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Although studies performed in vitro and with transfected cells in culture suggest a role for liver fatty acid binding protein (L-FABP) in regulating fatty acid oxidation and fat deposition, the physiological significance of this possibility is not completely clear. To begin to address this question, the effect of L-FABP gene ablation on phenotype of standard rodent chow-fed male mice was examined with increasing age up to 18 mo. While young (2-3 mo) L-FABP null mice displayed no visually obvious phenotype, with increasing age > 9 mo the L-FABP null mice were visibly larger, exhibiting increased body weight due to increased fat and lean tissue mass. Liver lipid concentrations were unaffected by L-FABP gene ablation with the exception of triacylglycerol, which was decreased by 74% in the livers of 3 mo old mice. Likewise, serum lipid levels were not altered in L-FABP null mice with the exception of triacylglycerol, which was increased in the serum of 18 mo old mice. Increased body weight, fat tissue mass, and lean tissue mass in 18 mo old L-FABP null mice were accompanied by increased hepatic levels of low density lipoprotein (LDL) receptor, peroxisome proliferator-activated receptor (PPAR) α, and PPARα-regulated proteins such as fatty acid transport protein (FATP), fatty acid translocase (FAT/CD36), carnitine palmitoyl transferase I (CPT I), and lipoprotein lipase (LPL). A key enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase was down-regulated in L-FABP null mice. These findings were consistent with a proposed role for L-FABP as an important physiological regulator of PPARα.
Long chain fatty acids (LCFAs) are not only biological membrane components, metabolic energy sources, and signaling molecule precursors, but also endogenous high-affinity ligands for nuclear receptors such as PPARs that regulate transcription of multiple genes involved in LCFA (β-oxidation, lipoprotein) and glucose metabolism [rev. in 1-3]. Abnormal PPAR activation contributes to lipotoxicity associated with obesity, insulin resistance, type 2 diabetes, and hyperlipidemia [rev. in 2,4]. Both LCFAs [5-7] and LCFA-CoAs [6-9] are high-affinity (i.e. nM Kds) endogenous PPARα ligands. LCFA and LCFA-CoA binding regulates PPARα conformation [6,7], cofactor recruitment [6-9], and transcriptional activity [2,6,7,10,11].
Despite the importance of LCFAs as endogenous physiologically-relevant ligands of PPARα, in vitro studies show LCFAs are poorly transported into purified nuclei and confocal imaging as well as other studies detect very low levels of LCFAs and LCFACoAs in nuclei of living cells, 39-68 nM and <10 nM, respectively [rev. in 1,12-15]. However, in the presence of L-FABP the bound LCFAs are rapidly co-transported into purified nuclei in vitro and L-FABP overexpression enhances LCFA distribution to nuclei of living cells [12,13,16]. Significant levels of L-FABP appear in nuclei of liver hepatocytes as well as other cell types expressing L-FABP [rev. in 1,13,17]. Furthermore, confocal microscopy, coimmunoprecipitation, two-hybrid, and transactivation assays demonstrate that L-FABP directly interacts with PPARα to potentially facilitate delivery of bound LCFA and thereby initiate transcription of proteins involved in LCFA metabolism [rev. in 1]. Interestingly, LCFA-mediated PPARα activation also enhances transcription of L-FABP—the primary intracellular LCFA transport protein [rev. in 1,18]. Taken together, these studies performed in vitro and in cell culture are consistent with a potential role for L-FABP in mediating transfer of bound LCFA into nuclei for regulating PPARα transcriptional activity [rev. in 1].
It has been proposed that PPARα acts as a LCFA nutrient sensor and plays a role in hepatic steatosis and obesity [rev. in 2,19]. Although PPARα gene-ablated mice do not exhibit an overt phenotype during the first 6 months of life [20-22], with increasing age PPARα null mice experience delayed onset of obesity without hepatic steatosis under conditions of stable caloric intake (standard rodent chow) [19,23]. Interestingly, PPARα gene ablation reduces L-FABP expression and LCFA oxidation , while increased LFABP expression is associated with increased LCFA β-oxidation [rev. in 1,20,24]. This suggested that L-FABP reduction might itself contribute in part to an obese phenotype in older mice. However, almost all other previous reports with L-FABP null mice examined the lipid metabolic phenotype only in relatively young (2-5 mo old) mice [25-30]. Further, these earlier studies did not examine the effect of age on obesity-related parameters. The data presented herein show for the first time that L-FABP gene ablation elicited an age-dependent increase in body weight without hepatic steatosis in standard chow-fed male mice. These data indicate that, with respect to age-dependent bodyweight gain, L-FABP gene-ablated and PPARα gene-ablated mice share significant phenotypic features—consistent with L-FABP being involved in regulating PPARα transcriptional activity of genes involved in LCFA oxidation.
