Liver and intestinal fatty acid-binding proteins obtain fatty acids from phospholipid membranes by different mechanisms

Intestinal enterocytes contain high concentrations of two cytosolic fatty acid-binding proteins (FABP), liver FABP (L-FABP) and intestinal FABP (I-FABP), which are hypothesized to play a role in cellular fatty acid trafficking. The mechanism(s) by which fatty acids move from membranes to each of these proteins is not known. Here we demonstrate that fluorescent anthroyloxy fatty acid analogues (AOFA) are transferred from phospholipid vesicles to L-FABP versus I-FABP by different mechanisms. For L-FABP a diffusion-mediated transfer process is demonstrated. The AOFA transfer rate from phosphatidylcholine-containing vesicles (POPC) to L-FABP is similar to that observed with another diffusional process, namely inter-membrane AOFA transfer. Furthermore, the AOFA transfer rate was modulated by buffer ionic strength and AOFA solubility, while the transfer rate remained relatively unchanged by the presence of anionic phospholipids in vesicles. In contrast, the data for I-FABP suggest that a transient collisional interaction of I-FABP with the phospholipid membrane occurs during AOFA extraction from the vesicles by the protein. In particular, the presence of the anionic phospholipid cardiolipin in donor vesicles increased the rate of AOFA transfer to I-FABP by 15-fold compared with transfer to POPC vesicles. The effects of ionic strength on transfer suggest that the interaction of I-FABP with cardiolipin-containing vesicles is likely to contain an electrostatic component. Finally, based on the regulation of AOFA transfer to I-FABP compared with transfer from I-FABP, it is hypothesized that apo- and holo-I-FABPs adopt conformations which may differentially promote I-FABP-membrane interactions.In summary, the results suggest that I-FABP, but not L-FABP, can directly extract fatty acids from membranes, supporting the concept that I-FABP may increase the cytosolic flux of fatty acids via intermembrane transfer.     

Expression of rat L-FABP in mouse fibroblasts: role in fat absorption

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose level is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigatedin vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accomodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtainedin vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.Abbreviations FABP fatty acid-binding protein - L-FABP liver fatty acid-binding protein - I-FABP intestinal fatty acid-binding protein - H-FABP heart fatty acid-binding protein - A-FABP adipocyte fatty acid-binding protein - PMFABP plasma membrane fatty acid-binding protein - SCP-2 sterol carrier protein-2 - Dehydroergosterol (DHE) d-5,7,9(11),22-ergostatetraene-3b-ol - cis-parinaric acid-9Z, 11E, 13E, 15Z-octatetraenoic acid - trans parinaric acid, 9E, 11E, 13E, 14E-octatetraenoic acid - BSA bovine serum albumin - KRH Krebs-Ringer-Henseleit buffer     

Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.     

Expression of rat intestinal fatty acid-binding protein in Escherichia coli. Purification and comparison of ligand binding characteristics with that of Escherichia coli-derived rat liver fatty acid-binding protein

Rat intestinal fatty acid-binding protein (I-FABP) is an abundant, 15,124-Da polypeptide found in the cytosol of small intestinal epithelial cells (enterocytes). It is homologous to rat liver fatty acid-binding protein (L-FABP), a 14,273-Da cytosolic protein which is found in enterocytes as well as hepatocytes. It is unclear why the small intestinal epithelium contains two abundant fatty acid-binding proteins. A systematic comparative analysis of the ligand binding characteristics of the two FABPs has not been reported. To undertake such a study we expressed the coding region of a full length I-FABP cDNA in Escherichia coli and purified large quantities of the protein. We also purified rat L-FABP from a similar, previously described expression system (Lowe, J. B., Strauss, A. W., and Gordon, J. I. (1984) J. Biol. Chem. 259, 12696-12704). Analysis of fatty acids associated with each of the homogeneous E. coli-derived FABPs suggested that the two proteins differed in their ligand binding specificity and capacity. All of the fatty acids associated with I-FABP were saturated while 30% of the E. coli fatty acids bound to L-FABP were unsaturated (16:1, 18:1, 18:2). We directly analyzed the ability of I- and L-FABP to bind fatty acids of different chain length and degree of saturation using a hydroxyalkoxypropyl dextran-based assay. Scatchard analysis revealed that each mole of L-FABP can bind up to 2 mol of long chain fatty acid while each mole of I-FABP can bind only 1 mole of fatty acid. L-FABP exhibited a relatively higher affinity for unsaturated fatty acids (oleate, arachidonate) than for saturated fatty acid (palmitate). By contrast, we were not able to detect a significant difference in the affinity of I-FABP for palmitate, oleate, and arachidonate. Neither protein exhibited any appreciable affinity for fatty acids whose chain length was less than C16. The observed differences in ligand affinities and capacities suggest that these proteins may have distinct roles in metabolism and/or compartmentalization of fatty acids within enterocytes.     

