From the
Department of Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4467,
¶ Department of Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4467,
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, Berlin-Buch D-13092, Germany
Received for publication, January 10, 2003
, and in revised form, March 27, 2003.
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ABSTRACT |
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INTRODUCTION |
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However, in vitro studies of FABPs have inherent limitations. The only firmly established function of FABPs is the reversible binding of hydrophobic ligands, and these proteins do not exhibit any enzymatic function or energy requirement. This suggests that these proteins play passive (facilitative) roles that, almost by definition, are strongly dependent on the cellular context. One context of the highly expressed L-FABP is the highly differentiated hepatocyte, a cell type featuring an intense lipid metabolism that is not easily modeled in cell-free systems or transfected cells. Thus, an in vivo approach is probably needed to elucidate the physiologically relevant roles of this protein. Within this context, a deletional approach is likely to be more revealing than an overexpression approach, because L-FABP is extremely highly expressed even under basal conditions. It is these considerations that have brought us to believe that the in vitro assays need to be complemented by targeted deletion of the L-FABP gene in vivo to reveal its function(s) and mode(s) of action. We have therefore decided to create L-FABP null mice by homologous recombination in embryonic stem cells, and the present paper reports their initial analysis. We have focused this analysis on the impact of this mutation on lipid composition, lipid binding, and the expression of other known fatty acid-binding proteins.
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EXPERIMENTAL PROCEDURES |
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Superdex 75 Prep Grade gel filtration medium was purchased from Amersham Biosciences (Piscataway, NJ). [9,10-3H(N)]oleic acid (15 Ci/mmol) and [1-14C]oleic acid (50 mCi/mmol) were obtained from PerkinElmer Life Sciences (Boston, MA). Silica gel G TLC plates were purchased from Analtech, Inc. (Newark, DE). Reference lipids were obtained from Nu-Chek-Prep, Inc. (Elysian, MN). cis-Parinaric acid and NBD-stearic acid (18-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)octadecanoic acid) were purchased from Molecular Probes (Eugene, OR). All other reagents were of the highest grade available.
Standard ProteinsMouse albumin (Fraction V), rat liver glutathione S-transferase (GST), and porcine heart glutamic-oxaloacetic transaminase (aspartate aminotransferase, Type II-A) were purchased from Sigma-Aldrich. Mouse L-FABP, SCP-2, and SCP-x were purified as described previously (12, 13).
AntibodiesRabbit anti-mouse albumin (IgG fraction) was purchased from Accurate Chemical and Scientific Corp. (Westbury, NY). Rabbit anti-rat glutathione S-transferase antiserum was purchased from Alpha Diagnostic International (San Antonio, TX), and its cross-reactivity with mouse GST was verified by enzyme-linked immunosorbent assay and Western analysis. Rabbit anti-rat L-FABP (14), anti-mouse SCP-2 (5), and anti-mouse SCP-x (13) were obtained and purified as described in the cited papers; rabbit polyclonal anti-porcine aspartate aminotransferase was generated and purified in the same way; its cross-reactivity with mouse aspartate aminotransferase was confirmed by enzyme-linked immunosorbent assay and Western analysis. Rabbit anti-mouse caveolin-1 (IgG fraction) was obtained from Affinity Bioreagents, Inc. (Golden, CO). Rabbit anti-mouse fatty acid transport protein (FATP) was a generous gift from Dr. Jean Schaffer (Washington University, St. Louis, MO). Goat anti-mouse fatty acid translocase (FAT, CD36) (IgG fraction) was purchased from Research Diagnostics, Inc. (Flanders, NJ). Alkaline phosphatase-conjugate goat anti-rabbit IgG and alkaline phosphatase-conjugate rabbit anti-goat IgG were from Sigma.
Creation of L-FABP-deficient MicePrimers 5'-gacctcatccagaaagggaag and 5'-cttttccccagtcatggtctc were designed to amplify from mouse DNA a 156-bp exon 2 fragment. The amplicon was used as a probe in Southern blotting and to screen a P1129/Ola genomic library (Genome Systems). Extensive restriction mapping and southern blotting of mouse genomic and P1 DNA demonstrated the identity of the P1 clones with the respective genomic regions and the absence of pseudogenes. Mouse L-FABP cDNA (15) was used to confirm the identity of the P1 clones by southern hybridization and PCR for exons 1 and 4.
An 8-kb EcoRV fragment containing the 5' gene flank plus exons 1 and 2 was subcloned from a P1 clone and used to isolate a 3.9-kb end-filled EcoRV/EcoRI fragment ("long arm") that in turn was ligated into vector pTVO (cut with XhoI and end-filled), a plasmid carrying the neomycin resistance marker, to create an intermediate long arm construct. A 10-kb SacI fragment overlapping the above EcoRV fragment and containing the whole gene as well as its 3' flank was subcloned from P1 and used to isolate a 1.3-kb BamHI/SalI fragment from the 3' flank ("short arm"). The short arm was then ligated into the long arm construct (opened with BamHI and SalI), resulting in the targeting vector. The SalI/EcoRI fragment just 3' of the short arm was partially sequenced to design primers.
