Showing papers on "Fatty acid-binding protein published in 2001"
TL;DR: In localization studies using laser-scanning microscopy, it is shown that L-FABP and PPARα colocalize in the nucleus of mouse primary hepatocytes and it is demonstrated that the observed interaction of both proteins is independent of ligand binding.
Abstract: Peroxisome proliferator-activated receptor alpha (PPARalpha) is a key regulator of lipid homeostasis in hepatocytes and target for fatty acids and hypolipidemic drugs. How these signaling molecules reach the nuclear receptor is not known; however, similarities in ligand specificity suggest the liver fatty acid binding protein (L-FABP) as a possible candidate. In localization studies using laser-scanning microscopy, we show that L-FABP and PPARalpha colocalize in the nucleus of mouse primary hepatocytes. Furthermore, we demonstrate by pull-down assay and immunocoprecipitation that L-FABP interacts directly with PPARalpha. In a cell biological approach with the aid of a mammalian two-hybrid system, we provide evidence that L-FABP interacts with PPARalpha and PPARgamma but not with PPARbeta and retinoid X receptor-alpha by protein-protein contacts. In addition, we demonstrate that the observed interaction of both proteins is independent of ligand binding. Final and quantitative proof for L-FABP mediation was obtained in transactivation assays upon incubation of transiently and stably transfected HepG2 cells with saturated, monounsaturated, and polyunsaturated fatty acids as well as with hypolipidemic drugs. With all ligands applied, we observed strict correlation of PPARalpha and PPARgamma transactivation with intracellular concentrations of L-FABP. This correlation constitutes a nucleus-directed signaling by fatty acids and hypolipidemic drugs where L-FABP acts as a cytosolic gateway for these PPARalpha and PPARgamma agonists. Thus, L-FABP and the respective PPARs could serve as targets for nutrients and drugs to affect expression of PPAR-sensitive genes.
462 citations
TL;DR: It is concluded that αS has biochemical properties and a structural motif that suggest it is a novel member of the fatty acid-binding protein family and may thus transport fatty acids between the aqueous and membrane phospholipid compartments of the neuronal cytoplasm.
Abstract: alpha-Synuclein (alphaS) is a 140-residue neuronal protein that forms insoluble cytoplasmic aggregates in Parkinson's disease (PD) and several other neurodegenerative disorders. Two missense mutations (A53T and A30P) are linked to rare forms of familial PD. The normal function of alphaS is unknown, and cultured cell systems that model its modification from soluble monomers to aggregated forms have not been reported. Through a systematic centrifugal fractionation of mesencephalic neuronal cell lines and transgenic mouse brains expressing wild-type or A53T human alphaS, we observed unusual, previously unrecognized species of alphaS that migrate well above the 17-kDa monomeric form in denaturing gels. Incubation at 65 degrees C of high-speed cytosols from cells or brains revealed a modified alphaS species migrating at approximately 36 kDa and an extensive higher molecular mass alphaS-reactive smear. Extraction of the cytosols with chloroform/methanol or with a resin (Lipidex 1000) that binds fatty acids resulted in a similar pattern of higher molecular mass alphaS forms. On the basis of this effect of delipidation, we reexamined the primary structure of alphaS and detected a motif at the N and C termini that is homologous to a fatty acid-binding protein signature. In accord, we found that purified human alphaS binds oleic acid, with an apparent K(d) of 12.5 microM. We also observed an enhanced association of A53T alphaS with microsomal membranes in both mesencephalic cells and transgenic mouse brains. We conclude that alphaS has biochemical properties and a structural motif that suggest it is a novel member of the fatty acid-binding protein family and may thus transport fatty acids between the aqueous and membrane phospholipid compartments of the neuronal cytoplasm.
297 citations
TL;DR: Overall, fatty acid uptake represents a continuous flow involving the following: dissociation from albumin by membrane proteins with high affinity for fatty acids; passive flip-flop across the phospholipid bilayer; binding to FABPc and caveolin-1 at the cytosolic plasma membrane; and intracellular trafficking via FABpc and/or caveolae to sites of metabolic disposition.
