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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Nutr. Author manuscript; available in PMC Mar 9, 2010.
Published in final edited form as:
PMCID: PMC2835297
NIHMSID: NIHMS178599

Liver Fatty Acid Binding Protein Gene-Ablated Female Mice Exhibit Increased Age-Dependent Obesity1,2,3

Abstract

Previous work done in our laboratory suggested a role for liver fatty acid binding protein (L-FABP) in obesity that develops in aging female L-FABP gene-ablated (−/−) mice. To examine this possibility in more detail, cohorts of wild-type (+/+) and L-FABP (−/−) female mice were fed a standard low-fat nonpurified rodent diet for up to 18 mo. Various obesity-related parameters were examined including body weight and fat and lean tissue mass. Obesity in (−/−) mice was associated with increased expression of nuclear receptors that induce peroxisome proliferator-activated receptor α (PPARα) (e.g., hepatocyte nuclear factor 1α, genotype effectα and of PPARα-regulated proteins involved in uptake of free (lipoprotein lipase and fatty acid transport protein, genotype and/or age effect) and esterified (scavenger receptor class B type 1, genotype effect) long chain fatty acids (LCFAs). Hepatic total lipid and neutral lipid levels were not affected by age or genotype, consistent with absence of gross and histologic steatosis. There was increased mRNA expression of liver proteins involved in LCFA oxidation [mitochondrial 3-oxoacyl-CoA thiolase (genotype effect) and butyryl-CoA dehydrogenase (genotype and/or age effect)], increased expression of LCFA esterification enzymes [glycerol-3-phosphate acyltransferase (age × genotype effect) and acyl-CoA:cholesterol acyltransferase-2 (genotype and/or age effect)], and increased expression of proteins involved in intracellular transfer and secretion of esterified LCFA [liver microsomal triacylglycerol transfer protein (genotype effect), serum apolipoprotein B (genotype or age effect), and liver apolipoprotein B (age and age × genotype effect)]. The data support a working model in which obesity development in these mice results from shifts toward reduced energy expenditure and/or more efficient energy uptake in the gut.

INTRODUCTION

Liver fatty acid binding protein (L-FABP)6, a small 14 kDa protein, is present at a high level (3–5% of soluble protein) in tissues active in long chain fatty acid (LCFA) uptake and metabolism, i.e. liver and intestine (rev. in 1). Although its physiological function is not completely clear, studies in vitro and with transfected cells indicate that L-FABP has both direct and indirect roles in LCFA uptake and metabolism.

Earlier in vitro studies of L-FABP function focused on structure, ligand binding characteristics, and L-FABP activity in long chain fatty acid (LCFA) uptake, diffusion, esterification, and oxidation—suggesting involvement (210) in both anabolic and catabolic LCFA pathways (rev. in 1). These putative functions were recently confirmed with cultured primary hepatocytes and livers from L-FABP null mice (1116) suggesting that L-FABP may be important in regulating lipid metabolism and affecting the obese state in mice.

L-FABP also indirectly influences cellular LCFA metabolism by transporting LCFA to the nucleus for interaction with peroxisome proliferator activated receptor α (PPARα), a nuclear receptor that regulates transcription of multiple enzymes involved in LCFA uptake, transport, esterification, and oxidation (rev. in 17–19). Both L-FABP (rev. in 1, 2) and PPARα (2022) exhibit acyl chain-dependent high affinity (i.e. nmol/L Kd) for LCFA and LCFA-CoA, both of which alter conformation of these proteins. Studies in vitro and with purified nuclei (23) as well as with intact living cells (2426) show L-FABP enhances LCFA and LCFA-CoA distribution to nuclei, directly binds PPARα and induces PPARα transcription of LCFA oxidative enzymes. Consequently, L-FABP may regulate its own expression via PPARα (rev. in 27).

