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The present study examined the expression and role of the thiazolidinedione (TZD)-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARgamma), in human bladder cancers. In situ hybridization shows that PPARgamma mRNA is highly expressed in all human transitional epithelial cell cancers (TCCa's) studied (n=11). PPARgamma was also expressed in five TCCa cell lines as determined by RNase protection assays and immunoblot. Retinoid X receptor alpha (RXRalpha), a 9-cis-retinoic acid stimulated (9-cis-RA) heterodimeric partner of PPARgamma, was also co-expressed in all TCCa tissues and cell lines. Treatment of the T24 bladder cancer cells with the TZD PPARgamma agonist troglitazone, dramatically inhibited 3H-thymidine incorporation and induced cell death. Addition of the RXRalpha ligands, 9-cis-RA or LG100268, sensitized T24 bladder cancer cells to the lethal effect of troglitazone and two other PPAR- activators, ciglitazone and 15-deoxy-delta(12,14)-PGJ2 (15dPGJ(2)). Troglitazone treatment increased expression of two cyclin-dependent kinase inhibitors, p21(WAF1/CIP1) and p16(INK4), and reduced cyclin D1 expression, consistent with G1 arrest. Troglitazone also induced an endogenous PPARgamma target gene in T24 cells, adipocyte-type fatty acid binding protein (A-FABP), the expression of which correlates with bladder cancer differentiation. In situ hybridization shows that A-FABP expression is localized to normal uroepithelial cells as well as some TCCa's. Taken together, these results demonstrate that PPARgamma is expressed in human TCCa where it may play a role in regulating TCCa differentiation and survival, thereby providing a potential target for therapy of uroepithelial cancers.

We describe the crystal structure of human epidermal-type fatty acid binding protein (E-FABP) that was recently found to be highly upregulated in human psoriatic keratinocytes. To characterize E-FABP with respect to ligand-binding properties and tertiary structure, we cloned the respective cDNA, overexpressed the protein in Escherichia coli and purified it to homogeneity by a combination of ion-exchange and size-exclusion chromatographic steps with a yield of 30 mg/L broth. The purified protein revealed a 5-fold higher affinity for stearic acid than for oleic and arachidonic acids. The crystal structure of recombinant human E-FABP was determined to 2.05 A and refined to an R(factor) of 20.7%. The initial residual electron density maps clearly showed the presence of a ligand, which was identified as endogenous bacterial fatty acid. Within a central cavity of 252 A(3), this ligand is bound in a U-shaped conformation, its carboxyl group interacting with tyrosine 131 and arginines 129 and 109, the latter via an ordered water molecule. The E-FABP crystal structure is unique in the FABP family because of the presence of a disulfide bridge between cysteines 120 and 127 that may be physiologically as well as pathophysiologically relevant. Cysteines 67 and 87 are also in close vicinity but in contrast do not form a disulfide bridge. We postulate that this protein belongs to a particular FABP subfamily whose members share common structural as well as functional features.

The human breast cancer cell line MCF7 does not express heart-type fatty acid binding protein (H-FABP), a marker protein for differentiated mammary gland. MCF7 cells transfected with the bovine H-FABP cDNA expressed the corresponding protein and were characterized by growth inhibition and lower tumorgenicity in nude mice [22]. By enzyme linked immunoassay we now determined the amount of bovine H-FABP in these cells as 638 +/- 80 ng/mg protein and used the transfected cells to study the role of H-FABP in fatty acid metabolism. Compared to control cells the uptake of radioactively labelled palmitic acid and oleic acid into MCF7 cells after 30 or 60 min was increased by 67% in H-FABP expressing transfectants, demonstrating a stimulatory role for this FABP-type in fatty acid metabolism. However, preferential targeting of [14C]oleic acid into neutral or phospholipid classes was not observed by the criterion of high performance thin layer chromatography followed by autoradiography. A reason for the modest increase of fatty acid uptake in H-FABP transfected MCF7 cells may be the basal expression of epidermal-type FABP, which was detected for the first time in these cells. It appears that the small amount of E-FABP expressed in MCF7 cells fulfils the need of the cells for a cytosolic fatty acid carrier under culture conditions and that even high concentrations of another FABP do only slightly increase the uptake due to limitations of fatty acid transport through the plasma membrane or of metabolism.

Fatty acid binding proteins (FABPs) are abundantly present in tissues that actively metabolize fatty acids (FA). While their precise physiological function is not known, FABPs have been shown to play a role in the uptake and/or utilization of FA within the cell. FA metabolism is markedly altered during the host response to infection and inflammation. Previous studies have demonstrated that endotoxin or bacterial lipopolysaccharide (LPS) enhances hepatic FA synthesis and re-esterification while inhibiting FA oxidation in liver, heart and muscle. Now, we have examined the in vivo effects of LPS and cytokines on FABPs in liver (L-FABP), heart and muscle (H-FABP). Syrian hamsters were injected with LPS, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) and the mRNA and protein content for L-FABP and H-FABP were analyzed. 16 h after administration, LPS (100 microg/100 g body weight) produced a 72% decrease in L-FABP mRNA levels in liver and this effect was sustained for 24 h. LPS also produced a 41% decrease in the protein content of L-FABP in liver after 24 h of treatment. TNF-alpha and IL-1beta decreased L-FABP mRNA levels in liver by 30 and 45%, respectively. LPS decreased H-FABP mRNA levels in skeletal muscle by 60% and in heart by 65%. LPS also produced a 49% decrease in H-FABP protein content in muscle. Neither TNF-alpha nor IL-1beta had any significant effect on H-FABP mRNA expression in heart and muscle. Taken together, these results indicate that LPS decreases FABP mRNA and protein levels in liver, heart and muscle, tissues that normally utilize FA as their primary fuel, whereas the inhibitory effect of cytokines is limited to the liver. The LPS-induced decrease in L-FABP and H-FABP may be an additional mechanism contributing to the decrease in FA oxidation that is associated with the host response to infection and inflammation.

The fluorescent probe anilinonaphthalene-8-sulfonate binds to adipocyte lipid binding protein at a site that competes with normal physiological ligands, such as fatty acids. Binding to the protein is accompanied by a relatively large increase in fluorescent intensity. To correlate the major change in optical properties and to determine the mechanism of competitive inhibition with fatty acids, the crystal structure of the protein with the bound fluorophore has been determined. In addition, the thermodynamic contributions to the binding reaction have been studied by titration calorimetry. Because the binding site is in a relatively internal position, kinetic studies have also been carried out to determine k(on). The results indicate that binding is not accompanied by any major conformational change. However, the negatively charged sulfonate moiety is not positioned the same as the carboxylate of fatty acid ligands as determined in previous studies. Nonetheless, the binding reaction is still driven by enthalpic effects. As judged by the crystallographic structure, a significant amount of the surface of the fluorophore is no longer exposed to water in the bound state.

