Fatty acid oxidation capacity and fatty acid-binding protein content of different cell types isolated from rat heart

Summary Heart tissue contains appreciable amounts of fatty acid-binding protein (FABP). FABP is thought to play a crucial role in the transport of fatty acids from the cellular membrane to the intracellular site of oxidation and also, in case of endothelial cells, in the transfer of fatty acids from the vascular to the interstitial compartment through the endothelial cytoplasm. The present study was designed to delineate a possible quantitative relationship between the capacity of different cell types in the heart to oxidize fatty acids and the presence of FABP. Palmitate oxidation capacity, measured in homogenates of cells isolated from adult rat hearts, was 2 nmol/min per mg tissue protein in freshly isolated cardiomyocytes (CMC), but only 0.09 and 0.31 nmol/min per mg tissue protein in cultivated endothelial (CEC) and fibroblast-like cells (CFLC), respectively. Palmitate oxidation rates were closely related to the cytochrome C oxidase activity and, hence, to the mitochondrial density in the cells under investigation. In CMC the content of cytosolic H-FABP (H-FABP_c) was about 4.51 µg/mg tissue protein. However, in CEC and CFLC the FABP content was less than 0.01 and 0.004 µg/mg tissue protein, respectively, corresponding to at maximum 0.2% of the FABP content of CMC. These findings indicate a marked difference between CMC and non-myocytal cells in the heart regarding their capacity to oxidize fatty acids, and a marked disproportion between the fatty acid oxidation capacity and immunochemically determined FABP content in both CEC and CFLC. The functional implication of these observations remains to be elucidated.     

Release of fatty acid-binding protein and long chain fatty acids from isolated rat heart after ischemia and subsequent calcium paradox

To obtain insight into the relation between the release of heart-type fatty acid-binding protein (H-FABP_c) and of long-chain fatty acids (FA) from injured cardiac tissue, rat hearts were Langendorff perfused according to the following scheme: 30 min normoxia, 60 min ischemia, 30 min reperfusion, 10 min Ca²⁺ free perfusion and finally 10 min Ca²⁺ repletion. During this protocol right ventricular (Q _rv ) and interstitial effluent samples (Q _i ) were collected at regular intervals. During reperfusion a total of 0.8±0.1 nmol H-FABP_c but no FA were detected in the effluents. However, during Ca²⁺ readmission, 45±4 nmol H-FABP_c (80–90% of total tissue content) was released with an initial (first 3 min) simultaneous release of FA (FA/H-FABP_c ratio 0.90±0.07 mol/mol). Thereafter, FA release continued at 10–15 nmol per min mainly inQ _rv while the rate of H-FABP_c release decreased. During Ca²⁺ repletion, tissue FA content raised rapidly from 168±20 to 1918±107 nmol/g dry weight. These findings suggest that after severe cardiac damage initially FA is released bound to H-FABP_c, whereas further FA release occurs in a non-protein bound manner.     

Revision of the amino acid sequence of human heart fatty acid-binding protein

Summary Cardiac-type fatty acid-binding protein (cFABP) from human heart muscle of three individuals was isolated and characterized as pI 5.3-cFABP. The proteins were structurally analyzed by tryptic peptide mapping, application of plasma desorption time-of-flight mass spectrometry and amino acid sequencing. All three preparations of human heart FABP, having 132 amino acids, differed from the published sequence [Offner et al. Biochem J 251: 191–198, 1988] in position 104, where Leu is found instead of Lys, and in position 124, where Cys is found instead of Ser.     

Type-specific immunodetection of human heart fatty acid-binding protein with polyclonal anti-peptide antibodies

Summary In order to develop specific antibodies against human heart cytoplasmic fatty acid-binding protein (HFABP_c), four oligo-peptides of 15–20 amino-acids each and corresponding with different antigenic parts of the human H-FABP_c molecule, were synthesized. Polyclonal antibodies against these synthetic peptides were raised in mice (Balb/C) and rabbits (Flemish giant). When tested in enzyme linked immunosorbent assays (ELISA, antibody-capture assay), antisera against three of the four peptides showed a high immunoreactivity with the synthetic peptide selected for immunization as well as with the native human H-FABP_c. Some cross-reactivity with the other synthetic peptides was observed for the rabbit antisera but not for those from mice. Polyclonal antibodies against synthetic peptides can be applied for the specific detection of the native protein in biological preparations containing proteins that show a high degree of homology with the protein to be assayed.     