Previous studies in our laboratory (12) demonstrated that under conditions of stable energy intake (standard nonpurified diet), L-FABP (−/−) mice exhibit similar, but less severe, age-dependent obesity without morphologically evident hepatic steatosis as observed in PPARα null mice (28, 29). To further explore the mechanistic basis of obesity in older female L-FABP (−/−) mice, the current study was undertaken to determine the age-dependence of hepatic lipid distribution, serum lipid distribution, and expression of select proteins involved in LCFA metabolism. These studies provided new insights into the mechanistic basis of hepatic lipid metabolism and obesity in L-FABP gene-ablated female mice and support a working model in which the age-dependent obesity that develops in the (−/−) females is a result of physiological shifts in these animals toward reduced energy expenditure and/or more efficient intestinal energy uptake.

MATERIALS AND METHODS

Materials

Protease inhibitor cocktail (Cat. # P8340) was from Sigma-Aldrich (St. Louis, MO). Protein was quantified by Protein Assay Dye Reagent (Bio-Rad Laboratories, Hercules, CA). Rabbit polyclonal antisera directed against recombinant rat L-FABP, mouse acyl-CoA binding protein (ACBP), and mouse sterol carrier protein-2 (SCP-2) were produced as described in (30). A rabbit polyclonal antibody directed against mouse SCP-x, which recognizes all SCP-x/SCP-2 gene products (58 kDa SCP-x, 46 kDa thiolase, 15 kDa pro-SCP-2, and 13.2 kDa SCP-2), was made as in (31). Rabbit polyclonal anti-human antibodies against PPARα, sterol regulatory element binding protein-1 (SREBP-1), caveolin-1, goat polyclonal anti-human antibodies against fatty acid transport protein-4 (FATP-4), fatty acid translocase (FAT/CD36), carnitine palmitoyltransferase I (CPT I), lipoprotein lipase (LPL), apolipoprotein (apo) B, microsomal triacylglycerol transfer protein (MTP), mitochondrial 3-hydroxy-3-methylglutaryl Coenzyme A (mHMG-CoA) synthase, hepatocyte nuclear factor-1α (HNF-1α), and HNF-4α, and goat polyclonal anti-mouse antibodies against LDL receptor and apo AI were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-human acyl-Coenzyme A:cholesterol acyltransferase-2 (ACAT-2) was from Cayman Chemical (Ann Arbor, MI). Rabbit polyclonal anti-mouse scavenger receptor class B type 1 (SRB-1) was from Novus Biologicals (Littleton, CO). Rabbit anti-human HMG-CoA reductase was from Upstate Cell Signaling Solutions (Lake Placid, NY). Rabbit polyclonal anti-mouse glutathione S-transferase (GST) and anti-Pseudomonas 3α-hydroxysteroid dehydrogenase (3α-HSD) were from US Biological (Swampscott, MA). Rabbit polyclonal anti-glycerol-3-phosphate acyltransferase (GPAT) was a generous gift of Dr. Rosalind Coleman (Department of Nutrition, University of North Carolina, Chapel Hill, NC). Alkaline phosphatase-conjugated goat anti-rabbit IgG and rabbit anti-goat IgG were from Sigma-Aldrich. All other reagents and solvents used were of the highest grade available and were cell-culture tested.

Animal Studies

C57Bl/6 mice were obtained from the National Cancer Institute (Frederick Cancer Research and Development Center, Frederick, MD). L-FABP gene-ablated (−/−) C57Bl/6 mice were generated by targeted disruption of the L-FABP gene through homologous recombination (12) and bred to generation 6. The Animal Care and Use Committee of Texas A&M University approved all animal protocols. Mice were housed individually in ventilated microisolator cages in a temperature-controlled (25 °C) facility on a constant 12-h light/dark cycle and were allowed to consume food and water ad libitum. All mice were fed a commercial, standard low-fat (5% of energy from fat) pelleted nonpurified diet (Supplemental Table 1; Harlan Teklad Rodent Diet 8604, Harlan Teklad, Madison, WI). The current aging study was initiated using thirty age-matched (age 2 mo) (−/−) female mice. Thirty age-matched (2 mo) (+/+) C57Bl/6 female cohorts were used as controls. Every two days, each mouse was weighed and the amount of food consumed by each mouse was measured (14). At age 3 mo, 15 (+/+) and 15 (−/−) mice were deprived of food overnight (12 h) and anesthetized (ketamine, 100 mg/kg; xylazine, 10 mg/kg). Blood was collected by cardiac puncture and processed to serum for storage at −80 °C and subsequent lipid and protein analysis. After killing the mice, livers were removed and weighed prior to further processing (14). At the end of the study (age 18 mo), the remaining (+/+) and (−/−) mice (n = 12–13/genotype) were food-deprived overnight (12 h) and killed as described above. Blood and liver were collected and processed as described above. At the time of analysis, liver (0.1–0.2 g) was minced and homogenized (motor-driven Teflon pestle) on ice in 0.4–0.5 mL of PBS (pH 7.4) with protease inhibitor cocktail.

