Decreased keratinocyte motility in skin wound on mice lacking the epidermal fatty acid binding protein gene

Fatty acids are shown to be important in various skin functions. Fatty acid binding protein (FABP) is postulated to serve as a lipid shuttle, solubilizing hydrophobic fatty acids and delivering them to the appropriate metabolic system. Among the FABP family proteins, epidermal-type FABP (E-FABP) is solely expressed in keratinocyte but its specific role in skin is not yet fully established. We found an elevated expression of E-FABP in regenerative keratinocytes of healing wounds. However, E-FABP null mice showed no marked differences compared to wild type mice in the process of wound closure, in vivo. On the other hand, in keratinocyte culture, E-FABP gene disruption decreased the cell motility, but did not affect the cell proliferation. E-FABP deletion may be compensated for in vivo by the microenvironment comprised of various cells such as fibroblasts and endothelial cells around the wound. Our analyses suggest that the E-FABP elevation may be necessary for the activation of cell motility within regenerative epidermis during wound healing.     

Expression of liver fatty acid binding protein alters growth and differentiation of embryonic stem cells

Although expression of liver fatty acid binding protein (L-FABP) modulates cell growth, it is not known if L-FABP also alters cell morphology and differentiation. Therefore, pluripotent embryonic stem cells were transfected with cDNA encoding L-FABP and a series of clones expressing increasing levels of L-FABP were isolated. Untransfected ES cells, as well as ES cells transfected only with empty vector, spontaneously differentiated from rounded adipocyte-like to fibroblast-like morphology, concomitant with marked reduction in expression of stage-specific embryonic antigen (SSEA-1). These changes in morphology and expression of SSEA-1 were greatest in ES cell clones expressing L-FABP above a threshold level. Immunofluorescence confocal microscopy revealed that L-FABP was primarily localized in a diffuse-cytosolic pattern along with a lesser degree of punctate L-FABP expression in the nucleus. Nuclear localization of L-FABP was preferentially increased in clones expressing higherlevels of L-FABP. In summary, L-FABP expression altered ES cell morphology and expression of SSEA-1. Taken together with the fact that L-FABP was detected in the nucleus, these data suggested that L-FABP may play a more direct, heretofore unknown, role in regulating ES cell differentiation by acting in the nucleus as well as cytoplasm.     

Effect of branched-chain fatty acid on lipid dynamics in mice lacking liver fatty acid binding protein gene

Although a role for liver fatty acid protein (L-FABP) in the metabolism of branched-chain fatty acids has been suggested based on data obtained with cultured cells, the physiological significance of this observation remains to be demonstrated. To address this issue, the lipid phenotype and metabolism of phytanic acid, a branched-chain fatty acid, were determined in L-FABP gene-ablated mice fed a diet with and without 1% phytol (a metabolic precursor to phytanic acid). In response to dietary phytol, L-FABP gene ablation exhibited a gender-dependent lipid phenotype. Livers of phytol-fed female L-FABP–/– mice had significantly more fatty lipid droplets than male L-FABP–/– mice, whereas in phytol-fed wild-type L-FABP+/+ mice differences between males and females were not significant. Thus L-FABP gene ablation exacerbated the accumulation of lipid droplets in phytol-fed female, but not male, mice. These results were reflected in the lipid profile, where hepatic levels of triacylglycerides in phytol-fed female L-FABP–/– mice were significantly higher than in male L-FABP–/– mice. Furthermore, livers of phytol-fed female L-FABP–/– mice exhibited more necrosis than their male counterparts, consistent with the accumulation of higher levels of phytol metabolites (phytanic acid, pristanic acid) in liver and serum, in addition to increased hepatic levels of sterol carrier protein (SCP)-x, the only known peroxisomal enzyme specifically required for branched-chain fatty acid oxidation. In summary, L-FABP gene ablation exerted a significant role, especially in female mice, in branched-chain fatty acid metabolism. These effects were only partially compensated by concomitant upregulation of SCP-x in response to L-FABP gene ablation and dietary phytol. gene targeting; phytanic acid     

Intestinal fatty acid binding protein may favor differential apical fatty acid binding in the intestine

The intestinal mucosa metabolizes fatty acids differently when presented to the lumenal or basolateral membrane. Expression of both liver and intestinal fatty acid binding proteins (L- and I-FABPs) uniquely in the enterocyte offers a possible explanation of this phenomenon. An organ explant system was used to analyze the relative binding of fatty acids to each protein. More fatty acid was bound to L-FABP than to I-FABPs (28% vs. 6% of cytosolic radioactivity), no matter on which side the fatty acid was added. However, a 2-3-fold increase in fatty acid binding to the intestinal paralog was noted after apical addition of palmitic or oleic acid in mucosa from chow fed rats. When oleic acid was added apically, a 1.4-fold increase in binding to I-FABP was observed in mucosa derived from chronically fat fed rats, consistent with the previously observed 50% increase in the content of that protein. Immunocytochemical localization of both FABPs in vivo demonstrated an apical cytoplasmic localization in the fasting state, and redistribution to the entire cytoplasm after fat feeding. These data are consistent with the hypothesis that I-FABP may contribute to the metabolic compartmentalization of apically presented fatty acids in the intestine.     