Brain membrane lipid fatty acid composition and consequently membrane fluidity change with increasing age. Intracellular fatty acid binding proteins (FABPs) such as heart H-FABP and the brain specific B-FABP, detected by immunoblotting of brain tissue, are thought to be involved in fatty acid uptake, metabolism, and differentiation in brain. Yet, almost nothing is known regarding the effect of age on the expression of the cytosolic fatty acid binding proteins (FABPs) or their content in brain subfractions. Electrophoresis and quantitative immunoblotting were used to examine the content of these FABPs in synaptosomes in brains from 4, 15, and 25 month old C57BL/6NNia male mice. Brain H-FABP and B-FABP were differentially expressed in mouse brain subcellular fractions. Brain H-FABP was highly concentrated in synaptosomal cytosol. The level of brain H-FABP in synaptosomes, synaptosomal cytosol, and intrasynaptosomal membranes was decreased 33, 35, and 43%, respectively, in 25 month old mice. B-FABP was detected in lower quantity than H-FABP. More important, B-FABP decreased in synaptosomes, synaptic plasma membranes, and synaptosomal cytosol from brains of 25 month old mice. In contrast to H-FABP, B-FABP was not detectable in the intrasynaptosomal membranes in any of the three age groups of mice. In conclusion, expression of both H-FABP and B-FABP was markedly reduced in aged mouse brain. Age differences in brain H-FABP and B-FABP levels in synaptosomal plasma membranes and synaptosomal cytosol may be important factors modulating neuronal differentiation and function.

Background: Dust mites are a major cause of allergic disease worldwide. The dust mite Acarus siro is an inducer of occupational allergy among farmers, but sensitisation has also been found in non-farming populations., Methods: A degenerate primer was designed to the N-terminal amino acid sequence of a 15-kD IgE-binding protein in A. siro extract. The cDNA sequence was obtained by using reverse transcriptase polymerase chain reaction, standard cloning and sequencing techniques. The protein was expressed in Escherichia coli with a 6-histidine tag at its C-terminus. Immunoblotting of the recombinant protein and whole extract was performed using patient sera., Results and Conclusion: 15 and 17-kD allergens were identified in a fraction of A. siro extract. The cDNA of the 15-kD allergen was isolated, cloned and sequenced and the allergen was expressed as a recombinant protein. The calculated molecular weight of the cDNA-encoded protein is 14.2 kD. The predicted amino acid sequence has one potential N-glycosylation site at position 4-6 and a cytosolic fatty acid-binding protein signature at position 5-22. The protein has 64% sequence identity with Blo t 13, an allergen from the dust mite Blomia tropicalis, as well as homology with several other fatty acid-binding proteins (FABPs) from different organisms. The allergen was named Aca s 13 and was recognised strongly by 3 of 13 (23%) of the subjects investigated. The amino acid sequence of the 17-kD protein was partly determined and it also showed high sequence homology with Blo t 13 and FABPs.

A variety of circulating fuels can support the work of the teleost gill. Previous work indicates, however, that unlike other aerobic tissues from teleosts, the gill may have a limited capacity to oxidize fatty fuels. We determined capacities for catabolism of carbohydrate, fatty acids, and amino acids in four species of temperate marine or euryhaline teleosts representing distinct lineages. In addition, we assessed the capacity for fatty acid oxidation in the gill from an Antarctic species. Activities of rate-limiting or regulatory enzymes from pathways of energy metabolism were measured at physiological temperatures (15 degrees or 1 degrees C). In the temperate species, ATP yields from glucose are 3- to 30-fold greater (varying with species) than ATP yields from a monounsaturated fatty acid, while ATP generation from glutamate is 2-50 times greater than similar capacities for the lipid fuel. Like the temperate species, capacity for beta-oxidation of fatty acids is limited in the Antarctic species. A positive linear correlation between activities of citrate synthase (central pathway of oxidative metabolism) and hexokinase (glycolysis) adds further support to the hypothesis that glucose is a preferred metabolic fuel in gill. Our results also demonstrate that fatty acid-binding protein is present in the gill of teleost fishes. It is likely that this protein plays a more important role facilitating anabolic pathways in lipid metabolism rather than fatty acid oxidation in the gill of teleost fishes., (Copyright 1999 Wiley-Liss, Inc.)

While aspects of cellular fatty acid uptake have been studied as early as 50 years ago, recent developments in this rapidly evolving field have yielded new functional insights on the individual mechanistic steps in this process. The extremely low aqueous solubility of long chain fatty acids (LCFA) together with the very high affinity of serum albumin and cytoplasmic fatty acid binding proteins for LCFA have challenged the limits of technology in resolving the individual steps of this process. To date no single mechanism alone accounts for regulation of cellular LCFA uptake. Key regulatory points in cellular uptake of LCFA include: the aqueous solubility of the LCFA; the driving force(s) for LCFA entry into the cell membrane; the relative roles of diffusional and protein mediated LCFA translocation across the plasma membrane; cytoplasmic LCFA binding protein-mediated uptake and/or intracellular diffusion; the activity of LCFA-CoA synthetase; and cytoplasmic protein mediated targeting of LCFA or LCFA-CoAs toward specific metabolic pathways. The emerging picture is that the cell has multiple, overlapping mechanisms that assure adequate uptake and directed intracellular movement of LCFA required for maintenance of physiological functions. The upcoming challenge is to take advantage of new advances in this field to elucidate the differential interactions between these pathways in intact cells and in tissues.

Background: The kidney metabolizes actively lipophilic molecules. Several species of lipid-binding proteins (LBPs) have been well characterized, including fatty acid-binding proteins (FABPs), acyl-CoA binding protein (ACBP), sterol carrier protein 2 (SCP2), cellular retinol binding protein (CRBP), and phosphatidylinositol transfer protein (PITP)., Methods: To clarify which LBPs are expressed in isolated rat glomeruli (RG), cultured rat mesangial cells (RMC) and human kidney, RT-PCR, immunoblot analysis and immunohistochemistry were performed., Results: Protein and mRNA expression of heart type (H-) FABP was found in RMC, but not in RG. Immunohistochemistry using antihuman H-FABP antibody revealed that an H-FABP like protein was present in the capillary wall and distal tubules of human glomeruli. Immunoblot analysis using the antibody showed that a 110-kDa protein related to H-FABP was present in human isolated glomeruli but not in any other tissues tested including blood, liver, and heart, and that the 14-kDa protein, H-FABP itself was localized in the distal tubules of human kidney. mRNA for SCP2, ACBP and PITP was detected in RG and RMC. CRBP and mRNA was detected in RG but not RMC., Conclusions: A variety of lipid-binding proteins are present in rat glomeruli. In human glomeruli, a novel 110-kDa H-FABP-related protein is localized specifically in the capillary wall.

To evaluate the effects of endurance training in rats on fatty acid metabolism, we measured the uptake and oxidation of palmitate in isolated rat hindquarters as well as the content of fatty acid-binding proteins in the plasma membranes (FABP(PM)) of red and white muscles from 16 trained (T) and 18 untrained (UT) rats. Hindquarters were perfused with 6 mM glucose, 1,800 microM palmitate, and [1-(14)C]palmitate at rest and during electrical stimulation (ES) for 25 min. FABP(PM) content was 43-226% higher in red than in white muscles and was increased by 55% in red muscles after training. A positive correlation was found to exist between succinate dehydrogenase activity and FABP(PM) content in muscle. Palmitate uptake increased by 64-73% from rest to ES in both T and UT and was 48-57% higher in T than UT both at rest (39.8 +/- 3.5 vs. 26.9 +/- 4. 4 nmol. min(-1). g(-1), T and UT, respectively) and during ES (69.0 +/- 6.1 vs. 43.9 +/- 4.4 nmol. min(-1). g(-1), T and UT, respectively). While the rats were resting, palmitate oxidation was not affected by training; palmitate oxidation during ES was higher in T than UT rats (14.8 +/- 1.3 vs. 9.3 +/- 1.9 nmol. min(-1). g(-1), T and UT, respectively). In conclusion, endurance training increases 1) plasma free fatty acid (FFA) uptake in resting and contracting perfused muscle, 2) plasma FFA oxidation in contracting perfused muscle, and 3) FABP(PM) content in red muscles. These results suggest that an increased number of these putative plasma membrane fatty acid transporters may be available in the trained muscle and may be implicated in the regulation of plasma FFA metabolism in skeletal muscle.