Whole-Body Phenotype Analysis

Whole body phenotype was analyzed longitudinally throughout the current study in mice at age 2, 3, 6, 9, and 18 mo by dual-energy X-ray absorptiometry (DEXA) utilizing a Lunar PIXImus densitometer (Lunar Corp., Madison, WI) to determine fat tissue mass (FTM) and bone-free lean tissue mass (LTM) according to a previously published procedure (31). Prior to PIXImus analysis the mouse was anesthetized by an intraperitoneal injection of a mixture of ketamine and xylazine (0.01 mL/g body weight; 10 mg ketamine/mL and 1 mg xylazine/mL in 0.9% saline solution). Following the procedure the mouse was injected with yohimbine (0.11 μg/g body weight) to facilitate recovery. The mouse was injected with warm saline solution for rehydration, kept warm during recovery with heat pads to minimize heat loss, and the mouse was checked every 30 min until recovery was complete. Determination of body composition was performed by exposing the entire animal, minus the head region, to sequential beams of low- and high-energy X-rays with an image taken of the X-rays impacting a luminescent panel. Separation of bone mass from soft tissue mass was accomplished by measurement of the ratios of signal attenuation at the different energy levels. Soft tissue mass was further separated into lean and fat tissue mass to provide accurate values of body composition. Instrument calibration was performed utilizing a phantom mouse of known bone mineral density and fat tissue mass, followed by correlation to chemical extraction techniques.

Lipid Analysis

Lipid, protein, and RNA data were generated from age-matched cohorts of female mice at age 3 mo and 18 mo. Lipids were extracted from liver homogenates (5 mg protein) and analyzed as described (12, 16). Serum lipids and apolipoproteins were quantified using commercially available kits: total cholesterol, Wako # 276-64909; free cholesterol, Wako # 274-47109; nonesterified fatty acid, Wako # 994-75409; triacylglycerol, Wako # 998-40391/# 994-40491; phospholipid, Wako # 990-54009; apo A1, Wako # 991-27201; and apo B, Wako # 993-27401 (Wako Diagnostics, Richmond, VA). Serum cholesteryl ester was determined by subtracting free cholesterol from total cholesterol.

Western Blotting Analysis

Protein expression levels in liver homogenates were measured by Western blotting of equivalent amounts of protein loaded in each lane (15, 16), quantified by densitometry (14, 31), and expressed in arbitrary units relative to that in 3 mo-old (+/+) mice defined as 1.0. For each Western blot the following procedure was used to control for potential blot-to-blot variability. Aliquots of each 3-mo old (+/+) liver homogenate containing equivalent amounts of total protein were pooled. The pooled sample was gently mixed, divided into 50-μL aliquots, and stored at −80 °C for subsequent SDS-PAGE/Western blot analysis. An aliquot of the pooled 3-mo old (+/+) homogenate that contained an amount of total protein equivalent to that of the individual liver homogenate samples to be examined by SDS-PAGE and subsequent Western blotting was removed and loaded onto each gel. After color development and densitometry, the integrated density value of the pooled 3-mo old (+/+) sample was defined as 1.0; the integrated density values of the individual liver homogenate samples on the blot of interest were normalized to this value.

Mouse Liver mRNA Quantification

Mouse liver mRNA levels were quantified as previously described (32). The amount of mRNA detected by RT-PCR was expressed in arbitrary units, with that present in the samples from (+/+) mice defined to be 1.0.