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 . 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 proSCP-2, and 13.2 kDa SCP-2), was prepared as described . Rabbit polyclonal anti-human antibodies against PPARα, sterol regulatory element binding protein-1 (SREBP-1), caveolin-1, goat polyclonal anti-human antibodies against FATP-4, FAT/CD36, CPT I, LPL, apolipoprotein (apo) B, microsomal triacylglycerol transfer protein (MTP), mitochondrial HMG-CoA synthase, hepatocyte nuclear factor-1α (HNF-1α), and HNF-4α were from Santa Cruz Biotechnology (Santa Cruz, CA). 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.
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  and bred to generation N6 (98.4% genetic homogeneity). 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 diet (Harlan Teklad Rodent Diet 8604, Harlan Teklad, Madison, WI). The aging study was initiated using thirty age-matched (age 2 mo) L-FABP (−/−) male mice and thirty age-matched (2 mo) L-FABP (+/+) C57Bl/6 male cohorts as controls. Every two days, each mouse was weighed and the amount of food consumed by each mouse was measured . At age 3 mo, 15 L-FABP (+/+) and 15 L-FABP (−/−) 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 the mice were euthanized, livers were removed and weighed prior to further processing . At the end of the study (age 18 mo), the remaining L-FABP (+/+) and L-FABP (−/−) mice were food-deprived overnight (12 h), euthanized, and 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 (Cat. # P8340, Sigma-Aldrich).
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 . Prior to PIXImus analysis each 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 mice were injected with yohimbine (0.11 μg/g body weight) to facilitate recovery, injected with warm saline solution for rehydration, kept warm during recovery with heat pads to minimize heat loss, and checked every 30 min until recovery was complete. Determination of body composition was performed by exposing each 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.
Liver histology was examined  and liver lipid volume was determined as described previously , but modified as follows for EM: liver tissue samples were incubated in 1% osmium tetroxide/2.5% potassium dichromate for 8 h, dehydrated in a graded ethanol series, and embedded in Spurr's epoxy resin. Semithin sections (0.75 μm thick) were mounted on glass slides, coverslipped, and examined without counterstaining. Liver sections were imaged with a 40x light microscope objective and images were recorded with a charge-coupled device (CCD) camera. Four representative images were randomly selected from each liver for a total image area of 0.6 mm2 per group per mouse. The relative area of lipid droplets in the image field was analyzed with the program ImageJ [developed at the National Institutes of Health (NIH) and available on the Internet at http://rsb.info.nih.gov/ij/].
Mouse serum leptin and insulin were quantified utilizing a mouse serum adipocytokine LINCOplex kit (# MADPK-71K) and a mouse adipocyte LINCOplex kit (# MADPCYT-72K) from LINCO Research (St. Charles, MO). The analyses were carried out essentially as described by the manufacturer. Each microtiter plate was read utilizing a Luminex 100IS microsphere analyzer (Luminex Corp., Austin, TX). Data analysis was accomplished using 5-parameter data reduction in the Luminex 100 version 2.1 software package as supplied by the manufacturer.
Mouse serum glucose was quantified utilizing a Glucose C2 kit (# 994-90902) that was purchased from Wako Diagnostics (Richmond, VA). The kit was used according to the manufacturer's directions; glucose concentration (mg/dL) was determined utilizing a glucose standard curve. Mouse serum β-hydroxybutyrate was quantified utilizing a kit (#2440-058) supplied by Stanbio Laboratory (Boerne, TX). The colorimetric assay kit was used according to the manufacturer's directions; β-hydroxybutyrate concentration (mg/dL) was determined using a sodium D-3-hydroxybutyrate standard curve.