Lipocalin-type prostaglandin D synthase is a major protein of the cerebrospinal fluid and was originally known as beta-trace. We investigated the binding ability of prostaglandin D synthase toward bile pigments, thyroid hormones, steroid hormones, and fatty acids in this present study. We found that the recombinant enzyme binds bile pigments and thyroid hormones, resulting in quenching of the intrinsic tryptophan fluorescence, the appearance of induced circular dichroism of the lipophilic ligands, and a red shift of the absorption spectra of bilirubin and biliverdin. The binding of prostaglandin D synthase to lipophilic ligands was also demonstrated by the resonant mirror technique and surface plasmon resonance detection. The dissociation constants were calculated to be 33 nM, 37 nM, 660 nM, 820 nM, and 2.08 microM for biliverdin, bilirubin, L-thyroxine, 3,3',5'-triiodo-L-thyronine, and 3,3', 5-triiodo-L-thyronine, respectively. Biliverdin and bilirubin underwent a shift in their absorption peaks from 375 to 380 nm and from 439 to 446 nm, respectively, after binding to prostaglandin D synthase. Bilirubin bound to the enzyme showed a bisignate CD spectrum with a (-) Cotton effect at 422 nm and a (+) Cotton effect at 472 nm, indicating a right-handed chirality. The ligands also inhibited prostaglandin D synthase activity noncompetitively in a concentration-dependent manner, with IC50 values between 3.9 and 10. 9 microM. Epididymal retinoic acid-binding protein and beta-lactoglobulin, two other lipocalin proteins that bind retinoids such as prostaglandin D synthase, did not show any significant interaction with bile pigments or thyroid hormones. These results show that prostaglandin D synthase binds small lipophilic ligands with a specificity distinct from that of other lipocalins.

Giant sarcolemmal vesicles were isolated from rat heart and hindlimb muscles for a) characterization of long-chain fatty acid transport in the absence of metabolism and b) comparison of fatty acid transport protein expression with fatty acid transport. Giant vesicles contained cytosolic fatty acid binding protein. Palmitate uptake was completely divorced from its metabolism. All palmitate taken up was recovered in the intravesicular cytosol as unesterified FA. Palmitate uptake by heart vesicles exhibited a K m of 9.7 nm, similar to that of muscle (K m = 9.7 nm). Vmax (2.7 pmol/mg protein/s) in heart was 8-fold higher than in muscle (0.34 pmol/mg protein/s). Palmitate uptake was inhibited in heart (55-80%) and muscle (31-50%) by trypsin, phloretin, sulfo-N-succinimidyloleate (SSO), or a polyclonal antiserum against the 40 kDa plasma membrane fatty acid binding protein (FABPpm). Palmitate uptake by heart and by red and white muscle vesicles correlated well with the expression of fatty acid translocase (FAT/CD36) and fatty acid binding protein FABPpm, which may act in concert. The expression of fatty acid transport protein (FATP), was 10-fold lower in heart vesicles than in white muscle vesicles. It is concluded that long-chain fatty acid uptake by heart and muscle vesicles is largely protein-mediated, involving FAT/CD36 and FABPpm. The role of FATP in muscle and heart remains uncertain.

Information on the interconversion potential of adipocytes and other end cells characteristic of the stromal fibroblastic cell lineages, key in the understanding of bone turnover in metabolic diseases such as osteoporosis, is limited. The object of the present study was: i) to isolate relatively pure populations of adipocytes from human bone marrow; ii) to clone single adipocytes from these populations; and iii) to examine in vitro the interconversion potential of the progeny of these single-cloned adipocytes between the osteogenic and adipogenic phenotypes. Adipogenic colonies were isolated from the low-density floating fraction of normal bone marrow cells cultured in adipogenic media for 4 days. Single adipocytes were isolated and cloned by limiting dilution. Cloned adipocytes were found to dedifferentiate into fibroblast-like cells, and subsequently to differentiate into two morphologically distinct cell types: osteoblasts and adipocytes in appropriate culture systems. The adipocytic phenotype was confirmed by morphology, oil red O staining, and immunocytochemistry using antiserum to aP2. The osteogenic phenotype was confirmed by alkaline phosphatase, osteocalcin immunostaining using specific osteocalcin antiserum, and formation of mineralized cell aggregates. These findings demonstrate the extent of plasticity between the differentiation of adipocytic and osteogenic cells in human bone marrow stromal cell cultures. We have shown the ability of isolated clonal adipogenic cells to redifferentiate into cells of the osteogenic and adipogenic lineage and the interconversion potential of human marrow stromal cells in vitro. These results provide further evidence that the osteogenic and adipogenic cells share a common multipotential precursor.

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that binds fatty acids, retinoids, and other hydrophobic ligands. The interaction was demonstrated in vitro by the binding of ALBP to HSL translated in vitro, to HSL in extracts of HSL overexpressing Chinese hamster ovary (CHO) cells, and to HSL in extracts of rat adipose tissue. Finally, the presence of ALBP was documented in immune complexes from rat adipose tissue immunoprecipitated with anti-HSL antibodies. The HSL-ALBP interaction was mapped to an N-terminal 300-aa region of HSL that is distinct from the C-terminal catalytic domain. These results suggest that HSL-derived fatty acids are bound by ALBP to facilitate intracellular trafficking of hydrophobic lipids.

The availability of mice containing an adipocyte lipid-binding protein (ALBP/aP2) gene disruption allowed for a direct examination of the presumed role of lipid-binding proteins in the mobilization and trafficking of intracellular fatty acids. Total body and epididymal fat pad weights, as well as adipose cell morphology, were unaltered in male ALBP/aP2 disrupted mice when compared to their wild-type littermates. Analysis of adipocytes isolated from wild-type and ALBP/aP2 null mice revealed that a selective 40- and 13-fold increase in the level of the keratinocyte lipid-binding protein (KLBP) mRNA and protein, respectively, accompanied the ALBP/aP2 gene disruption. Although KLBP protein was significantly up-regulated, the total lipid-binding protein level decreased 8 -fold as a consequence of the disruption. There was no appreciable difference in the rate of fatty acid influx or esterification in adipocytes of wild-type and ALBP/aP2 null animals. To the contrary, basal lipolysis decreased approximately 40% in ALBP/aP2 nulls as compared to wild-type littermates. The glycerol release from isproterenol-stimulated ALBP/aP2 null fat cells was similarly reduced by approximately 35%. Consistent with a decrease in basal efflux, the non-esterified fatty acid (NEFA) level was nearly 3-fold greater in adipocytes from ALBP/aP2 nulls as compared to wild-type animals. The significant decrease in both basal and isoproterenol-stimulated lipolysis in adipose tissue of ALBP/aP2 null mice supports the model whereby intracellular lipid-binding proteins function as lipid chaperones, facilitating the movement of fatty acids out of the fat cell.

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.

