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.     

Induction of hepatic cytosolic fatty acid binding protein with clofibrate accelerates both membrane and cytoplasmic transport of palmitate

The role of liver cytosolic fatty acid binding protein (L-FABP) in fatty acid transport and metabolism is unclear. Female liver contains substantially more L-FABP than male liver. Female liver also has a different fatty acid transport phenotype, including more rapid uptake, efflux and cytoplasmic transport. However, it is not known if the greater levels of L-FABP are responsible for these differences. We therefore determined whether increasing L-FABP using clofibrate causes male liver to acquire a female transport phenotype. The multiple indicator dilution (MID) method was used to estimate the rate constants for influx, efflux and cytoplasmic diffusion of palmitate in isolated perfused rat livers. Clofibrate treatment increased cytosolic concentrations of L-FABP 4.2+/-0.8-fold, the rate of cytoplasmic diffusion of palmitate 4.3+/-1.7-fold, and the steady-state palmitate extraction 1.5+/-0.3-fold (mean+/-S.E.). Influx and efflux constants were both increased (by 44% and 79%, respectively) to levels typical of female livers. These data suggest that clofibrate-induced elevation of cytosolic L-FABP not only stimulates intracellular diffusion but also influx and efflux of fatty acids. Possible mechanisms include reducing fatty acid binding to cytoplasmic membranes, induction of membrane fatty acid carriers, and catalyzing fatty acid exchange between aqueous cytoplasm and the plasma membrane.     

Structural and biochemical characterization of toad liver fatty acid-binding protein

Two paralogous groups of fatty acid-binding proteins (FABPs) have been described in vertebrate liver: liver FABP (L-FABP) type, extensively characterized in mammals, and liver basic FABP (Lb-FABP) found in fish, amphibians, reptiles, and birds. We describe here the toad Lb-FABP complete amino acid sequence, its X-ray structure to 2.5 A resolution, ligand-binding properties, and mechanism of fatty acid transfer to phospholipid membranes. Alignment of the amino acid sequence of toad Lb-FABP with known L-FABPs and Lb-FABPs shows that it is more closely related to the other Lb-FABPs. Toad Lb-FABP conserves the 12 characteristic residues present in all Lb-FABPs and absent in L-FABPs and presents the canonical fold characteristic of all the members of this protein family. Eight out of the 12 conserved residues point to the lipid-binding cavity of the molecule. In contrast, most of the 25 L-FABP conserved residues are in clusters on the surface of the molecule. The helix-turn-helix motif shows both a negative and positive electrostatic potential surface as in rat L-FABP, and in contrast with the other FABP types. The mechanism of anthroyloxy-labeled fatty acids transfer from Lb-FABP to phospholipid membranes occurs by a diffusion-mediated process, as previously shown for L-FABP, but the rate of transfer is 1 order of magnitude faster. Toad Lb-FABP can bind two cis-parinaric acid molecules but only one trans-parinaric acid molecule while L-FABP binds two molecules of both parinaric acid isomers. Although toad Lb-FABP shares with L-FABP a broad ligand-binding specificity, the relative affinity is different.     

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.     

Fatty acid binding protein is a major determinant of hepatic pharmacokinetics of palmitate and its metabolites

Disposition kinetics of [(3)H]palmitate and its low-molecular-weight metabolites in perfused rat livers were studied using the multiple-indicator dilution technique, a selective assay for [(3)H]palmitate and its low-molecular-weight metabolites, and several physiologically based pharmacokinetic models. The level of liver fatty acid binding protein (L-FABP), other intrahepatic binding proteins (microsomal protein, albumin, and glutathione S-transferase) and the outflow profiles of [(3)H]palmitate and metabolites were measured in four experimental groups of rats: 1) males; 2) clofibrate-treated males; 3) females; and 4) pregnant females. A slow-diffusion/bound model was found to better describe the hepatic disposition of unchanged [(3)H]palmitate than other pharmacokinetic models. The L-FABP levels followed the order: pregnant female > clofibrate-treated male > female > male. Levels of other intrahepatic proteins did not differ significantly. The hepatic extraction ratio and mean transit time for unchanged palmitate, as well as the production of low-molecular-weight metabolites of palmitate and their retention in the liver, increased with increasing L-FABP levels. Palmitate metabolic clearance, permeability-surface area product, retention of palmitate by the liver, and cytoplasmic diffusion constant for unchanged [(3)H]palmitate also increased with increasing L-FABP levels. It is concluded that the variability in hepatic pharmacokinetics of unchanged [(3)H]palmitate and its low-molecular-weight metabolites in perfused rat livers is related to levels of L-FABP and not those of other intrahepatic proteins.     