Polyene fatty acid interactions with recombinant intestinal and liver fatty acid-binding proteins. Spectroscopic studies

Binding and proximity relationships of fatty acids with recombinant rat liver fatty acid-binding protein (L-FABP) and intestinal fatty acid-binding protein (I-FABP) were studied with absorption and fluorescence spectroscopy. Protein aromatic amino acids were examined in the absence and presence of bound fatty acid. Second derivative absorbance spectroscopy of the apo- and holoproteins suggested that fatty acid binding altered the conformation of L-FABP, but not of I-FABP. Fatty acid binding also blocked the accessibility of L-FABP tyrosine and I-FABP tryptophan to Stern-Volmer quenching by acrylamide, indicating that these amino acids were present in the fatty acid-binding pocket. Forster energy transfer from I-FABP tryptophan to bound cis-parinaric acid resulted in quenching of tryptophan lifetime and appearance of sensitized lifetime of bound cis-parinaric acid. The calculated donor-acceptor distances were 16.9 +/- 0.6 and 19.2 +/- 0.3 A for I-FABP and L-FABP, respectively. Absorbance spectral shifts and ratios of fluorescence excitation maxima indicated that the parinaric acid microenvironment in the fatty acid-binding site of I-FABP was much less polar than that of L-FABP. Parinaric acids displayed similar rotational correlation time and limiting anisotropy when bound to I-FABP and to L-FABP. These results are consistent with a close proximity of bound fatty acids to the tyrosine and tryptophan residues and with immobilization of the polyene fatty acids in the fatty acid-binding site(s) of L-FABP and I-FABP. The two proteins differ in that only L-FABP has two fatty acid-binding sites and appears to undergo significant conformational change upon fatty acid binding.     

Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum

The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.     

Specific binding of the endocytosis tracer horseradish peroxidase to intestinal fatty acid-binding protein (I-FABP) in apical membranes of carp enterocytes

In a previous study we had demonstrated that a 15-kDa protein present in carp intestinal brush-border membrane vesicles (BBMV) was able to bind the endocytosis tracer horseradish peroxidase (HRP) with high specificity. Here we show that this protein corresponds to a peripheral membrane protein, identified by partial amino acid sequence analysis as the intestinal fatty acid-binding protein (I-FABP), a member of the small cytosolic fatty acid binding protein family (FABPs). The presence of I-FABP and its HRP-binding activity was demonstrated both in the cytosolic and membrane-associated fractions of intestinal mucosa by Western and ligand blot analyses, respectively. Also, both fractions displayed significant capacity to bind [(3)H]palmitic acid, a known ligand for I-FABP. Immunohistochemical analysis showed that I-FABP localizes both in the cytosol and in the brush-border membranes of epithelial cells. Taken together the unusual extra-cellular localization of I-FABP as well as its ability to interact with HRP suggests a novel function for this protein in the intestinal mucosa.     

Ligand specificity and conformational stability of human fatty acid-binding proteins.

Fatty acid binding proteins (FABPs) are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids. At least eight different types of FABP occur, each with a specific tissue distribution and possibly with a distinct function. To define the functional characteristics of all eight human FABPs, viz. heart (H), brain (B), myelin (M), adipocyte (A), epidermal (E), intestinal (I), liver (L) and ileal lipid-binding protein (I-LBP), we studied their ligand specificity, their conformational stability and their immunological crossreactivity. Additionally, binding of bile acids to I-LBP was studied. The FABP types showed differences in fatty acid binding affinity. Generally, the affinity for palmitic acid was lower than for oleic and arachidonic acid. All FABP types, except E-FABP, I-FABP and I-LBP interacted with 1-anilinonaphtalene-8-sulphonic acid (ANS). Only L-FABP, I-FABP and M-FABP showed binding of 11-((5-dimethylaminonaphtalene-1-sulfonyl)amino)undecanoic acid (DAUDA). I-LBP showed increasing binding of bile acids in the order taurine-conjugated>glycine-conjugated>unconjugated bile acids. A hydroxylgroup of bile acids at position 7 decreased and at position 12 increased the binding affinity to I-LBP. The fatty acid-binding affinity and the conformation of FABP types were differentially affected in the presence of urea. Our 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. The preference of I-LBP (but not L-FABP) for conjugated bile acids is in accordance with a specific role in bile acid reabsorption in the ileum.     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