Expression of epidermal-type fatty acid-binding protein (E-FABP) and S100A7 has previously been shown to be elevated in psoriatic skin, a disease characterized by abnormal keratinocyte differentiation. However, no causal relationship between the up-regulation of these proteins and the disease has been shown. E-FABP is thought to be involved in cytosolic fatty acid (FA) transport, whereas the role of S100A7 is still unknown. In this report, we show by overlay assays that E-FABP, immobilized on nitrocellulose, is able to capture S100A7 from cytosolic psoriatic protein extracts and vice versa, suggesting the formation of a complex between the two proteins. Using purified E-FABP and S100A7, the complex can be reconstituted only in presence of EDTA. Moreover, we show that increased EDTA concentrations in psoriatic cytosolic protein extracts enhance complex formation. Partial complex disruption was obtained by the addition of physiological concentrations of Zn2+ (0.1 mM), whereas Ca2+ at 5 mM and Mg2+ at 30 mM had no effect. On the other hand, high Ca2+ concentrations (30 mM) resulted in partial complex disruption. Oleic acid-binding properties were observed for free E-FABP and the complex E-FABP-S100A7, but not for free S100A7. By using confocal microscopy we show that S100A7 and E-FABP are co-localized in the cytoplasm of differentiating keratinocytes from lesional psoriatic skin. These data indicate that formation of the E-FABP-S100A7 complex and its FA-binding function might be regulated at least by bivalent cations.

Branched-chain phytanic acid is metabolized in liver peroxisomes. Sterol carrier protein 2/sterol carrier protein x (SCP2/SCPx) knockout mice, which develop a phenotype with a deficiency in phytanic acid degradation, accumulate dramatically high concentrations of this fatty acid in serum (Seedorf at al. 1998. Genes Dev. 12: 1189-1201) and liver. Concomitantly, a 6.9-fold induction of liver fatty acid binding protein (L-FABP) expression is observed in comparison to wild-type animals fed standard chow, possibly mediated by the peroxisome proliferator-activated receptor alpha (PPARalpha). Cytosolic transport of phytanic acid to either peroxisomal membranes or to the nucleus for activation of PPARalpha may be mediated by L-FABP, which gives rise to the question whether phytanic acid is a transactivator of this protein. Here we show first that phytanic acid binds to recombinant L-FABP with high affinity. Then the increase of the in vivo phytanic acid concentration by phytol feeding to mice results in a 4-fold induction of L-FABP expression in liver, which is in the order of that attained with bezafibrate, a known peroxisome proliferator. Finally to test in vitro whether this induction is conferred by phytanic acid, we cotransfected HepG2 cells with an expression plasmid for murine PPARalpha and a CAT-reporter gene with 176 bp of the murine L-FABP promoter, containing the peroxisome proliferator responsive element (PPRE). After incubation with phytanic acid, we observed a 3.2-fold induction of CAT expression. These findings, both in vivo and in vitro, demonstrate that phytanic acid is a transcriptional activator of L-FABP expression and that this effect is mediated via PPARalpha.

The effect was investigated of treadmill training of moderate intensity on the fatty acid-binding protein (FABP) content in relation to parameters of oxidative and glycolytic metabolism. To this end, the cytoplasmic FABP content and the activity of beta-hydroxyacyl-coenzyme A dehydrogenase (HAD), citrate synthase (CS), and 6-phosphofructokinase (PFK) were measured in heart, fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles (SOL) of male Wistar rats. To investigate the influence of the amount of training (defined as the product of exercise duration, intensity and frequency), two training groups were created that differed in training frequency (HF, high frequency 5 days x week(-1), n = 9; LF, low frequency 2 days x week(-1), n = 9; the exercise being 20 m x min(-1) for 2 h with no gradient, over 6 weeks) and compared with SC, sedentary controls (n = 7). In heart muscle, the cytoplasmic FABP content was 34% higher in HF than in SC but was the same as in LF. The CS and HAD activities were no different in the three groups, suggesting that the capacity to oxidize fatty acids (FA) was not affected by training. The PFK activity was higher (43%) in HF, suggesting a shift towards carbohydrate utilization. The FABP content and HAD activity did not change in SOL and EDL after training whereas the CS activity increased (27%) in SOL and decreased (21%) in EDL in both training groups. In addition, PFK activity in EDL was much higher (113%) in the HF than in SC group. The HF training was associated with a fine-tuning of FA availability and use in heart muscle, and with a more efficient energy production. It is suggested therefore that cytoplasmic FABP could be an early marker of muscle adaptation to training in heart but not in skeletal muscle. The training reinforced the metabolic profile of the skeletal muscles, in particular that of the fast-twitch glycolytic muscle. We concluded that a large amount of training is needed when the effect on both oxidative and glycolytic parameters is to be studied.

The long-distance migratory flights of birds are predominantly fueled by the oxidation of fatty acids, which are sourced primarily from extracellular adipose stores. These fatty acids have to be transported, via the circulatory system, to the mitochondria of the active muscles. An important facilitator of fatty acid transport within the cytoplasm of muscle cells is fatty acid binding protein (FABP), which serves as an intracellular carrier of long-chain fatty acids. In mammals, the muscular FABP content is related to the fatty acid oxidation capacity of the tissue. The aim of this study was to measure FABP in samples taken from the cardiac, pectoralis, and semimembranosus muscles of a long-distance avian migrant, the barnacle goose (Branta leucopsis), at various stages of development. Western blot analysis identified a single goose muscle protein of 15 kDa that was able to bind fatty acids and showed a 66% cross-reactivity with antibodies against human heart-type FABP. Captive goslings showed no significant changes in FABP content of either the heart (62.6 +/- 10.6 microgram/g wet wt) or the semimembranosus muscle (8.4 +/- 1.9 microgram/g wet wt) during development. However, in both peripheral and deep sites within the pectoralis muscle, FABP content of samples taken from captive goslings were approximately 10-fold higher throughout development and reached values of 30-40 microgram/g wet wt in fledging goslings at 7 wk of age. A further twofold higher value was seen in wild but not in captive goslings immediately before migration (12 wk of age). Similarly, FABP content was significantly higher in pectoralis samples taken from wild adults (94.3 +/- 3.6 microgram/g wet wt) compared with those from captive adults (60.5 +/- 3.6 micro/g wet wt). These results suggest that the experience of flight activity may be of critical importance in achieving maximal expression of FABP in the pectoralis muscles of postfledging and mature geese immediately before migration.

Epidermal growth factor (EGF) has been reported to stimulate carbohydrate, amino acid, and electrolyte transport in the small intestine, but its effects on lipid transport are poorly documented. This study aimed to investigate EGF effects on fatty acid uptake and esterification in a human enterocyte cell line (Caco-2). EGF inhibited cell uptake of [14C]palmitate and markedly reduced its incorporation into triglycerides. In contrast, the incorporation in phospholipids was enhanced. To elucidate the mechanisms involved, key steps of lipid synthesis were investigated. The amount of intestinal fatty acid-binding protein (I-FABP), which is thought to be important for fatty acid absorption, and the activity of diacylglycerol acyltransferase (DGAT), an enzyme at the branch point of diacylglycerol utilization, were reduced. EGF effects on DGAT and on palmitate esterification occurred at 2-10 ng/ml, whereas effects on I-FABP and palmitate uptake occurred only at 10 ng/ml. This suggests that EGF inhibited palmitate uptake by reducing the I-FABP level and shifted its utilization from triglycerides to phospholipids by inhibiting DGAT. This increase in phospholipid synthesis might play a role in the restoration of enterocyte absorption function after intestinal mucosa injury.