The targeting construct was opened with NotI and electroporated into HM1 cells (16). After selection with G418, colonies were screened by PCR for homologous recombination, using primers 5'-ccttctatcgccttcttgacgag and 5'-agcctccagggattggaatg and an annealing temperature of 63 °C. These primers correspond to the neo resistance marker and the 3' genomic flank outside of the construct, respectively. A fragment of the expected size of 1.4-kb was amplified in 5 out of 200 clones, and 2 clones were expanded and injected into C57Bl/6 blastocysts to create chimeric mice by standard procedures (17). Heterozygous mice were obtained by breeding the chimeras with C57Bl/6 wild type mice, and experimental wild type and L-FABP null mice were created by interbreeding of the first generation heterozygous mice.
Some of these mice were used to verify the gene deletion as follows. Liver DNA was purified by standard procedures (18) and used as a template for long PCR to amplify an 8-kb fragment from wild type and a 2.5-kb fragment from L-FABP null DNA (see Fig. 1A). Cycling conditions were 96 °C, 30 s; 32x (94 °C, 30 s; 63 °C, 30 s; 68 °C, 8 min). One primer (5'-ttcaagcctccagggattggaatg) corresponded to a sequence located immediately 3' of the short arm homology region (i.e. outside of the recombination construct), the other primer (5'-cctggactgagacttgcctggattg) to a sequence located at the 3'-end of the long homology arm. The long PCR products (Fig. 1B) were further verified by nested PCR for a 157-bp fragment of exon 2, using primers 5'-ccgaggacctcatccagaaag and 5'-tccccagtcatggtctccag at an annealing temperature of 60 °C (Fig. 1C). With the same exon 2 primers, absence of exon 2 was also verified directly on genomic DNA (Fig. 1C). In addition, absence of exons 3 and 4 in knockout DNA was also directly confirmed by PCR with genomic DNA (not shown). After verification of the targeted gene deletion, another PCR assay was designed for routine single-tube genotyping of tail biopsies. Primers 5'-caagggggtgtcagaaatcgtgc and 5'-ccagtcatggtctccagttcgca amplify 123 bp from exon 2 of the wild type allele, and 5'-aagagcttggcggcgaatgg and 5'-tggccatttgtggctgtgctc amplify 227 bp from the neomycin resistance marker into the 3' flank of both alleles. An annealing temperature of 68 °C was used.
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RT-PCRTotal liver RNA was isolated with the TRIzol reagent from Invitrogen (Carlsbad, CA), and reverse transcription was performed with random hexamer primers and Moloney murine leukemia virus reverse transcriptase (Invitrogen) according to a standard procedure (18). Aliquots of the RT reaction were used for PCR, using L-FABP primers (5'-ctcattgccaccatgaacttctc and 5'-agccttgtctaaattctcttgctgact, amplifying 404 bp) and hypoxanthine phosphoribosyl transferase (HPRT) primers (5'-gcttgctggtgaaaaggacctct and 5'-ggaaatcgagagcttcagactcgtc, amplifying 584 bp) at an annealing temperature of 62 °C.
AnimalsChimeric mice were bred with C57/Bl6 mice, and the resulting heterozygous offspring were interbred to produce the L-FABP null (/) and wild type (+/+) littermate control mice used for this study. Mice used for the present study were female and 1315 months of age, except for those used for the in vivo labeling experiment that were from the next generation and 5 months of age. Mice were fed a pelleted Teklad Rodent Diet (W8604) obtained from Harlan Teklad (Madison, WI). The animals were maintained in a temperature-controlled (25 °C) facility on a 12-h light/dark cycle and were allowed free access to food and water. The experimental protocols for the use of laboratory animals were approved by the University Lab Animal Care Committee.
Animal Sacrifice and Tissue CollectionFor determination of serum parameters and liver fatty acid binding capacity and lipid distribution, female mice (13-15 months old), were food-deprived for 12 h, weighed, and anesthetized with Avertin. Blood was collected via cardiac puncture and immediately processed to serum. The animals were euthanized by cervical dislocation, and tissues of interest were removed, flash-frozen with dry ice, and stored at 80 °C for further analysis. The liver was excised and weighed, and a small portion of the liver was used immediately for histological analysis. The remainder of the liver was divided into small portions, flash-frozen with dry ice, and stored at 80 °C for further analysis.
Serum ParametersSerum metabolites were determined with kits (triglycerides, Sigma #336; glucose, Sigma #315; nonesterified fatty acids, half micro kit from Roche Applied Science).
Liver Tissue Homogenization and FractionationAll procedures were performed on ice or at 4 °C. 0.1 g of fresh, minced mouse liver was homogenized in 0.5 ml of phosphate-buffered saline (PBS, pH 7.4) containing protease inhibitor mixture (Sigma, St. Louis, MO) by 20 strokes in a Potter-Elvehjem homogenizer. After centrifugation at 600 x g for 10 min, the resulting post nuclear supernatant was further centrifuged at 105,000 x g for 90 min., yielding a pelleted fraction ("membranes") and a 105,000 x g supernatant ("cytosol"). Protein was quantified by the Bradford protein assay (Bio-Rad, Richmond, CA) (19).
SDS-PAGE16.5% SDS-PAGE was performed using the system of Schagger and von Jagow (20), with minor modifications (21). Protein samples were reduced with 2-mercapthoethanol before loading. The gels were stained with 0.1% Coomassie Brilliant Blue R-250, and the stained proteins were quantified by densitometry, utilizing a single-chip charge-coupled device video camera FluorChemimager and accompanying FluorChem image analysis software (version 2.0) from Alpha Innotech (San Leandro, CA).