Abstract: Fatty acids are the main structural and energy sources of the human body. Within the organism, they are presented to cells as fatty acid:albumin complexes. Dissociation from albumin represents the first step of the cellular uptake process, involving membrane proteins with high affinity for fatty acids, e.g., fatty acid translocase (FAT/CD 36) or the membrane fatty acid-binding protein (FABPpm). According to the thus created transmembrane concentration gradient, uncharged fatty acids can flip-flop from the outer leaflet across the phospholipid bilayer. At the cytosolic surface of the plasma membrane, fatty acids can associate with the cytosolic FABP (FABP(c)) or with caveolin-1. Caveolins are constituents of caveolae, which are proposed to serve as lipid delivery vehicles for subcellular organelles. It is not known whether protein (FABP(c))- and lipid (caveolae)-mediated intracellular trafficking of fatty acids operates in conjunction or in parallel. Channeling fatty acids to the different metabolic pathways requires activation to acyl-CoA. For this process, the family of fatty acid transport proteins (FATP 1-5/6) might be relevant because they have been shown to possess acyl-CoA synthetase activity. Their variable N-terminal signaling sequences suggest that they might be targeted to specific organelles by anchoring in the phospholipid bilayer of the different subcellular membranes. At the highly conserved cytosolic AMP-binding site of FATP, fatty acids are activated to acyl-CoA for subsequent metabolic disposition by specific organelles. Overall, fatty acid uptake represents a continuous flow involving the following: dissociation from albumin by membrane proteins with high affinity for fatty acids; passive flip-flop across the phospholipid bilayer; binding to FABP(C) and caveolin-1 at the cytosolic plasma membrane; and intracellular trafficking via FABP(c) and/or caveolae to sites of metabolic disposition. The uptake process is terminated after activation to acyl-CoA by the members of the FATP family targeted intracellularly to different organelles.
207 citations
TL;DR: New evidence from in-vitro and whole-animal studies supports the existence of protein-mediated transmembrane transport of FAs, which is likely to coexist with passive diffusional uptake.
Abstract: Cellular long-chain fatty acid (FA) transport and metabolism are believed to be regulated by membrane-associated and soluble proteins that bind and transport FAs. Several different classes of membrane proteins have been proposed as FA acceptors or transmembrane FA transporters. New evidence from in-vitro and whole-animal studies supports the existence of protein-mediated transmembrane transport of FAs, which is likely to coexist with passive diffusional uptake. The trafficking of FAs by intracellular fatty acid-binding proteins may involve their interaction with specific membrane or protein targets. Evidence is also emerging for concerted actions between the membrane and cytoplasmic fatty acid-binding proteins that allow for efficient regulation of FA transport and metabolism.
152 citations
TL;DR: Binding studies with a range of physiological FA showed no large differences between recombinant proteins of the four human FABP types in binding specificity and affinity, also not for polyunsaturated FA (PUFA).
Abstract: The cytosol (105,000 xg supernatant) of nearly all tissues investigated appears to contain one or more proteins with a molecular weight of approximately 14,000 that bind long-chain fatty acids or their CoA and carnitine esters with a rather high affinity. The concentration of these fatty acid-binding proteins (FABPs) is high (30–50 μg mg -1 cytosolic protein) in tissues with a high capacity for fatty acid oxidation (liver, heart) or lipid metabolism (intestinal epithelium). Most investigations have been concentrated on the FABPs from these tissues (reviewed in Bass 1985; Glatz and Veerkamp 1985; Glatz et al. 1985a). FABPs occur in the rat in at least three tissue-specific forms (liver, intestinal and heart FABP), but two distinct forms may be present in one tissue (e.g. liver and intestinal forms in intestine). Liver FABP was also named Z-protein or protein A.
126 citations
TL;DR: Not only does Gp-FAR-1 function as a broad-spectrum retinol- and fatty-acid-binding protein, but it was found to inhibit the lipoxygenase-mediated modification of these substrates in vitro, consistent with the idea that G p-Far-1 is involved in the evasion of primary host plant defence systems.