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, plays an essential role in the mediation of the actions of antidiabetic drugs known as thiazolidinediones (TZDs). PPARgamma activates many target genes involved in lipid anabolism including the adipocyte fatty acid binding protein (aP2). In this study, induction of aP2 gene expression by PPARgamma agonists was examined in both cultured cells and diabetic mice using branched DNA (bDNA)-mediated mRNA quantitation. bDNA technology allows for the direct measurement of a particular mRNA directly within cellular lysate using a 96-well plate format in a time frame comparable to a reporter gene assay. In cultured human subcutaneous preadipocytes, the TZDs, troglitazone and BRL-49653, both rapidly induced aP2 mRNA as detected with the bDNA method. In these cells, the effect of BRL-49653 on aP2 mRNA levels was detectable as early as 30 min after treatment (47% increase) and was maximal after 24 h of treatment (12-fold increase). The effects of troglitazone on aP2 mRNA induction were similar to those of BRL-49653 except that the maximal level of induction was consistently lower (e.g. 24 h treatment = 4-fold increase). Dose-response relationships for both of the TZDs were also determined using the 24-h treatment time point. EC50s for both BRL-49653 and troglitazone were estimated to be 80 nM and 690 nM, respectively. A natural PPARgamma ligand, 15-deoxy-delta12,14-PGJ2, was also active in this assay with a maximal induction of aP2 mRNA of approximately 5-fold when tested at 1 microM. Since the PPARgamma:retinoid X receptor (RXR) heterodimer has been characterized as a permissive heterodimer with respect to RXR ligands, the ability of 9-cis-retinoic acid (9-cis-RA) to induce aP2 mRNA was examined. Although 9-cis-RA had very low efficacy (2-fold induction), the maximal effect was reached at 100 nM. No synergism or additivity in aP2 mRNA induction was detected when 9-cis-RA was included with either of the TZDs used in this study. Significant induction of aP2 mRNA in bone marrow of db/db mice treated with either troglitazone or BRL-49653 was also detected, indicating that the bDNA assay may be a simple method to monitor nuclear receptor target gene induction in vivo.

It has been suggested that specific membrane-associated and cytoplasmic proteins cooperate in the uptake of long-chain fatty acids by cardiac and skeletal muscle cells. A prerequisite for this hypothesis would be the co-occurrence of these proteins in muscle. Thus, we studied the possible co-expression in rat muscles of the genes coding for the integral membrane proteins fatty acid transport protein (FATP) and fatty acid translocase (FAT), the membrane-associated plasmalemmal fatty acid-binding protein (FABPpm) and the cytoplasmic heart-type fatty acid-binding protein (H-FABPc). The transcripts of the four proteins were assessed in heart and skeletal muscles of adult Wistar rats, in isolated cells and cell lines from rat heart and also in rat heart during development and upon streptozotocin-induced diabetes. All four genes showed high expression levels in heart, somewhat lower in red skeletal muscle (soleus) and appreciably lower in white skeletal muscle (extensor digitorum longus). FATP, FAT and H-FABPc showed a 3- to 5-fold increase in mRNA expression during maturational growth of the heart, while the FABPpm expression remained virtually constant. In the heart, streptozotocin-diabetes induced a slight, but statistically not significant, increase in the expression of all four genes. In conclusion, this study shows the co-expression of FATP, FAT, FABPpm and H-FABPc in rat muscles. This finding supports the possible cooperation of these proteins in the uptake of long-chain fatty acids by muscle cells.

The insolubility of fatty acids in cellular environments requires that specific trafficking mechanisms be developed to vectorally orient and deliver lipids for cellular needs. The roles of putative membrane bound fatty acid transporters and soluble carrier proteins are discussed in terms of mechanisms of fatty acid trafficking. The numerous roles for fatty acids as an energy source, as structural elements for membrane synthesis, as bioregulators and as prohormones with the potential to regulate gene expression, are discussed in terms of the necessity to regulate their intracellular location and concentration.

The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that forWT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37 degrees C is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.

The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common beta-barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited 'portal' region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.

The overexpression of E-FABP and S100A7 in lesional psoriatic skin suggests a possible link with this hyperproliferative skin disease. In order to investigate a role for the proteins in this disease, the purifications for both proteins were re-analyzed. Moreover, a specific antiserum directed against purified human S100A7 was generated. By SDS-PAGE immunoblotting we show that E-FABP and S100A7 are expressed in cultured human differentiating keratinocytes and confirm their overexpression in psoriatic scales. Gel filtration and non-denaturing PAGE revealed that S100A7 co-purified with E-FABP, indicating an association between the two proteins. Ion-exchange chromatography resulted in the dissociation of the complex. Finally, immunoprecipitations using antiserum against E-FABP revealed that S100A7 co-immunoprecipitated with E-FABP from protein extracts of psoriatic scales. These data indicate that E-FABP and S100A7 might form a complex in the cytosol of human keratinocytes.

Diabetes mellitus generally results in an increased systemic fatty acid mobilization which can be associated with an increase in mitochondrial and peroxisomal beta-oxidation of fatty acids in selected tissues. The latter is usually accompanied by a concomitant increase in the tissue content of cytoplasmic fatty acid-binding protein (FABP) which functions in the intracellular translocation of fatty acids. It was previously found that in liver clofibrate-induced proliferation of peroxisomes and increase in FABP expression each are dependent on the induction by cytochrome P4504A1 -mediated (CYP4A1) formation of dicarboxylic acids. We studied whether peroxisome proliferation and an increase of FABP contents in liver, heart and kidney of streptozotocin-induced diabetic rats are also accompanied by an increase of CYP4A1 activity, as this would indicate a possible regulatory role for dicarboxylic acids in peroxisome proliferation and FABP induction in diabetic organs other than liver. In livers of the diabetic rat, a concomitant increase was observed of the activities of CYP4A1 and the peroxisomal key enzyme fatty acyl-CoA oxidase (FACO) and of the FABP content. In the diabetic heart FACO activity and FABP content also increased, but there was no induction of CYP4A1 activity. Conversely, in diabetic kidney there was no increase in FACO activity nor FABP content in spite of a marked induction of CYP4A1 activity. It is concluded that streptozotocin-induced diabetes leads to increased peroxisome proliferation and increased levels of FABP in both liver and heart, which only in liver is accompanied by an induction of the cytochrome P450 system. Consequently, it is not likely that dicarboxylic acids are involved in the induction of peroxisome proliferation in the heart.

Liver fatty acid binding protein (L-FABP) appears to contain several different forms that may result from post-translational modification or bound ligand. To further assess this possibility, L-FABP was purified from rat liver homogenate and two putative isoforms separated using a sulfonyl column, a strong cation exchange resin. Fraction I eluted at 0.2 M NaCl, had a pI of 7.59, and following a final size exclusion step contained > 98% L-FABP. Fraction II eluted at 1.0 M NaCl, had a pI of 7.59, and following a final size exclusion step contained > 99% L-FABP. Both fractions contained approx. 0.15 moles of endogenous bound fatty acid per mole of protein, while L-FABP not subjected to the cation exchange step contained 0.75 moles of fatty acid per mole of protein. Fractions I and II had a greater proportion of saturated and monounsaturated fatty acids with a large reduction in polyunsaturated fatty acids compared to L-FABP not fractionated by cation exchange. Mass spectral analysis indicated the molecular mass of Fraction I was 14,315.02 +/- 0.35 Da and Fraction II was 14,315.86 +/- 0.34 Da. The peptide map for each fraction was determined by limited digestion of each fraction with either trypsin, Asp-N, or chymotrypsin to yield overlapping peptide fragments. Mass spectral analysis of these digests indicated the two proteins had identical amino acid fragments and that Cys69 was reduced and there were no Asn to Asp exchanges. Hence, these two forms of L-FABP were not isoforms and were not the result of differences in bound fatty acid. It is proposed that these two distinct forms of rat L-FABP were structural conformers based on two alternative folding pathways.