Lipid, protein, and mRNA data were generated from age-matched cohorts of male mice at age 3 mo and 18 mo. Lipids were extracted from liver homogenates (5 mg protein) and analyzed as described [30,33]. 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.
Protein expression levels in liver homogenates were measured by Western blotting of equivalent amounts of protein loaded in each lane [28,30], quantified by densitometry [29,32], and expressed in arbitrary units relative to that in 3 mo-old L-FABP (+/+) 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 L-FABP (+/+) 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 LFABP (+/+) 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 L-FABP (+/+) 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 levels were quantified as previously described . The amount of mRNA detected by real time-polymerase chain reaction (RT-PCR) was expressed in arbitrary units, with that present in the samples from 3 mo old L-FABP (+/+) mice defined to be 1.0.
The current investigation used cohorts of male mice in which all L-FABP (−/−) mice were of the N6 generation (98.4% genetic homogeneity). 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).
The mean body weight of 2 mo old L-FABP (−/−) male mice did not differ from that of L-FABP (+/+) cohorts (Fig. 1a). By ≥ 9 mo of age weight differences were significant (Fig. 1a) and L-FABP (−/−) mice were significantly (4 g, P < 0.05) heavier than L-FABP (+/+) mice by 18 mo (Fig. 1a). There were no obvious differences in physical activity by subjective observation and no significant differences in food consumption between LFABP (+/+) and L-FABP (−/−) mice at any age examined (Fig. 1b).
Although there was no difference in body weight between L-FABP (−/−) and LFABP (+/+) mice at age 2 mo, DEXA analysis revealed that L-FABP (−/−) males had 14% less (P < 0.05) FTM than did their L-FABP (+/+) cohorts (Fig. 1c, d and 2a, b) at this age. By contrast, 2 mo old L-FABP (−/−) mice had 9% more (P < 0.05) LTM than did 2 mo L-FABP (+/+) mice (Fig. 1e, f). While FTM (Fig. 1c) and LTM (Fig. 1d) increased with age for all mice, the increases were more extensive for L-FABP (−/−) mice. By age 9 mo the L-FABP (−/−) mice had 29% more (P < 0.05) FTM than did L-FABP (+/+) mice at this age (Fig. 1c). The quantity of LTM in 9 mo old L-FABP (−/−) was also greater (10%, P < 0.05) than in L-FABP (+/+) mice (Fig. 1e). Finally, 18 mo old L-FABP (−/−) had 3.3 and 1.8 g more FTM and LTM (33% and 9% more, P < 0.05), respectively, than did their 18 mo old L-FABP (+/+) cohorts (Fig. 1c, e and 2c, d). Consequently, when expressed as %, the % FTM increased (Fig. 1d) while that of % LTM (Fig. 1f) correspondingly decreased. Thus, the age-dependent increase in body weight in L-FABP (−/−) male mice was associated with significantly more FTM than LTM.
Since obesity can be accompanied by steatosis [rev. in 19], liver was examined by gross measurements, histopathology, and EM. Gross liver weight of L-FABP (−/−) male mice did not differ significantly from that of L-FABP (+/+) mice at 3 mo of age (not shown). With aging, liver weight increased concomitantly with body weight in both groups of mice (not shown). Quantification of total homogenate protein/g liver also showed no significant differences between L-FABP (−/−) and L-FABP (+/+) counterparts at any age (not shown).
Histologic examination of the livers from 3 mo old L-FABP (−/−) mice showed a similar appearance to that of livers from age-matched L-FABP (+/+) mice (Fig. 3a, b). Very little hepatic fatty vacuolation was evident in any group. At 18-mo, livers from LFABP (+/+) mice (Fig. 3c) or L-FABP (−/−) mice (Fig. 3d) showed little evidence of fatty vacuolation or any other consistent histologic abnormalities. Light microscopy of osmium tetroxide/potassium dichromate-stained livers showed no differences in lipid droplet area between L-FABP (−/−) and (+/+) mice at either age 3 mo (Fig. 4a, b) or 18 mo (Fig. 4c, d); however, there was a 2-4-fold increase in lipid droplet area in older mice when compared with their 3-mo old counterparts.