Role of fatty acid-binding protein in lipid metabolism of insect flight muscle

Since insect flight muscles are among the most active muscles in nature, their extremely high rates of fuel supply and oxidation pose interesting physiological problems. Long-distance flights of species like locusts and hawkmoths are fueled through fatty acid oxidation. The lipid substrate is transported as diacylglycerol in the blood, employing a unique and efficient lipoprotein shuttle system. Following diacylglycerol hydrolysis by a flight muscle lipoprotein lipase, the liberated fatty acids are ultimately oxidized in the mitochondria. Locust flight muscle cytoplasm contains an abundant fatty acid-binding protein (FABP). The flight muscle FABP ofLocusta migratoria is a 15 kDa protein with an isoelectric point of 5.8, binding fatty acids in a 1:1 molar stoichiometric ratio. Binding affinity of the FABP for longchain fatty acids (apparent dissociation constant K_d=5.21±0.16 M) is however markedly lower than that of mammalian FABPs. The NH₂-terminal amino acid sequence shares structural homologies with two insect FABPs recently purified from hawkmoth midgut, as well as with mammalian FABPs. In contrast to all other isolated FABPs, the NH₂ terminus of locust flight muscle FABP appeared not to be acetylated. During development of the insect, a marked increase in fatty acid binding capacity of flight muscle homogenate was measured, along with similar increases in both fatty acid oxidation capacity and citrate synthase activity. Although considerable circumstantial evidence would support a function of locust flight muscle FABP in intracellular uptake and transport of fatty acids, the finding of another extremely well-flying migratory insect, the hawkmothAcherontia atropos, which employs the same lipoprotein shuttle system, however contains relatively very low amounts of FABP in its flight muscles, renders the proposed function of FABP in insect flight muscles questionable.     

Nomenclature of fatty acid-binding proteins

Summary A variety of designations is currently being used to refer to cellular fatty acid-binding proteins (FABPs). Besides from the use of other general names (e.g. Z protein), confusion mostly arises from the application of various abbreviations and symbols to denote the tissue(s) of origin and cellular localization (cytoplasm, plasma membrane) of a specific FABP. In order to minimize confusion a more unified and rational nomenclature is proposed, which is based on application of the formula X-FABPy. The prefix X is a capital letter indicating the tissue of greatest abundance, the suffix Y similarly denotes the (sub)cellular localization of the protein. The general and functional name fatty acid-binding protein (FABP) is preferred for the cellular proteins with the property to bind fatty acids, unless future research reveals that the binding of fatty acids is not the primary biological property or physiological role of (some of) these proteins.     

The membrane fatty acid-binding protein is not identical to mitochondrial glutamic oxaloacetic transaminase (mGOT)

Summary For evaluation whether the membrane fatty acid-binding protein is related to mGOT, studies on the structure and function of both purified proteins were performed. Physicochemical characterization revealed that both proteins are different: the membrane fatty acid-binding protein has a molecular weight of 40 kD and a pI of 8.5–9.0, whereas rat mGOT has a molecular weight of 44 kD and a pI of 9.5–10.0. According to this distinct differences, they migrated separately on 2-dimensional electrophoresis. Furthermore, monospecific antibodies against the membrane fatty acid binding protein did not react with rat mGOT. In co-chromatography studies only the membrane fatty acid-binding protein showed affinity for long chain fatty acids, but not mGOT. Moreover, membrane binding studies were performed with the monospecific antibody to the membrane fatty acid binding protein. The inhibitory effect of this antibody on plasma membrane binding of oleate was reversed after preabsorption of the antibody with the membrane fatty acid binding protein, but was not affected after preabsorption with mGOT. These results indicate that the membrane fatty acid binding protein and mGOT are structurally and functionally not related. The data also support the significance of this membrane protein in the plasma membrane binding process of long chain fatty acids.     