Liver fatty acid-binding protein (L-FABP) is a small cytoplasmic molecule highly expressed in the liver. Since L-FABP exhibits affinities for several biliary components, its presence in bile was explored by Western blotting and competitive ELISA in various mammalian species. A L-FABP-like immunoreactivity was consistently found in both hepatic and gallbladder bile. A close molecular identity between this 14 kDa biliary protein and the purified L-FABP was assessed by immunological analyses and high performance capillary electrophoresis. Pharmacological induction of hepatic L-FABP biosynthesis led to a similar increase in biliary L-FABP levels showing a close relationships between the cytosolic and biliary contents of this protein. Finally, a correlation between the presence of L-FABP in bile and both bile flow and bile acid release was found. These data suggest an output of L-FABP in bile in normal conditions which might be coupled with the physiological release of biliary components.

The hyperlipidemia associated with obesity and type 2 diabetes is caused by an increase in hepatic triglyceride synthesis and secretion that is secondary to an increase in de novo lipogenesis, a decrease in fatty acid (FA) oxidation, and an increase in the flux of peripherally derived FA to the liver. The uptake of FA across the plasma membrane may be mediated by three distinct proteins--FA translocase (FAT), plasma membrane FA binding protein (FABP-pm), and FA transport protein (FATP)--that have recently been characterized. Acyl-CoA synthetase (ACS) enhances the uptake of FAs by catalyzing their activation to acyl-CoA esters for subsequent use in anabolic or catabolic pathways. In this study, we examine the mRNA levels of FAT, FABP-pm, FATP, and ACS in the liver and adipose tissue of genetically obese (ob/ob) mice and their control littermates. FAT mRNA levels were 15-fold higher in liver and 60-80% higher in adipose tissue of ob/ob mice. FABP-pm mRNA levels were twofold higher in liver and 50% higher in adipose tissue of ob/ob mice. FATP mRNA levels were not increased in liver or adipose tissue. ACS mRNA levels were higher in adipose tissue but remained unchanged in liver. However, the distribution of ACS activity associated with mitochondria and microsomes in liver was altered in ob/ob mice. In control littermates, 61% of ACS activity was associated with mitochondria and 39% with microsomes, whereas in ob/ob mice 34% of ACS activity was associated with mitochondria and 66% with microsomes; this distribution would make more FA available for esterification, rather than oxidation, in ob/ob mouse liver. Taken together, our results suggest that the upregulation of FAT and FABP-pm mRNAs may increase the uptake of FA in adipose tissue and liver in ob/ob mice, which, coupled with an increase in microsomal ACS activity in liver, will enhance the esterification of FA and support the increased triglyceride synthesis and VLDL production that characterizes obesity and type 2 diabetes.

Despite the importance of long-chain fatty acids (FA) as fuels for heart and skeletal muscles, the mechanism of their cellular uptake has not yet been clarified. There is dispute as to whether FA are taken up by the muscle cells via passive diffusion and/or carrier-mediated transport. Kinetic studies of FA uptake by cardiac myocytes and the use of membrane protein-modifying agents have suggested the bulk of FA uptake is due to a protein component. Three membrane-associated FA-binding proteins were proposed to play a role in FA uptake, a 40-kDa plasma membrane FA-binding protein (FABPpm), an 88-kDa FA translocase (FAT/CD36), and a 60-kDa FA transport protein (FATP). In cardiac and skeletal myocytes the intracellular carrier for FA is cytoplasmic heart-type FA-binding protein (H-FABP), which likely transports FA from the sarcolemma to their intracellular sites of metabolism. A scenario is discussed in which FABPpm, FAT/CD36, and H-FABP, probably assisted by an albumin-binding protein, cooperate in the translocation of FA across the sarcolemma.

Muscular overuse after high force eccentric muscle action is associated with structural damage of the contractile apparatus that can be observed as Z-line steaming and myofibrillar disruption. Mechanical stress is the major contributing factor for inducing muscle injury, which initiates a cascade of processes resulting in skeletal muscle damage. Disturbances in Ca2+ homeostasis with elevated intracellular [Ca2+] activates the nonlysomal cysteine protease, calpain. Calpain is assumed to play an important role in triggering the response of skeletal muscle protein breakdown, of inflammatory changes, and of regeneration processes in response to eccentric muscle action. The inflammatory response is attributed to changes in hormone and cytokine levels in blood and skeletal muscle. To assess the amount of skeletal muscle damage, plasma CK activity and plasma myoglobin levels have been widely used as markers for muscle injury. As the cytosolic proteins do not necessarily reflect the amount of structural damage, structurally bound proteins such as myosin heavy chains and troponin have been investigated. This paper briefly reviews the cascade of events causing muscle cell injury after unaccustomed eccentric muscle action and the potential of muscle proteins as markers of skeletal muscle damage.

Up until now, the primary structure of fatty-acid-binding proteins (FABPs) from the livers of four mammalian (rat, human, cow and pig) and three nonmammalian (chicken, catfish and iguana) species has been determined. Based on amino acid sequence comparisons, it has been suggested that mammalian and nonmammalian liver FABPs may be paralogous proteins that originated by gene duplication, rather than as a consequence of mutations of the same gene. In this paper we report the isolation and amino acid sequence determination of two FABPs from axolotl (Ambistoma mexicanum) liver. One of them is similar to mammalian liver FABPs (L-FABPs) and the other to chicken, catfish and iguana liver FABPs (Lb-FABPs). The finding of both L-FABP and Lb-FABP in a single species, as reported here, indicates that they are paralogous proteins. The time of divergence of these two liver FABP types is estimated to be of approximately 694 million years ago. The ligand-binding properties of axolotl liver FABPs were studied by means of parinaric-acid-binding and parinaric-acid-displacement assays. L-FABP binds two fatty acids per molecule but Lb-FABP displays a fatty-acid-conformation-dependent binding stoichiometry; L-FABP shows a higher affinity for fatty acids, especially oleic acid, while Lb-FABP has a higher affinity for other hydrophobic ligands, especially retinoic acid. In addition, the tissue-expression pattern is different, L-FABP is present in liver and intestinal mucosa while the expression of Lb-FABP is restricted to liver. Data indicate distinct functional properties of both liver FABP types.

Although a morphologically distinct population of lipid-laden interstitial cells (lipofibroblasts, LF), has been identified, the origins and functions of this population during lung development and disease remain undefined. Illumination of the developmental and functional characteristics of two other populations of lipid-laden mesenchymal cells, namely adipocytes and hepatic lipocytes, has fashioned tools that can be used to explore similar properties in pulmonary LFs. As the LF is transiently a very abundant cell in the perinatal lung, we elected to study the perinatal ontogeny of the expression of several genes that are involved in the acquisition of lipids by adipocytes, and may be involved in promoting the triglyceride accumulation that is the morphologic hallmark of the pulmonary LF. We found that the maximal expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma), at gestational day 21 in the LF, precedes the rise at birth, in the expression of genes that are involved in the hydrolysis of triglycerides at the plasma membrane (lipoprotein lipase, LPL), transport of fatty acids across the plasma membrane (fatty acid transporter, FAT) and in the cytoplasm (adipocyte lipid binding protein, ALBP). The steady-state levels of LPL, FAT, and ALBP mRNAs that were isolated from whole lung tissue showed a similar temporal pattern. The levels of the protein products of the LPL and ALBP genes changed in tandem with those of their precursor mRNAs in the LF, suggesting that these gene products are under pre-translational control. These findings indicate that characteristic adipocyte genes are also expressed in lipid-laden pulmonary fibroblasts and may participate in triglyceride accumulation and metabolism by these cells.