In some mouse models, obesity is accompanied by increased serum levels of the adipocytokine leptin and decreased leptin transport across the blood-brain barrier [35,36]. L-FABP gene ablation resulted in 59% lower serum leptin levels in 3 mo L-FABP (−/−) mice as compared to their L-FABP (+/+) cohorts (Fig. 5a, P < 0.01). While serum leptin levels were 2.4- to 7.4-fold higher in 18 mo old mice compared with their young counterparts, there was no difference in serum leptin concentration between L-FABP (−/−) and L-FABP (+/+) mice aged 18 mo (Fig. 5a, P < 0.001). Thus, the decreased serum leptin concentration in young mice may have contributed to preventing the observation of increased body weight and FTM in young mice—an effect overcome by greatly increased serum leptin concentrations for both groups at increasing age (18 mo).
Although serum glucose and insulin concentrations were significantly increased in 18 mo old males compared with their 3 mo old counterparts, L-FABP gene ablation had no effect on serum glucose or insulin levels at any age examined (Fig. 5b, c). While serum β-hydroxybutyrate levels were 43-75% lower in 18 mo old (+/+) and (−/−) mice compared with their 3 mo old cohorts, there was no significant effect of L-FABP gene ablation on serum β-hydroxybutyrate concentration in these mice (Fig. 5d).
Deletion of the L-FABP gene and subsequent absence of L-FABP protein had no effect on liver levels of free cholesterol, cholesteryl ester, total cholesterol, or nonesterified fatty acid in either 3 mo or 18 mo old male mice (Table 1). By contrast, 3 mo old LFABP (−/−) mice exhibited 74% lower triacylglycerol concentration than did their LFABP (+/+) counterparts (P < 0.01, Table 1). Aging eliminated this L-FABP gene-ablation effect such that 18 mo old L-FABP (+/+) and L-FABP (−/−) mice exhibited similar liver triacylglycerol levels that were statistically equivalent to the liver triacylglycerol concentration measured in 3 mo old L-FABP (+/+) mice (Table 1). L-FABP gene decreased liver phospholipid levels slightly, i.e. 14-19%, at both ages examined (P < 0.05, Table 1). Finally, the decreased triacylglycerol concentration in 3 mo old (−/−) mouse liver resulted in reduced neutral and total lipid levels (53% and 34%, respectively) in these animals whereas neutral and total lipid concentrations were unchanged in 18 mo old L-FABP (−/−) mice when compared with 18 mo old L-FABP (+/+) animals (Table 1). In summary, the increased body weight observed in 18 old L-FABP (−/−) as compared to 18 mo old L-FABP (+/+) mice was not associated with increased hepatic lipid concentrations.
In 3 mo old mice L-FABP gene ablation had no effect on the serum concentrations of any lipids examined (Table 2). Although L-FABP gene ablation did not alter the liver concentrations of free cholesterol, cholesteryl ester, total cholesterol, nonesterified fatty acid, or phospholipid in 18 mo old mice (Table 2), serum levels of triacylglycerol increased by 81% in L-FABP (−/−) mice compared with their L-FABP (+/+) counterparts (P < 0.01, Table 2). The increased serum triacylglycerol observed in 18 mo old L-FABP (−/−) mice was the major factor contributing to a 55% increase (P < 0.01) in neutral lipids and a 27% increase (P < 0.05) in total lipids in these animals when compared with their 18 mo old L-FABP (+/+) cohorts (Table 2). Thus, the increased body weight in 18 mo old L-FABP (−/−) as compared to L-FABP (+/+) mice was associated with increased serum triacylglycerol, neutral lipid and total lipid—consistent with increased levels of triglyceride-rich lipoproteins.
In order to investigate whether concomitant upregulation of other known lipid binding/metabolism proteins present in mouse liver might contribute to the age-dependent obese phenotype in L-FABP (−/−) mice, Western blotting was performed to determine the levels of intracellular fatty acid/cholesterol transporters (SCP-2, caveolin-1) as well as a key nuclear receptor (68 kDa nuclear form of SREBP-1) known to be involved in cholesterol and fatty acid metabolism [37-39].