Brain arachidonic acid incorporation is decreased in heart fatty acid binding protein gene-ablated mice

Heart fatty acid binding protein (H-FABP) is expressed in neurons, but its role in brain fatty acid incorporation and metabolism is poorly defined. We examined the effect of H-FABP gene ablation on brain incorporation of arachidonic ([1-(14)C]20:4n-6) or palmitic ([1-(14)C]16:0) acid in vivo. Analysis of brain mRNA confirmed gene ablation and demonstrated no compensatory changes in the levels of other FABP mRNA in the gene-ablated mice. In brains from H-FABP gene-ablated mice, the incorporation coefficient for [1-(14)C]20:4n-6 was reduced 24%, while that for [1-(14)C]16:0 was unaffected. Within the organic and aqueous fractions, significantly more [1-(14)C]20:4n-6 was distributed into the aqueous fraction, suggesting a disruption in the metabolic targeting of 20:4n-6 in these mice. There was less incorporation of [1-(14)C]20:4n-6 into total phospholipids and a marked reduction (51%) in the level of incorporation into the choline glycerophospholipids (ChoGpl). Because FABP can influence steady-state lipid mass, brain individual lipid masses were measured. The brain total phospholipid mass was reduced 17% by gene ablation, ascribed to a 27% and 32% reduction in the masses of ChoGpl and sphingomyelin, respectively. Plasmalogen subclass masses were also reduced, suggesting that H-FABP may augment brain plasmalogen synthesis. In gene-ablated mice, the phosphatidylinositol 20:4n-6 level was reduced 25%, while the proportion of total n-6 fatty acids was reduced in the major phospholipid classes. Thus, these results demonstrate for the first time that H-FABP expression influences brain 20:4n-6 uptake and trafficking as well as steady-state brain lipid levels.     

Localization of epidermal-type fatty acid binding protein in the thymic epithelial cells of mice

The immunoreactivity for epidermal-type fatty acid binding protein of epidermis type (E-FABP) was selectively localized in the epithelial cells of both cortex and medulla of mouse thymus. The cortical epithelial cytoreticulum was clearly visible with the intense immunoreactivity and the immunoreactive cytoreticulum extended intricately throughout the thymic cortex to enclose thymocytes. In the thymic medulla, the immunoreactivity was variable in intensity among the epithelial cells and there was a tendency that epithelial cells containing more numerous tonofilament bundles were less immunoreactive. Considering the possibility that FABPs function as intracellular carriers for unsaturated long chain fatty acids, the present finding suggests that E-FABP in the thymic epithelial cells, especially the cortical ones because of their extensive location, are intimately involved in the metabolic processes of fatty acids including production of bioactive substances, such as prostaglandin and leukotriene, which are known to exert some regulation of thymic immune responses.     

The involvement of fatty acid binding protein in peroxisomal fatty acid oxidation

Rat liver fatty acid-binding protein (FABP) can function as a fatty acid donor protein for both peroxisomal and mitochondrial fatty acid oxidation, since 14C-labeled palmitic acid bound to FABP is oxidized by both organelles. FABP is, however, not detected in peroxisomes and mitochondria of rat liver by ELISA. Acyl-CoA oxidase activity of isolated peroxisomes was not changed by addition of FABP or flavaspidic acid, an inhibitor of fatty acid binding to FABP, nor by disruption of the peroxisomal membranes. These data indicate that FABP may transfer fatty acids to peroxisomes, but is not involved in the transport of acyl-CoA through the peroxisomal membrane.     