Differential Regulation of Intestinal and Liver Fatty Acid-Binding Proteins in Human Intestinal Cell line (Caco-2): Role of Collagen

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 α-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.     

Diversity of fatty acid-binding protein structure and function: studies with fluorescent ligands

The mammalian fatty acid-binding proteins (FABP) are localized in many distinct cell types. They bind long chain fatty acidsin vitro, however, their functions and mechanisms of actionin vivo remain unknown. The present studies have sought to understand the relationships among these proteins, and to address the possible role of FABP in cellular fatty acid traffic. A series of anthroyloxy-labeled fluorescent fatty acids have been used to examine the physicochemical properties of the fatty acid-binding sites of different members of the FABP family. The fatty acid probes have also been used to study the rate and mechanism of fatty acid transfer from different FABP types to phospholipid membranes. The results of these studies show a number of interesting and potentially important differences between FABP family members. An examination of adipocyte and heart FABP (A- and H-FABP) shows that their fatty acid-binding sites are less hydrophobic than the liver FABP (L-FABP) site, and that the bound ligand experiences less motional constraint within the A- and H-FABP binding sites than within the L-FABP binding site. In keeping with these differences in structural properties, it was found that anthroyloxy-fatty acid transfer from A- and H-FABP to membranes is markedly faster than from L-FABP. Moreover, the mechanism of fatty acid transfer was found to be similar for the highly homologous logous A- and H-FABP, whereby transfer to phospholipid membranes appears to occur via transient collisional interactions between the FABP and membranes. Transfer of fatty acids from L-FABP, in contrast, occurs via an aqueous phase diffusion mechanism. Other studies utilized fluorescent fatty acid and monoacylglycerol derivatives to compare how the two FABP which are present in high abundance in the proximal small intestine interact with the two major products of dietary triacylglycerol hydrolysis. The results showed that whereas L-FABP binds both fatty acid and monoacylglycerol derivatives, intestinal FABP (I-FABP) appears to bind fatty acid but not monoacylglycerol. In summary, studies with fluorescent ligands have demonstrated unique properties for different FABP family members. A number of these differences appear to correlate with the degree of primary sequence homology between the proteins, and suggest functional diversity within the FABP family.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Liver FABP - H-FABP Heart FABP - A-FABP Adipocyte FABP - I-FABP Intestinal FABP - AOffa n-(9-anthroyloxy)fatty acid - MG Monoacylglycerol - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine     

Liver fatty acid-binding protein gene ablation inhibits branched-chain fatty acid metabolism in cultured primary hepatocytes

Whereas the role of liver fatty acid-binding protein (L-FABP) in the uptake, transport, mitochondrial oxidation, and esterification of normal straight-chain fatty acids has been studied extensively, almost nothing is known regarding the function of L-FABP in peroxisomal oxidation and metabolism of branched-chain fatty acids. Therefore, phytanic acid (most common dietary branched-chain fatty acid) was chosen to address these issues in cultured primary hepatocytes isolated from livers of L-FABP gene-ablated (-/-) and wild type (+/+) mice. These studies provided three new insights: First, L-FABP gene ablation reduced maximal, but not initial, uptake of phytanic acid 3.2-fold. Initial uptake of phytanic acid uptake was unaltered apparently due to concomitant 5.3-, 1.6-, and 1.4-fold up-regulation of plasma membrane fatty acid transporter/translocase proteins (glutamic-oxaloacetic transaminase, fatty acid transport protein, and fatty acid translocase, respectively). Second, L-FABP gene ablation inhibited phytanic acid peroxisomal oxidation and microsomal esterification. These effects were consistent with reduced cytoplasmic fatty acid transport as evidenced by multiphoton fluorescence photobleaching recovery, where L-FABP gene ablation reduced the cytoplasmic, but not membrane, diffusional component of NBD-stearic acid movement 2-fold. Third, lipid analysis of the L-FABP gene-ablated hepatocytes revealed an altered fatty acid phenotype. Free fatty acid and triglyceride levels were decreased 1.9- and 1.6-fold, respectively. In summary, results with cultured primary hepatocytes isolated from L-FABP (+/+) and L-FABP (-/-) mice demonstrated for the first time a physiological role of L-FABP in the uptake and metabolism of branched-chain fatty acids.     

