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Originally published In Press as doi:10.1074/jbc.M309377200 on October 8, 2003

J. Biol. Chem., Vol. 278, Issue 51, 51664-51672, December 19, 2003
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Decreased Hepatic Triglyceride Accumulation and Altered Fatty Acid Uptake in Mice with Deletion of the Liver Fatty Acid-binding Protein Gene*

Elizabeth P. Newberry{ddagger}, Yan Xie{ddagger}, Susan Kennedy{ddagger}, Xianlin Han{ddagger}, Kimberly K. Buhman{ddagger}, Jianyang Luo{ddagger}, Richard W. Gross{ddagger}§, and Nicholas O. Davidson{ddagger}§

From the Departments of {ddagger}Internal Medicine and §Pharmacology and Molecular Biology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, August 25, 2003 , and in revised form, October 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Liver fatty acid-binding protein (L-Fabp) is an abundant cytosolic lipid-binding protein with broad substrate specificity, expressed in mammalian enterocytes and hepatocytes. We have generated mice with a targeted deletion of the endogenous L-Fabp gene and have characterized their response to alterations in hepatic fatty acid flux following prolonged fasting. Chow-fed L-Fabp–/– mice were indistinguishable from wild-type littermates with regard to growth, serum and tissue lipid profiles, and fatty acid distribution within hepatic complex lipid species. In response to 48-h fasting, however, wild-type mice demonstrated a ~10-fold increase in hepatic triglyceride content while L-Fabp–/– mice demonstrated only a 2-fold increase. Hepatic VLDL secretion was decreased in L-Fabp–/– mice suggesting that the decreased accumulation of hepatic triglyceride was not the result of increased secretion. Fatty acid oxidation, as inferred from serum {beta}-hydroxybutyrate levels, was increased in response to fasting, although the increase in L-Fabp–/– mice was significantly reduced in comparison to wild-type controls, despite comparable induction of PPAR{alpha} target genes. Studies in primary hepatocytes revealed indistinguishable initial rates of oleate uptake, but longer intervals revealed reduced rates of uptake in fasted L-Fabp–/– mice. Oleate incorporation into cellular triglyceride and diacylglycerol was reduced in L-Fabp–/– mice although incorporation into phospholipid and cholesterol ester was no different than wild-type controls. These data point to an inducible defect in fatty acid utilization in fasted L-Fabp–/– mice that involves targeting of substrate for use in triglyceride metabolism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A major function for differentiated mammalian enterocytes and hepatocytes centers on their ability to direct intracellular trafficking of long chain fatty acids, both in connection with the processing of exogenous dietary lipid and in the complex metabolic regulation of endogenous lipid homeostasis. Key to such intricate regulation is the ability to direct delivery of fatty acids to sites of complex lipid synthesis and to provide a physiological buffer in order to accommodate fluxes that accompany changes in fatty acid delivery. In regard to hepatic and intestinal fatty acid metabolism, there is an obvious need to accommodate large periodic fluxes, such as might occur in connection with dietary lipid ingestion or alternatively in association with changes in hepatic fatty acid uptake. An important component of such accommodation likely occurs following fatty acid uptake across the plasma membrane, where tissue-specific regulation of fatty acid binding has been attributed to distinct members of a large multigene family of small cytosolic lipid-binding proteins, among them liver (L-Fabp),1 heart (H-Fabp), adipocyte (A-Fabp), and intestinal fatty acid-binding protein (I-Fabp) (1, 2).

L-Fabp is expressed as an abundant gene product in differentiated enterocytes and hepatocytes, with lower levels of expression in the kidney and colon (1, 3). L-Fabp binds a broad range of ligands, including fatty acids, with a preference for unsaturated versus saturated fatty acids, branched-chain fatty acids, cholesterol, and bile acids (4–8). Extensive study using in vitro binding and crystallographic analyses indicate that each molecule of L-Fabp binds two molecules of fatty acid (9, 10), with binding facilitated through diffusional interactions (11, 12). These data, coupled with studies from both transfected cells (13) and also from quantitative estimates of endogenous hepatic fatty acid binding activity (14), suggest that L-Fabp is likely the most important single source of such binding in mammalian liver.

In addition to fatty acid binding and sequestration, emerging evidence suggests that L-Fabp may be involved in regulating the activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) through its ability to shuttle ligand (polyunsaturated fatty acids and peroxisome proliferators) to the nucleus (15, 16). Further study has revealed that L-Fabp interacts physically with PPAR{alpha} both in vitro and in vivo, providing a plausible basis for the possibility that ligand delivery via L-Fabp may represent a potential regulatory restriction point in this metabolic cascade (15).