Statistics

The current investigation used cohorts of female mice in which all (−/−) mice were of the N = 6 generation. Repeated measures analysis of variance (ANOVA) was used to analyze body weight, food intake, and fat and lean tissue mass. Tukey’s post hoc test was done when the interaction was significant. Data from the separate groups of 3-mo and 18-mo old mice were analyzed by 2-way ANOVA (genotype x age). Tukey’s post hoc test was used to compare age within genotype or genotype within age when the interaction was found to be significant (GraphPad Prism Version 3.02, San Diego, CA). Data are expressed as means ± SEM. Differences with P < 0.05 were considered statistically significant. Graphical analysis was accomplished using SigmaPlot 2002 for Windows version 8.02 (SPSS, Chicago, IL).

RESULTS

Mouse Body Weight

The initial body weight of 2 mo old L-FABP (−/−) mice did not differ from that of L-FABP WT cohorts (Fig. 1A). By 9 mo of age weight differences were significant (P < 0.05) and L-FABP (−/−) mice were 4 g heavier than the WT cohorts by 18 mo (Fig. 1A). There were no obvious differences in physical activity by subjective observation and no significant differences in food consumption between (+/+) and (−/−) mice at any age examined (Fig. 1B).

FIGURE 1
Body weight (A), food intake (B), fat tissue mass (C), and lean tissue mass (D) in L-FABP (−/−) vs. (+/+) female mice. Values are means ± SEM, n = 10. * Different from 2-mo old for that genotype, P < 0.01. # Different from ...

There was no significant difference in body fat tissue mass (FTM) between 2 mo old (−/−) and (+/+) mice (Fig. 1C). (+/+) mice at 2 mo had 13.1 ± 0.6% of total tissue mass (TTM) as FTM; this value was not significantly different from (−/−) mice at 2 mo that had 14.3 ± 0.7% of total tissue mass as FTM. In contrast, at 18 mo, FTM in (−/−) mice was 58% greater than in (+/+) mice (Fig. 1C). The amount of body weight gained as lean tissue mass (LTM) did not differ significantly throughout the study as a result of genotype (Fig. 1D). As a result, the amount of FTM as a percent of (TTM) increased to 31.5 ± 0.9% in (−/−) mice by the end of the aging study (18 mo); this was significantly (P < 0.05) higher than the amount of FTM as a percent of TTM measured in (+/+) mice (22.2 ± 0.7%).

Hepatic Lipid Concentrations

L-FABP gene ablation significantly increased the concentration of hepatic cholesterol, cholesteryl ester, and phospholipid in female mice (Table 1). For primarily neutral storage lipids, hepatic triacylglycerol declined while cholesteryl ester increased in old mice (Table 1). L-FABP gene ablation did not further alter triacylglycerol concentration; however, L-FABP gene ablation resulted in an increased cholesteryl ester concentration in both young and old mice. Finally, liver nonesterified fatty acid (LCFA) increased with genotype or age in mice (Table 1).

TABLE 1
Liver lipid concentrations in 3-mo and 18-mo old (+/+) and (−/−) mice1

Serum Lipid Concentrations

Serum cholesterol, cholesteryl ester, phospholipid, and triacylglycerol were not altered in 18-mo (+/+) mice as compared with 3-mo (+/+) animals; however, there was a significant decrease in serum concentration of nonesterified fatty acids in the older (+/+) vs. younger (+/+) mice (Table 2). L-FABP gene ablation alone resulted in a significant increase in serum triacylglycerol levels in 3 mo old mice (Table 2). Serum cholesterol concentration was increased in 18 mo (−/−) mice (genotype × age); however, serum cholesteryl ester was decreased in these mice (Table 2). In summary, the greater obesity observed in older (−/−) mice was reflected in elevated serum total lipids, especially lipid classes typically enriched in VLDL (triacylglycerol, cholesterol).

TABLE 2
Serum lipid concentrations in 3-mo and 18-mo old (+/+) and (−/−) mice1

Effects of L-FABP Gene Ablation and Aging on Proteins Important in LCFA Delivery to Hepatocytes as Esterified Lipids in Lipoproteins

Serum apoB (high in VLDL, LDL) levels were increased in 3-mo (−/−) mice and in 18-mo (+/+) mice (Table 3). Hepatic apo B concentration was unaltered by L-FABP gene ablation in younger mice, while apoB was significantly increased in older (+/+) and (−/−) mice (Table 3). Serum and liver levels of apo A1 in these mice were unaffected by L-FABP gene ablation and/or aging (not shown). The hepatic scavenger receptor B1 (SR-B1) was upregulated in young and old (−/−) mice (Table 3).