Western AnalysisProteins separated by SDS-PAGE were transferred to nitrocellulose membranes with a Miniprotean II transblot apparatus (Bio-Rad) at 40 V/gel constant voltage and 4 °C for 2 h. After transfer, the nitrocellulose membranes were rinsed in 10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween 20 (TBST) and blocked by incubation in TBST plus 3% gelatin for 30 min at room temperature. The membranes were then washed 3 x 5 min with TBST and incubated with primary antibody (1:1000 dilution in 10 mM Tris, pH 8.0, 150 mM NaCl (TBS), 1% gelatin) for several hours at room temperature with gentle shaking. Then the membranes were washed 2 x 5 min with TBST, 2 x 5 min with TBS, and incubated with alkaline phosphatase-conjugated secondary antibody (1:3000 dilution in TBS/1% gelatin) for 2 h at room temperature with gentle shaking. The membranes were then washed 2 x 5 min with TBST, 2 x 5 min with TBS, and 1 x 5 min with alkaline phosphatase buffer (100 mM Tris, pH 9.0, 100 mM NaCl, 5 mM MgCl2). Color development was initiated by the addition of alkaline phosphatase substrate (5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium, Sigma, St. Louis, MO) and stopped by washing the membranes with doubly distilled water. Membrane photography and protein quantification were accomplished utilizing the imaging system described above. For the quantification of L-FABP, SCP-2, SCP-x, albumin, and glutathione S-transferase, standard curves were produced with the appropriate purified proteins that had been processed under identical conditions.
Gel Permeation Chromatography of Liver 105,000 x g SupernatantA 1.5 x 30 cm Superdex G75 column was equilibrated with PBS, pH 7.4, and calibrated using a protein molecular mass kit (Sigma, St. Louis, MO), including aprotinin (6.5 kDa), cytochrome c (12. 4 kDa), carbonic anhydrase (29 kDa), and albumin (66 kDa). The molecular weight standard curve was generated by plotting the log of the protein molecular weight versus Ve/V0, where Ve is the elution volume and V0 is the void volume (determined by applying 1 mg of blue dextran to the column). 1 mg of 105,000 x g supernatant protein was incubated with [3H]oleic acid (30 nmol, 1 x 107 dpm) for 5 min at 4 °C and loaded onto the column. Fractions were eluted at 4 °C with PBS at a flow rate of 1.0 ml/min using a Model P-1 peristaltic pump (Amersham Biosciences, Piscataway, NJ). Absorbance (280 nm) was monitored using a Model 2238 Uvicord SII in-line detector (Amersham Biosciences, Piscataway, NJ) coupled with a Model 2210 single-channel recorder (Amersham Biosciences). 1-ml fractions were collected utilizing a SuperFrac fraction collector (Amersham Biosciences). 100-µl aliquots were used to measure 3H content by liquid scintillation counting (Packard 1600 TR, Meriden, CT), and 5.0 µl aliquots were used for Western blotting.
Determination of Fatty Acid Binding Capacity and Binding ParametersThe assay has been previously described (22). Briefly, in a final volume of 2 ml the incubation mixture contained an aliquot of Superdex 75 column fraction III (15 µg of protein/ml) or murine recombinant L-FABP in 10 mM potassium phosphate, pH 7.4. The protein sample was titrated with small amounts of cis-parinaric acid using a 100 µM stock solution prepared in 10 mM NaOH. Upon addition of cis-parinaric acid, the mixture was allowed to equilibrate at 24 °C for 5 min prior to spectroscopic analysis. Fluorescence intensities were measured in a 1-cm quartz cuvette utilizing a PC1 photon-counting spectrofluorometer (ISS Instruments, Champaign, IL). For each cis-parinaric acid concentration, a control fluorescence intensity was measured in the absence of protein and subtracted. cis-Parinaric acid was excited at 324 nm, whereas fluorescence emission was monitored at 410 nm. Excitation and emission monochromator bandwidths were 4 nm. To avoid the inner filter artifact, absorbance at the wavelength of excitation was maintained at ≤0.15 absorbance units. The dissociation constant, Kd, and the binding stoichiometry, n, were calculated as described previously (22).
Lipid QuantificationAll glassware was washed with sulfuric acid/chromate and rinsed several times with doubly distilled water prior to use. Lipid analysis was performed as previously described (9, 21). Each homogenate (5 mg of protein) and lipid standard sample (see below) was extracted two times with a total of 10 ml of hexane/2-propanol (3:2, v/v), i.e. 5 ml per extraction. The organic phases were collected after centrifugation (1500 rpm, 4 °C) and combined, then dried under N2, resuspended in 100 µl of chloroform, and spotted onto silica gel G thin-layer chromatography plates. After running the plates with petroleum ether/diethyl ether/methanol/acetic acid (180:14:4:1, v/v), the separated lipids were visualized in an iodine chamber and scraped into acid-washed glass test tubes. Lipid content was determined by the method of Marzo et al. (23). To this end, each TLC scraping was extracted two times with 2 ml of chloroform/methanol/hydrochloric acid (100:50:0.375, v/v) per sample (i.e. 4 ml total). The TLC scrapings were removed by centrifugation at 1000 rpm, 4 °C, 10 min. The two extracts from each sample were pooled, vortexed with 2 ml of ddH2O, and centrifuged (1500 rpm, 4 °C, 10 min). The organic phase was dried under N2, and the residue was resuspended in 1 ml of sulfuric acid and incubated at 200 °C for 15 min in screw-cap glass test tubes. After removing debris by centrifugation at 1000 rpm, 4 °C, 10 min, absorbance was measured at 375 nm using a Lambda 2 UV-visible spectrophotometer (PerkinElmer Life Sciences, Shelton, CT). The standard curve was generated using 5, 10, 20, 50, and 100 µl (6.7, 13.4, 26.8, 67.0, and 134 µg of individual lipid, respectively) of a lipid reference mixture (Nu-Chek Prep, Elysian, MN).