Abstract: Parasitic nematodes produce at least two structurally novel classes of small helix-rich retinol- and fatty-acid-binding proteins that have no counterparts in their plant or animal hosts and thus represent potential targets for new nematicides. Here we describe a protein (Gp-FAR-1) from the plant-parasitic nematode Globodera pallida, which is a member of the nematode-specific fatty-acid- and retinol-binding (FAR) family of proteins but localizes to the surface of this species, placing it in a strategic position for interaction with the host. Recombinant Gp-FAR-1 was found to bind retinol, cis-parinaric acid and the fluorophore-tagged lipids 11-(dansylamino)undecanoic acid and dansyl-D,L-alpha-amino-octanoic acid. The fluorescence emission characteristics of the dansylated analogues indicated that the entire ligand enters the binding cavity. Fluorescence competition experiments showed that Gp-FAR-1 binds fatty acids in the range C(11) to C(24), with optimal binding at C(15). Intrinsic fluorescence analysis of a mutant protein into which a tryptophan residue had been inserted supported computer-based predictions of the position of this residue at the protein's interior and possibly also at the binding site. Of direct relevance to plant defence systems was the observation that Gp-FAR-1 binds two lipids (linolenic and linoleic acids) that are precursors of plant defence compounds and the jasmonic acid signalling pathway. Moreover, Gp-FAR-1 was found to inhibit the lipoxygenase-mediated modification of these substrates in vitro. Thus not only does Gp-FAR-1 function as a broad-spectrum retinol- and fatty-acid-binding protein, the results are consistent with the idea that Gp-FAR-1 is involved in the evasion of primary host plant defence systems.
125 citations
TL;DR: The distinct tissue expression pattern and binding specificity suggest that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse and is a novel FABP family member, cellular retinol-binding protein, type III.
124 citations
TL;DR: It is shown that FA adsorb to the plasma membrane of adipocytes and diffuse through the membrane by the flip-flop mechanism within the time resolution of the authors' measurements, which shows that passive diffusion is a viable mechanism, although it did not evaluate its exclusivity.
Abstract: The transport of fatty acids (FA) across membranes can be described by three fundamental steps: adsorption, transmembrane movement, and desorption In model membranes, these steps are all rapid and spontaneous for most fatty acids, suggesting that FA can enter cells by free diffusion rather than by protein-mediated mechanisms Here we present new fluorescence approaches that measure adsorption and transmembrane movement of FA independently We show that FA adsorb to the plasma membrane of adipocytes and diffuse through the membrane by the flip-flop mechanism within the time resolution of our measurements (approximately 5 s) Thus we show that passive diffusion is a viable mechanism, although we did not evaluate its exclusivity Important implications of the diffusion mechanism for neural cells are that all types of FA could be available and that selectivity is controlled by metabolism Studies of FA uptake into brain endothelial cells and other brain cell types need to be performed to determine mechanisms of uptake, and metabolism of FA must be separated in order to understand the role of membrane transport in the overall uptake process
122 citations
TL;DR: The results demonstrate significant differences in ligand binding, conformational stability and surface properties between different FABP types which may point to a specific function in certain cells and tissues.
104 citations
TL;DR: A role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution is supported and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein.
99 citations
TL;DR: Carnitine palmitoyltransferase 1 activity increased during differentiation and was higher in perirenal than in epididymal preadipocytes, particularly the muscle isoform.
Abstract: Regional differences in free fatty acid (FFA) handling contribute to diseases associated with particular fat distributions. As cultured rat preadipocytes became differentiated, FFA transfer into preadipocytes increased and was more rapid in single perirenal than in epididymal cells matched for lipid content. Uptake by human omental preadipocytes was greater than uptake by abdominal subcutaneous preadipocytes. Adipose-specific fatty acid binding protein (aP2) and keratinocyte lipid binding protein abundance was higher in differentiated rat perirenal than in epididymal preadipocytes. This interdepot difference in preadipocyte aP2 expression was reflected in fat tissue in older animals. Carnitine palmitoyltransferase 1 activity increased during differentiation and was higher in perirenal than in epididymal preadipocytes, particularly the muscle isoform. Long-chain acyl-CoA levels were higher in perirenal than in epididymal preadipocytes and isolated fat cells. These data are consistent with interdepot differences in fatty acid flux ensuing from differences in fatty acid binding proteins and enzymes of fat metabolism. Heterogeneity among depots results, in part, from distinct intrinsic characteristics of adipose cells. Different depots are effectively separate miniorgans.