TABLE 3
Serum apoB and relative liver concentrations of key proteins important in hepatocyte LCFA transport in 3-mo and 18-mo old (+/+) and (−/−) mice1

Effects of L-FABP Gene Ablation and Aging on Proteins that Transport LCFA into Hepatocytes, Release LCFA from Lipoproteins for Transport into Hepatocytes, and Transport LCFA Within Hepatocytes

Albumin-bound LCFA is delivered to plasma membrane LCFA transporters [caveolin-1, fatty acid translocase/CD36 (FAT/CD36), fatty acid transport protein (FATP)] for translocation into the cell. While neither FAT/CD36 nor caveolin-1 was increased by L-FABP gene ablation or aging (not shown), FATP increased in (−/−) mice and increased with age regardless of genotype (Table 3). At the surface of hepatic endothelial cells, lipoprotein lipase (LPL) also releases LCFA from lipoproteins (e.g. VLDL). As with FATP expression, LPL concentration was increased in (−/−) mice and increased with age regardless of genotype (Table 3). With regards to the intracellular LCFA and LCFA-CoA transport proteins, L-FABP gene ablation resulted in the complete absence of L-FABP but without concomitant upregulation of SCP-2 or ACBP (not shown).

Effects of L-FABP Gene Ablation and Aging on Liver Proteins of LCFA Anabolic Metabolism: Esterification and Intracellular Transport for Secretion of Esterified LCFA

The activity of glycerol-3-phosphate acyltransferase (GPAT), the rate limiting enzyme in glyceride (phospholipid, triacylglycerol) synthesis, is age- and L-FABP-expression dependent. L-FABP gene ablation had no effect on liver GPAT concentration in young or old mice; however, there was a significant decrease in GPAT concentration in 18-mo (+/+) mice (Table 4). Aging did not affect the expression of microsomal triglyceride transfer protein (MTP), a protein that transports triglycerides and cholesteryl esters in cells for secretion as VLDL (Table 4). In contrast, L-FABP gene ablation increased liver MTP concentration in young and old mice (Table 4).

TABLE 4
Relative liver concentrations of key proteins/mRNAs important in hepatocyte LCFA/cholesterol metabolism in 3-mo and 18-mo old (+/+) and (−/−) mice1

In terms of the acyl-CoA cholesterol acyltransferase enzymes, L-FABP gene ablation or aging had very little effect on the liver concentration of ACAT-1 (not shown). In contrast, ACAT-2 (predominant form of ACAT in liver) levels were age and L-FABP gene-ablation dependent (Table 4). L-FABP gene ablation or aging resulted in increased hepatic ACAT-2 levels in mice (Table 4). At least two proteins compete with ACAT for cholesterol substrate by enhancing active transport of bile salts and cholesterol within the hepatocyte and across the hepatocyte plasma membranes: glutathione-S-transferase (GST) and 3α-hydroxysteroid dehydrogenase (3α-HSD). Neither L-FABP gene ablation nor aging had any effect on GST or 3α-HSD levels in mice (not shown). L-FABP gene ablation and/or aging had no effect on the level of SCP-x, a key enzyme in oxidation of the branched side chain of cholesterol to form bile acids (not shown).

HMG-CoA synthase is involved in de novo synthesis of cholesterol (precursor of cholesteryl ester), but expression of this protein was not affected by age or L-FABP ablation (not shown). Age and/or L-FABP gene ablation also did not alter HMG-CoA reductase levels in females (not shown).

Effects of L-FABP Gene Ablation and Aging on Liver Proteins Important for LCFA Catabolic Metabolism: Mitochondrial and Peroxisomal Oxidation

Carnitine palmitoyl transferase I (CPT I, rate limiting enzyme in mitochondrial LCFA oxidation) increased with age or L-FABP gene ablation in mice (Table 4). L-FABP gene ablation resulted in increased hepatic levels of mitochondrial 3-oxoacyl-CoA thiolase mRNA in 3-mo and 18-mo old mice (Table 4). Butyryl-CoA dehydrogenase mRNA concentration significantly increased in older mice regardless of genotype (Table 4).