The porcine A-FABP gene (FABP4) was isolated and sequenced to study the role of A-FABP in the differentiation of intramuscular fat (IMF) accretion in pigs. The coding sequence of the porcine A-FABP gene is highly conserved across human, mouse, and rat. Moreover, all the functionally important amino acids are conserved. This high similarity extends into the first 270 bp of the 5' upstream region. Within this region, a 56-bp nucleotide sequence was completely identical with the corresponding sequence in the mouse A-FABP gene, which contains the transcription factor binding sites for C/EBP and AP-1, and is implicated in the differentiation-dependent regulation of A-FABP. The A-FABP gene was assigned to porcine Chromosome (Chr) 4 by a porcine sequence-specific PCR on a cell hybrid panel, fully consistent with comparative mapping data with human and mouse. In the first intron of the porcine A-FABP gene, a microsatellite sequence was detected that was polymorphic for all six pig breeds tested. This genetic variation within the A-FABP gene was associated with differences in IMF content and possibly growth in a Duroc population, whereas no effect on backfat thickness and drip loss of the meat were detected. A considerable and significant contrast of approximately 1% IMF was observed between certain genotype classes. We conclude that the A-FABP locus is involved in the regulation of intramuscular fat accretion in Duroc pigs.

Background: Skeletal muscle has the ability to adapt as result of dietary, hormonal or pharmacological interventions affecting energy metabolism. The aim of the present study was to investigate the effects of energy restriction on skeletal muscle metabolic characteristics in obese women., Methods: The effects of 8 weeks' energy restriction on body composition, energy expenditure and skeletal muscle characteristics were investigated in 28 healthy obese women. Subjects were aged 37.9 +/- 1.5 years and had a body mass index of 32.0 +/- 0.8 kg m-2., Results: Energy restriction (2800 kJ day-1) resulted in a 10.8 +/- 0.5 kg weight loss consisting of 8. 6 +/- 0.5 kg of fat mass and 2.2 +/- 0.3 kg of fat-free mass. Basal respiratory exchange ratio, sleeping metabolic rate and exercise-induced thermogenesis significantly declined in response to the diet. These changes were accompanied by an increase (P = 0.038) in the skeletal muscle content of cytosolic fatty acid-binding protein (H-FABP), whereas no changes occurred in fibre type distribution or activities of enzymes reflecting beta-oxidation and mitochondrial density (3-hydroxyacyl-CoA dehydrogenase and citrate synthase respectively)., Conclusion: The results suggest that increased capacity of intracellular fatty acid transport in skeletal muscle cells is involved in the physiological adaptations of fat metabolism to energy restriction in obese female subjects.

We studied the effect of a high physiologic concentration of palmitate (1mM) on in vitro 2-deoxy-D-glucose (2DG) uptake by flexor digitorum brevis (FDB) muscle from ad libitum fed rats (AL) and rats fed 60% of ad libitum intake (CR) for 20 days. CR did not alter muscle 2DG uptake in the absence of insulin, but relative to AL, CR significantly (p<0.01) increased 2DG uptake in the presence of 20,000 microU/ml insulin. This effect of CR persisted in the presence of 1mM palmitate. The presence of 1mM palmitate significantly (p<0.01) impaired 2DG glucose uptake, both in the presence and absence of insulin, to the same extent in AL and CR muscle, despite an 18% decrease in FABPpm expression with CR. Thus, although CR profoundly affects insulin-mediated muscle glucose uptake, it does not alter the ability of extracellular fatty acid to modulate glucose utilization by skeletal muscle., (Copyright 1998 Academic Press.)

Intestinal fatty acid binding protein (IFABP) undergoes a reversible thermal transition between 35 and 50 degreesC, as revealed by circular dichroism spectroscopy in the near-UV region. For the apoprotein, the molar ellipticity measured at 254 nm (possibly implicating the environment around F17 and/or F55) decreases significantly in this temperature range, while in the holoprotein (bound to oleic acid), this phenomenon is not observed. Concomitantly, an increase in the activity of binding to [14C]oleic acid occurs. Nevertheless, other spectroscopic evidence indicates that the beta-barrel structure, the major motif of this protein, is highly stable up to 70 degreesC. No changes associated with conformation were detected for both structures by fourth-derivative analysis of the UV absorption spectra, circular dichroism in the far-UV region, and intrinsic fluorescence measurements. Further structural information arises from experiments in which binding to the anionic fluorescent probes 1-anilinonaphthalene-8-sulfonic acid (ANS) and its dimer bisANS was examined. The fluorescence intensity of bound ANS diminishes monotonically, whereas that of bisANS increases slightly in the temperature range of 35-50 degreesC. Given the different size of these probes, model building suggests that ANS would be able to sense regions located deeply inside the cavity, while bisANS could also reach the vicinity of the small helical domain of this protein. In light of these results, we believe that this subtle conformational transition of IFABP, which positively influences the binding activity, would involve fluctuations at the peripheral "entry portal" region for the ligand. This interpretation is compatible with the discrete disorder observed in this place in apo-IFABP, as evidenced by NMR spectroscopy [Hodsdon, M. E., and Cistola, D. P. (1997) Biochemistry 36, 1450-1460].

Fatty acid-binding proteins (FABP) are small cytosolic proteins which are thought to play a key role in fatty acid metabolism. The intestine contains the intestinal (I-FABP) and the liver (L-FABP) isoforms, but their regulation is still poorly documented. In order to find suitable conditions for studying the regulation of the two FABP isoforms in Caco-2 cells, we investigated the effects of the presence of collagen during cell proliferation or differentiation. When collagen was present only during cell proliferation on culture dishes, I-FABP expression was enhanced, whereas sucrase-isomaltase was unaffected and L-FABP expression was merely accelerated. In contrast, when collagen was present during cell differentiation on filter inserts, both I-FABP and sucrase-isomaltase were strongly reduced, but L-FABP was not affected. Under the former conditions (the more suitable for studying FABP regulation), the peroxysome proliferator-activated receptor (PPAR) activators, clofibrate and alpha-bromopalmitate, enhanced the two isoforms. This study, which is the first one providing a quantitative protein analysis of I-FABP and L-FABP in Caco-2 cells, demonstrates different time courses of expression of these proteins during cell differentiation. It also shows that I-FABP is specifically regulated by collagen and that, under conditions optimal for their expression, both isoforms are modulated by metabolic factors., (Copyright 1998 Academic Press.)

In equine hyperkalemic periodic paralysis (HyperPP), there is evidence suggesting that the primary defect in the sodium channel is associated with a secondary alteration in triacylglycerol-associated fatty acid metabolism (TAFAM) in skeletal muscle. Furthermore, TAFAM may be involved in the therapeutic action of phenytoin. The effects of phenytoin treatment on the transcript levels of three key proteins in TAFAM, hormone sensitive lipase (HSL), carnitine palmitoyltransferase (CPT), and fatty acid binding protein (FABP), were examined. These transcripts were quantitated by competitive reverse transcription polymerase chain reaction in undifferentiated and differentiated primary cultures of equine skeletal muscle from control, heterozygous HyperPP, and homozygous-affected HyperPP horses. There was a 10-fold lower level of HSL transcript in both undifferentiated and differentiated cultures from homozygous-affected horses than from horses of the other genotypes. Phenytoin selectively increased the HSL transcript in homozygous-affected differentiated cultures to levels similar to those of the other genotypes. The levels of CPT and FABP transcripts were unaffected by genotype, differentiation, and phenytoin treatment. These results suggest that the primary defect in HyperPP may secondarily decrease HSL transcript levels and that the therapeutic action of phenytoin may include regulation of mRNA transcripts in skeletal muscle., (Copyright 1998 Academic Press.)