However, despite the observation that L-Fabp represents one of the most abundant gene products in enterocytes and hepatocytes, details of its physiological functions in vivo have yet to be elucidated. Studies in HepG2 cells implicated L-Fabp in fatty acid uptake following antisense RNA expression (13), while gain-of-function experiments revealed increased fatty acid uptake and augmented lipoprotein secretion from rat hepatoma cells (17), suggesting that L-Fabp may play an important role in fatty acid delivery and metabolic utilization. In order to study the role of L-Fabp in a more representative physiological context, we have generated a mouse line with a targeted deletion in the L-Fabp gene. These mice were used to examine elements of the physiological adaptations associated with prolonged fasting, a well established model of altered hepatic fatty acid flux in which mobilization of adipose tissue triglyceride (TG) stores results in the release of large amounts of fatty acids for delivery and uptake by hepatocytes (18).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Serum- and tissue-free fatty acid, total cholesterol, and triglyceride concentrations were determined using NEFA C, Cholesterol E, and L-Type TG-H kits, respectively, from Wako Chemicals. Serum glucose concentrations were measured using a kit from Sigma. Serum {beta}-hydroxybutyrate levels were measured by RIA and serum insulin levels were determined by ELISA using a kit from Crystal Chem (Chicago, IL). Anti CD-36 IgG was obtained from Cascade Bioscience (catalog ABM-5525), anti-GFP antiserum was obtained from Santa Cruz Biotechnology (sc-8334), anti-albumin antiserum was from ICN. Anti-L-Fabp and I-Fabp antisera were a generous gift from Dr. Jeff Gordon. The anti-FATP1 antiserum was kindly provided by Dr. Jean Schaffer. Rabbit polyclonal antisera to mouse FATP2, FATP4, and FATP5 were generated by Zymed Laboratories Inc. (San Francisco, CA) by injecting peptides corresponding to the sequence of each FATP isoform: FATP2, CRRVRSYRQRRPVR; FATP4, CLDQEAYTRIQAGEEKL; FATP5, KPDVYQAVCEGTWNL. The resulting antisera were affinity purified by incubation with the immunizing peptide coupled to resin. Control experiments demonstrated that each FATP antiserum specifically recognized only the appropriate family member. Western blotting used the ECL reagent kit from Amersham Biosciences.

Generation of L-Fabp–/– Mice—A genomic BAC clone was obtained by screening a 129/SvJ genomic BAC library (Incyte, St. Louis, MO) with a full-length mouse L-Fabp cDNA clone. The BAC was digested with EcoRI, and the fragments subcloned into pUC19. Clones containing the L-Fabp gene were identified by colony hybridization and mapped by restriction digest and partial sequencing. To create the 5'-arm, a fragment containing 2.5 kb of DNA upstream of the initiator methionine was amplified by PCR using the BAC as a template. To create a "GFP-knock in" construct, the initiator ATG of L-Fabp was replaced with the ATG of green fluorescent protein by overlapping PCR, using pEGFP-N1 (Clontech) as a template for the coding sequence and polyadenylation signal of GFP. This arm was then subcloned into the Kpn/Bam sites of p1339 (accession number AF335420 [GenBank] , a kind gift from Dr. Tim Ley), using restriction sites introduced by PCR. The 3kb 3'-arm, which includes exons 3 and 4 of the L-Fabp gene, was also amplified by PCR using the BAC clone as a template, and subcloned into the XhoI/ApaI sites of p1339. The targeting construct was electroporated into the RW-4 subclone of 129X1/SvJ embryonic stem (ES) cells. Genomic DNA was isolated from G418-resistant colonies and Southern blot analysis was performed on XbaI-digested DNA using a 32P-labeled 300 nucleotide probe corresponding to sequence upstream of the 5'-arm (Fig. 1). In 2 out of 65 clones, a 5-kb band was detected in addition to the 11-kB wild-type band, indicating homologous recombination of one of the L-Fabp alleles. Additional Southern blots were performed to confirm complete integration of the targeting construct. ES clones were individually injected into C57BL/6 blastocysts, yielding two high percentage chimeras, one from each ES cell line. Both chimeric founders produced offspring containing the targeted allele when crossed with C57BL/6 females. Intercrosses of the heterozygotes produced viable and fertile homozygous L-Fabp–/– mice at the expected Mendelian frequency in both sexes. The two lines of mice have been maintained separately on a C57BL/6 129/SvJ background and appear to display an identical phenotype. Mice were housed in a full barrier facility with a 12-hour light/dark cycle, and were maintained on standard chow (Picolab Rodent Diet) with free access to food and water unless otherwise noted. Age-matched, male mice between the ages of 8 and 14 weeks were used for all experiments, with L-Fabp+/+ mice on a mixed 129/SvJ C57BL/6 background used as controls. All animal protocols were approved by the Washington University Animal Studies Committee.



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  		FIG. 1.
Creation and characterization of L-Fabp-deficient mice. A, endogenous mouse L-Fabp gene and gene-targeting vector. Homologous recombination of the 3'- and 5'-arms of the targeting vector (bottom) with the L-Fabp locus (top) should result in the replacement of exons 1 and 2 with the coding sequence of GFP and a LoxP-flanked neomycin (Neo) cassette. Southern blot analysis of XbaI-digested genomic DNA should produce an 11-kB band in the wild-type allele and a 5-kB band in the targeted allele, using a probe upstream of the 5'-arm. Arrowheads denote the approximate position of the initiator methionines of L-Fabp and GFP; the arrow indicates that the Neo cassette is in the opposite orientation with respect to GFP. X, XbaI restriction site. B, Northern blot Analysis. Total RNA was extracted from the proximal small intestine (SI), colon, liver, and kidney of L-Fabp+/+ (lanes 1–4) and L-Fabp–/– (lanes 5–8) mice, resolved by electrophoresis, and hybridized with 32P-labeled DNA probe corresponding to the cDNA sequence of L-Fabp. The blot was stripped and reprobed with a 32P-labeled Gapdh probe to demonstrate the presence of intact RNA in every lane. C, Western blot analysis. Equal amounts of protein from proximal small intestine, liver, kidney and colon of L-Fabp+/+ (lanes 1–4) and L-Fabp–/– (lanes 5–8) mice were resolved by SDS-PAGE and probed with antisera to L-Fabp, intestinal fatty acid-binding protein (I-Fabp), and GFP. Note that there is no up-regulation of I-Fabp expression in the intestine of L-Fabp–/– mice (compare lanes 1 and 5). The expression pattern of GFP in L-Fabp–/– mice mirrors the expression pattern of endogenous L-Fabp in L-Fabp+/+ mice (lanes 1–4).

