role, especially in female mice, on branched-chain FA metabolism. The loss of L-FABP in these mice was only partially compensated by concomitant upregulation of SCP-x in response to dietary phytol. These findings were supported by earlier studies with transfected cells overexpressing L-FABP showing that L-FABP enhances the peroxisomal oxidation of low levels of phytanic acid (4). Conversely, oxidation of low levels of phytanic acid was reduced in cultured hepatocytes from L-FABP gene-ablated mice (1). On the basis of these observations it may be postulated that without L-FABP present, and despite upregulation of SCP-x, the ability of peroxisomes to oxidize branched-chain FAs is saturated, thereby resulting in enhanced accumulation of phytanic acid metabolites and increased toxicity. Consistent with this prediction, liver necrosis and accumulation of phytanic acid were highest in 1% phytol-fed female L-FABP−/− mice. Together, these results indicate that L-FABP functions to enhance peroxisomal oxidation of branched chain FAs by 1) enhancing transport of bound phytanic acid to peroxisomes and/or 2) enhancing transport of bound phytanic acid to the nucleus for stimulating transcriptional activity of PPARα-regulated proteins (including L-FABP) involved in peroxisomal branched-chain FA oxidation.

GRANTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41402.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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View Abstract