Fatty acid interactions with rat intestinal and liver fatty acid-binding proteins expressed in Escherichia coli. A comparative 13C NMR study

Enterocytes in the small intestinal mucosa contain abundant quantities of two homologous cytosolic proteins known as intestinal and liver fatty acid-binding proteins (I- and L-FABP, respectively). To elucidate structure-function relationships for these proteins, the interactions between 13C-enriched palmitate and oleate and Escherichia coli-expressed rat I- and L-FABP were systematically compared using 13C NMR spectroscopy. NMR spectra of samples containing fatty acids (FA) and I-FABP at different molar ratios (all at pH 7.2 and 37 degrees C) exhibited a single carboxyl resonance corresponding to FA bound to I-FABP (181.4 ppm, peak I) and an additional carboxyl resonance corresponding to unbound FA in a bilayer phase (179.6 ppm). Peak I reached a maximum intensity corresponding to 1 mol of bound FA/mol of I-FABP under all sample conditions examined. NMR spectra for samples containing FA and L-FABP also exhibited a single carboxyl resonance corresponding to FA bound to L-FABP but at a different chemical shift value (182.2 ppm, peak L). Its maximum intensity varied depending on the physical state of the unbound FA (liquid crystalline or crystalline), the FA used (palmitate or oleate), and the sample pH. In the presence of a liquid crystalline (bilayer) phase, up to 1 (oleate) or 2 (palmitate) mol of FA were bound/mol of L-FABP, but in the presence of a crystalline phase (1:1 acid-soap), up to 3 mol of palmitate were bound/mol of L-FABP (all at pH 7.2). Peak I exhibited little or no ionization shift over a wide pH range (pH 3.0-11.0), and its chemical shift was unaffected by the ionization of Lys and His residues. Hence, the carboxylate group of FA bound to I-FABP was solvent inaccessible and most likely involved in an ion-pair electrostatic interaction with the delta-guanidinium moiety of an Arg residue. In contrast, peak L exhibited an ionization shift and an estimated apparent pKa value similar to that obtained for monomeric FA in water, suggesting that the carboxylate groups of FA bound to L-FABP were solvent accessible and located at or near the protein solvent interface. With decreasing pH, FA dissociated from L-FABP but not I-FABP, as monitored by NMR peak intensities. Concurrently, a large decrease in circular dichroism molar ellipticity was observed with L-FABP but not I-FABP. In conclusion, I-FABP and L-FABP are distinct with regards to their FA-binding stoichiometries, binding mechanisms, and sensitivity to pH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus

Our studies were conducted to explore the role of hepatic fatty acid-binding protein (L-FABP) in fatty acid transport to the nucleus. Purified rat L-FABP facilitated the specific interaction of [(3)H]oleic acid with the nuclei. L-FABP complexed with unlabeled oleic acid decreased the nuclear association of [(3)H]oleic acid:L-FABP; however, oleic acid-saturated bovine serum albumin (BSA) or fatty acid-free L-FABP did not. The peroxisome-proliferating agents LY171883, bezafibrate, and WY-14,643 were also effective competitors when complexed to L-FABP. Nuclease treatment did not affect the nuclear association of [(3)H]oleic acid:L-FABP; however, proteinase treatment of the nuclei abolished the binding. Nuclei incubated with fluorescein-conjugated L-FABP in the presence of oleic acid were highly fluorescent whereas no fluorescence was observed in reactions lacking oleic acid, suggesting that L-FABP itself was binding to the nuclei. The nuclear binding of FABP was concentration dependent, saturable, and competitive. LY189585, a ligand for L-FABP, also facilitated the nuclear binding of fluorescein-conjugated L-FABP, although it was less potent than oleic acid. A structural analog that does not bind L-FABP, LY163443, was relatively inactive in stimulating the nuclear binding. Potential interactions between L-FABP and nuclear proteins were analyzed by Far-Western blotting and identified a 33-kDa protein in the 500 mm NaCl extract of rat hepatocyte nuclei that bound strongly to biotinylated L-FABP. Oleic acid enhanced the interaction of L-FABP with the 33-kDa protein as well as other nuclear proteins.We propose that L-FABP is involved in communicating the state of fatty acid metabolism from the cytosol to the nucleus through an interaction with lipid mediators that are involved in nuclear signal transduction.     

