Decreased Liver Fatty Acid Binding Capacity and Altered Liver Lipid Distribution in Mice Lacking the Liver Fatty Acid-binding Protein Gene

Although liver fatty acid-binding protein (L-FABP) is an important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2- to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected [14C]oleate was markedly reduced, showing altered lipid pool turnover. An increase of ∼75% of soluble SCP-2 but little or no change of other soluble (glutathione S-transferase, albumin) and membrane (fatty acid transport protein, CD36, aspartate aminotransferase, caveolin) fatty acid transporters was measured. These results (i) provide for the first time a quantitative assessment of the contribution of L-FABP to cytosolic fatty acid binding capacity, (ii) establish L-FABP as an important determinant of hepatic lipid composition and turnover, and (iii) suggest that SCP-2 contributes to the accumulation of cholesterol in L-FABP null liver. Although liver fatty acid-binding protein (L-FABP) is an important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2- to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected [14C]oleate was markedly reduced, showing altered lipid pool turnover. An increase of ∼75% of soluble SCP-2 but little or no change of other soluble (glutathione S-transferase, albumin) and membrane (fatty acid transport protein, CD36, aspartate aminotransferase, caveolin) fatty acid transporters was measured. These results (i) provide for the first time a quantitative assessment of the contribution of L-FABP to cytosolic fatty acid binding capacity, (ii) establish L-FABP as an important determinant of hepatic lipid composition and turnover, and (iii) suggest that SCP-2 contributes to the accumulation of cholesterol in L-FABP null liver. Liver fatty acid-binding protein (L-FABP), 1The 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.1The 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. a member of the genetically related cytosolic fatty acid-binding protein (FABP) family (1McArthur M.J. Atshaves B.P. Frolov A. Foxworth W.D. Kier A.B. Schroeder F. J. Lipid Res. 1999; 40: 1371-1383Abstract Full Text Full Text PDF PubMed Google Scholar, 2Storch J. Thumser A.E. Biochim. Biophys. Acta. 2000; 1486: 28-44Crossref PubMed Scopus (421) Google Scholar, 3Zimmerman A.W. Veerkamp J.H. Cell. Mol. Life Sci. 2002; 59: 1096-1116Crossref PubMed Scopus (405) Google Scholar), is found in the liver, intestine, and kidney, but only in liver is it not co-expressed with other members of its family. L-FABP is known to bind fatty acids and various other hydrophobic molecules, although its actual contribution to the lipid-binding capacity of liver cytosol is not known. Given that L-FABP is expressed at very high levels (2–5% of cytosolic protein) in the differentiated hepatocyte (4Bordewick U. Heese M. Borchers T. Robenek H. Spener F. Biol. Chem. Hoppe-Seyler. 1989; 370: 229-238Crossref PubMed Scopus (104) Google Scholar, 5Starodub O. Jolly C.A. Atshaves B.P. Roths J.B. Murphy E.J. Kier A.B. Schroeder F. Am. J. Physiol. 2000; 279: C1259-C1269Crossref PubMed Google Scholar) and that these levels correlate well with lipid metabolism (2Storch J. Thumser A.E. Biochim. Biophys. Acta. 2000; 1486: 28-44Crossref PubMed Scopus (421) Google Scholar), it can be speculated that L-FABP contributes considerably to hepatic lipid-binding and lipid metabolism. Work with cell-free systems and transfected cells has further strengthened this view. For example, in cell-free preparations L-FABP was shown to stimulate the esterification of oleic acid while inhibiting that of palmitic acid (6Jolly C.A. Wilton D.A. Schroeder F. Biochim. Biophys. Acta. 2000; 1483: 185-197Crossref PubMed Scopus (64) Google Scholar). L cells overexpressing L-FABP show increased rates of fatty acid uptake and esterification (7Murphy E.J. Prows D.R. Jefferson J.R. Schroeder F. Biochim. Biophys. Acta. 1996; 1301: 191-198Crossref PubMed Scopus (122) Google Scholar) as well as increased contents of phospholipid and cholesterol esters (8Jefferson J.R. Powell D.M. Rymaszewski Z. Kukowska-Latallo J. Schroeder F. J. Biol. Chem. 1990; 265: 11062-11068Abstract Full Text PDF PubMed Google Scholar, 9Murphy E.J. Prows D.R. Stiles T. Schroeder F. Lipids. 