 
Hepatocyte Isolation and Uptake Assay—Mice were studied following ad libitum feeding or following a 48-h fast. Hepatocytes were isolated by liver perfusion as previously described (19). Viable hepatocytes were by identified by trypan blue exclusion and counted. For the uptake assay, 200 µl of assay buffer (40 µM sodium oleate, 10 µM fatty acid-free bovine serum albumin, 5 µCi/ml [3H]oleate in phosphate-buffered saline (PBS) with 1 mM MgCl2 and 1.2 mM CaCl2) was added to 200 µl of the cell suspension and incubated for various times at 37 °C. Assays were stopped by the addition of 5 ml of ice-cold stop solution (PBS containing 1 mM MgCl2, 1.2 mM CaCl2, 0.1% fatty acid-free bovine serum albumin, and 500 µM phloretin). Cells were pelleted by centrifugation at 500 x g, resuspended in 200 µl of water, and analyzed for cell associated-radioactivity by scintillation counting. All assays were performed in duplicate and corrected for the number of viable cells per assay. Nonspecific binding was determined by simultaneous addition of uptake assay buffer and stop solution to 200 µl of the cell suspension on ice. This background value was obtained for each cell preparation and subtracted from all time points. Lipids were extracted with chloroform/methanol, dried under nitrogen, and resolved by thin layer chromatography (70 ml of hexane/30 ml of ethyl ether/1 ml of acetic acid).

Isolation of RNA and Protein—Tissue was collected, washed in PBS, and flash frozen in liquid nitrogen and stored at –80 °C until needed. For isolation of RNA, tissues were homogenized in Trizol (Invitrogen) and 20 µg of total RNA used for Northern blot analyses. For preparation of protein extracts, tissues were homogenized in 25 mM Hepes (pH7.9), 150 mM sodium chloride, 0.5 mM EDTA containing 1% Triton, 0.1% SDS, and protease inhibitors (1 Complete-Mini tablet (Roche Diagnostics, Indianapolis, IN) per 10 ml of solution). Samples were incubated on ice for 15 min, centrifuged at 12,000 rpm for 10 min to remove insoluble material, and assayed for protein concentration (BioRad DC Protein Assay, BioRad) prior to Western blotting.

Real Time PCR—Total RNA was treated with DNaseI using the DNA-free kit (Ambion) and incubated with SuperScript II Reverse Transcriptase (Invitrogen) to prepare cDNA. Real time PCR reactions were performed on SDS 7000 (Applied Biosystems) using 2x Sybr Green Master Mix (Applied Biosystems) as directed by the manufacturer. Relative gene expression was determined using the comparative Ct method (User Bulletin 2, Applied Biosystems). PCR primers used were as follows (5' -> 3'): Fatp2, GGT ATG GGA CAG GCC TTG CT and GGG CAT TGT GGT ATA GAT GAC ATC; Scp2, GGC TTT GAT GAC TGG AAA AAT GA and CCC ATG TTA CCA GCA ATC TTC A; HMGS, TGG TGG ATG GGA AGC TGT CTA and TTC TTG CGG TAG GCT GCA TAG; MCAD, TGA CGG AGC AGC CAA TGA and ATG GCC GCC ACA TCA GA; 18S, CGG CTA CCA CAT CCA AGG AA and GCT GGA ATT ACC GCG GCT; ACO, GGA TGG TAG TCC GGA GAA CA and AGT CTG GAT CGT TCA GAA TCA AG.

VLDL Secretion—Hepatic VLDL production was determined as described (20). Mice were weighed and injected intravenously with 20 mg of Tyloxapol (Sigma T-8761) in 100 µl of PBS. Serum was collected prior to injection of tyloxapol and at 30-min intervals thereafter up to 2 h, and triglyceride content determined.