TL;DR: The affinity of H-FABP for EETs suggests that this family of intracellular proteins may modulate the metabolism, activities, and targeting of these potent eicosanoid biomediators.
Abstract: Arachidonic acid metabolism by lipoxygenases and cytochrome P450 monooxygenases produces regioisomeric hydroperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs), which serve as components of cell signaling cascades. Intracellular fatty acid-binding proteins (FABPs) may differentially bind these nonprostanoid oxygenated fatty acids, thus modulating their metabolism and activities. Vascular cells, which express heart FABP (H-FABP), utilize oxygenated fatty acids for regulation of vascular tone. Therefore, the relative affinities of H-FABP for several isomeric series of these compounds were measured by fluorescent displacement of 1-anilinonaphthalene-8-sulfonic acid (ANS). In general, H-FABP rank order affinities (arachidonic acid > EETs > HETEs > DHETs) paralleled reversed-phase high-performance liquid chromatography retention times, indicating that the differences in H-FABP affinity were determined largely by polarity. H-FABP displayed a similar rank order of affinity for compounds derived from linoleic acid. H-FABP affinity for 20-HETE [apparent dissociation constant (K(d)') of 0.44 microM] was much greater than expected from its polarity, indicating unique binding interactions for this HETE. H-FABP affinity for 5,6-EET and 11,12-EET (K(d)' of approximately 0.4 microM) was approximately 20-fold greater than for DHETs (K(d)' of approximately 8 microM). The homologous proteins, liver FABP and intestinal FABP, also displayed selective affinity for EET versus DHET. Thus, FABP binding of EETs may facilitate their intracellular retention whereas the lack of FABP affinity for DHETs may partially explain their release from cells. The affinity of H-FABP for EETs suggests that this family of intracellular proteins may modulate the metabolism, activities, and targeting of these potent eicosanoid biomediators.
TL;DR: The more effective binding of the FA metabolite, 13-HODE, than its precursor 18:2 by FABP may help protect cellular membranes from potential damage by monohydroxy fatty acids and may contribute a pathway for entry of 13- HODE into the nucleus.
TL;DR: In this article, the authors quantified skeletal muscle mRNA levels of a number of genes involved in fatty acid metabolism, including uncoupling protein-2, carnitine palmitoyl transferase-1β and fatty acid binding proteins, and transporters.
TL;DR: Evidence is presented that in skeletal muscle, fatty acid uptake is subject to short-term regulation by translocation of FAT/CD36 from intracellular stores to the plasma membrane, analogous to the regulation of muscular glucose uptake by GLUT-4 translocation.
Abstract: The transport of long-chain fatty acids across cellular membranes most likely occurs to some extent by passive diffusion and additionally is facilitated by a number of membrane-associated and cytoplasmic proteins. In this overview we focus on the involvement of the membrane proteins fatty acid translocase (FAT/CD36), plasma membrane fatty acid-binding protein (FABPpm) and fatty acid-transport protein (FATP). Newly obtained evidence is presented that in skeletal muscle, fatty acid uptake is subject to short-term regulation by translocation of FAT/CD36 from intracellular stores to the plasma membrane, analogous to the regulation of muscular glucose uptake by GLUT-4 translocation. These new findings establish a significant role of membrane-associated proteins in the cellular fatty acid-uptake process. Possible implications for the uptake and transport of long-chain fatty acids by the brain are discussed.
TL;DR: The threonine-encoding allele of the FABP2 gene is associated with an increased postprandial response of 14-18-carbon fatty acids but not with changes in the relative amounts of individual fatty acids introduced to chylomicron triacylglycerols.
Journal Article•
TL;DR: The results suggest that the Ala54Thr substitution in the FABP2 gene is associated with increased fat oxidation and hyperinsulinemia in normal Korean men, but these effects are not mediated by an increase in the intestinal fatty acid absorption.