Unravelling the significance of cellular fatty acid-binding proteins

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.     

Modulation of intestinal and liver fatty acid-binding proteins in Caco-2 cells by lipids, hormones and cytokines

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.     

Effects of intestinal fatty acid-binding protein overexpression on fatty acid metabolism in Caco-2 cells

Intestinal fatty acid-binding protein (I-FABP) is a cytosolic protein expressed at high levels (up to 2% of cytosolic proteins) in the small intestine epithelium. Despite cell transfection studies, its function is still unclear. Indeed, different effects on fatty acid metabolism depending on the cell type and the amount of I-FABP expressed have been reported. Furthermore, a decrease in fatty acid incorporation has been unexpectedly obtained when I-FABP reached 0. 72% of cytosolic proteins in fibroblasts (Prows et al. 1997. Arch. Biochem. Biophys. 340: 135). In the present study, the effect of a high level of I-FABP similar to amounts present in the small intestine was investigated in the human colon adenocarcinoma cell line, Caco-2. After transfection with human I-FABP cDNA, a clone expressing 1.5% I-FABP and unchanged level of liver FABP was selected. These cells, which had a lower rate of proliferation as compared with mock-transfected cells, developed the typical morphological characteristics of differentiated enterocytes. Incubation of differentiated cells with [(14)C]palmitate showed a 34% reduction (P < 0.01) of fatty acid incorporation, whereas the relative distribution of radiolabel into triglycerides was not affected. A nonsignificant 21% reduction of fatty acid incorporation was observed with another clone expressing 10-fold less I-FABP. In conclusion, a high level of I-FABP expressed in a differentiated enterocyte model inhibited fatty acid incorporation, by a mechanism which remains to be defined.     

Intracellular sterol distribution in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein

The potential role of liver fatty acid binding protein (L-FABP) in modulating cellular sterol distribution was examined in mouse L-cell fibroblasts transfected with cDNA encoding L-FABP. L-cells were chosen because they contain only a small amount of endogenous FABP which does not bind [3H]cholesterol, does not enhance intermembrane sterol transfer, and whose content is unaltered by the expression of L-FABP. Transfected L-cells expressed 0.34% of cytosolic protein as L-FABP. Transfection alone with low expression of L-FABP (0.008% of cytosolic protein) had no effect on any of the parameters tested. Three aspects of cellular sterol transfer were examined. First, cellular sterol uptake, monitored by [3H]cholesterol and the fluorescent sterol, delta-5,7,9(11),22-ergostatetraen-3 beta-ol, was increased 21.5 +/- 2.6% (p less than 0.001) in L-cells expressing L-FABP. This increase was not accounted for by increased sterol esterification in the cells expressing L-FABP. Inhibition of both cholesterol transfer and esterification with 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide from Sandoz abolished the L-FABP related enhancement of both [3H]cholesterol uptake and esterification. Second, plasma membrane transbilayer distribution of sterol, determined by fluorescence methods indicated that the majority of sterol was in the inner leaflet of the plasma membrane. In transfected cells expressing L-FABP, twice as much sterol (28 +/- 4%) was present in the exofacial leaflet of the plasma membrane as compared to that of control cells (15 +/- 2%). Third, expression of L-FABP enhanced sterol transfer from the plasma membrane to microsomes in intact cells. Treatment of [3H]cholesterol or [3H]oleate-loaded cells with sphingomyelinase resulted in increased formation of radiolabeled cholesterol ester, consistent with enhanced microsomal esterification of plasma membrane derived cholesterol. Concomitantly, plasma membrane [3H]cholesterol became less accessible to oxidation by cholesterol oxidase. Sphingomyelinase-stimulated cholesterol esterification was 21 +/- 3% greater in transfected cells. Concomitantly, accessibility of plasma membrane [3H]cholesterol to cholesterol oxidase was decreased 18 +/- 3% in cells expressing L-FABP. These differences are consistent with the ability of L-FABP to influence sterol transport and plasma membrane transbilayer sterol distribution in intact cells.     