Lipid Extraction and Analysis—Livers were homogenized in PBS and protein concentration determined. 300 µl of homogenate was extracted with 5 ml of chlorofom/methanol (2:1) and 0.5 ml of 0.1% sulfuric acid (21). An aliquot of the organic phase was collected, dried under nitrogen, and resuspended in 2% Triton X-100. Hepatic FFA, TG, PL, and cholesterol content were determined using commercially available kits. Data were normalized for differences in protein concentration. For mass spectrometry, lipids were extracted using LiCl as detailed (22) and analyzed using both positive ion and negative ion electrospray ionization/mass spectrometry (23). Oil red O staining of frozen liver samples was conducted using established methods.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of L-Fabp–/– Mice—The targeting construct was designed to replace the initiator methionine and coding sequence of exon 1 and 2 with the ATG and coding sequence of green fluorescent protein (GFP) (Fig. 1A). Clones demonstrating homologous recombination were identified and injected into C57BL/6 blastocysts. Intercrosses of the resultant L-Fabp+/– mice yielded viable L-Fabp–/– mice at the expected Mendelian frequency. Expression of L-Fabp mRNA (Fig. 1B) and protein (Fig. 1C) were undetectable in L-Fabp–/– mice. GFP protein was expressed in a tissue specific manner, its distribution paralleling the expression of endogenous L-Fabp in wild-type mice (Fig. 1C). No compensatory up-regulation of I-Fabp protein or mRNA was detected in the small intestine or the liver of L-Fabp–/– mice (Fig. 1C). There was also no compensatory change in mRNA abundance of epidermal, adipocyte, brain, or heart FABPs as inferred from semi-quantitative RT PCR (data not shown). Findings in relation to SCP2 mRNA and protein abundance will be presented in a later section of this report.

Phenotype of L-Fabp–/– Mice—There was no difference in postnatal growth or body weight of either male or female mice (Fig. 2), or in liver or epididymal fat pat weights of male wild-type (WT) or L-Fabp–/– mice at 10–14 weeks of age (Table I). Furthermore, there were no differences in the equivalent parameters in female mice (data not shown). No difference was observed in serum TG, cholesterol, FFA, or glucose levels in WT versus L-Fabp–/– mice (Table I), nor was there a difference in serum ApoB100 or B48 levels as detected by Western blotting (data not shown). Serum insulin levels were significantly decreased in fed male L-Fabp–/– mice (1200 versus 737 pg/ml) in the setting of indistinguishable levels of free fatty acids (Table I). These findings are of interest in view of the demonstration that mice lacking intestinal fatty acid-binding protein (I-Fabp) display hyperinsulinemia when fed either a low or high fat diet (24). There was no difference in hepatic TG, FFA, phospholipid, or cholesterol content in L-Fabp–/– mice compared with WT controls (Table I), indicating that the absence of L-Fabp does not dramatically alter the abundance of complex lipid species within the liver. These findings are at variance with the recent report of Martin et al. (14), who demonstrated a 3-fold increase in hepatic cholesterol content in L-Fabp–/– mice; this point will be discussed below. Because L-Fabp binds a wide range of saturated and unsaturated fatty acids, we examined the fatty acid species present in hepatic free fatty acids, TG, and phospholipid of ad libitum fed WT and L-Fabp–/– mice. Overall, no dramatic differences were observed in fatty acid species present in free fatty acids, TG, phosphatidylcholine, or phosphatidylethanolamine, in terms of either mass (data not shown) or species distribution (Fig. 3). Thus, the absence of L-Fabp does not dramatically alter complex lipid abundance or the individual component fatty acid species present in these lipids within the liver.



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  		FIG. 2.
Growth of wild type and L-Fabp–/– mice. Offspring generated from heterozygous intercrosses of L-Fabp+/– mice were weighed 9–10 days after birth and at 2–3 day intervals thereafter, until ~6 weeks of age. Data shown include only L-Fabp+/+ and L-Fabp–/– mice, with male and female mice grouped separately. n = 5–8 mice per group.

 


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  		TABLE I
Fed male mice aged 10–14 weeks were used for all analyses

n indicates the number of mice analyzed for each parameter. Data are expressed as the mean ± S.E. Significance was determined using a Student's t test.

 



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  		FIG. 3.
Electrospray ionization mass spectrometric analysis of lipids from wild type and L-Fabp–/– mice. Liver tissue was obtained from male L-Fabp+/+ and L-Fabp–/– mice fed ad libitum. Lipids were extracted with chloroform:methanol and analyzed by mass spectrometry as described under "Experimental Procedures." Fatty acid species present in free fatty acids (panel A), triglyceride (panel B), phosphatidylcholine (panel C), and phosphatidylethanolamine (panel D) were quantitated and are expressed as a percent of the total amount of lipids in each class. Gray bars represent data from three L-Fabp+/+ mice; open bars represent data from three L-Fabp–/– mice. Results are shown as the mean ± S.D.