Fatty acid-binding proteins in the heart

Long-chain fatty acids are important fuel molecules for the heart, their oxidation in mitochondria providing the bulk of energy required for cardiac functioning. The low solubility of fatty acids in aqueous solutions impairs their cellular transport. However, cardiac tissue contains several proteins capable of binding fatty acids non-covalently. These fatty acid-binding proteins (FABPs) are thought to facilitate both cellular uptake and intracellular transport of fatty acids. The majority of fatty acids taken up by the heart seems to pass the sarcolemma through a carrier-mediated translocation mechanism consisting of one or more membrane-associated FABPs. Intracellular transport of fatty acids towards sites of metabolic conversion is most likely accomplished by cytoplasmic FABPs. In this review, the roles of membrane-associated and cytoplasmic FABPs in cardiac fatty acid metabolism under (patho)physiological circumstances are discussed.     

Fasting increases plasma membrane fatty acid-binding protein (FABPPM) in red skeletal muscle

The present study was designed to investigate the presence of the fatty acid-binding protein (FABPPM) in the plasma membranes of skeletal muscles with different oxidative capacities for free fatty acid (FFA) oxidation during conditions of normal (fed) or increased (fasted) FFA utilization in the rat. Female Sprague-Dawley rats were either fed or fasted for 12, 24, or 48 h and, plasma membranes (PM) fractions from red and white skeletal muscles were isolated. Short-term fasting significantly decreased body weight by 11% and blood glucose concentration by 42% (6.6 ± 0.2-3.8 ± 0.4 mmol/l) and increased plasma FFA concentration by 5-fold (133 ± 14-793 ± 81 µmol/l). Immunoblotting of PM fractions showed that FABPPM protein content was 83 ± 18% higher in red than in white skeletal muscle and correlated with oxidative capacity as measured by succinate dehydrogenase activity (r = 0.78, p < 0.05). Short-term fasting significantly increased FABPPM protein content by 60 ± 8% in red skeletal muscle but no change was measured in white skeletal muscle. These results show that FABPPM protein content in skeletal muscle is related to oxidative potential and can be increased during a physiological condition known to be associated with an increase in FFA utilization, suggesting that cellular expression of FABPPM may play a role in the regulation of FFA metabolism in skeletal muscle. (Mol Cell Biochem 166: 153-158, 1997)     

Solution structure of bovine heart fatty acid-binding protein (H-FABPc)

Fatty acid-binding protein (FABP) from bovine heart, a 15 kDa cytoplasmic protein has been investigated by multidimensional homonuclear and heteronuclear NMR-spectroscopy. Perdeuterated palmitic acid has been used as fatty acid ligand. The tertiary structure has been determined from distance geometry calculations with the variable target functions algorithm (DIANA) [1] utilizing 1027 interproton distance constraints, which were obtained from¹H-homo-nuclear NOESY spectra. Overlapping NOE crosspeaks were assigned by heteronuclear multidimensional NMR-experiments with a¹⁵N-labelled sample. The tertiary structure resembles a -barrel (-clam) consisting of ten anti-parallel -strands and a short helix-turn-helix motif. The -strands are arranged in two nearly orthogonal -sheets composed of 5 strands each. The solution structure is compared with the x-ray cyrstal structure of bovine heart [4] and rat intestinal FABPs.Abbreviations DOF-COSY Double Quantum Filtered Correlated Spectroscopy - TOCSY Total Correlated Spectroscopy - NOE Nuclear Overhauser Enhancement - NOESY Nuclear Overhauser Enhancement and Exchange Spectroscopy - HMQC Heteronuclear Multiple Quantum Coherence - FABP Fatty Acid-Binding Protein - FABP_c Cellular Fatty Acid-Binding Protein - H-FABP_c Cellular Heart Fatty Acid-Binding Protein - I-FABP_c Cellular Intestinal Fatty Acid-Binding Protein     

A comparison of heart and liver fatty acid-binding proteins: interactions with fatty acids and possible functional differences studied with fluorescent fatty acid analogues

Summary Fatty acid-binding proteins (FABP) are distinct but related gene products which are found in many mammalian cell types. They are generally present in high abundance, and are found in those tissues where free fatty acid (ffa) flux is high. The function(s) of FABP is unknown. Also not known is whether all FABP function similarly in their respective cell types, or whether different FABP have unique functions. The purpose of these studies was to assess whether different members of the FABP family exhibit different structural and functional properties. Two fluorescent analogues of ffa were used to compare the liver (L-FABP) and heart (H-FABP) binding proteins. The propionic acid derivative of diphenylhexatriene (PADPH) was used to examine the physical properties of the ffa binding site on L- and H-FABP, as well as the relative distribution of ffa between FABP and membranes. An anthroyloxy-derivative of palmitic acid, 2AP, was used to monitor the transfer kinetics of ffa from liver or heart FABP to acceptor membranes, using a resonance energy transfer assay. The results demonstrate that the ffa binding sites of both FABP are hydrophobic in nature, although the L-FABP site is more nonpolar than the H-FABP site. Equilibration of PADPH between L-FABP and phosphatidylcholine (PC) bilayers resulted in a molar partition preference of > 20: 1, L-FABP : PC. Similar studies with H-FABP resulted in a PADPH partition preference of only 3:1, H-FABP : PC. Finally, the transfer of 2AP from H-FABP to acceptor membranes was found to be 50-fold faster than transfer from L-FABP. These studies demonstrate that important structural and functional differences exist between different members of the FABP family, and therefore imply that the roles of different FABP may be unique.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Liver FABP - H-FABP Heart FABP - SUV Small Unilamellar Vesicle - PADPH 3-[p-(6-Phenyl)-1,3,5-Hexatrienyl]-phenylpropionic acid - 2AP 2-(9-Anthroyloxy)Palmitic acid - Q Quantum yield - _F Fluorescence lifetime     

Fatty acid-binding protein from human heart localized in native and denaturing two-dimensional gels

Summary A group of low molecular weight fatty acid-binding cytosolic proteins, FABP_c with high abundance in heart, liver, skeletal muscle, intestine and adipose tissue, are anticipated to play a role in long-chain fatty acid metabolism in these tissues. Recently, a FABP_c with M_T 15 kDa has been purified from human heart muscle and found to be present in levels 2–4% of cytosolic proteins of human heart myocytes. In the present study two-dimensional gel electrophoresis under native and denaturing conditions has been used to characterize FABP_c from human heart and this protein is found to be a major protein of human heart myocytes. The pI of FABP_c from human heart was found to be about 5.3 under native conditions and about 6.5 in the presence of 9 M urea.     

Similar mechanisms of fatty acid transfer from human anal rodent fatty acid-binding proteins to membranes: Liver,intestine, heart muscle,and adipose tissue FABPs

The mammalian fatty acid-binding proteins (FABPs) are thought to be important for the transport and metabolism of fatty acids in numerous cell types. The transfer of FA from different members of the FABP family to membranes has been shown to occur by two distinct mechanisms, an aqueous diffusion-based mechanism and a collisional mechanism, wherein the FABP interacts directly with membrane acceptors. Much of the work that underlies this concept comes from efforts using rodent FABPs. Given the increasing awareness of links between FABPs and several chronic diseases in humans, it was important to establish the mechanisms of FA transfer for human FABPs. In the present studies, we examined the rate and mechanism of fatty acid transfer from four pairs of human and rodent (rat or mouse, as specified) FABPs: hLFABP and rLFABP, hIFABP and rIFABP, hHFABP and rHFABP, and hAFABP and mAFABP. In the case of human IFABP, both the Ala⁵⁴ and Thr⁵⁴ forms were examined. The results show clearly that for all FABPs examined, the mechanisms of ligand transfer observed for rodent proteins hold true for their human counterparts. Moreover, it appears that the Ala to Thr substitution at residue 54 of the human IFABP does not alter the fundamental mechanism of ligand transfer to membranes, but nevertheless causes a consistent decrease in the rate of transfer.     

Involvement of arginine in the binding of heme and fatty acids to fatty acid-binding protein from bovine liver

Fatty acid-binding protein from bovine liver but not from bovine heart binds hematin in a saturable manner with high affinity. This property is not confined to a particular isoform as both, pI 6.0- and pI 7.0 L-FABP, bind hematin similarly. In competition experiments hematin and oleic acid could replace each other demonstrating that they share at least parts of the same binding site. Common structural features, i.e. the presence of carboxylic groups and of hydrophobic carbon chains led to the hypothesis that both ligands interact similarly with L-FABP. This was supported by the decrease of binding affinity for either ligand upon modification with phenylglyoxal. Modification in the presence of fatty acid revealed the protection of one of the two arginines of L-FABP. By peptide mapping and Edman degradation Arg¹²² was identified as the counterpart of the fatty acids carboxylic group.     

Quantitation of plasma membrane fatty acid-binding protein by enzyme dilution and monoclonal antibody based immunoassay

Summary A plasma membrane fatty acid-binding protein (h-FABPPm) has been isolated from rat hepatocytes. Analogous proteins have also been identified in adipocytes, jejunal enterocytes and cardiac myocytes, all cells with high transmembrane fluxes of fatty acids. These 43 kDa, highly basic (pl = 9.1) FABP_pm 's appear unrelated to the smaller, cytosolic FABP's (designated FABP's) identified previously in the same tissues. h-FABP_pm appears closely related to the mitochondrial isoform of glutamic-oxaloacetic transaminase (mGOT), and both the purified protein and liver cell plasma membranes (LPM) possess GOT enzymatic activity. From their relative GOT specific activities it is estimated that h-FABP_pm constitutes approximately 2% of LPM protein, or about 0.7 × 10⁷ sites per cell. A monoclonal antibody-based competitive inhibition enzyme immunoassay (CIEIA) for h-FABP_pm is described; it yields an estimate of 3.4 x 10⁷ h-FABP_pm sites per hepatocyte. Quantitated by either method, h-FABPPm appears to be a highly abundant protein constituent of LPM.     

Serum adipocyte-specific fatty acid-binding protein is associated with nonalcoholic fatty liver disease in apparently healthy subjects

Adipocyte-specific fatty acid-binding protein (A-FABP) is a cytoplasmic protein that is expressed in adipocytes and is closely associated with insulin resistance, metabolic syndrome, and Type 2 diabetes. We investigated the relationship between A-FABP as a surrogate marker of metabolic syndrome and non-alcoholic fatty liver disease (NAFLD) in apparently healthy subjects. We assessed clinical and biochemical metabolic parameters and measured serum levels of A-FABP, high-sensitivity C-reactive protein and tumor necrosis factor-α (TNF-α) in 494 subjects who were divided into two groups according to the presence of NAFLD by abdominal ultrasonography. All parameters associated with metabolic syndrome were significantly higher in patients with NAFLD (P<.001). A-FABP showed positive correlation with TNF-α, homeostasis model assessment index of insulin resistance (HOMA-IR), and metabolic syndrome (P<.001) when adjusted for age and sex. The odds ratio for the risk of NAFLD in the highest tertile of A-FABP compared with the lowest tertile was 7.36 (CI 3.80-14.27, P<.001) after adjustment for age and sex; 4.52 (CI 2.22-9.20, P<.001) after adjustment for age, sex, HOMA-IR and metabolic syndrome and 2.86 (CI 1.11-7.35, P<.05) after further adjustment for all metabolic parameters including TNF-α. The serum level of A-FABP was independently associated with NAFLD and showed significant correlation with TNF-α, HOMA-IR, and metabolic syndrome.     

Fatty acid-binding proteinuria diagnoses myocardial infarction in the rat

Cytoplasmic heart-type fatty acid-binding protein (H-FABP_c) is a low molecular weight protein with abundant presence in the myocardium. Upon ischemia it is released from the heart and can subsequently be detected in plasma and urine. In this study, the value of measurement of H-FABP_c excreted into urine for the diagnosis of myocardial infarction (MI) is investigated in the rat. To this end, firstly the kinetic behaviour of H-FABP_c in plasma was examined and its release into urine quantified.After injection of purified H-FABP_c in normal animals, a net recovery in urine of 14–29% was found. The kinetic behaviour of H-FABP_c in plasma was characterized by a total clearance of 0.33 ml/min and a half-life value of total elimination of about 270 min.Knowing these plasma characteristics of H-FABP_c, a comparison was made between the cumulative amounts of H-FABP_c released in the 24-hour urine of MI rats and of sham-operated animals. In MI rats, with a mean morphometric MI size of 43%, the mean total amount of H-FABP_c excreted into urine was 79 g, while in sham-operated rats this was 23 g. This difference between both groups is significant (p<0.001)It is concluded that urinary H-FABP_c can be used as a noninvasive marker for MI in the rat.     