Loss of regulation of lipogenesis in the Zucker diabetic rat. II. Changes in stearate and oleate synthesis

De novo lipogenesis and dietary fat uptake are two major sources of fatty acid deposits in fat of obese animals. To determine the relative contribution of fatty acids from these two sources in obesity, we have determined the distribution of c16 and c18 fatty acids of triglycerides in plasma, liver, and epididymal fat pad of Zucker diabetic fatty (ZDF) rats and their lean littermates (ZL) under two isocaloric dietary fat conditions. Lipogenesis was also determined using the deuterated water method. Conversion of palmitate to stearate and stearate to oleate was calculated from the deuterium incorporation by use of the tracer dilution principle. In the ZL rat, lipogenesis was suppressed from 70 to 24%, conversion of palmitate to stearate from 86 to 78%, and conversion of stearate to oleate from 56 to 7% in response to an increase in the dietary fat-to-carbohydrate ratio. The results suggest that suppression of fatty acid synthase and stearoyl-CoA desaturase activities is a normal adaptive mechanism to a high-fat diet. In contrast, de novo lipogenesis, chain elongation, and desaturation were not suppressed by dietary fat in the ZDF rat. The lack of ability to adapt to a high-fat diet resulted in a higher plasma triglyceride concentration and excessive fat accumulation from both diet and de novo synthesis in the ZDF rat.     

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)     

Enhanced expression of cytosolic fatty acid binding protein and fatty acid uptake during liver regeneration in rats

BACKGROUND/AIMS: Cytoplasmic liver fatty acid binding protein (L-FABP) has been suggested to be associated with cellular mitotic activity but the changes in L-FABP mRNA and protein levels during liver regeneration following partial hepatectomy (PHx) are not clear. METHODS: In the present study, we determined the time course of L-FABP mRNA expression and L-FABP levels following 70% PHx using Northern and Western blot, respectively. To elucidate one of the roles for L-FABP in PHx, [3H]-palmitic acid clearance in hepatocytes isolated from 24 h post-PHx and control animals was assessed. RESULTS: L-FABP mRNA increased at 30 min, peaked at approximately 1 h (163 +/- 17%; mean +/- SE, n = 5), and returned to control levels 6 h post-PHx. L-FABP level also increased at 1 h but peaked at 24-h (219 +/- 41%; mean +/- SE, n = 5). Hepatocyte [3H]-palmitic acid clearance increased by 29% at 24-h post-PHx, suggesting an increased intracellular transport (or binding) function by L-FABP. Pre-treatment with dexamethasone statistically reduced L-FABP levels (29%) and suppressed the regenerative process (mitotic activity). CONCLUSIONS: L-FABP mRNA increased sharply in response to PHx but the increase was short lived, while L-FABP level increased at a later stage. Both L-FABP content and fatty acid uptake increased significantly during liver regeneration induced by PHx in rats. It is likely that L-FABP is one of the factors responsible for hepatic regeneration.     

Effect of copper deficiency and Sodium intake upon liver lipid and mineral composition in the rat

The effect of copper and sodium intake upon liver cholesterol concentrations, fatty acid profile, and mineral concentrations were studied in the Long-Evans rat. Forty-eight male weaning rats were divided into three groups of 16 each and fed a semipurified diet containing either 0, 3, or 8 mg of added copper/kg of diet. At 100 d of age, half of the animals in each group were given 1% NaCl as drinking water and the other half was given deionized-distilled water for 12 wk. Copper deficiency in rats produced elevations in liver palmitate and oleate concentrations, but decreases in linoleate concentrations. The ratio of oleate:stearate was higher in copper deficient rats. Liver copper levels were decreased, but liver iron concentrations were elevated in copper deficient rats. Sodium intake did not have an effect on any of the parameters studied. These results suggested that dietary copper deficiency alters both liver mineral and fatty acid composition.     

The effect of sex on the quantity and properties of the very low density lipoprotein secreted by the liver in vitro.