In Vivo Studies with Radiolabeled Fatty AcidMice were anesthetized with Avertin, the abdomen was opened, and 0.1 ml of [14C]oleate (5 µCi/ml in 0.9% NaCl/4% fatty acid-free bovine serum albumin) was injected evenly over 1 min into the vena cava inferior. 10 min after start of injection, liver was taken and blood was drawn by cardiac puncture for measurement of fatty acid levels. Liver pieces (5080 mg) were quickly rinsed with cold isotonic saline, blotted dry, weighed, and dissolved in hyamine for scintillation counting. Tissue radioactivities (per milligram of liver) were corrected for serum fatty acid levels to represent true tissue deposition. Depositions are given in arbitrary units rather than moles, because blood volumes are unknown (but assumed similar because body weights did not differ (+/+ versus /): 25.8 ± 5g versus 26.6 ± 4.5 g (fasted mice); 32.9 ± 3.2 g versus 32.1 ± 4.2 g (fed mice)).
Statistical AnalysisData are presented as the mean ± S.E. with n and p indicated under "Results." Statistical analysis was performed using the unpaired Student's t test (GraphPad Prism, San Diego, CA).
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RESULTS |
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General CharacterizationL-FABP null mice did not show any obvious abnormalities in appearance, behavior, sex ratio, or fertility. Under the standard chow, the body weight of LFABP null (/) mice (38.8 ± 5.2 g) used for the present study did not differ significantly from that of their wild type (+/+) littermates (37.4 ± 3.8 g). No significant alterations were noticed in the fasting serum levels of triglycerides (1.01 ± 0.17 mM (/) versus 0.92 ± 0.15 mM (+/+)) and free fatty acids (0.93 ± 0.1 mM (/) versus 0.81 ± 0.18 mM (+/+)), but a small reduction (p < 0.05) in glucose levels was seen (6.06 ± 0.58 mM (/) versus 9.07 ± 0.61 mM (+/+)). Liver weights of FABP (/) mice were normal (1.59 ± 0.44 g (/) versus 1.52 ± 0.39 g (+/+)). Gross histology of L-FABP (/) livers was also essentially normal.
Protein DistributionBecause L-FABP is normally one of the most abundant proteins in liver cytosol, its absence might visibly alter the protein pattern. When equal amounts of protein were loaded onto SDS-polyacrylamide gels, a conspicuous decrease of band intensity was noticed in the 14-kDa region of both homogenate (Fig. 2A) and 105,000 x g supernatant (Fig. 2C) from L-FABP (/) mice. By densitometry, this decrease was about 29% (p < 0.001) in homogenates (Fig. 2B) and about 50% (p < 0.001) in the 105,000 x g supernatants (Fig. 2D). In contrast to this significant selective decrease, no statistically significant (p < 0.05) changes were noticed in the total protein contents (in milligrams/g of liver) of homogenates (463 ± 40 (/) versus 451 ± 51 (+/+)), 105,000 x g supernatants (213 ± 40 (/) versus 155 ± 29 (+/+)), and 105,000 x g pellets (154 ± 20 (/) versus 183 ± 21 (+/+)).
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Potential Compensation by Soluble Fatty Acid Binding ProteinsNext, we investigated whether other known lipid-binding proteins might be up-regulated to compensate for the absence of L-FABP. The most abundant of these is the soluble low molecular mass (25 kDa) glutathione S-transferase (GST), a protein that represents up to 5% of cytosolic proteins (24). Recombinant human GST A11 binds cis-parinaric acid with a Kd near 0.2 µM,2 which is in the similar range as that reported for L-FABP (22). Furthermore, liver expresses sterol carrier protein-2 (SCP-2), a 13-kDa protein found in peroxisomes and cytosol (25, 26), as well as the 58-kDa sterol carrier protein-x (SCP-x) (27). Finally, serum albumin (68 kDa) is also an abundant liver product, although it is not thought to accumulate in the cytosol.
Using Western analysis, no statistically significant changes in the levels of albumin (Fig. 3B) and glutathione S-transferase (Fig. 3C) were measured in liver homogenate and 105,000 x g supernatants of L-FABP (/) livers, although a minor increase was uniformly seen.
In contrast, SCP-2 levels were increased significantly (1.4-fold, p < 0.05) in L-FABP (/) homogenates (27.2 ± 1.3 µg/g liver (/) versus 19.4 ± 1.8 µg/g liver (+/+)). This increase was even more pronounced (1.8-fold, p < 0.05) when expressed per microgram of homogenate protein (1.00 ± 0.10 ng/µg (/) versus 0.57 ± 0.19 ng/µg (+/+)) (Fig. 3D). Concomitantly, SCP-2 levels in 105,000 x g supernatants were also significantly (1.6-fold, p < 0.05) increased (3.01 ± 0.41 ng/µg supernatant protein (/) versus 1.88 ± 0.44 ng/µg (+/+)) (Fig. 3D). Despite these genotypic differences, SCP-2 was enriched similarly (3-fold) in the 105,000 x g supernatant over homogenate (Fig. 3D).