Effects of L-FABP Gene Ablation and Aging on Key Nuclear Receptors of LCFA and Cholesterol Metabolism

Expression of certain key nuclear receptors involved in regulating transcription of enzymes/proteins participating in LCFA uptake, transport, esterification, and oxidation were examined. Neither L-FABP gene ablation nor aging had any effect on levels of the active 68 kDa nuclear form of SREBP-1, involved in cholesterol and fatty acid metabolism (not shown). PPARα, which regulates expression of proteins participating in LCFA transport into the cell, LCFA oxidation, and glucose metabolism, was increased by age regardless of genotype (Table 4). HNF1α and HNF4α regulate expression of multiple proteins in LCFA metabolism, including PPARα. HNF1α expression was increased by L-FABP gene ablation in young mice (Table 4). L-FABP gene ablation had no effect on levels of HNF4α in either young or old mice (not shown).

DISCUSSION

L-FABP may regulate liver LCFA metabolism not only directly, but also through nuclear transcription mechanisms including enhanced fatty acid distribution to nuclei (24, 25), interaction with PPARα in nuclei (25, 26), and regulation of PPARα transcriptional activity of genes involved in LCFA uptake, oxidation, lipoprotein transport, and glucose metabolism (17, 2426). If, as suggested by in vitro and cultured cell studies, L-FABP contributes significantly to physiological regulation of PPARα, then L-FABP null mice should exhibit phenotypic similarities to PPARα null mice, which show age-dependent obesity without hepatic steatosis but with increased serum triacylglycerols (28, 37). L-FABP (−/−) female male mice exhibit age-dependent obesity without hepatic steatosis, in a phenotype similar, but less severe, than that observed in PPARα null mice (28, 37). The data presented herein yielded several mechanistic insights into age-dependent obesity in L-FABP null mice.

First, obesity in older (−/−) mice was associated with select elevation in serum triacylglycerol and cholesterol and serum apo B, while serum cholesteryl ester decreased. This pattern of lipid and apolipoprotein changes was consistent with, but does not prove, increased VLDL (rev. in 38, 39) similar to that observed in older PPARα (−/−) female mice (28) suggesting that changes in L-FABP may regulate PPARα-related pathways.

Second, obesity in older (−/−) mice was associated with selective increases in liver cholesterol, cholesteryl ester, and phospholipid (but not triacylglycerol) as well as apo B. In contrast, older PPARα (−/−) female mice had not only higher liver cholesterol, but also higher cholesteryl ester and triacylglycerol (28). These chemical analyses were consistent with lack of morphological evidence of overt steatosis in livers of older (−/−) female mice. Although steatosis was also not morphologically evident in livers of older PPARα (−/−) female mice, chemical analysis indicated that older female PPARα (−/−) mice exhibited increased liver lipid levels when compared to older L-FABP (−/−) female mice—consistent with the fact that L-FABP is only one (26) of several proteins (rev. in 17–19) that activate PPARα transcriptional activity. The absence of overt steatosis in PPARα (−/−) female mice and in L-FABP (−/−) females suggests more efficient packaging and export of lipids from the livers of these animals.

Third, as shown by Western blotting and/or RT-PCR, the age-dependent lipid changes observed prominently in old obese (−/−) female mice were associated with increased expression of select liver proteins LPL and FATP involved in LCFA tissue release/uptake. The increased hepatic level of LPL is especially significant in view of LPL not being synthesized in liver, but rather in adipose and muscle tissue from where it must be transported to endothelial cells in liver as well as adipose, muscle, and other tissues. The finding of increased LPL and FATP in older obese L-FABP (−/−) females indicates increased LCFA hepatic uptake and suggests, but does not prove, an increased level of LPL in adipose tissue which could contribute to the adiposity noted in older obese (−/−) mice. Whether these proteins are elevated in older obese PPARα (−/−) females is not known.