Western blotting demonstrated the absence of any detectable L-FABP protein in liver homogenates from either 3 mo or 18 mo old males (Table 3). L-FABP gene ablation resulted in a small (17%, P < 0.01) decrease in the concentration of SCP-2 in 3 mo old male mice; however, there was no gene-ablation effect observed in liver homogenates in 18 mo old male mice (Table 3). Neither caveolin-1 nor the active nuclear form of SREBP-1 was affected by the absence of L-FABP at either age examined (Table 3).
Western blotting was performed to examine the possibility that L-FABP gene ablation-related obesity in older mice was related to the content of certain key proteins involved in cholesterol esterification/storage (ACBP, ACAT-2), uptake (LDL receptor), efflux (SRB-1), and synthesis (HMG-CoA reductase). L-FABP gene ablation had no effect on the liver concentrations of ACBP, ACAT-2 (major form of ACAT in liver), SRB-1 (Table 3), or ACAT-1 (not shown). Liver LDL receptor levels were increased by 33-41% in both 3 and 18 mo old L-FABP (−/−) mice as compared with their L-FABP (+/+) cohorts (Table 3). Consistent with increased LDL-receptor concentration and, presumably, enhanced LDL-cholesterol uptake , L-FABP gene ablation resulted in a 25-35% decrease (P < 0.01) in HMG-CoA reductase in both 3 and 18 mo old mice as compared with L-FABP (+/+) counterparts (Table 3).
L-FABP expression is regulated by PPARα and L-FABP directly interacts with PPARα to regulate transcriptional activity [rev. in 1]. Therefore, Western blotting and RT-PCR were performed to examine whether age-dependent obesity in L-FABP (−/−) mice was associated with alterations in the level of PPARα and/or PPARα-regulated proteins.
Western blotting showed that L-FABP gene ablation resulted in a 57% and 26% increase (P < 0.01) in hepatic PPARα in 3 mo and 18 mo old mice, respectively, as compared to their L-FABP (+/+) counterparts (Table 4). Liver levels of the plasma membrane fatty acid transporter FATP were increased by 18% and 10% (P < 0.05), respectively, in 3 mo old and 18 mo old L-FABP (−/−) animals as compared with their LFABP (+/+) counterparts (Table 4). While the liver concentration of another plasma membrane fatty acid transporter FAT was increased by 39% in 3 mo old L-FABP (−/−) mice as compared with 3 mo old L-FABP (+/+) counterparts, L-FABP gene ablation did not affect FAT levels in 18 mo old mice (Table 4). Loss of L-FABP resulted in a 63% and 44% increase, respectively, in hepatic levels of the rate limiting enzyme in mitochondrial fatty acid oxidation CPT-I in 3 mo old and 18 mo old L-FABP (−/−) mice as compared with their L-FABP (+/+) cohorts (Table 4). Hepatic concentrations of lipoprotein lipase were increased by 56% and 31%, respectively, as a result of L-FABP gene ablation in 3 mo and 18 mo old mice (Table 4). Finally, L-FABP gene ablation had no effect on liver levels of HMG-CoA synthase in either mouse age group examined (Table 4).
RT-PCR of liver homogenates from 3 mo old mice showed that L-FABP gene ablation increased levels of key enzymes of peroxisomal fatty acid oxidation, acyl-CoA oxidase (Table 5) and peroxisomal bifunctional enzyme (Table 5), by 1.2-fold (P < 0.05) and 2.7-fold (P < 0.01), respectively. Likewise, L-FABP gene ablation also increased by 70% (P < 0.05) the level of mitochondrial 3-oxoacyl-CoA thiolase in 3 mo old mice (Table 5). By contrast, liver levels of acyl-CoA oxidase and peroxisomal bifunctional enzyme mRNA were decreased by 60% (P < 0.05, Table 5) and 52% (P < 0.05, Table 5), respectively, in 18 mo old L-FABP (−/−) mice as compared with their L-FABP (+/+) cohorts. Liver concentration of mitochondrial 3-oxoacyl-CoA thiolase mRNA was unaffected by L-FABP gene ablation in 18 mo old animals (Table 5). Mitochondrial butyryl-CoA dehydrogenase mRNA levels were unaffected by L-FABP gene ablation in 3 mo old mice; however, there was a 78% decrease in the liver concentration of this mRNA in 18 mo old L-FABP (−/−) mice as compared with their L-FABP (+/+) counterparts (Table 5).