 
L-Fabp–/– Mice: Response to Metabolic Challenge of Prolonged Fasting—Fasting stimulates the release of free fatty acids from adipose tissue, triggering increased uptake and oxidation of fatty acids by the liver. As shown in Fig. 4A, livers of WT mice contain abundant TG droplets following a 48-h fast. By contrast, livers of fasted L-Fabp–/– mice accumulate significantly less neutral lipid (Fig. 4B). Quantitative determination revealed that hepatic TG content was ~4-fold lower in fasted L-Fabp–/– mice compared with WT controls (Fig. 4C), with no difference in the distribution of fatty acid species present in these triglycerides (data not shown). Consistent with decreased hepatic steatosis, liver weight in fasted L-Fabp–/– mice was significantly reduced compared with fasted controls (Table II). As shown in Table II, no significant differences were observed in serum FFA, glucose, insulin, or cholesterol levels between fasted WT and L-Fabp–/– mice. Additionally, there was no difference in hepatic concentrations of cholesterol or non-esterified fatty acids between fasted WT and L-Fabp–/– mice (Table II) indicating that fatty acids are not being redirected into cholesterol or cholesterol ester synthesis rather than being stored as TG. Moreover, there was no significant difference in the profile of fatty acid species present in the livers of fasted WT or L-Fabp–/– mice when examined by mass spectrometry (data not shown). Hepatic phospholipid content was significantly decreased in fasted L-Fabp–/– mice compared with ad libitum fed controls (48.4 ± 4.0 (n = 8) versus 65.3 ± 4.4 (n = 12), p = 0.013), yet no difference was observed between fasted and fed WT mice. Despite a trend toward lower hepatic PL content in fasted L-Fabp–/– mice versus fasted controls (Table II), these differences were not statistically significant. To further pursue the possibility that subtle differences may have been overlooked, we examined PL content of hepatocytes isolated from fasted mice of both genotypes. These data again revealed indistinguishable PL content between the groups (WT: 123.4 ± 13.1 µg/mg protein; L-Fabp–/–: 103.3 ± 12.0 µg/mg protein; n = 4). Bile collected from the gall bladders of fasted wild type and L-Fabp–/– mice revealed no difference in biliary cholesterol or phospholipid concentration in fasted wild-type versus fasted L-Fabp–/– mice (data not shown). These findings indicate that hepatic TG accumulation is decreased in L-Fabp–/– mice in response to prolonged fasting, without obvious redistribution of fatty acids into other complex lipid species or accumulation in either the plasma or liver.



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  		FIG. 4.
Neutral lipid accumulation in livers of fasted wild type and L-Fabp–/– mice. Panels A and B, Oil Red O-stained liver tissue from male wild-type (A) and L-Fabp–/– mice (B) fasted for 48 h. Representative photomicrographs are shown. C, hepatic triglyceride content. Lipids were extracted from liver tissue obtained from male wild-type or L-Fabp–/– mice fed ad libitum (black bars) or fasted for 48 h (gray bars) and analyzed by mass spectrometry. Data are expressed as nmoles of triglyceride per milligram of protein and represent the means ± S.E. Number of animals per group: Fed WT, n = 3; Fed L-Fabp–/–, n = 3; Fasted WT, n = 5; Fasted L-Fabp–/–, n = 7. The asterisk indicates p <= 0.001 versus fasted WT mice.

 


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  		TABLE II
Fasted male mice aged 10–14 weeks were used for all analyses

n indicates the number of mice analyzed for each parameter. Data are expressed as the mean ± S.E. Liver triglyceride content of fasted mice is shown in Fig. 4C. Significance was determined using a Student's t test.

 
Hepatic Fatty Acid Utilization in L-Fabp–/– Mice—Additional experiments were performed to investigate the mechanism(s) responsible for decreased TG accumulation in fasted L-Fabp–/– mice. One possibility was that TG is preferentially incorporated into VLDL particles for secretion versus storage, resulting in decreased accumulation of hepatic TG. However, hepatic TG secretion rates in fasted L-Fabp–/– mice were significantly lower than in fasted WT controls (Fig. 5). Thus, the decreased neutral lipid accumulation in fasted L-Fabp–/– mice cannot be explained by increased TG secretion.



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  		FIG. 5.
VLDL secretion in wild type and L-Fabp–/– mice. Male WT and L-Fabp–/– mice were fasted for 48 h, then injected intravenously with Tyloxapol. Serum was obtained prior to injection and at 30-min intervals thereafter. Serum triglyceride levels were determined and are expressed as mg/dL. Values shown represent the means ± S.E. of data obtained from 6–7 mice per group. Open squares, L-Fabp+/+ mice; closed circles, L-Fabp–/– mice. An asterisk denotes p <= 0.05 when compared with fasted L-Fabp+/+ mice.

 
A second possibility was that decreased accumulation of hepatic TG in fasted L-Fabp–/– mice reflects increased rates of {beta}-oxidation, resulting in less fatty acid available for storage as TG. To examine this possibility we measured ketone body production using the surrogate of serum {beta}–hydroxybutyrate. This revealed a ~9-fold increase in WT mice following prolonged fasting (Fig. 6A), compared with a 3.5-fold induction in L-Fabp–/– mice, suggesting that induction of ketogenesis is significantly reduced in fasted male L-Fabp–/– mice. We further examined the expression of several PPAR{alpha} target genes, including medium chain acyl-CoA dehydrogenase (25), HMG-CoA synthetase (26), acyl-CoA oxidase (25), and fatty acid transport protein 2 (27) (also known as very long chain acyl-CoA synthetase and believed to be involved in peroxisomal {beta}-oxidation) in WT and L-Fabp–/– mice following prolonged fasting. As shown in Fig. 6B, there was no significant difference in the induction of any of these genes in WT versus L-Fabp–/– mice after fasting. These data suggest that L-Fabp is not required for activation of PPAR{alpha} in response to fasting, and that the decreased induction of ketogenesis in fasted L-Fabp–/– mice is not due to decreased expression of genes involved in fatty acid oxidation. In addition, we observed no difference in oxygen consumption or carbon dioxide production levels in fed or fasted wild-type and L-Fabp–/– mice (data not shown). Finally, expression of UCP2 mRNA was similar in the livers of fed and fasted wild-type and L-Fabp–/– mice (data not shown). Together, these data suggest that fatty acids are not being utilized for heat production and that there is no overall change in energy utilization in L-Fabp–/– mice.