Abstract: The alanine to threonine substitution at codon 54 (Ala54Thr) of the fatty acid binding protein 2 (FABP2) gene has been reported to be associated with increased fat oxidation and insulin resistance in several populations. It has been hypothesized that Ala54Thr substitution results in enhanced intestinal uptake of fatty acids and thereby an impairment of insulin action, but this hypothesis has not been proven in vivo. We studied the association between the Ala54Thr polymorphism of the FABP2 gene and intestinal 3 H-oleic acid absorption, as well as basal insulin level, basal metabolic rate, and fat oxidation rate in 96 healthy young Korean men. Among our subjects, the allele frequency of the Ala54Thr substitution was 0.34. Subjects with Thr54-encoding allele were found to have a higher mean fasting plasma insulin concentration and a higher basal fat oxidation rate compared with the subjects who were homozygous for the Ala54-encoding allele. However, there was no significant difference in basal metabolic rate or 3 H-oleic acid absorption according to the FABP2 gene polymorphism. These results suggest that the Ala54Thr substitution in the FABP2 gene is associated with increased fat oxidation and hyperinsulinemia in normal Korean men, but these effects are not mediated by an increase in the intestinal fatty acid absorption.
TL;DR: In a new model system (giant vesicles), it is demonstrated that LCFA transport rates are scaled with the oxidative capacity of heart and muscle andLCFA transport can be increased by increasing the FAT/CD36 protein of muscle.
Abstract: While it has long been assumed that long chain fatty acids (LCFA) can freely diffuse across the plasma membrane, recent work has shown that LCFA uptake also involves a protein-mediated mechanism. Three putative LCFA transporters have been identified (FABPpm, FATP, and FAT/CD36), and all are expressed in rodent and human muscles. In a new model system (giant vesicles), we have demonstrated that (a) LCFA transport rates are scaled with the oxidative capacity of heart and muscle, (b) only FABPpm and FAT/CD36, but not FATP1, correlate with vesicular LCFA transport, and (c) LCFA transport can be increased by increasing (1) the FAT/CD36 protein of muscle (chronic adaptation) or (2) via the translocation of FAT/CD36 from an intracellular pool to the plasma membrane during muscle contraction (acute adaptation).
TL;DR: It is demonstrated that I‐FABP expression is limited to fully differentiated Caco‐2 cells and can be more easily regulated than L‐fABP by lipids, hormones, and cytokines.
Abstract: Intestinal and liver fatty acid binding proteins (I- and L-FABP) are thought to play a role in enterocyte fatty acid (FA) trafficking. Their modulation by cell differentiation and various potential effectors was investigated in the human Caco-2 cell line. With the acquisition of enterocytic features, Caco-2 cells seeded on plastic progressively increased L-FABP quantities, whereas I-FABP was not detectable even very late in the maturation process. On permeable filters that improved differentiation markers (sucrase, alkaline phosphatase, transepithelial resistance), Caco-2 cells furthered their L-FABP content and expressed I-FABP. Western blot analysis showed a significant increase in I- and L-FABP expression following an 8-hour incubation period with butyric acid, oleic acid, and phosphatidylcholine. However, in all cases, I-FABP levels were higher than L-FABP concentrations regardless of the lipid substrates added. Similarly, hydrocortisone and insulin enhanced the cellular content of I- and L-FABP whereas leptin triggered I-FABP expression only after an 8-hour incubation. Finally, tumor necrosis factor-alpha was more effective in increasing the cytosolic amount of I-FABP levels. In conclusion, our data demonstrate that I-FABP expression is limited to fully differentiated Caco-2 cells and can be more easily regulated than L-FABP by lipids, hormones, and cytokines.
TL;DR: GDF-3/Vgr-2 is identified as an age- and fat-regulated, adipose-derived cytokine suggesting a linkage between adipocyte fatty acid metabolism and the expression of the bone morphogenetic family of differentiation regulators.
TL;DR: Almost all alveolar macrophages in the mouse lung were strongly immunoreactive for epidermal-type fatty acid binding protein, while a substantial number of the type II cells were immunonegative.
Abstract: Almost all alveolar macrophages in the mouse lung were strongly immunoreactive for epidermal-type fatty acid binding protein. At the electron microscope level, the immunoreactive material was localized diffusely in the cytoplasm but not within the nucleus. A certain number of alveolar type II epithelial cells were also immunoreactive for the protein with variable immuno-intensity, while a substantial number of the type II cells were immunonegative. No immunoreactive interstitial fibroblasts were encountered. Based on the present findings, possible roles of epidermal-type fatty acid binding protein in the host-defence mechanism played by alveolar macrophages are suggested.