The muscle fatty acid binding protein of spadefoot toad (Scaphiopus couchii)

Fatty acid binding protein was purified from skeletal muscle of the spadefoot toad (Scaphiopus couchii), an estivating species. While estivating, this animal relies on the fatty acid oxidation for energy. Hence we were interested in the behaviour of fatty acid binding protein under conditions of elevated urea (up to 200 mM) and potassium chloride such as exist during estivation. Also we examined whether there were interactions between glycolytic intermediates and the binding ability of the protein. The amount of bound fatty acid (a fluorescence assay using cis-parinarate) was not affected (P < 0.05) by glucose, fructose 6-phosphate or phosphoenolpyruvate at physiological concentrations. By contrast, glucose 6-phosphate increased the amount of bound cis-parinarate but the apparent dissociation constant was not different from the control. Fructose 1,6-bisphosphate but not fructose 2,6-phosphate decreased cis-parinarate binding by 40%, commensurate with doubling the apparent dissociation constant (1.15-2.62 microM). Urea, guanidinium and trimethylamine N-oxide at 200 mM increased cis-parinarate binding 60% over controls. Urea (1 M) and KCl (200 mM) did not affect cis-parinarate binding compared to controls. The interaction of this fatty acid transporter with fructose 1,6-bisphosphate is discussed in terms of reciprocal interaction with phosphofructokinase since fatty acid is also an inhibitor of phosphofructokinase.     

Expression of epidermal growth factor in transgenic mice causes growth retardation

The epidermal growth factor (EGF) family of peptides signals through the erbB family of receptor tyrosine kinases and plays important roles in development and tumorigenesis. Both EGF and transforming growth factor (TGF)-alpha only bind to erbB1 and activate it. The precursor of EGF is distinct from that of TGF-alpha in having eight additional EGF-like repeats. We have recently shown that the EGF precursor without these repeats is biologically active and leads to hypospermatogenesis in transgenic mice. Here we present evidence that the growth of transgenic mice widely expressing this engineered EGF precursor is also stunted. These mice were consistently born at half the normal weight and reached almost 80% of normal weight at adulthood. The mechanism involved a reduction of serum insulin-like growth factor-binding protein-3. Chondrocyte development in the growth plate was affected, and osteoblasts accumulated in the endosteum and periosteum. Besides these novel findings on the in vivo effects of EGF on bone development, we observed no sign of tumor formation in our transgenic animals. In contrast to previous reports on TGF-alpha transgenic mice, we show that the biological functions of EGF and TGF-alpha are clearly distinct.     

Structure and dynamics of the fatty acid binding cavity in apo rat intestinal fatty acid binding protein.

The structure and dynamics of the fatty acid binding cavity in I-FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I-FABP, the probe occupied cavity volume and surface are 539+/-8 A3 and 428 A2, respectively (1.4 A probe). A total of 31 residues contact the cavity with their side chains. The side-chain cavity surface is partitioned according to the residue type as follows: 36-39% hydrophobic, 21-25% hydrophilic, and 37-43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity-with the exception of Asp74-are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I-FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I-FABP showed global conformational changes of I-FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnanosecond time scale, which are capable of facilitating exchange of I-FABP internal water.     

Effect of liver fatty acid binding protein (L-FABP) gene ablation on lipid metabolism in high glucose diet (HGD) pair-fed mice

Liver fatty acid binding protein (L-FABP) is the major fatty acid binding/”chaperone” protein in hepatic cytosol. Although fatty acids can be derived from the breakdown of dietary fat and glucose, relatively little is known regarding the impact of L-FABP on phenotype in the context of high dietary glucose. Potential impact was examined in wild-type (WT) and Lfabp gene ablated (LKO) female mice fed either a control or pair-fed high glucose diet (HGD). WT mice fed HGD alone exhibited decreased whole body weight gain and weight gain/kcal food consumed—both as reduced lean tissue mass (LTM) and fat tissue mass (FTM). Conversely, LKO alone increased weight gain, lean tissue mass, and fat tissue mass while decreasing serum β-hydroxybutyrate (indicative of hepatic fatty acid oxidation)—regardless of diet. Both LKO alone and HGD alone significantly altered the serum lipoprotein profile and increased triacylglycerol (TG), but in HGD mice the LKO did not further exacerbate serum TG content. HGD had little effect on hepatic lipid composition in WT mice, but prevented the LKO-induced selective increase in hepatic phospholipid, free-cholesterol and cholesteryl-ester. Taken together, these findings suggest that high glucose diet diminished the effects of LKO on the whole body and lipid phenotype of these mice.     

Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization.

Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long-chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart-type FABP (H-FABP) exhibit a severe defect of peripheral (nonhepatic, non-fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and beta-hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H-FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H-FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.     

Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes

Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates.     

