Michaël M. Vork

Cytoplasmic fatty acid binding protein: Significance for intracellular transport of fatty acids and putative role on signal transduction pathways

The cellular transport of long-chain fatty acid moieties is thought to be mediated by a plasmalemmal and a cytoplasmic fatty acid binding protein (FABPPM and FABPC, respectively) and a cytoplasmic acyl-coenzyme A binding protein (ACBP). Their putative main physiological significance is the assurance that long-chain fatty acids and derivatives, either in transit through membranes or present in intracellular compartments, are largely complexed to proteins. FABPC distinguishes from the other proteins in that distinct types of FABPC exist and that these are found in a variety of tissues in remarkable abundance, with some cells containing more than one type In addition, liver type FABPC binds not only fatty acids, but also several other hydrophobic ligands, including heme, bilirubin, prostaglandin E1 and lipoxygenase metabolites of arachidonic acid. Calculations made for rat cardiomyocytes reveal that the presence of FABPC substantially enhances the cytoplasmic solubility as well as the maximal diffusional flux of fatty acids in these cells. Apart from this putative function in the bulk transport of ligands, FABPC may also function in the fine-tuning of cellular events by modulating the metabolism of hydrophobic compounds implicated in the regulation of cell growth and differentiation.

Modulation of fatty-acid-binding protein content of rat heart and skeletal muscle by endurance training and testosterone treatment

The effects of training and/or testosterone treatment and its aromatization to oestradiol on fatty-acid-binding protein (FABP) content and cytochrome c oxidase activity in heart, soleus and extensor digitorum longus (EDL) muscles were studied in intact adult female rats. One group of rats remained sedentary, whereas the others were trained for 7 weeks. Thereafter the trained rats were divided into control and testosterone-treated groups, with or without an aromatase inhibitor. Testosterone was administered by a silastic implant. Training was continued for 2 weeks. In untreated sedentary rats the immunochemically assayed FABP contents were 497±28, 255±49 and 58±17 μg/g wet weight for the heart, soleus, and EDL respectively. In the heart the FABP content was increased after training (29%), testosterone treatment (33%) or both manipulations (53%). In soleus muscle FABP increased only after testosterone treatment (16%), whereas in EDL no changes were found. Inhibiting the aromatase enzyme complex abolished the testosterone-induced effect on FABP content in soleus (suggesting an oestradiol effect) but not in heart muscle. Among the three muscles studied the FABP content was found to be related to the cytochrome c oxidase activity in a non-linear way. In conclusion, it is shown that the FABP contents and mitochondrial activities of heart and skeletal muscle are affected by training and sex hormones and that these effects are different for heart and skeletal muscles.

Assay of the binding of fatty acids by proteins: evaluation of the Lipidex 1000 procedure

SummaryFatty acid (FA) binding by fatty acid-binding protein (FABP) is frequently Monitored with the so-called Lipidex 1000 assay, in which protein associated and non-protein bound FA are separated by selectively binding the latter to Lipidex 1000. Careful evaluation of this assay showed that the use of aqueous FA solutions resulted in a Marked decrease (60 to 70%) of FA concentration due to their aspecific binding to the surface of the test-tube used. In addition, solutions of rat heart FABP in the μMolar range also showed a concentration decrease up to 80% due to protein binding to the surface of the test-tube. Introduction of detergents, Triton X-100 or Tween 20, limited the FA loss to less than 20% and totally eliminated FABP adsorption. Kinetic parameters for the binding of [1-14C]oleic acid by purified rat heart FABP, assayed in the presence of Triton X-100, were found to be similar to those assayed in the absence of detergent, when adequate corrections were Made for losses of FA and FABP due to surface adsorption. Use of Tween 20 resulted in a substantial increase of the dissociation constant. The addition of 100 μM Triton X-100 to the assay medium considerably facilitates the determination of kinetic parameters of fatty acid-binding by proteins.

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

SummaryHeart 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-FABPc) 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.

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 Kd=5.21±0.16 μM) 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.

Modelling intracellular fatty acid transport: possible mechanistic role of cytoplasmic fatty acid-binding protein

A computer model is presented in which the role of cytoplasmic fatty acid-binding protein (FABP) in the intracellular translocation of fatty acids (FA) from one membrane to an opposite membrane is studied. The model consists of a cubical space, in which FABP and FA are allowed to diffuse at random. The amount of FA released from the donor membrane and reaching an opposite acceptor membrane is calculated in a variety of conditions. The data provided by the various simulations suggest that FABP can play a significant role in intracellular FA transport only if FABP is able to take up FA directly from FA containing membranes and to directly deliver FA to an acceptor membrane, thus preventing the unfavourable thermodynamical situation in which FA must solubilize in an aqueous environment prior to binding to FABP.

The beneficial effect of intermediate normothermic perfusion during cold storage of ischemically injured kidneys. A study of renal nucleotide homeostasis during hypothermia in the dog.