Fatty acid binding proteins (FABPs) exhibit a beta-barrel topology, comprising 10 antiparallel beta-sheets capped by two short alpha-helical segments. Previous studies suggested that fatty acid transfer from several FABPs occurs during interaction between the protein and the acceptor membrane, and that the helical domain of the FABPs plays an important role in this process. In this study, we employed a helix-less variant of intestinal FABP (IFABP-HL) and examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from this protein to model membranes in comparison to the wild type (wIFABP). In marked contrast to wIFABP, IFABP-HL does not show significant modification of the AOFA transfer rate as a function of either the concentration or the composition of the acceptor membranes. These results suggest that the transfer of fatty acids from IFABP-HL occurs by an aqueous diffusion-mediated process, i.e., in the absence of the helical domain, effective collisional transfer of fatty acids to membranes does not occur. Binding of wIFABP and IFABP-HL to membranes was directly analyzed by using a cytochrome c competition assay, and it was shown that IFABP-HL was 80% less efficient in preventing cytochrome c from binding to membranes than the native IFABP. Collectively, these results indicate that the alpha-helical region of IFABP is involved in membrane interactions and thus plays a critical role in the collisional mechanism of fatty acid transfer from IFABP to phospholipid membranes.

Bietti crystalline dystrophy (BCD) is an autosomal recessive retinal degeneration characterized by intraretinal lipid inclusions with degeneration of the retina and sclerosis of the choroidal vessels, resulting clinically in progressive night blindness and constriction of the visual fields. Characterization of fatty acid metabolism in Bietti crystalline dystrophy suggested that BCD might result from abnormalities in lipid-binding proteins or one or more enzymes active in fatty acid elongation and desaturation. To further investigate the first possibility, the docosahexaenoic acid-binding proteins (DHABPs) of human lymphocytes from patients with Bietti crystalline dystrophy were studied and compared with those of normal controls. For fatty acid-binding protein (FABP) identification, lymphocyte cytosol was first subjected to Lipidex 1000 chromatography. FABPs were then cross-linked with [14C]22:6n-3 and identified by HPLC and SDS-PAGE. Ten major peaks corresponding to calculated molecular weights of 13, 14, 32, 43, 45, 50, 64, 96, 105, and 186 kDa exhibit high-affinity binding of fatty acids. Significantly, peaks corresponding to two fatty acid-binding proteins of 32 and 45 kDa present in age-matched controls are absent from lymphocytes of patients with BCD. The 32-kDa fatty acid-binding protein present in normal individuals but absent from patients with BCD was isolated from cultured control human lymphocytes, its fatty acid-binding properties were characterized, and its amino acid composition was analyzed. It shows specific binding of 3n-3 fatty acids, consistent with the pattern of abnormalities of lipid metabolism demonstrated in patients with BCD. These results suggest that the 32- and 43-kDa FABPs are reasonable candidates for causing BCD., (Copyright 1998 Academic Press.)

The purpose of this study was to examine the effects of bis[4-[2,4-dioxo-5-thiazolidinyl)methyl]phenyl]methane (YM-268), a thiazolidinedione derivative, on glucose uptake, adipocyte differentiation through peroxisome proliferator-activated receptor gamma(PPARgamma), and phosphatidylinositol 3-kinase (PI 3-kinase) activity in cultured cells. YM268 and pioglitazone dose-dependently increased the 2-deoxyglucose uptake in 3T3-L1 cells. YM268 facilitated the insulin-stimulated triglyceride accumulation in 3T3-L1 adipocytes and increased the mRNA expression of fatty acid-binding protein. YM268, with and without insulin, increased the mRNA expression of glucose transporter isoforms such as GLUT1 and GLUT4, indicating enhancement of adipocyte differentiation. Additionally, YM268 and pioglitazone showed activity of the PPARgamma ligand, a member of the nuclear receptor superfamily responsible for adipogenesis. To examine the possible involvement of the increased activity of PI 3-kinase in YM268-stimulated glucose uptake, the enzyme activity was estimated by measuring the phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) concentration in human monocytic cells. Insulin dose-dependently increased the PI-3,4,5-P3 production but YM268 had no significant effect on the insulin-dependent and -independent PI 3-kinase activation. These results indicate that the mechanism by which YM268 increased glucose uptake, may be accounted for in part by the enhancement of GLUT1 and GLUT4 expression through PPARgamma activation.

We performed studies 1) to investigate the kinetics of palmitate transport into giant sarcolemmal vesicles, 2) to determine whether the transport capacity is greater in red muscles than in white muscles, and 3) to determine whether putative long-chain fatty acid (LCFA) transporters are more abundant in red than in white muscles. For these studies we used giant sarcolemmal vesicles, which contained cytoplasmic fatty acid binding protein (FABPc), an intravesicular fatty acid sink. Intravesicular FABPc concentrations were sufficiently high so as not to limit the uptake of palmitate under conditions of maximal palmitate uptake (i.e., 4.5-fold excess in white and 31.3-fold excess in red muscle vesicles). All of the palmitate taken up was recovered as unesterified palmitate. Palmitate uptake was reduced by phloretin (-50%), sulfo-N-succinimidyl oleate (-43%), anti-plasma membrane-bound FABP (FABPpm, -30%), trypsin (-45%), and when incubation temperature was lowered to 0 degrees C (-70%). Palmitate uptake was also reduced by excess oleate (-65%), but not by excess octanoate or by glucose. Kinetic studies showed that maximal transport was 1.8-fold greater in red vesicles than in white vesicles. The Michaelis-Menten constant in both types of vesicles was approximately 6 nM. Fatty acid transport protein mRNA and fatty acid translocase (FAT) mRNA were about fivefold greater in red muscles than in white muscles. FAT/CD36 and FABPpm proteins in red vesicles or in homogenates were greater than in white vesicles or homogenates (P < 0.05). These studies provide the first evidence of a protein-mediated LCFA transport system in skeletal muscle. In this tissue, palmitate transport rates are greater in red than in white muscles because more LCFA transporters are available.

The importance of phenylalanine 57, an adipocyte lipid-binding protein (ALBP) portal residue, to ligand affinity and specificity has been investigated using a series of ALBP position 57 mutants. In wild-type ALBP, phenylalanine 57 undergoes a side chain rotation upon ligand binding, moving from an inwardly oriented, ligand-exclusive position in apoprotein structures to an outwardly oriented position in the holoprotein. To examine the role of F57 side chain rotation in the apoprotein-holoprotein transition and in ligand selectivity, ALBP site-specific mutants F57A, F57G, F57H, and F57W were expressed in Escherichia coli and purified to homogeneity. Mutants were analyzed for binding characteristics and stability toward chemical denaturation, and energy-minimized models of each mutant were constructed using apo, oleate-, and arachidonate-bound ALBP crystallographic coordinates. The stability of ALBP forms (wtALBP approximately F57G > F57A > F57W > F57H) was unrelated to the affinity of ALBP forms (wtALBP approximately F57W > F57H > F57G > F57A) for various lipids and did not vary between fatty acids. Since ligand selectivity was maintained between wild type and all mutants while ligand affinity was grossly diminished, we conclude that phenylalanine 57 is critical to the formation of the fatty acid/ALBP complex, but is uninvolved in determination of selectivity over the range of physiological ligands tested.