In summary, in young mice L-FABP gene ablation increased the level of PPARα and many key PPARα-regulated proteins involved in fatty acid uptake, peroxisomal fatty acid oxidation, and mitochondrial oxidation—consistent with the absence of hepatic steatosis in young L-FABP (−/−) male mice. While in 18 mo old mice L-FABP gene ablation also increased the level of PPARα and CPT1, the effects on other PPARα regulated proteins was diminished or abolished.
Both GST and 3α-HSD have been shown to be involved in the active transport of bile salts within the hepatocyte [rev. in 30]. Liver GST levels were increased by 18% (P < 0.05) in 3 mo old mice and by 43% (P < 0.01) in 18 mo old animals as a result of LFABP gene ablation (Table 5). In young mice the loss of L-FABP resulted in a 38% increased (P < 0.01) level of 3α-HSD, but not in 18 mo old animals (Table 5). Finally, liver homogenate SCP-x concentration was unaffected by L-FABP gene ablation in either young or old mice (Table 5). Thus, L-FABP gene ablation increased the level of bile acid binding/transporting proteins in young 3 mo old mice, but this effect was diminished in 18 mo old mice.
Liver fatty acid binding protein (L-FABP) is a member of a large family of nonenzymatic, lipid-binding proteins that is present in high concentration (0.2-0.4mM) in liver tissue [rev. in 40]. L-FABP has a high affinity for fatty acids and much research to date has focused on the physiological role of L-FABP in intracellular fatty acid transport and metabolism. Evidence has accumulated for both non-genomic and genomic contributions of L-FABP to regulating liver lipid metabolism. Non-genomic roles of LFABP include: increased fatty acid uptake [rev. in 33,40], faster intracellular transport [27,40-43], and increased LCFA utilization by catabolic (mitochondrial and peroxisomal oxidation) [27,44] and anabolic (endoplasmic esterification) [45-47] lipid metabolic pathways. Recently, L-FABP has also been identified as a key liver protein involved in intracellular bile acid transport and secretion as well as in the regulation of cholesterol accumulation and metabolism in young (2-4 mo) mice in response to dietary cholesterol [28,30,48,49]. Genomic roles of L-FABP involve: enhanced fatty acid distribution to nuclei [12,13], interaction with PPARα in nuclei [rev. in 1,13], and regulation of PPARα transcriptional activity [rev. in 1]. Thus, either by enhancing fatty acid transport to the nucleus, channeling fatty acid to PPARα in the nucleus, or directly interacting with PPARα itself to regulate transcription, L-FABP may influence the PPARα-mediated transcription of numerous genes involved in fatty acid oxidation and glucose metabolism [rev. in 1]. If, as suggested by these studies, L-FABP contributes significantly to physiological regulation of PPARα, then L-FABP (−/−) mice should exhibit significant similarities to PPARα (−/−) mice, such as an age- and gender-dependent phenotype. For example, similar to young chow-fed PPARα gene-ablated mice [20,23], young chow-fed L-FABP gene-ablated mice also do not exhibit an overt phenotype (i.e. body weight, obesity, hepatic total lipids, serum triacylglycerols). Only with increasing age do chow-fed PPARα (−/−) mice exhibit increased body weight, obesity without hepatic steatosis, and increased serum triacylglycerols [19,23,50]. The results presented herein showed for the first time that with increasing age L-FABP (−/−) male mice also exhibited an increased body weight phenotype with many similarities to that of older PPARα (−/−) male mice. These findings were not noted in earlier studies of L-FABP (−/−) male mice, which utilized only young (2-4 mo old) mice that were genetically more variable—i.e. less back-crossed (N1 or N4 vs. N6 in the current study) [25-29]. The results presented herein provide the following new insights regarding the role of L-FABP gene ablation in body weight gain of aged, chow-fed male mice and the relevance of this phenotype to that elicited by PPARα gene ablation.