TL;DR: A fluorescence resonance energy transfer (FRET) assay is used to examine the rate and mechanism of transfer of a fluorescent long-chain fatty acid from B-FABP to phospholipid vesicles, and the rate of transfer is shown to be independent of buffer ionic strength and dramatically enhanced by the presence of specific anionicospholipids.
Abstract: Mammalian fatty acid-binding proteins (FABP) are a family of intracellular proteins (approx 15 kDa) that bind long-chain fatty acids (FA) with high affinity. They are believed to serve as cytoplasmic transporters of FA and to target FA to specific cellular sites of utilization. Several different FABPs are expressed in neural tissue, including brain FABP (B-FABP), myelin FABP (M-FABP), and heart FABP (H-FABP). We have previously shown that H-FABP transfers FA via direct collisional interactions with acceptor model membranes. In the present studies, we use a fluorescence resonance energy transfer (FRET) assay to examine the rate and mechanism of transfer of a fluorescent long-chain fatty acid from B-FABP to phospholipid vesicles. The rate of transfer is shown to be independent of buffer ionic strength and dramatically enhanced by the presence of specific anionic phospholipids. These results are consistent with a mechanism by which FA are transferred from B-FABP to phospholipid membranes by a transient collision-based mechanism.
TL;DR: The Echinococcus FABP is the unique member of the H-FABP group able to bind two long chain fatty acid molecules with high affinity and structure-function relationships and putative roles for EgFABp1 in E. granulosus metabolism are discussed.
TL;DR: The three-dimensional solution structure of human heart-type FABP is determined by multi-dimensional heteronuclear NMR spectroscopy and shows that different fatty acids induce distinct conformational states of the protein backbone in this portal region, depending on the chain length of the fatty acid ligand.
Abstract: Recent advances in the characterization of fatty acid-binding proteins (FABPs) by NMR have enabled various research groups to investigate the function of these proteins in aqueous solution. The binding of fatty acid molecules to FABPs, which proceeds through a portal region on the protein surface, is of particular interest. In the present study we have determined the three-dimensional solution structure of human heart-type FABP by multi-dimensional heteronuclear NMR spectroscopy. Subsequently, in combination with data collected on a F57S mutant we have been able to show that different fatty acids induce distinct conformational states of the protein backbone in this portal region, depending on the chain length of the fatty acid ligand. This indicates that during the binding process the protein accommodates the ligand molecule by a "selected-fit" mechanism. In fact, this behaviour appears to be especially pronounced in the heart-type FABP, possibly due to a more rigid backbone structure compared with other FABPs, as suggested by recent NMR relaxation studies. Thus differences in the dynamic behaviours of these proteins may be the key to understanding the variations in ligand affinity and specificity within the FABP family.
TL;DR: The results suggest that acyl-CoA oxidase, L-FABP and the other PPAR-dependent genes in the small intestine may be coordinately modulated during postnatal development by the disproportional expression of PPARalpha over PPARdelta.
TL;DR: A role for ACBP is suggested in the partitioning of fatty acids between esterification reactions leading to the formation of neutral lipids and β-oxidation, and ACBP may play a regulatory role by influencing this important branch point in intermediary lipid metabolism.
Abstract: Overexpression of acyl-CoA binding protein (ACBP) was induced in a rat hepatoma cell line (McA-RH 7777) by stable integration of rat ACBP cDNA. The transfected cells (ACBP-27) had 3.5-fold higher concentrations of ACBP than control cells (14 vs. 4 ng/μg DNA). Both ACBP-27 and control cells were cultured in the presence of various concentrations of radiolabeled palmitic acid; and the effects of ACBP on lipogenesis and β-oxidation were studied. Incubation of the cells with 100 μM palmitic acid resulted in 42% greater incorporation of the fatty acid in ACBP-27 cells as compared to that in the control cells. This increased incorporation of the fatty acid was observed predominanly in the triglyceride fraction. Higher concentrations of palmitic acid (200 to 400 μM) were associated with a significant decrease in the production of 14CO2 in the ACBP-27 cell line than in the control cells, while lower concentrations had not effect. Our data suggest a role for ACBP in the partitioning of fatty acids between esterification reactions leading to the formation of neutral lipids and β-oxidation. ACBP may play a regulatory role by influencing this important branch point in intermediary lipid metabolism.