Fourth, obesity in older (−/−) mice was associated with increased LCFA esterification (ACAT-2) and intracellular transfer/secretion of esterified LCFA (MTP, apoB). Interestingly, coordinate repression of MTP (MTP inhibitor 8aR) and L-FABP (gene ablation) in standard nonpurified diet-fed young mice resulted in decreased VLDL secretion without hepatosteatosis (40). However, L-FABP gene ablation alone increased MTP expression in female mice.

The age-dependent obesity observed in L-FABP (−/−) mice was unique as compared to that of other known fatty acid binding protein targeted mice including adipocyte fatty acid binding protein (A-FABP) (−/−) mice, which exhibited increased body weight gain as compared with their wild-type counterparts only when fed a high-fat diet (41); I-FABP (−/−) mice, which displayed no differences in body weight gain when fed either a control diet or a high-fat, high-cholesterol diet as compared with their wild-type counterparts (42); epidermal fatty acid binding protein (E-FABP) (−/−) mice, which were phenotypically normal (43).

Ultimately, increased body weight and increased fat tissue mass observed in L-FABP (−/−) female mice must be due to an energy imbalance in these animals. This energy imbalance can arise from increased food intake, change in metabolic rate, and/or differences in feed efficiency. Although there were no obvious differences between (+/+) and (−/−) mice with respect to food intake or physical activity these conclusions were based on subjective observations and need to be examined in more detail.

In summary, the net effect of the lipid and protein changes in older obese (−/−) mice was to shift the balance of hepatic LCFA metabolism toward a net increase in LCFA uptake (LPL, FATP), esterification (ACAT-2, GPAT), transport/secretion as VLDL (MTP, apoB), and utilization by adipose tissue for storage. This phenotype shared many, but not all, attributes with those reported for PPARα (−/−) mice, supporting the view that L-FABP and PPARα influence some common pathways that regulate lipid metabolism. The data reported herein support a working model in which loss of L-FABP results in obesity without obvious steatosis. The absence of steatosis could result from more efficient packaging and export of lipids from the liver. Since food consumption was equivalent in wild-type and L-FABP gene-ablated mice, obesity development in the knockout animals must have resulted from shifts toward a reduced energy expenditure and/or more efficient energy uptake in the gut.

Supplementary Material

Footnotes

1This work was supported by the U.S. Public Health Service National Institutes of Health grants DK41402 and GM31651.

2 Author disclosures: G. G. Martin, B. P. Atshaves, A. L. McIntosh, J. T. Mackie, A. B. Kier, and F. Schroeder, no conflicts of interest.

3Supplemental Table 1 is available as Online Supporting Material with the online posting of this paper at http://jn.nutrition.org.

Supplementary online material has been submitted.

6Abbreviations used: ACAT-2, acyl-Coenzyme A:cholesterol acyltransferase-2; ACBP, acyl-Coenzyme A binding protein; A-FABP, adipocyte FABP; apo, apolipoprotein; CPT I, carnitine palmitoyl transferase I; DEXA, dual-energy X-ray absorptiometry; E-FABP, epidermal FABP; FABP, fatty acid binding protein; FAT/CD36, fatty acid translocase; FATP-4, fatty acid transport protein-4; FTM, fat tissue mass; GPAT, glycerol-3-phosphate acyltransferase; GST, glutathione S-transferase; HMG-CoA, 3-hydroxy-3-methylglutaryl Coenzyme A; HNF, hepatocyte nuclear factor; 3α-HSD, α3-hydroxysteroid dehydrogenase; I-FABP, intestinal FABP; LCFA, long chain fatty acid; L-FABP, liver FABP; LPL, lipoprotein lipase; LTM, lean tissue mass; MTP, microsomal triacylglyerol transfer protein; PPARα, peroxisome proliferator-activated receptor α;SCP-2, sterol carrier protein-2; SCP-x, sterol carrier protein-x/3-ketoacyl-CoA thiolase; SR-B1, scavenger receptor class B type 1; SREBP-1, sterol regulatory element binding protein-1; TTM, total tissue mass; (+/+), liver fatty acid binding protein wild-type mice; (−/−), liver fatty acid binding protein gene-ablated mice.

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