Liver fatty acid binding protein expression enhances branched-chain fatty acid metabolism

Although liver fatty acid binding protein (L-FABP) is known to enhance uptake and esterification of straight-chain fatty acids such as palmitic acid and oleic acid, its effects on oxidation and further metabolism of branched-chain fatty acids such as phytanic acid are not completely understood. The present data demonstrate for the first time that expression of L-FABP enhanced initial rate and average maximal oxidation of [2,3-3H] phytanic acid 3.5- and 1.5-fold, respectively. This enhancement was not due to increased [2,3-3H] phytanic acid uptake, which was only slightly stimulated (20%) in L-FABP expressing cells after 30 min. Similarly, L-FABP also enhanced the average maximal oxidation of [9,10-3H] palmitic acid 2.2-fold after incubation for 30 min. However, the stimulation of L-FABP on palmitic acid oxidation nearly paralleled its 3.3-fold enhancement of uptake. To determine effects of metabolism on fatty acid uptake, a non-metabolizable fluorescent saturated fatty acid, BODIPY-C16, was examined by laser scanning confocal microscopy (LSCM). L-FABP expression enhanced uptake of BODIPY-C16 1.7-fold demonstrating that L-FABP enhanced saturated fatty acid uptake independent of metabolism. Finally, L-FABP expression did not significantly alter [2,3-3H] phytanic acid esterification, but increased [9,10-3H] palmitic acid esterification 4.5-fold, primarily into phospholipids (3.7-fold) and neutral lipids (9-fold). In summary, L-FABP expression enhanced branched-chain phytanic acid oxidation much more than either its uptake or esterification. These data demonstrate a potential role for L-FABP in the peroxisomal oxidation of branched-chain fatty acids in intact cells.     

肝型脂肪酸结合蛋白研究进展

肝型脂肪酸结合蛋白（liver fatty acid binding protein,L-FABP）是脂肪酸结合蛋白（fatty acid binding proteins,FABPs）家族重要的成员,在肝脏、小肠、肾脏等组织中均有表达。L-FABP在不饱和脂肪酸、饱和脂肪酸、胆固醇、胆汁酸等转运过程中扮演重要角色。目前研究显示L-FABP在脂肪肝、肝硬化以及肝癌发生发展中起到重要作用,并有望作为肝损伤的早期检测指标。此外,新近研究发现尿中L-FABP水平还可以用于预测1型糖尿病患者的临床结局。在2型糖尿病中,尿中L-FABP与糖尿病性肾病的病程有密切关系。主要就L-FABP的特性、结构及其与疾病的关系做一综述。     

Liver fatty acid binding protein gene ablation enhances age-dependent weight gain in male mice

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 months. While young (2–3 months old) L-FABP null mice displayed no visually obvious phenotype, with increasing age >9 months 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-month-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-month-old mice. Increased body weight, fat tissue mass, and lean tissue mass in 18-month-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.     

Antibodies against mammary derived growth inhibitor (MDGI) react with a fibroblast growth inhibitor and with heart fatty acid binding protein

A polypeptide growth inhibitor, designated as mammary derived growth inhibitor, has previously been purified from lactating bovine mammary glands. Polyclonal rabbit antiserum raised against mammary derived growth inhibitor cross-reacts with bovine heart fatty acid binding protein and bovine peripheral myelin P2 protein. These results are consistent with the observation that the amino acid sequence of mammary derived growth inhibitor showed homology to the sequences of these proteins. In a parallel series of immunoblotting experiments, rabbit anti-mammary derived growth inhibitor also reacted specifically with a fibroblast growth inhibitor purified from the conditioned medium of cultured mouse 3T3 fibroblasts. These data suggest that bovine mammary derived growth inhibitor and mouse fibroblast growth inhibitor may share common structural features and raise the possibility that these growth inhibitors may together define a new family of growth regulatory molecules.     

Fasciola gigantica fatty acid binding protein (FABP) as a prophylactic agent against Schistosoma mansoni infection in CD1 mice

Although schistosomicidal drugs and other control measures exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study, native fatty acid binding protein (FABP) from Fasciola gigantica was purified from the adult worm's crude extract by saturation with ammonium sulphate followed by separation on DEAE-Sephadex A-50 anion exchange chromatography and gel filtration using Sephacryl HR-100, respectively. CD1 mice were immunized with the purified, native F. gigantica FABP in Freund's adjuvant and challenged subcutaneously with 120 Schistosoma mansoni cercariae. Immunization of CD1 mice with F. gigantica FABP has induced heterologous protection against S. mansoni, evidenced by the significant reduction in mean worm burden (72.3%), liver and intestinal egg counts (81.3% and 80.8%, respectively), and hepatic granuloma counts (42%). Also, it elicited mixed IgG(1)/IgG(2b) immune responses with predominant IgG1 isotype, suggesting that native F. gigantica FABP is mediated by a mixed Th1/Th2 response. However, it failed to induce any significant differences in the oogram pattern or in the mean granuloma diameter. This indicated that native F. gigantica FABP could be a promising vaccine candidate against S. mansoni infection.