TL;DR: The data do not provide evidence for a cytoprotective role of FABP in this kidney cell model and it is suggested that glycolytic ATP could substitute for uncoupled oxidative phosphorylation.
Abstract: Intracellular accumulation of fatty acids (FAs) is a well-described consequence of renal ischaemia and may lead to lethal cell injury. Fatty-acid-binding proteins (FABPs) are small cytosolic proteins with high affinity for FAs. They may protect vital cellular functions by binding to and promoting the metabolism of FAs, thereby reducing their intracellular concentration. In this study we investigated the putative cytoprotective role of FABPs in a Madin-Darby canine kidney (MDCK) cell model for renal damage. We studied the effects of transfection with cDNA encoding heart FABP, adipocyte FABP or liver FABP on cytotoxicity induced by chemical anoxia or FAs. Transfection of MDCK type II cells with these cDNA types caused a 5-20-fold increase in FABP content, but did not change the rate or extent of palmitate uptake. After 1 h of incubation with KCN, all cell types showed reduced viability and cellular ATP content and an intracellular accumulation of non-esterified FAs. High extracellular concentrations of oleate, but not palmitate, caused a markedly decreased cell viability and cellular ATP content. Oleate accumulated in non-esterified form in these cells. Simultaneous addition of glucose ameliorated the damaging effects of KCN or oleate, indicating that glycolytic ATP could substitute for uncoupled oxidative phosphorylation. No significant differences in the effects of chemical anoxia or oleate were observed between non-transfected, mock-transfected and FABP-cDNA-transfected cells. Non-esterified FA accumulation was not reduced in any of the FABP-cDNA-transfected cell lines. In conclusion, our data do not provide evidence for a cytoprotective role of FABP in this kidney cell model.
TL;DR: Evidence is provided suggesting that this step is driven by intracellular metabolism that supports a NEFA gradient across the cell membrane, and that for very long chain NEFA, the former is more likely.
Abstract: Transport of nonesterified fatty acids (NEFA) is an important component of whole-body energy metabolism, and derangements in NEFA transport have been linked to several diseases. NEFA are transferred from their sites of production to cells in hepatic and peripheral tissues by mechanisms that are regulated in part by cell status and as determined by the covalent structure of the NEFA species. Major barriers to physical transport are transfer from the hydrophobic surfaces on cell membranes and NEFA-binding proteins, such as albumin, into the surrounding aqueous phase and translocation across a membrane that contains a very hydrophobic interior; this process could be purely diffusive or require specific protein cofactors. Herein evidence is provided suggesting that this step is driven by intracellular metabolism that supports a NEFA gradient across the cell membrane. According to current models of NEFA transfer, the rate-limiting step is likely to be desorption of NEFA from the inner leaflet of the cell membrane or intracellular metabolism; for very long chain NEFA, the former is more likely.
TL;DR: A lipid analysis of nFH12 was undertaken using gas chromatography–mass spectrometry to demonstrate that the nFh12 protein complex is, in fact, a complex of fatty acid binding proteins.
Abstract: Fasciola hepatica adult flukes have a native protein complex denoted nFh12 and consisting of fatty acid binding proteins that comprise at least 8 isoforms. It is a potent immunogen because in several animal hosts it induces an early antibody response to F. hepatica infection. It is also a potent cross-protective immunogen because it induces a protective immune response in mice to challenge infection with Schistosoma mansoni cercariae. The gene encoding this protein has been cloned and sequenced. It produces a polypeptide of 132 amino acids with a predicted molecular mass of 14.7 kDa and is denoted rFh15. It also has a significant homology to a 14-kDa S. mansoni fatty acid binding protein (Sm14). In the present study, nFh12 was delipidated with charcoal treatment and then studied by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Additionally, a lipid analysis of nFh12 was undertaken using gas chromatography–mass spectrometry to demonstrate that the nFh12 protein complex is, in fact, a complex o...