Livers from normally fed male and female rats were perfused in vitro with different amounts of oleate, and the production and properties of the very low density lipoprotein (VLDL) were studied. The mobility of the VLDL in the zonal ultracentrifuge was dependent on the uptake of free fatty acid and on the sex of the animal from which the liver was obtained. A higher proportion of the VLDL secreted by livers from females displayed a more rapid mobility in the zonal ultracentrifuge and, in addition, contained less phospholipid and cholesterol per mole triglyceride than the VLDL from the male, suggestive of larger size of the VLDL secreted by livers from the female rats. Such differences were diminished when the VLDL was compared at equal output of triglyceride but unequal uptake of free fatty acid. These data suggest that the properties of the VLDL are only secondarily modulated by sex, and primarily result from differences in the capacities of livers from either male or female rats to synthesize triglyceride for transport as VLDL. The quantity of triglyceride secreted, regardless of sex, may be an important determinant of both size and number of the VLDL particles. The incorporation of endogenous hepatic fatty acid into VLDL triglyceride was diminished in livers from both sexes by increased uptake of oleate. The greater output of VLDL triglyceride by livers from female animals was dependent on both exogenous and endogenous fatty acids when relatively small quantities of exogenous oleate were available for uptake by the liver. The proportion of palmitate and oleate in the phospholipid of the VLDL secreted by livers from male rats decreased and the content of arachidonate increased with increasing uptake of oleate; no differences were observed in the composition of the phospholipid fatty acids among the various experimental female groups, although these contained more stearate and less oleate and linoleate compared to the male groups. The change of fatty acid composition of the VLDL phospholipid may reflect inclusion of specific types of phospholipid in the VLDL structure for transport of triglyceride from the liver under particular conditions.     

Comparison of metabolism of free fatty acid by isolated perfused livers from male and female rats.

Livers from normal, fed male and female rats were perfused with different amounts of [1-14C]oleate under steady state conditions, and the rates of uptake and utilization of free fatty acid (FFA) were measured. The uptake of FFA by livers from either male or female rats was proportional to the concentration of FFA in the medium. The rate of uptake of FFA, per g of liver, by livers from female rats exceeded that of the males for the same amount of FFA infused. The incorporation by the liver of exogenous oleic acid into triglyceride, phospholipid, and oxidation products was proportional to the uptake of FFA. Livers from female rats incorporated more oleate into triglyceride (TG) and less into phospholipid (PL) and oxidation products than did livers from male animals. Livers from female rats secreted more TG than did livers from male animals when infused with equal quantities of oleate. The incorporation of endogenous fatty acid into TG of the perfusate was inhibite) by exogenous oleate. At low concentrations of perfusate FFA, however, endogenous fatty acids contributed substantially to the increased output of TG by livers from female animals. Production of 14CO2 and radioactive ketone bodies increased with increasing uptake of FFA. The partition of oleate between oxidative pathways (CO2 production and ketogenesis) was modified by the availability of the fatty acid substrate with livers from either sex. The percent incorporation of radioactivity into CO2 reached a maximum, whereas incorporation into ketone bodies continued to increase. The output of ketone bodies was dependent on the uptake of FFA, and output by livers from female animals was less than by livers from male rats. The increase in rate of ketogenesis was dependent on the influx of exogenous FFA, while ketogenesis from endogenous sources remained relatively stable. The output of glucose by the liver increased with the uptake of FFA, but no difference due to sex was observed. The output of urea by livers from male rats was unaffected by oleate, while the output of urea by livers from females decreased as the uptake of FFA increased. A major conclusion to be derived from this work is that oleate is not metabolized identically by livers from the two sexes, but rather, per gram of liver, livers from female rats take up and esterify more fatty acid to TG and oxidize less than do livers from male animals; livers from female animals synthesize and secrete more triglyceride than do livers from male animals when provided with equal quantities of free fatty acid.     