2000; 35: 729-738Crossref PubMed Scopus (33) Google Scholar). HepG2 hepatoma cells expressing an L-FABP antisense RNA showed a dose-dependent reduction of fatty acid uptake (10Wolfrum C. Buhlman C. Rolf B. Borchers T. Spener F. Biochim. Biophy. Acta. 1999; 1437: 194-201Crossref PubMed Scopus (80) Google Scholar). Furthermore, overexpression of L-FABP in McA-RH7777 hepatoma cells incubated with palmitic acid decreased the synthesis and secretion of triglycerides while increasing beta oxidation and the secretion of apolipoprotein B100 (11Linden D. Lindberg K. Oscarsson J. Claesson C. Asp L. Li L. Gustafsson M. Boren J. Olofsson S.O. J. Biol. Chem. 2002; 277: 23044-23053Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Thus, the various in vitro systems have allowed researchers to propose specific functions of L-FABP in vivo. 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. Materials—REDTaq DNA polymerase (Sigma-Aldrich, St. Louis, MO) was used for all standard PCR reactions, whereas JumpStart REDAccuTaq polymerase (Sigma-Aldrich) was used for long PCR. Protease inhibitor mixture for mammalian tissues, ultra-low range color markers for SDS-PAGE, oleic acid (∼99%), gel filtration molecular mass markers (range, 6,500–66,000 Da), and 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium for Western analysis were also from Sigma-Aldrich. Protein Assay Dye Reagent Concentrate was obtained from Bio-Rad Laboratories (Richmond, CA). 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 Proteins—Mouse 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 (12Pu L. Foxworth W.B. Kier A.B. Annan R.S. Carr S.A. Edmondson R. Russell D. Wood W.G. Schroeder F. Prot. Expr. Purif. 1998; 13: 337-348Crossref PubMed Scopus (18) Google Scholar, 13Atshaves B.P. Petrescu A. Starodub O. Roths J. Kier A.B. Schroeder F. J. Lipid Res. 1999; 40: 610-622Abstract Full Text Full Text PDF PubMed Google Scholar). Antibodies—Rabbit 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 (14Schroeder F. Atshaves B.P. Starodub O. Boedeker A.L. Smith R. Roths J.B. Foxworth W.B. Kier A.B. Mol. Cell. Biochem. 2001; 219: 127-138Crossref PubMed Scopus (30) Google Scholar), anti-mouse SCP-2 (5Starodub O. Jolly C.A. Atshaves B.P. Roths J.B. Murphy E.J. Kier A.B. Schroeder F. Am. J. Physiol. 2000; 279: C1259-C1269Crossref PubMed Google Scholar), and anti-mouse SCP-x (13Atshaves B.P. Petrescu A. Starodub O. Roths J. Kier A.B. Schroeder F. J. Lipid Res. 1999; 40: 610-622Abstract Full Text Full Text PDF PubMed Google Scholar) 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 Mice—Primers 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 P1–129/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 (15Wolfrum C. Ellinghaus P. Fobker M. Seedorf U. Assmann G. Borchers T. Spener F. J. Lipid Res. 1999; 40: 708-714Abstract Full Text Full Text PDF PubMed Google Scholar) 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 (16Magin T.M. McWhir J. Melton D.W. Nucleic Acids Res. 1992; 20: 3795-3796Crossref PubMed Scopus (208) Google Scholar). 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 (17Bradley A. Robertson E.J. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford/IRL Press, New York1987: 113-152Google Scholar). 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 (18Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar) 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; 32× (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. RT-PCR—Total 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 (18Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). 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. Animals—Chimeric 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 13–15 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 Collection—For 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 Parameters—Serum 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 Fractionation—All 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 × g for 10 min, the resulting post nuclear supernatant was further centrifuged at 105,000 × g for 90 min., yielding a pelleted fraction ("membranes") and a 105,000 × g supernatant ("cytosol"). Protein was quantified by the Bradford protein assay (Bio-Rad, Richmond, CA) (19Bradford M. Anal. Biochemistry. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). SDS-PAGE—16.5% SDS-PAGE was performed using the system of Schagger and von Jagow (20Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10410) Google Scholar), with minor modifications (21Atshaves B.P. Storey S. McIntosh A.L. Petrescu A.D. Lyuksyutova O.I. Greenberg A.S. Schroeder F. J. Biol. Chem. 2001; 276: 25324-25335Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). 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 Analysis—Proteins 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 × 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 × 5 min with TBST, 2 × 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 × 5 min with TBST, 2 × 5 min with TBS, and 1 × 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 × g Supernatant—A 1.5 × 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 × g supernatant protein was incubated with [3H]oleic acid (30 nmol, 1 × 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 Parameters—The assay has been previously described (22Frolov A. Cho T.H. Murphy E.J. Schroeder F. Biochemistry. 1997; 36: 6545-6555Crossref PubMed Scopus (93) Google Scholar). 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 (22Frolov A. Cho T.H. Murphy E.J. Schroeder F. Biochemistry. 1997; 36: 6545-6555Crossref PubMed Scopus (93) Google Scholar). Lipid Quantification—All 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 (9Murphy E.J. Prows D.R. Stiles T. Schroeder F. Lipids. 2000; 35: 729-738Crossref PubMed Scopus (33) Google Scholar, 21Atshaves B.P. Storey S. McIntosh A.L. Petrescu A.D. Lyuksyutova O.I. Greenberg A.S. Schroeder F. J. Biol. Chem. 2001; 276: 25324-25335Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). 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. (23Marzo A. Ghirardi P. Sardini D. Meroni G. Clin. Chem. 1971; 17: 145-147Crossref PubMed Scopus (73) Google Scholar). 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 Acid—Mice 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 (50–80 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 Analysis—Data 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). Creation of L-FABP Null Mice—The entire L-FABP gene was deleted by homologous recombination (Fig. 1A) in embryonic stem cells. The gene deletion was verified by long PCR (Fig. 1B), specificity of this reaction was confirmed by nested PCR (Fig. 1C, lanes 2–4), and specificity of the nested primers was in turn confirmed on genomic DNA (Fig. 1C, lanes 5–7). As expected from these results, RT-PCR failed to amplify L-FABP cDNA from total RNA of L-FABP (–/–) livers, whereas the control HPRT cDNA was readily amplified (Fig. 1D, lanes 2 and 3); in contrast, L-FABP cDNA was easily amplified from wild type RNA (Fig. 1D, lanes 4 and 5). To confirm the absence of L-FABP (14.2. kDa) on the protein level, Western blotting followed by quantitative densitometry was performed. The results (see Fig. 3A below) showed that L-FABP was present at 18.1 ± 3.0 ng/μg in homogenates and at 45.2 ± 3.3 ng/μg in 105,000 × g supernatants prepared from wild type livers. These data confirm earlier findings that L-FABP constitutes 2–3% of cytosolic proteins and is largely soluble (4Bordewick U. Heese M. Borchers T. Robenek H. Spener F. Biol. Chem. Hoppe-Seyler. 1989; 370: 229-238Crossref PubMed Scopus (104) Google Scholar, 5Starodub O. Jolly C.A. Atshaves B.P. Roths J.B. Murphy E.J. Kier A.B. Schroeder F. Am. J. Physiol. 2000; 279: C1259-C1269Crossref PubMed Google Scholar). In contrast, immunoreactivity was nondetectable in liver homogenates and 105,000 × g supernatants from the L-FABP (–/–) mice (Fig. 3A). This result demonstrates not only the absence of L-FABP but also of any potential close relatives that previously might have gone undetected. Thus, the L-FABP gene and its product are eliminated in the L-FABP (–/–) mice. General Characterization—L-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