First, L-FABP gene ablation elicited significant weight gain in older (≥ 9 mo) chow-fed male mice. The increased weight gain was not due to altered food intake. Similarly elevated weight gain occurs in PPARα (−/−) mice, albeit at slightly earlier age of onset (i.e. 6 mo) .
Second, the age-dependent increase in body weight of chow-fed L-FABP (−/−) mice was due to increased fat (FTM) and lean (LTM) tissue mass, with the effect being nearly 2-fold greater for FTM than LTM. The finding of increased FTM was also observed in chow-fed PPARα male mice, albeit at slightly younger age of onset (i.e. 6 mo), as indicated by 1.5-fold increased total white adipose tissue weight .
Third, the increased body weight and obesity observed in old L-FABP (−/−) male mice were not due to hepatic abnormalities (e.g. morphology, steatosis, inflammation). Liver weights and total lipid were unaltered in chow-fed old (18 mo) L-FABP (−/−) mice. Likewise, histological examination and staining for lipid droplets revealed no visually obvious inflammation (associated with increased liver lipids) or increased hepatic lipid accumulation as a result of L-FABP gene ablation. Finally, the increased body weight gain observed in old chow-fed L-FABP (−/−) mice was not associated with alterations in the levels of insulin, or leptin, which were not increased in L-FABP (−/−) mice. Obesity is often associated with development of fatty liver (steatosis) . Although obesity and hepatic steatosis were observed in 6-10 mo old PPARα (−/−) male mice , livers of 18 mo old chow-fed L-FABP (−/−) male mice did not exhibit steatosis. Likewise, although serum levels of triacylglycerols, cholesterol, and phospholipids were all elevated in 8 mo old male chow-fed PPARα (−/−) mice , serum of 18 mo old male chow-fed L-FABP (−/−) mice was less severely affected—i.e. triacylglycerols, but not cholesterol or phospholipids, were elevated. These differences in hepatic and serum lipid profile between old chow-fed PPARα (−/−) or L-FABP (−/−) mice were noteworthy. While ablating either PPARα or L-FABP significantly reduces fatty acid β-oxidation, only PPARα gene ablation resulted in fatty liver in male mice. Taken together these results suggest that L-FABP may be only one of several factors that regulate PPARα transcriptional activity in the nucleus as has been suggested earlier [rev. in 1]. Consequently, complete loss of PPARα results in a more severe phenotype in old male mice.
Fourth, the increased body weight observed in old L-FABP (−/−) male mice was not associated with altered serum or hepatic cholesterol phenotype, but instead correlated with increased triacylglycerol level in serum concomitant with unaltered hepatic triacylglycerol in 18 mo old mice. In contrast, there is some ambiguity regarding the lipid phenotype of young 2-3 mo old male L-FABP (−/−) mice. The current study with 3-mo old male L-FABP (−/−) of N6 backcross generation showed that L-FABP gene ablation did not alter serum triacylglycerols or cholesterol, but decreased hepatic triacylglycerol without altering hepatic cholesterol—findings consistent with an earlier report with 3-mo old male L-FABP (−/−) of N4 backcross generation . In contrast, comparison of these studies with an earlier report using an independently generated L-FABP (−/−) male mouse has raised concern regarding potential ambiguity of the role of L-FABP in serum and hepatic lipid accumulation . The latter study showed that 2.5-3.5 mo old male LFABP (−/−) mice did not exhibit increased serum triacylglycerol, but did show decreased hepatic triacylglycerol accumulation without altered hepatic cholesterol . While the basis for the differences in serum triacylglycerol phenotype exhibited by young male LFABP (−/−) mice observed herein vs. that reported by others  is not known, it is important to note that the two studies differed markedly in: (i) fasting regimen (12 vs 48h); (ii) extent of back-crossing to obtain genetic homogeneity (N6 vs N1 generation), and (iii) major differences in the constructs used to ablate L-FABP. The construct used to delete L-FABP in the present investigation deleted not only much of the promoter region, but most importantly all four exons of the L-FABP gene—thereby precluding the possibility of expressing L-FABP fragments . In contrast, the L-FABP (−/−) study reporting unaltered serum triacylglycerol used a construct that left intact the 5′ noncoding region, 5′ portion of exon 1, portion of intron 2 (between exons 1 and 2), all of exons 3 and 4, and the 3′ noncoding region—thereby not precluding the possibility of expressing L-FABP fragment(s) . The importance of different construct strategies on subsequent L-FABP KO phenotype cannot be underestimated in view of reports from multiple labs demonstrating that different knock-out construct strategies can yield mice sharing some phenotypic aspects but differing significantly in others as shown for four different hormone sensitive lipase knockouts , two different perilipin knockouts [52,53], and different knockouts of other proteins . Thus, differences in serum triacylglycerol phenotype arising at least in part from different construct strategies illustrate the complexity of biological systems and are a reminder that controversy stimulates continued seeking out and resolution of these intricacies.