In contrast to the increased levels of SCP-2, levels of its precursor protein, SCP-x, were dramatically reduced (p < 0.01) in liver of L-FABP (/) mice (5.8 ± 0.8 µg of SCP-x/g of liver (/) versus 12.4 ± 1.1 µg/g (+/+)). As in the case of SCP-2, the difference was more striking (4-fold, p < 0.001) when expressed per microgram of homogenate protein (0.08 ng of SCP-x/µg of homogenate protein (/) versus 0.33 ± 0.05 ng of SCP-x/µg of homogenate protein (+/+)) (Fig. 3E). SCP-x was not detected in 105,000 x g supernatant (Fig. 3E).
Importantly, the total increase in SCP-2 levels in livers from L-FABP (/) mice appeared to match the decrease in SCP-x levels. Thus, total (i.e. SCP-2 plus SCP-x) values (33.0 ± 2.0 µg of protein/g of liver) were not significantly different from those in wild type mice (30.8 ± 1.6 µg of protein/g of liver) (Fig. 3F). However, because SCP-x was absent from the 105,000 x g supernatant fraction, the increase in SCP-2 levels in livers from L-FABP (/) mice resulted in an increase in total (SCP-2 plus SCP-x) in 105,000 x g supernatant from L-FABP (/) mice (Fig. 3F).
In summary, livers of L-FABP (/) mice showed an increase of SCP-2 levels and a matching decrease of SCP-x levels, whereas albumin and glutathione S-transferase were not significantly altered. However, the increment of SCP-2 levels (by 1.13 ng of SCP-2/µg of supernatant protein) was not nearly as large as the decrement of L-FABP levels (by nearly 50 ng/µg of supernatant protein).
Potential Compensation by Plasma Membrane-associated Fatty Acid-binding ProteinsA number of membrane-bound proteins are known to be involved in cellular lipid transport and metabolism. These include the caveolins, fatty acid transport protein (FATP), fatty acid translocase (FAT/CD) (36), and aspartate aminotransferase (glutamic-oxalacetic transaminase) (2831). Western blotting was performed in homogenates of L-FABP (/) and wild type livers to determine whether L-FABP gene ablation might result in compensatory up-regulation of these proteins. No significant changes in the levels of caveolin, fatty acid transport protein, and aspartate aminotransferase were found, but a slight (20%, p < 0.05) increase in fatty acid translocase levels was noticed (Table I).
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Gel Permeation ChromatographyThe absence of major compensation by other fatty acid-binding proteins predicts a significantly reduced fatty acid binding of L-FABP null cytosol. To investigate this possibility in more detail, cytosols (105,000 x g supernatants) from wild type and L-FABP (/) livers were incubated with [3H]oleic acid and loaded onto a Superdex 75 gel filtration column. The eluted fractions were examined for radioactivity, total protein, and presence of LFABP, SCP-2, and albumin. Radioactivity added to wild type cytosol resolved into four distinct regions designated as Fractions IIV (Fig. 4A). Highest levels of total protein (Fig. 4A, closed circles) and albumin (Fig. 4C) were detected along with significant levels of [3H]oleic acid in Fractions I and II (Fig. 4A, open circles). Fraction III contained the least amount of total protein (Fig. 4A, closed circles) but the largest amount of [3H]oleic acid (Fig. 4A, open circles). Western blot analysis of Fraction III showed the presence of both L-FABP and SCP-2 (Fig. 4C). In the gel filtration medium, L-FABP migrated with an apparent molecular mass of 10 kDa; its hydrodynamic volume was smaller than the hydrodynamic volume of the 12.4-kDa column calibration protein, cytochrome c. Amino acid analysis and SDS-PAGE confirmed that this protein was intact L-FABP (data not shown). Thus, Fraction III from the gel permeation column comprised the soluble fatty acid-binding proteins that appear in liver cytosol (primarily L-FABP; less so SCP-2).
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The gel permeation chromatographic profile of L-FABP (/) mouse liver 105,000 x g supernatant proteins (Fig. 4B) differed markedly from that of L-FABP (+/+) mice (Fig. 4A) in that [3H]oleic acid binding of Fraction III was reduced by >95%. Western blot analysis confirmed that L-FABP was absent throughout (Fig. 4C) and that SCP-2 was present in Fraction III (Fig. 4C).
Relative Contribution of L-FABP to the Maximal Fatty Acid Binding Capacity of Fraction III-soluble ProteinsBecause it is not obtained under equilibrium conditions, the radioactivity profile of a gel filtration experiment may not correctly reflect binding capacity of the eluted fractions. To measure the binding capacity of column fraction III, a fluorescent ligand, saturation-binding assay was performed with cis-parinaric acid. This fatty acid shows low quantum yield and fluorescence intensity in aqueous environment (22) but high quantum yields upon binding to proteins such as L-FABP (22) or SCP-2 (25). Aliquots of fraction III (Fig. 4), equivalent to 0.2 µM L-FABP in the wild type fraction III, were therefore titrated with increasing amounts of cis-parinaric acid. Wild type Fraction III showed a biphasic binding curve (Fig. 5A), consistent with the known presence of two cis-parinaric acid ligand-binding sites in L-FABP (22, 32). Mathematical analysis revealed the presence of a high affinity (Kd1 = 130 ± 17 nM) and a low affinity (Kd2 = 691 ± 111 nM) binding site (Table II). When the assay was repeated with pure recombinant L-FABP, a similar biphasic titration curve (Fig. 5B) and similar binding constants were obtained (Table II).