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  		FIG. 6.
Induction of fatty acid oxidation in fasted wild-type and L-Fabp–/– mice. A, levels of serum {beta}-hydroxybutyrate in fed and fasted wild type and L-Fabp–/– mice. Male mice were either fed ad libitum (black bars) or fasted for 48 h (gray bars). Serum was collected and assayed for levels of {beta}-hydroxybutyrate. Values shown represent the means ± S.E. of data obtained from 6–10 animals per group. The asterisk denotes p < 0.001 versus fasted WT mice. B, alterations in gene expression in L-Fabp+/+ and L-Fabp–/– mice after fasting. RNA was obtained from the livers of ad libitum fed or fasted, WT or L-Fabp–/– mice, 6 mice per group. Expression of medium chain acyl coA dehydrogenase (MCAD), HMG-CoA synthetase (HMGS), fatty acid transport protein 2 (FATP2), and Acyl-CoA oxidase (ACO) was examined by real time PCR. Data from L-Fabp+/+ (gray bars) and L-Fabp–/– (open bars) mice represent the average fold induction of each gene after a 48-h fast, as calculated using the Comparative Ct method (User Bulletin 2, Applied Biosystems). Differences between WT and L-Fabp–/– mice are not significant.

 
Alterations in Fatty Acid Uptake and Distribution in L-Fabp–/– Mice—Fasted L-Fabp–/– mice display decreased hepatic secretion of TG, decreased ketone body synthesis, and decreased accumulation of hepatic TG. Together these data suggest that there may be decreased hepatic free fatty acid availability in these mice, perhaps due to a defect in fatty acid import/uptake or trafficking. To examine this possibility, hepatocytes were isolated from fed and fasted WT and L-Fabp–/– mice and incubated with radiolabeled oleate. Hepatocytes from fasted L-Fabp–/– mice displayed significantly less cell-associated radioactivity at 45 s and 120 s compared with fasted WT mice (Fig. 7A). Note that cell-associated radioactivity was similar at 15 and 30 s, suggesting no difference in the initial rates of fatty acid uptake. Oleate uptake by hepatocytes isolated from ad libitum-fed mice demonstrated values intermediate between fasted WT and L-Fabp–/– mice with no significant differences between the groups (Fig. 7A). To examine incorporation of fatty acid into complex lipids, isolated hepatocytes from fasted mice were incubated with radiolabeled oleate for 10 min, washed, and extracted with chloroform/methanol. Total radioactivity incorporated into complex lipid was decreased more than 50% in cells isolated from fasted L-Fabp–/– mice (Fig. 7B). Analysis of individual lipids shows a decrease in radiolabel incorporation into TG and diacylglycerol, while incorporation into phospholipid and cholesterol ester was unchanged (Fig. 7B). These findings are most consistent with the possibility that the early phase of fatty acid uptake is normal but that the absence of L-Fabp compromises the ability of hepatocytes from fasted mice to retain and target fatty acids for TG synthesis and {beta}-oxidation. These findings suggest that the adaptation(s) required to accommodate the increased flux of fatty acids associated with prolonged fasting at least partially require L-Fabp.



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  		FIG. 7.
Uptake and incorporation of [3H]oleate into hepatocytes from fed and fasted wild type and L-Fabp–/– mice. A, short term uptake of [3H]oleate. Hepatocytes were isolated from wild-type (solid lines) and L-Fabp–/– mice (dashed lines) fed ad libitum (squares) or following a 48-h fast (triangles). Cells were incubated with [3H]oleate for 15, 30, 45, or 120 s. Assays were stopped, and cell-associated radioactivity was determined. All assays were performed in duplicate and results shown represent the means ± S.E. of data obtained from 4–5 male mice per group. An asterisk indicates p <= 0.05 between fasted L-Fabp–/– and fasted L-Fabp+/+ mice. B, incorporation of [3H]oleate into complex lipids. Hepatocytes were isolated from fasted L-Fabp+/+ (open bars) and L-Fabp–/– (black bars) mice and incubated with [3H]oleate for 10 min. Lipids were extracted from the cells with chloroform/methanol and separated by thin layer chromatography. The position of radiolabeled TG, DAG, PL, and CE on the TLC plates was determined based on the migration of standards. Total incorporation of [3H]oleate into complex lipids is also shown (far left, labeled Total). Values shown represent the means ± S.E. of data obtained from 4–5 mice per group, assayed in duplicate. Asterisks indicate p <= 0.02 versus fasted WT mice.