Fifth, the age-dependent increases in fat and lean tissue mass observed in L-FABP (−/−) male mice were accompanied by concomitant increases in protein expression levels of hepatic FATP, FAT/CD36, and LPL. These proteins are involved in LCFA uptake and release. Increased hepatic LPL levels are especially interesting because LPL is synthesized in adipose and muscle tissue and must be transported to endothelial cells in liver and other tissues. These findings suggest increased hepatic LCFA uptake as well as increased adipose LPL that could contribute to the increased adiposity observed in older L-FABP (−/−) males.
In summary, there is overall agreement between studies from our and other laboratories that young L-FABP (−/−) mice exhibit reduced hepatic fatty acid oxidation, concomitant with unaltered or increased levels of PPARα and PPARα-mediated transcription of proteins involved in fatty acid uptake and oxidation [25-30,33,55]. Old male L-FABP (−/−) mice exhibited a milder increase in PPARα and PPARα-mediated transcription of proteins involved in fatty acid uptake and oxidation. Concomitantly, serum triacylglycerols were elevated without hepatic accumulation of esterified fatty acids (steatosis). Taken together, these findings suggest the fatty acids taken up by the liver were preferentially diverted for secretion to storage in adipose tissue—consistent with the observed age-dependent weight gain in male L-FABP (−/−) mice. While ablation of some members of the fatty acid binding protein family (e.g. heart fatty acid binding protein, H-FABP) also reduces fatty acid oxidation in tissues wherein the respective protein is highly expressed [56,57], that is not the case for all members of the fatty acid binding protein family (e.g. intestinal fatty acid binding protein, I-FABP) . Furthermore, the age-dependent body weight gain observed in chow-fed 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) gene-ablated mice, which exhibited increased body weight gain as compared with their wild-type counterparts only when fed a high-fat diet ; H-FABP (−/−) mice [57,60]; and epidermal fatty acid binding protein (E-FABP) gene-ablated mice . By contrast, I-FABP (−/−) male mice gained significantly more body weight than their (+/+) counterparts when fed either a low-fat or a high-fat diet . Finally, the data presented herein showed for the first time that the age-related body-weight gain phenotype of L-FABP (−/−) male mice shares some, but not all similarities with that of old standard-chow fed male PPARα (−/−) mice since the latter exhibit hepatic steatosis while old standard-chow fed male L-FABP (−/−) mice did not. These data, taken together with findings obtained in cultured cells showing that L-FABP enhances fatty acid uptake [rev. in 40], fatty acid intracellular transport [rev. in 41,43,62], fatty acid distribution to nuclei [12,13], distributes partly into nuclei [rev. in 1,13,17], directly interacts with PPARα to stimulate PPARα transcriptional activity [rev. in 1], suggest that L-FABP may be an important physiological regulator of PPARα. A recent study of PPARγ with a mutation in the ligand binding domain (Q286P) demonstrates that a functional ligand-binding domain is not required for PPARγ mediated adipogenesis in vitro or in mice . This study suggests, but does not prove, that L-FABP-mediated delivery of lipidic ligands to PPARα may not be the only type of interaction between these two proteins. L-FABP may also act as a coactivator/corepressor of PPARα function and this potential function of L-FABP warrants further investigation.
This work was supported in part by the United States Public Health Service National Institutes of Health grants DK41402 (FS and ABK), GM31651 (FS and ABK), and DK70965 (BPA).