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In contrast, column Fraction III from L-FABP (/) mice exhibited a markedly (>80%) reduced cis-parinaric acid binding capacity and yielded a monophasic binding curve (Fig. 5A). Mathematical analysis of this curve revealed the presence of a single low affinity-binding site with a Kd of 2.78 ± 0.56 µM (Table II) and thus a considerably lower affinity for fatty acids than SCP-2 (Kd near 0.2 µM) (25). Thus, although SCP-2 was present in fraction III (see above), the residual fatty acid binding of this fraction seems to be largely caused by less specific proteins.
In summary, the above saturation binding experiments demonstrate for the first time that L-FABP constitutes the majority (8095%) of fatty acid binding capacity of all low molecular weight cytosolic proteins. The small increase in SCP-2 noted in liver of L-FABP (/) mice (see preceding sections) did not compensate for the loss of L-FABP in terms of fatty acid binding capacity. On this basis, it was predicted that liver fatty acid and esterified lipid levels might be altered in L-FABP gene-ablated mice.
Effect of L-FABP Gene Ablation on Liver Nonesterified Fatty Acid Pool SizeBecause of its abundance, high fatty acid binding affinity (22), and ability to enhance fatty acid uptake in transfected cells (7, 10, 33, 34), L-FABP might be expected to increase the nonesterified fatty acid pool size. However, as shown in Fig. 6D, there was no statistically significant difference in the amount of nonesterified fatty acid between wild type (24.6 ± 3.0 nmol/mg of protein) and L-FABP (/) (26.6 ± 2.5) livers.
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Effect of L-FABP Gene Ablation on Fatty Acids Esterified to Glycerides: Phospholipids and TriglyceridesL-FABP is known to stimulate microsomal fatty acid esterification to glycerides (4, 3537) in vitro and in transfected cells overexpressing L-FABP (5, 7, 9, 33). Therefore, deletion of the L-FABP gene might be expected to reduce the cellular levels of these compounds in vivo. To investigate this possibility, lipid was extracted from mouse liver homogenates, resolved into individual lipid classes, and quantified. Contrary to expectations, LFABP (/) liver contained about 35% (p < 0.05) more phospholipids (63.8 ± 4.1 nmol/mg of protein) than wild type liver (47.5 ± 3.2 nmol/mg of protein) (Fig. 6F). In contrast, the amount of triglycerides was not significantly altered (Fig. 6E).
Effect of L-FABP Gene Ablation on Fatty Acids Esterified to CholesterolL-FABP has been shown to stimulate microsomal fatty acid esterification to cholesterol (38) in vitro and in transfected cells overexpressing L-FABP (8, 39). Therefore, deletion of the L-FABP gene might reduce the cellular levels of cholesterol and cholesterol esters. To test this possibility, lipid was extracted from mouse liver homogenates, resolved into individual lipid classes, and cholesterol and cholesteryl esters were quantified. Contrary to expectations, L-FABP (/) livers contained about 63% (p < 0.001) more cholesteryl esters (21.5 ± 1.2 nmol/mg of protein) than wild type livers (13.2 ± 0.2 nmol of cholesterol esters/mg of protein) (Fig. 6C). The increase was even more marked (3-fold, p < 0.001) for nonesterified cholesterol (17.2 ± 1.5 nmol/mg of protein (/) versus 5.3 ± 0.8 nmol/mg of protein (+/+)) (Fig. 6B).
Effect of L-FABP Gene Ablation on Overall Distribution of Liver Lipid ClassesThe above data were used to calculate the change in total cholesterol contents and to describe the change of cholesterol contents in molar terms. Total cholesterol (cholesterol plus cholesteryl esters) increased 2-fold (p < 0.001) in livers of L-FABP (/) mice (38.7 ± 2.1 nmol/mg of protein) as compared with wild type mice (18.5 ± 0.8 nmol/mg of protein, Fig. 7A). The nonesterified cholesterol/cholesterol ester molar ratio increased 2-fold (p < 0.01) in the L-FABP (/) livers (0.87 ± 0.10 mol/mol) when compared with the wild type mice (0.40 ± 0.07 mol/mol) (Fig. 7B). The cholesterol ester/triglycerides molar ratio increased 66% (p < 0.05) in L-FABP (/) liver (0.48 ± 0.04 mol/mol) as compared with the wild type liver (0.29 ± 0.05 mol/mol) (Fig. 7C). Thus, absence of L-FABP favored esterification of fatty acids with cholesterol more than that of glyceride. Finally, L-FABP gene ablation caused a dramatic 2-fold increase (p < 0.05) of the cholesterol/phospholipid molar ratio (0.25 ± 0.04 mol/mol (/) versus 0.11 ± 0.01 mol/mol (+/+)) (Fig. 7D). As a result, the quantitative distribution of lipid classes in mouse liver was changed from the order, phospholipids and triglycerides > nonesterified fatty acids > cholesteryl esters > cholesterol (wild type), to the order, phospholipid > triglyceride > nonesterified fatty acids > cholesteryl esters > cholesterol (knockout).