Effect of dietary fatty acids on lipoprotein lipase gene expression in the liver and visceral adipose tissue of fed and starved red sea bream Pagrus major

Juvenile red sea bream Pagrus major were fed either a commercial diet (diet 1) or diets supplemented with 10% oleate (diet 2), 5% oleate+5% linoleate (diet 3) or 5% oleate+5% n-3 polyunsaturated fatty acid mixture (diet 4) for 4 weeks. Following the conditioning period, the effects of dietary fatty acids on lipoprotein lipase (LPL) gene expression in the liver and visceral adipose tissue of fed (5 h post-feeding) and starved (48 h post-feeding) fish were investigated by competitive polymerase chain reaction. Fish liver showed substantial LPL mRNA expression that is not found in adult rat liver. When compared with diet 1, diets 2-4 tended to increase the LPL mRNA level in the liver, but tended to decrease it in the visceral adipose tissue under the fed condition. The reciprocal regulation of the liver and visceral adipose LPL mRNA abundance by dietary fatty acids was comparable to that of rat brown and white adipose tissue, respectively. The change in the LPL mRNA level by fatty acids was not completely consistent with the degree of fatty acid unsaturation. Our results indicate that the regulatory effect of dietary fatty acids on LPL gene expression was tissue-specific and related to feeding conditions, but was not solely dependent on the degree of unsaturation of fatty acids.     

Effect of fatty acids on physical properties of microsomes from isolated perfused rat liver

A computer-centered spectrofluorimeter was used to examine the physicochemical properties of hepatic microsomes and microsomal lipids obtained from isolated rat livers perfused with medium containing palmitate or oleate. The fatty acid composition and degree of unsaturation of the liver microsomal lipids reflected that the fatty acid present in the perfusate. The absorption corrected fluorescence, relative fluorescence efficiency, polarization, and fluorescence anisotropy of several fluorescent probe molecules were measured to determine if their different microenvironments may be altered by the type of fatty acid infused. The probe molecules β-parinaric acid and 1.6-diphenyl-1,3,5-hexatriene had higher values for each of these parameters when incorporated into microsomes obtained from livers perfused with a medium containing palmitate than with oleate. The same parameters measured for cholesta-5,7,9(11)-trien-3β-ol and N-phenyl-1-naphthylamine were not altered. These differences appeared to be primarily due to alterations in microviscosity of the probe microenvironments since the rotational correlation time of 1,6-diphenyl-1,3,5-hexatriene was 25% lower in the microsomes from livers perfused with oleate as compared to livers perfused with palmitate. Thermal discontinuities in Arrhenius plots were noted in the intact microsomes but not in the isolated microsomal lipids with the fluorescence probe molecule β-parinaric acid. Break points occurred at 10°C and 26°C for microsomes from livers perfused with palmitate and at 12°C and 17°C for microsomes from livers perfused with oleate containing medium. These results suggest that the physicochemical properties of liver microsomes were determined in part by the fatty acid in the perfusate.     

The effect of endogenous essential and nonessential fatty acids on the uptake and subsequent agonist-induced release of arachidonate

We have demonstrated that the uptake and agonist-induced release of a pulse of arachidonate are influenced by the size and composition of preexisting endogenous fatty acid pools. EFD-1 cells, an essential fatty acid-deficient mouse fibrosarcoma cell line, were incubated with radiolabeled (14C or 3H] arachidonate, linoleate, eicosapentaenoate (EPA), palmitate, or oleate in concentrations of 0-33 microM for 24 h. After 24 h, the cells were pulsed with 0.67 microM radiolabeled (3H or 14C, opposite first label) arachidonate for 15 min and then stimulated with 10 microM bradykinin for 4 min. Because EFD-1 cells contain no endogenous essential fatty acids, we were able to create essential fatty acid-repleted cells for which the specific activity of the newly constructed endogenous essential fatty acid pool was known. Loading the endogenous pool with the essential fatty acids arachidonate, eicosapentaenoate, or linoleate (15-20 nmol of fatty acid incorporated/10(6) cells) decreased the uptake of a pulse of arachidonate from 200 to 100 pmol/10(6) cells but had no effect on palmitate uptake. The percent of arachidonate incorporated during the pulse which was released upon agonist stimulation increased 2-fold (4-8%) as the endogenous pool of essential fatty acids was increased from 0 to 15-20 nmol/10(6) cells. This 8% release was at least 3-fold greater than the percent release from the various endogenous essential fatty acid pools. In contrast, loading the endogenous pool with the nonessential fatty acids oleate or palmitate to more than 2-3 times their preexisting cellular level had no effect on the uptake of an arachidonate pulse. Like the essential fatty acids, increasing endogenous oleate increased (by 2-fold) the percent release of arachidonate incorporated during the pulse, whereas endogenous palmitate had no effect on subsequent agonist-induced release from this arachidonate pool. These studies show that preexisting pools of essential and nonessential fatty acids exert different effects on the uptake and subsequent releasability of a pulse of arachidonate.     