Liver fatty acid binding protein enhances sterol transfer by membrane interaction

Among the large family of fatty acid binding proteins, the liver L-FABP is unique in that it not only binds fatty acids but also interacts with sterols to enhance sterol transfer between membranes. Nevertheless, the mechanism whereby L-FABP potentiates intermembrane sterol transfer is unknown. Both fluorescence and dialysis data indicate L-FABP mediated sterol transfer between L-cell fibroblast plasma membranes occurs by a direct membrane effect: First, dansylated-L-FABP (DNS-L-FABP) is bound to L-cell fibroblast plasma membranes as indicated by increased DNS-L-FABP steady state polarization and phase resolved limiting anisotropy. Second, coumarin-L-FABP (CPM-L-FABP) fluorescence lifetimes were significantly increased upon interaction with plasma membranes. Third, dialysis studies with³H-cholesterol loaded plasma membranes showed that L-FABP added to the donor compartment of the dialysis cell stimulated³H-cholesterol transfer whether or not the dialysis membrane was permeable to L-FABP. However, L-FABP mediated intermembrane sterol transfer did require a sterol binding site on L-FABP. Chemically blocking the ligand binding site also inhibited L-FABP activity in intermembrane sterol transfer. Finally, L-FABP did not act either as an aqueous carrier or in membrane fusion. The fact that L-FABP interacted with plasma membrane vesicles and required a sterol binding site was consistent with a mode of action whereby L-FABP binds to the membrane prior to releasing sterol from the bilayer.Abbreviations ³H-CHO [1,2-³H(N)]-cholesterol - ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid - CF carboxyfluorescein - CHO cholesterol - CPM (coumarin maleimide) 7-diethylamino-3-(4-maleimidylphenyl)-4-methylcoumarin - cPNA cisparinaric acid - DHE (dehydroergosterol) ^5,7,9(11),22-ergostatetraen-3-ol - DMF dimethyl formamide - DMPOPOP 1,4-bis[4-methyl-5-phenyl-2-oxazolyl]benzene - DNS (dansyl chloride) 5-dimethylaminonaphthalene-1-sulfonylchloride - DPX p-xylene-bis-pyridinium bromide - FBS fetal bovine serum - fluorescamine 4-phenylspiro[furan-2(3H), 1 phthalan]-3,3-dione - L-FABP liver fatty acid binding protein - NPG p-nitrophenylglyoxal - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine - SUV small unilamellar vesicle(s) - TNM tetranitromethane This work was supported in part by the National Institutes of Health United States Public Health Service (GM31651 and DK41402) and the American Heart Association (Postdoctoral Fellowship to JKW). The helpful assistance of Dr. Scott M. Colles and Mr. Daniel R. Prows in isolating L-FABP was much appreciated.     

Human liver fatty acid binding protein cDNA and amino acid sequence. Functional and evolutionary implications

Human liver fatty acid binding protein (L-FABP) cDNA clones were identified in a liver cDNA library. The two longest clones were completely sequenced. The nucleotide sequence predicts a protein of 127 amino acid residues. Identity of the clones was confirmed by limited amino acid sequence analysis of purified human L-FABP peptides and Edman degradation of radiolabeled in vitro translated FABP. Statistical analysis of the amino acid and mRNA sequences of human L-FABP, rat L-FABP, rat intestinal (I-) FABP, and mouse 422 protein indicates that the human and rat L-FABPs are highly homologous and that L-FABP and I-FABP diverged a long time ago (approximately 650-690 million years ago), although they are more closely related to each other than either of them is to 422 protein. Secondary structure predictions from the primary sequence of human and rat L-FABP reveal a region (residues 12-30) that might be the putative fatty acid binding domain of the two L-FABPs. Knowledge of the primary amino acid sequence of L-FABP and possible functional domains will be pivotal in further defining and understanding the mechanism of ligand binding and transfer by this protein.