The effect of simple cold storage on ice with or without preceding warm ischemic injury on the energy metabolism and posttransplant viability of canine kidneys was examined in the present study. In addition, we investigated the possible beneficial effect of an intermediate normothermic perfusion half-way through the storage period on the preservation of ischemically injured kidneys. Thirty mongrel dogs were allocated to 5 experimental groups. In groups I and II kidneys were simply stored on ice for 24 and 48 hr, respectively. In groups III and IV kidneys were additionally subjected to 30 min warm ischemia before storage. In group 5 kidneys were treated as in group IV, but halfway through the storage period an intermediate normothermic ex-vivo perfusion was performed. The effect of these procedures on renal viability was tested by autologous reimplantation of the kidneys. During implantation the contralateral kidney was immediately removed. In group I all animals survived, whereas in group IV none of the animals survived. In groups II, III, and V, 2 of 6, 1 of 6, and 3 of 6 animals survived, respectively. The relationship, if any, between poststorage renal viability and the tissue levels of adenine nucleotides, guanine nucleotides, IMP, and purine degradation products was assessed by measuring the content of these metabolites in tissue specimen of the renal cortex, on which biopsies were done at various intervals during the experimental procedures. After an initial drop of about 30% in the content of adenine and guanine nucleotides and an increase in IMP, these values remained constant during 48 hr of cold storage. In contrast to kidneys stored for 24 hr, reimplantation of kidneys stored for 48 hr resulted in a significant decrease of adenine nucleotides following 60 min of in vivo reperfusion. Warm ischemia for 30 min prior to cold storage lead to lower initial nucleotide levels at the start of the storage period. During the first 24 hr nucleotide levels did not change, but a further decrease was observed during the following 24 hr of storage. Reimplantation after 24 hr of storage resulted in an additional decrease in the content of nucleotides. This poststorage decrease was absent after 48 hr of cold storage. Intermediate normothermic perfusion halfway through the storage for 48 hr significantly prevented the drop in the nucleotide content observed during the last 24 hr of storage in the corresponding control group. This nucleotide-sparing effect did not increase the level of nucleotides at the end of 60 min of reperfusion following reimplantation.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of warm ischemia time at donor nephrectomy.

To assess the warm ischemia time of kidneys with obscure donor histories we attempted to develop an index for the duration of ischemia by analysis of adenine nucleotides and their degradation products in cortical biopsies of canine kidneys. Two biopsy harvesting techniques were compared. The use of a laboratory technique (dentists drill) resulted in higher concentrations of adenosine triphosphate (ATP) in normoxic tissue specimens as compared with a clinical method of harvesting biopsies (wedge biopsy). However the sum of adenine nucleotides (AN) (ATP, adenosine diphosphate [ADP], and adenosine monophosphate [AMP]) was not significantly different in both groups (P less than 0.05). Therefore, wedge biopsies were used to study the degradation of AN following 0, 30, 60, 90, and 120 min of ischemia. Adenine nucleotides and their degradation products were assayed by high-performance liquid chromatography. Concentrations of individual adenine nucleotides did not show a consistent correlation with warm ischemia time. However, as the sum of the AN and the sum of their degradation products (DP) decreased and increased, respectively, the balance between these metabolites offered a good correlation with duration of warm ischemia. The ratio of DP to AN was significantly different at each interval (P less than 0.05). To study the influence of temperature on the degradation process, ischemia was induced at 37 degrees C and 32 degrees C. Lowering of the temperature reduced the catabolic rate of the AN. The ratio of DP to AN was significantly different from corresponding values at 37 degrees C. In biopsies of nonischemic human donor kidneys, concentrations of adenine nucleotides and their degradation products were measured. Biopsies weighing less than 0.01% of total renal mass were large enough to meet analytical demands. The ratio of DP to AN in human kidney biopsies was in the same range as in the corresponding dog kidney biopsies. These findings demonstrate that the ratio of DP to AN, as determined from concentrations of purine metabolites in canine cortical wedge biopsies, is a sensitive and potentially useful index of warm ischemia time.

Heterogeneous distribution of fatty acid-binding protein in the hearts of Wistar Kyoto and spontaneously hypertensive rats

In the present study we investigated the concentrations of cardiac cytoplasmic fatty acid-binding protein (H-FABPc) in various regions of the left and right ventricles of both Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). To this end, the ventricles of six WKY and six SHR hearts were cut in three slices, which were further dissected in one right ventricular piece and ten left ventricular pieces (five inner layer and five outer layer pieces). After homogenisation. H-FABPc was assessed using an Enzyme Linked Immuno Sorbent Assay (ELISA) of the sandwich type. It was found that, when expressed per gram wet tissue, the overall concentration of H-FABPc tended to be lower in SHR than in WKY hearts (874 +/- 53 micrograms/g and 955 +/- 51 micrograms/g, respectively; 0.1 less than P less than 0.2, means +/- S.E.M. for n = 6 animals in each group). However, due to a 30-35% higher ventricular heart mass in SHR than in WKY, the total H-FABPc content per heart turned out to be about 20% higher in SHR than in WKY rats. No concentration differences could be detected between right and left ventricles in WKY and SHR but a marked difference between the outer layer and the inner layer of the left ventricular wall was monitored in both groups. In general, the concentration in the outer layer was 5-15% higher than in the corresponding inner layer. These differences reached the level of significance (P less than 0.05) in regions close to the basis of the heart.

High-yield two-step chromatographic procedure for purification of fatty acid-binding protein from human heart

A high-yield procedure for the purification of cytoplasmic fatty acid-binding protein from human heart (H-FABP) is described. H-FABP was purified by gel permeation chromatography on a Sephacryl S-200 column followed by anion-exchange chromatography on a Sepharose Q fast-flow column at pH 7.0. At this pH H-FABP binds strongly to the column and can be selectively eluted with a salt gradient. The two-step procedure showed a high degree of reproducibility. On average 50 mg of H-FABP was obtained from 150 g of human heart tissue, which corresponds to a recovery of about 50%. Purity was confirmed by gel electrophoresis and isoelectric focusing. Binding of oleic acid to purified H-FABP, using the Lipidex 1000 assay, revealed a maximal binding of 0.75 +/- 0.01 mol fatty acid/mol protein and a dissociation constant of 0.19 +/- 0.01 microM.