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Effect of L-FABP Gene Ablation on Deposition of Blood-borne Long-chain Fatty AcidsFinally, we asked whether L-FABP influences the hepatic deposition of long-chain fatty acids or their metabolites. A bolus of [14C]oleic acid was injected intravenously, and tissue radioactivity was measured 10 min later. This was first done with mice that had been fasted overnight. Under this predominantly oxidative condition, the tissue radioactivity level was low in both wild type and L-FABP null liver, likely because [14C]CO2 (the main radioactive product of oxidation) is not retained. Nevertheless, a small (p < 0.05) reduction was seen in L-FABP null versus wild type liver (Fig. 8).
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However, the effect of the mutation was much more visible when this method was applied to fed mice. Under this predominantly lipogenic condition, retention of blood-borne fatty acid by both wild type and L-FABP null livers was high compared with fasting, but livers of L-FABP null mice showed a reduction of almost 50% compared with wild type mice (Fig. 8).
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DISCUSSION |
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The results of the present report add important new findings on the role of L-FABP in vivo and raise new interesting questions. First, the data presented here provided, for the first time, a quantitative assessment of the contribution of a fatty acid-binding protein to cytosolic fatty acid binding capacity in vivo. This assessment was facilitated by the fortuitous circumstance that no quantitatively significant compensation by other proteins occurred. Second, lack of L-FABP exerted an important influence on hepatic lipid composition. Some of these changes were quite unexpected, and, as discussed below, it remains to be elucidated whether they are direct or indirect consequences of L-FABP deficiency. Third, although the fatty acid-binding capacity of L-FABP null liver cytosol was reduced to very low levels, the specific increase of another lipid-binding protein, SCP-2, was observed, and, as discussed below, this may be relevant for the observed changes in lipid composition. Fourth, lack of L-FABP altered the turnover of the hepatic lipid pool.
The present data formally establish that L-FABP constitutes at least 80% of fatty acid-binding capacity of all low molecular weight proteins of cytosol (Fraction III of gel permeation chromatography). This finding automatically excludes the possibility of any significant compensatory expression of other members of the FABP gene family. With respect to larger molecular weight regions, changes in fatty acid binding capacity due to deletion of the L-FABP gene cannot be formally excluded yet; however, we found that the major known fatty acid-binding proteins present in this region, glutathione S-transferase and serum albumin, were not changed. Moreover, little or no change was found in the levels of several plasma membrane fatty acid transporters.
The absence of L-FABP may conceivably alter cellular lipid composition in several ways. First, absence of L-FABP might change lipid composition simply because of the absence of a quantitatively important lipid-binding site. Second, because lipid binding to L-FABP is reversible and thus contributes to cellular lipid fluxes, absence of L-FABP can be expected to alter the rates of both lipid synthesis and breakdown and establish a new steady state. Finally, secondary changes of other protein levels may affect the lipid profile. Thus, the absence of L-FABP may affect cellular lipid composition in complex ways that potentially vary under different physiological conditions. Here we have analyzed the main liver lipid classes of L-FABP null liver of fasted mice.
On one hand, we found that the hepatocytes maintained normal levels of total lipid, nonesterified fatty acid, and triglyceride. We note, however, that turnover of at least one of these pools must be changed, because the hepatic deposition of a pulse of blood-borne fatty acid was much lower in L-FABP null versus wild type mice under lipogenic conditions. Although the one or more responsible pathways remain to be identified, reduced influx or esterification rates of fatty acids are more likely explanations than increased secretion of newly esterified triglyceride because of the short time frame (10 min) of the experiment. Clearly, the impacts of the L-FABP gene deletion on the rates of fatty acid influx, esterification, hydrolysis, and oxidation deserve more detailed investigation.
On the other hand, a moderate increase of the phospholipid level in L-FABP null liver was seen, but this change was opposite to what was expected based on the increased phospholipid levels previously found in transfected cells overexpressing L-FABP (8, 9). The most striking finding of the present investigation was a dramatic increase of hepatocellular cholesterol levels in L-FABP null mice. Cholesterol esters were also increased, although somewhat less. Again, the opposite effect of L-FABP gene deletion was predicted, because overexpression of L-FABP in cell culture has previously been shown to increase the cholesterol pool as well as the cholesterol ester level (8, 9). This difference in cholesterol ester levels is more likely related to the change in cholesterol, rather than to alterations of free fatty acids, given that the free fatty acid pool was not changed.