Decreased liver fatty acid binding capacity and altered liver lipid distribution in mice lacking the liver fatty acid-binding protein gene

Although liver fatty acid-binding protein (L-FABP) is an important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2- to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected [14C]oleate was markedly reduced, showing altered lipid pool turnover. An increase of approximately 75% of soluble SCP-2 but little or no change of other soluble (glutathione S-transferase, albumin) and membrane (fatty acid transport protein, CD36, aspartate aminotransferase, caveolin) fatty acid transporters was measured. These results (i) provide for the first time a quantitative assessment of the contribution of L-FABP to cytosolic fatty acid binding capacity, (ii) establish L-FABP as an important determinant of hepatic lipid composition and turnover, and (iii) suggest that SCP-2 contributes to the accumulation of cholesterol in L-FABP null liver.     

Fatty acid composition and properties of the liver microsomal membrane of rats fed diets enriched with cholesterol.

Male rats were fed diets containing olive (OO) or evening primrose (EPO) oil (10% w/w), with or without added cholesterol (1% w/w). After 6-week feeding, the lipid and fatty acid compositions, fluidity, and fatty acid desaturating and cholesterol biosynthesis/esterification related enzymes of liver microsomes were determined. Both the OO and EPO diets, without added cholesterol, increased the contents of oleic and arachidonic acids, respectively, of rat liver microsomes. The results were consistent with the increases in delta 9 and delta 6 desaturation of n-6 essential fatty acids and the lower microviscosity in the EPO group. Dietary cholesterol led to an increase in the cholesterol content of liver microsomes as well as that of phosphatidylcholine (PC). The cholesterol/phospholipid and PC/PE (phosphatidylethanolamine) ratios were also elevated. Fatty acid composition changes were expressed as the accumulation of monounsaturated fatty acids, with accompanying milder depletion of saturated fatty acids in rat liver microsomes. In addition, the arachidonic acid content was lowered, with a concomitant increase in linoleic acid, which led to a significant decrease in the 20:4/18:2 ratio in comparison to in animals fed the cholesterol-free diets. Cholesterol feeding also increased delta 9 desaturase activity as well as membrane microviscosity, whereas it decreased delta 6 and delta 5 desaturase activities. There was a very strong correlation between fluidity and the unsaturation index reduction in the membrane. Furthermore, the activity of hydroxymethylglutaryl-CoA reductase increased and the activity of acyl-CoA:cholesterol acyltransferase decreased in liver microsomes from both cholesterol-fed groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation of liver-type fatty acid binding protein content in the human hepatoma cell line HepG2 by peroxisome proliferators and antisense RNA affects the rate of fatty acid uptake

The liver-type fatty acid binding protein (L-FABP), a member of a family of mostly cytosolic 14-15 kDa proteins known to bind fatty acids in vitro and in vivo, is discussed to play a role in fatty acid uptake. Cells of the hepatoma HepG2 cell line endogenously express this protein to approximately 0.2% of cytosolic proteins and served as a model to study the effect of L-FABP on fatty acid uptake, by manipulating L-FABP expression in two approaches. First, L-FABP content was more than doubled upon treating the cells with the potent peroxisome proliferators bezafibrate and Wy14,643 and incubation of these cells with [1-14C]oleic acid led to an increase in fatty acid uptake rate from 0.55 to 0.74 and 0.98 nmol/min per mg protein, respectively. In the second approach L-FABP expression was reduced by stable transfection with antisense L-FABP mRNA yielding seven clones with L-FABP contents ranging from 0.03% to 0.14% of cytosolic proteins. This reduction to one sixth of normal L-FABP content reduced the rate of [1-14C]oleic acid uptake from 0.55 to 0. 19 nmol/min per mg protein, i.e., by 66%. The analysis of peroxisome proliferator-treated cells and L-FABP mRNA antisense clones revealed a direct correlation between L-FABP content and fatty acid uptake.     

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.     

Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity

Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of ∼80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or α-bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABP''s antioxidant activity is through inactivation of the free radicals by L-FABP''s methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant.