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.     

Significance of cytoplasmic fatty acid-binding protein for the ischemic heart

Ischemia of the heart is accompanied by the tissue accumulation of long-chain fatty acids and their metabolic derivatives such as -hydroxy fatty acids and fatty acyl-CoA and acyl-L-carnitine esters. These substances might be detrimental for proper myocardial function. Previously, it has been suggested that intracellular lipid binding proteins like cytoplasmic fatty acid-binding protein (FABP) and acyl-CoA binding protein (ACBP) may bind these accumulating fatty acyl moieties to prevent their elevated levels from potentially harmful actions. In addition, the suggestion has been made that the abundantly present FABP may scavenge free radicals which are generated during reperfusion of the ischemic heart. However, these protective actions are challenged by the continuous physico-chemical partition of fatty acyl moieties between FABP and membrane structures and by the rapid release of FABP from ischemic and reperfused cardiac muscle. Careful evaluation of the available literature data reveals that at present no definite conclusion can be drawn about the potential protective effect of FABP on the ischemic and reperfused heart. Biochem123: 167–173, 1993)Abbreviations FABP Fatty Acid-Binding Protein - ACBP Acyl-CoA Binding Protein - MDGI Mammary-Derived Growth Inhibitor - CK Creatine Kinase - LDH Lactate Dehydrogenase     

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.     

Release of heart fatty acid-binding protein into plasma after acute myocardial infarction in man

The release of cytoplasmic heart fatty acid-binding protein (H-FABP) into the plasma of cardiac patients up to 38 hr after the onset of the first clinical symptoms of acute myocardial infarction (AMI) was studied, using a sensitive direct and noncompetitive Enzyme Linked Immunosorbent Assay of the antigen capture type (sandwich ELISA), newly developed for the measurement of small amounts of human H-FABP in plasma samples. Plasma levels of H-FABP were compared with plasma activity levels of the myocardial cytoplasmic enzymes creatine kinase MB (CK-MB) and alpha-hydroxybutyrate dehydrogenase (-HBDH). Upper normal levels of H-FABP (19g/l), CK-MB (10 U/l) and -HBDH (160 U/l) as determined in plasma from 72 blood donors served as threshold levels. H-FABP levels were significantly elevated above their threshold level within 3 hr after AMI. Peak levels of H-FABP, CK-MB and -HBDH were reached 4.1 ± 0.9 hr, 8.4 ± 1.4 hr and 25.0 ± 9.5 hr (means ± S.D., n = 10) after acute myocardial infarction, respectively. Serial time curves of the plasma contents of H-FABP reveal that after myocardial infarction H-FABP is released in substantial amounts from human hearts. In 18 out of 22 patients with established AMI the plasma FABP level was at or above the threshold level in blood-samples taken within 3.5 hr after the first onset of symptoms of AMI, while for CK-MB this applied to 9 patients and for -HBDH to 6 patients. These findings suggest that for an early indication of acute myocardial infarction in man cytoplasmic heart fatty acid-binding protein is more suitable than heart type creatine kinase MB and/or alpha-hydroxybutyrate dehydrogenase. (Mol Cell Biochem116: 155–162,1992)Abbreviations H-FABP (cytoplasmic) Heart Fatty Acid-Binding Protein - LDH Lactate Dehydrogenase, -HBDH--Hydroxybutyrate Dehydrogenase - CK-MB Creatine Kinase-MB - AMI Acute Myocardial Infarction - PBS Phosphate Buffered Saline - BSA Bovine Serum Albumin     

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.     

Expression of fatty acid-binding protein from bovine heart in Escherichia coli

Summary The coding part of the cDNA of cardiac fatty acid-binding protein (cFABP) from bovine heart was cloned into the vector pKK233-2. After induction with isopropyl--d-thiogalactopyranoside cFABP was found in a soluble form in the cytosol of plasmid transformed E. coli amounting up to 5.7% of the soluble protein. cFABP was detected after SDS-polyacrylamide gelelectrophoresis and/or isoelectric focusing and Western blot by immuno-staining and was determined quantitatively by a solid phase enzyme-linked immuno sorbent assay. The cFABP produced by bacteria binds oleic acid with high affinity as shown by comigration of protein and ligand in both gelfiltration and isoelectric focusing. cFABP was purified from bacterial lysates to near homogeneity and resolved into four isoproteins.     

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     

One-step purification of rat heart-type fatty acid-binding protein expressed in Escherichia coli

Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14–15 kDa lipid binding proteins which are believed to enhance intracellular transport of lipids by facilitating their cytoplasmic diffusion. To obtain sufficient amounts of protein for in vitro studies, we expressed rat H-FABP in Escherichia coli and compared its biochemical properties with the protein isolated from rat heart. An effective method was developed to purify recombinant rat H-FABP from cell lysates in a single step using anion-exchange chromatography. This method also proved to be applicable for purifying heterologously expressed human H-FABP. Recombinant rat H-FABP, which made up approximately 25% of the soluble proteins in E. coli, was obtained in a yield of 30–40 mg/l culture. Characterization showed that recombinant rat H-FABP was indistinguishable from the protein isolated from rat heart regarding molecular mass and oleic acid binding. Some heterogeneity upon isoelectric focusing was observed, presumably due to differences in N-terminal processing of the proteins. In conclusion, a method is presented for efficient high-yield production of recombinant rat H-FABP.     

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.     

Fatty acid-binding protein and its relation to fatty acid oxidation

A relation between fatty acid oxidation capacity and cytosolic FABP content was found in heart and various muscles of the rat. Other tissues do not show such a relation, since they are involved in more or other pathways of fatty acid metabolism. At postnatal development FABP content and fatty acid oxidation capacity rise concomitantly in heart and quadriceps muscle in contrast to in liver and kidney. A dietary fat content of 40 en. % increased only the FABP content of liver and adipose tissue. Peroxisomal proliferators increased fatty acid oxidation in both liver and kidney, but only the FABP content of liver, and had no effect on heart and skeletal muscle. The FABP content of muscle did not show adaptation to various conditions. Only it increased in fast-twitch muscles upon chronic electrostimulation and endurance training.