The one or more mechanisms causing these conspicuous alterations, especially of cholesterol homeostasis, remain to be elucidated. The first possibility to be considered is that the absence of L-FABP is directly responsible. Fluorescence and light scattering direct binding assays indicate that L-FABP binds cholesterol and a fluorescent sterol (dehydroergosterol) with low affinity (i.e. submicromolar Kd levels) (38, 44, 45). Although L-FABP poorly competes for binding, extracting, or transferring sterol from model membranes (4648), it nevertheless enhances transfer of sterol in vitro from purified plasma membrane vesicles, albeit not as strongly as sterol carrier protein-2 (46). Thus, L-FABP might affect cholesterol levels directly, although its potential cholesterol binding ability would not yet explain the direction (increase rather than decrease) of change observed in L-FABP null liver. We note that increased L-FABP gene expression seen in SCP-2/SCP-x knockout livers does not cause reduced unesterified cholesterol levels (49). Furthermore, although cholesterol ester levels are reduced in SCP null liver (in line with the increase seen in L-FABP null liver) (49), they are increased in L cells transfected with L-FABP (8), in contradiction to the increase we see in L-FABP null liver. Thus, absence of L-FABP per se does not provide a straightforward explanation for the increased cholesterol/cholesterol ester levels in L-FABP null liver. A second mechanism for the increased cholesterol in L-FABP null liver might be provided by our observation made here that the level of SCP-2 is also increased. This increase in SCP-2 was clearly not sufficient to prevent the dramatic reduction in total fatty acid binding capacity of L-FABP-deficient cytosol. However, it might have been sufficient to cause the observed alterations in cholesterol homeostasis. The ability of SCP-2 to bind cholesterol is well established (52). Overexpression of SCP-2 in L cells inhibited cholesterol efflux and increased the intracellular cholesterol pool (53), and adenoviral overexpression of SCP-2 was previously found to increase hepatic cholesterol contents (54). Conversely, liver cholesterol ester levels are reduced by 50% in SCP-2/SCP-x null mice (49) along with hypersecretion of cholesterol in bile (17, 50, 51). The findings in the latter two models are especially relevant, because they were obtained in vivo, although at least the second model (SCP null mice) is complicated by the concomitant alteration of L-FABP gene expression (49) mentioned above. A third formal explanation for the increased cholesterol contents in the L-FABP null liver is the decrease in the levels of SCP-x that we have found. However, this seems to be not likely, because the cholesterol content of SCP-x/SCP-2 null liver was not increased (49). Moreover, overexpression of full-length SCP-x in L cells led not only to higher (rather than lower) cholesterol contents but also to increased levels of 13-kDa SCP-2 (13). In summary, although the mechanism underlying the increased cholesterol content in L-FABP null liver remains to be resolved, the available data are more in support of a direct role of the increased content of SCP-2 as opposed to the absence of L-FABP or the reduction of SCP-X. In any case, the foregoing discussion suggests that an important strategy to conclusively resolve the relative contributions of these three proteins to hepatocellular cholesterol levels and the other shifts in lipid distribution will be the creation and analysis of L-FABP/SCP double-knockout mice.
Apart from their potential contributions to cholesterol metabolism, the reciprocal alterations in the levels of SCP-2 and SCP-x in the L-FABP null liver raise the interesting question as to what causes them. SCP-2 is encoded by the same gene as SCP-x but can be initiated from a separate codon (13, 27). Hence, there is potential for differential regulation at the level of gene transcription. However, SCP-x can also be proteolytically processed to SCP-2 (13). We have shown here that the decrease in total cellular SCP-x levels is approximately matched by the increase in SCP-2, and the simplest explanation for this observation is given by the hypothesis that, in the absence of L-FABP, proteolytic processing of SCP-x to SCP-2 is enhanced. Furthermore, considering that SCP-x exhibits enzymatic activity as a peroxisomal branched chain 3-ketoacyl-CoA thiolase, its reduction might be the cause or result of a general reduction in branched chain fatty acid oxidation in L-FABP null mice, possibly initiated by a reduction of substrate flux that is normally mediated by the much more abundant LFABP. Thus, L-FABP-deficient mice provide a system to learn more about the coordinate regulation of SCP-2/SCP-x. Finally, these mice will also be of use to resolve the mechanism behind the previously observed toxicity of phytanic acid in SCP-x/SCP-2 null mice (49), which may in fact be due to the increased levels of L-FABP (55).
Taken together, the results of the present report predict that the L-FABP null mice will be useful not only to further elucidate the physiological role of L-FABP but also to better understand the regulation and functions of SCP-2. In this, the LFABP null mouse exemplifies both the strength and the weakness of the gene knockout approach: compensatory mechanisms are a source of new insights (in this case potentially on SCP-2), but they complicate the investigation of the protein of primary interest. This has been seen before in the FABP gene family: Absence of A-FABP (56) and E-FABP (57) was each partially compensated by increased expression of other members of the FABP gene family, and, as mentioned above, deletion of SCP-2/SCP-x up-regulates L-FABP (49). The present report adds a further example to this list, although the level of regulation (transcriptional versus post-transcriptional) remains to be determined.
In summary, L-FABP gene ablation not only dramatically reduced cytosolic fatty acid-binding capacity, but also dramatically shifted liver lipid distribution in favor of cholesterol, cholesterol esters, and phospholipids. The increase in liver cholesterol was so large that, despite the concomitant increase in phospholipid content, the overall ratio of cholesterol/phospholipid still doubled. Because cholesterol and phospholipid are both primarily membrane lipids, this increase in cholesterol/phospholipid suggests that L-FABP gene ablation may also elicit major structural (e.g. fluidity) changes, which in turn can contribute to altered activities of proteins sensitive to fluidity. The present data allow the hypothesis that increased levels of SCP-2 contributed to this shift in lipid composition. Further experiments are needed to understand the mechanism and the functional consequences of the dramatic change in hepatocellular lipid distribution in the L-FABP null mice.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Dept. of Pathobiology, College of Veterinary Medicine, Texas A&M University, Raymond Stotzer Pkwy., College Station, TX 77843-4467. Tel.: 979-845-4207; Fax: 979-862-1088; E-mail: bbinas{at}cvm.tamu.edu.
1 The abbreviations used are: L-FABP, liver fatty acid-binding protein; SCP-2, sterol carrier protein-2; SCP-x, sterol carrier protein-x/3-ketoacyl-CoA thiolase; FATP, fatty acid transport protein; FAT/CD36, fatty acid translocase; PBS, phosphate-buffered saline; GST, glutathione S-transferase.
2 G. Martin and F. Schroeder, unpublished result.
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ACKNOWLEDGMENTS |
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REFERENCES |
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