Fatty acid-binding protein expression in the liver: its regulation and relationship to the zonation of fatty acid metabolism

Summary Liver fatty acid-binding protein (L-FABP) is expressed in a declining gradient between the portal and central zones of the liver acinus. This paper discusses the results of experimental studies which address the questions: (a) What factors regulate L-FABP expression in liver and produce its acinar gradient? (b) What is the relationship between the acinar gradient of L-FABP and acinar gradients in the transport and metabolism of long-chain fatty acids? Both high-fat diets and clofibrate-treatment increase L-FABP proportionally at both extremes of the liver acinus and the small intestine, with preservation of the L-FABP gradient in both tissues. Female rats differ from males, however, in showing a greater hepatic abundance of L-FABP which is expressed almost equally throughout the acinus. Dietary studies show that L-FABP is induced with increased fatty acid flux derived from dietary fat but not from de novo hepatic fatty acid synthesis. Studies of the synthesis and utilization of fatty acids by hepatocytes isolated from the periportal and pericentral zones of the liver acinus suggest that the acinar gradient of L-FABP is not associated with differences in the instrinsic capacity of zone 1 and zone 3 hepatocytes to utilize or synthesize fatty acids. In addition, studies of the acinar uptake pattern of a fluorescent fatty acid derivative by isolated perfused livers indicate that the acinar distribution of L-FABP does not determine the pattern of fatty acid uptake in the intact acinus. Rather, the acinar gradient of L-FABP is most likely to represent a response to physiological conditions existing in the intact acinus which may include gradients in the flux of fatty acids, fatty acid metabolites and hormones.Abbreviations ALT Alanine Aminotransferase - FABP Fatty Acid Binding Protein - I-FABP Intestinal-type Fatty Acid Binding Protein - L-FABP Liver-type Fatty Acid Binding Protein - 12-NBD-stearate 12-(N-methyl)-N-(7-nitrobenzo-2-oxa-1, 3,-diazol-4-yl)amino)-octadecanoic acid     

Effects of linoleic and gamma-linolenic acids (efamol evening primrose oil) on fatty acid-binding proteins of rat liver

Summary We have studied the effects of Efamol evening primrose oil (EPO) on fatty acid-binding proteins (L-FABP) of rat liver. EPO contains 72% cis-linoleic acid and 9% cis-gamma linolenic acid. EPO has been clinically used for treatment of a number of diseases in humans and animals. EPO is also known to lower cholesterol level in humans and animals. Feeding of an EPO supplemented diet to rats (n = 9) for 2 months decreases the oleate binding capacity of purified L-FABP of rat liver whereas the palmitate binding activity was increased by 38%. However, EPO feeding did not alter the L-FABP concentrations significantly as measured by using the fluorescence fatty acid probe, dansylamino undecanoic acid. Endogenous fatty acid analysis of L-FABPs revealed significant qualititative and quantitative changes in fatty acid pattern after EPO feeding. EPO feeding decreased the endogenous palmitate level by 53% and oleate level by 64% in L-FABPs and also EPO feeding decreased the total endogenous fatty acid content from 62 nanomole per mg of protein to 42 nanomole per mg of L-FABP (n = 3).     

Expression of testicular fatty acid-binding protein PERF 15 during germ cell apoptosis

PERF 15 is a testicular germ cell specific fatty acid-binding protein (FABP) isolated from rat. Indirect immunofluorescent analysis of juvenile rat testis showed that there were some strongly PERF 15-positive spermatocytes. These cells showed unclear nuclear structure and were predicted to undergo apoptosis. Apoptosis in germ cells is an important regulatory event to limit the number of germ cells in the seminiferous epithelium, but the physiological significance and molecular mechanisms of this testicular germ cell apoptosis are poorly understood. To determine whether PERF 15 participates in germ cell apoptosis, juvenile rat testis was examined by immunohistochemical and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) methods. Strongly PERF 15-positive cells and TUNEL-positive cells were co-localized in adjacent sections. Exposure to methoxyacetic acid (MAA), known to induce apoptosis in spermatocytes, increased the number of strongly PERF 15-positive cells in 25-day-old rats' testes. Therefore, it seems that PERF 15 is involved in both spermatogenesis and testicular germ cell apoptosis.