 
We considered the possibility that compensatory alterations in some of the candidate fatty acid transport proteins might contribute to the phenotype observed in fasting L-Fabp–/– mice, specifically in regard to fatty acid uptake. Accordingly, we examined the expression of several putative fatty acid transport proteins as well as sterol carrier protein x (SCPx) and SCP2 in livers of fasted WT and L-Fabp–/– mice. SCP2 was considered to be a particularly relevant target since a recent report by Martin et al. (14) demonstrated a significant increase in hepatic SCP2 expression in female L-Fabp–/– mice, coupled with a 75% decrease in the precursor form, SCPx. In contrast to the findings reported by these workers, our results indicate that the abundance of SCPx and SCP2 was indistinguishable in both fed and fasted WT and L-Fabp–/– mice, both in males (Fig. 8B) and females (data not shown). Furthermore, there were no significant differences between WT and L-Fabp–/– mice in regard to the abundance of any of the putative fatty acid transporters examined, including FATP1, FATP2, FATP4, FATP5 (Fig. 8A). These findings are of special interest with regard to FATP1 and FATP4, since these proteins are likely present on the plasma membrane (28, 29). The abundance of CD36 appears to be slightly increased in fed L-Fabp–/– mice, findings in accord with the results of Martin et al. (14). In addition, CD36 protein levels increased after fasting, with WT mice demonstrating a greater increase compared with L-Fabp–/– mice (Fig. 8A).



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  		FIG. 8.
Expression of fatty acid transport proteins and fatty acid-binding proteins in fasted wild-type and L-Fabp–/– mice. Tissue homogenates were prepared from the livers of fasted and ad libitum fed wild-type and L-Fabp–/– mice, with an n of 3 male mice per group. Equal amounts of protein were separated on either 10% (panel A) or 14% (panel B) SDS-PAGE gels and subjected to Western blot analysis using antisera to CD36, FATP1, FATP2, FATP4, and FATP5 (panel A) or SCP2, L-Fabp, and GFP (panel B). Blots were stripped and reprobed with anti-albumin antibody to demonstrate equal loading of protein. Lanes 1–3, fed L-Fabp+/+; lanes 4–6, fed L-Fabp–/–; lanes 7–9, fasted L-Fabp+/+; lanes 10–12, fasted L-Fabp–/–.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
L-Fabp is a member of a large multigene family of short, cytosolic lipid-binding proteins and represents one of the most abundant gene products in the mammalian enterocyte and hepatocyte (1, 2). These properties, coupled with its diverse ligand-binding activity, suggested several possible outcomes that might accompany targeted deletion of L-Fabp. Our conclusions, based upon the initial characterization reported herein, suggest that L-Fabp–/– mice, while consuming a low fat chow diet, demonstrate no obvious phenotype with respect to either cholesterol or complex lipid accumulation, and fail to exhibit alterations in their component fatty acid species in the liver. However, under circumstances when fatty acid flux to the liver is augmented, in this case following prolonged fasting, there is a detectable phenotype characterized by diminished hepatic fatty acid utilization and a specific decrease in targeting of oleate for triglyceride synthesis as well as for storage and secretion. These features suggest that L-Fabp plays an important role in fatty acid metabolism under conditions of metabolic stress.

Extensive evidence points to a broad range of ligands bound by L-Fabp including cholesterol as well as fatty acids, the latter representing the most avidly bound ligand class, with two molecules of fatty acid bound per molecule of L-Fabp (8, 10). Accordingly, our a priori hypothesis was that disruption of the murine L-Fabp gene would perturb important elements of fatty acid trafficking and complex lipid metabolism, as a result of altered triglyceride, phospholipid and cholesterol ester production. Our findings in chow fed L-Fabp–/– mice, however, revealed no obvious alterations in hepatic total lipid or cholesterol content, or in the distribution of fatty acid species within either the free fatty acid pool or in the triglyceride and phospholipid pools. Based upon extensive prior information pointing to the importance of L-Fabp in hepatic fatty acid binding, one interpretation of the current findings is that compensatory changes in fatty acid compartmentalization abrogate the effects of L-Fabp deficiency, presumably reflecting the existence of alternative mechanisms for intracellular fatty acid delivery and storage. This suggestion is consistent with the findings from a recent report (14) in which the fatty acid binding capacity of L-Fabp was demonstrated to account for >80% of all the low molecular weight cytosolic proteins in mouse liver, although the source(s) of the presumed residual activity is unclear. Nevertheless, our findings exclude at least one such candidate, SCP2 (Fig. 8B), and demonstrated no compensatory up-regulation of I-Fabp (Fig. 1C) or any other Fabp family members (data not shown). These latter findings are reminiscent of the findings of Agellon and co-workers (24) in which there was no compensatory up-regulation of L-Fabp gene expression in the intestines of I-Fabp–/– mice.

In regard to hepatic lipid content, several findings from the current report differ from the central findings reported recently by Martin et al. (14) in their characterization of hepatic lipid metabolism in L-Fabp–/– mice. Specifically, these workers demonstrated a 3-fold increase in hepatic free cholesterol content and significant increases in both cholesterol ester and phospholipid content, but no change in hepatic TG concentration (14). By contrast, we observed no significant differences in hepatic cholesterol, phospholipid or triglyceride content in our chow-fed L-Fabp–/– mice compared with WT controls. Martin et al. (14) also observed increased SCP2 and decreased SCPx protein expression in L-Fabp–/– mice, whereas we observed no change in the abundance of SCP2 or SCPx protein in either male (Fig. 8B) or female (data not shown) L-Fabp–/– mice, either ad libitum fed or fasted. Moreover, we detected no difference in SCP2 mRNA levels in WT and L-Fabp–/– mice (data not shown). The reasons for these discrepancies are not immediately apparent, but may include the fact that female mice aged 13–15 months were used in their study, compared with 2–4-month-old male mice in the current report. Subtle strain differences may also contribute to the discrepancy since both the current study and that of Martin et al. (14) used mice in a mixed genetic background. The finding that male L-Fabp–/– mice demonstrate no alteration in hepatic cholesterol content or in SCP2 gene expression is of particular interest in view of the compensatory, dramatic up-regulation of hepatic L-Fabp gene expression in SCP2 knockout mice, findings interpreted to support a role for L-Fabp in hepatic cholesterol trafficking (30, 31). Taken together, the strong presumptive evidence from both SCP2 knockout mice and from in vitro studies of cholesterol binding by L-Fabp (7), suggest that more detailed studies of hepatic cholesterol metabolism in an inbred genetic background will be required in order to evaluate the role of L-Fabp in hepatic sterol metabolism, and will likely include an evaluation of combined deletion of both SCP2 and L-Fabp genes.

The most striking phenotype emerging from the current characterization is the abrogation of TG accumulation in the livers of fasted L-Fabp–/– mice. In evaluating the mechanisms underlying this effect, several mechanisms were considered. First, we considered the possibility that increased fatty acid oxidation might account for the reduced TG storage observed in fasted L-Fabp–/– mice. However, fatty acid oxidation, as inferred by determining circulating {beta}-hydroxybutyrate concentrations, was reduced in L-Fabp–/– mice and observations of energy utilization through indirect calorimetry revealed no increased energy expenditure. Thus, while further examination of mitochondrial and peroxisomal fatty acid oxidation may be informative, we suspect that alterations in these pathways will not account for the decreased hepatic TG found in L-Fabp–/– mice upon fasting. In this regard, the observation that targets of PPAR{alpha} were induced to a comparable extent in both fasted WT and L-Fabp–/– mice suggests that the induction of enzymes involved in mitochondrial and peroxisomal fatty acid utilization is largely preserved in the absence of L-Fabp. Secondly, we considered the possibility that increased hepatic VLDL TG secretion might account for the reduced TG storage in fasted L-Fabp–/– mice. Again, examination of this parameter of hepatic TG metabolism revealed reduced rates of VLDL secretion in L-Fabp–/– mice. Our findings further reveal that isolated hepatocytes from fasted WT and L-Fabp–/– mice demonstrate comparable initial rates of oleate uptake. Nevertheless, hepatocytes from fasted L-Fabp–/– mice demonstrate a plateau of uptake that is significantly lower than that observed in WT mice and moreover reveal reduced incorporation of oleate into diacylglycerol and TG (Fig. 7). These findings are in agreement with the results of Martin et al. (14) who demonstrated reduced fatty acid uptake into the livers of L-Fabp–/– mice following intravenous bolus administration of oleic acid. Thus, we suspect that the absence of L-Fabp reduces hepatic fatty acid storage capacity leading to saturation of the pathways involved in trafficking of the bound fatty acids for oxidation, storage and VLDL secretion. While the finding of reduced incorporation of oleate into diacylglycerol and TG, but not other complex lipid species, is intriguing, understanding the mechanism of such an effect will clearly require detailed examination of substrate utilization and intracellular lipolytic-resynthesis pathways, studies that will form the basis of future investigation.

It bears emphasis also that L-Fabp is abundantly expressed in murine enterocytes (3). Accordingly, we considered the possibility of a defect in intestinal triglyceride transport secondary to alterations in fatty acid transport in L-Fabp–/– mice. Our findings revealed no obvious defect in intestinal triglyceride transport as evidenced by morphological examination of fat malabsorption and growth rate. Preliminary analysis of intestinal triglyceride secretion rates using tyloxapol revealed no differences between WT and L-Fabp–/– mice.2 However, we recognize that subtle defects may exist and that these will require formal evaluation in the future.


   FOOTNOTES
 
* These studies were supported by Grants HL-38180, DK-56260, and DDRC grant DK-P30-52574 (to N. O. D.), a pilot and feasibility grant (to E. P. N.) by CNRU Grant DK-P30-56341, and PO1 HL-57278 (to R. W. G. and X. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 314-362-2027; Fax: 314-362-8959; E-mail: nod{at}im.wustl.edu.

1 The abbreviations used are: Fabp, fatty acid-binding protein; VLDL, very low density lipoprotein particle; FATP, fatty acid transport protein; TG, triglyceride; PPAR, peroxisome proliferator-activated receptor; GFP, green fluorescent protein; BAC, bacterial artificial chromosome; ES cells, embryonic stem cells; PL, phospholipid; FFA, free fatty acids; SCP2, sterol carrier protein 2; SCPx, sterol carrier protein x; ELISA, enzyme-linked immunosorbent assay; WT, wild type; PBS, phosphate-buffered saline; CE, cholesterol ester; DAG, diacylglycerol. Back

2 E. Newberry, Y. Xie, K. Buhman, and N. O. Davidson, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We acknowledge the contributions of Dr. Fatiha Nassir, including development of the hepatocyte isolation protocol and helpful discussions. We acknowledge the input and reagents provided by Drs. David Alpers, Jeff Gordon, Tim Ley, and Jean Schaffer. In addition we acknowledge the services provided by the CNRU Animal Model Research Core (supported by DK-56341), the DDRC Morphology Core, and the Diabetes Research & Training Center Radioimmunoassay CORE laboratory.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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