Cytoplasmic Fatty Acid-binding Protein Research Articles

Fatty acid-binding proteinuria diagnoses myocardial infarction in the rat.

Cytoplasmic heart-type fatty acid-binding protein (H-FABPc) 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-FABPc excreted into urine for the diagnosis of myocardial infarction (MI) is investigated in the rat. To this end, firstly the kinetic behaviour of H-FABPc in plasma was examined and its release into urine quantified. After injection of purified H-FABPc in normal animals, a net recovery in urine of 14-29% was found. The kinetic behaviour of H-FABPc 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-FABPc, a comparison was made between the cumulative amounts of H-FABPc 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-FABPc excreted into urine was 79 micrograms, while in sham-operated rats this was 23 micrograms. This difference between both groups is significant (p < 0.001). It is concluded that urinary H-FABPc can be used as a noninvasive marker for MI in the rat.

Research of an in vitro model to study the expression of fatty acid-binding proteins in the small intestine.

In order to find an in vitro model for studying the regulation of the biosynthesis of the cytoplasmic Fatty Acid-Binding Proteins (FABPc) expressed in the small intestine, Intestinal- and Liver- (I- and L-) FABPc expressions were tested by Northern blotting in 8 normal or cancerous intestinal cell lines from man, mouse and rat and in organ culture of mouse jejunal explants. Neither I- nor L-FABPc mRNA was detected in any cell strains tested except in the highly differentiated human enterocyte-like intestinal cell line Caco-2. In this line, Northern blot analysis revealed a single messenger of about 0.7 kb corresponding to the L-FABPc. A two-fold increase in mRNA L-FABPc occurred in differentiated Caco-2 cells treated for 7 days with 0.05 mM bezafibrate, a peroxisome-proliferating hypolipidemic drug. The lack of I-FABPc messengers in this strain led us to seek another in vitro model. I- and L-FABPc messengers were found using an organ culture of mouse jejunal explants. A clear rise in I- and, especially, L-FABPc mRNA levels occurred 6 and 24 hr after the addition of 0.05 mM bezafibrate in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty acid homeostasis in the normoxic and ischemic heart

Fatty acid homeostasis in the normoxic and ischemic heart

Immunocytochemical localization of rat intestinal 15 kDa protein, a member of cytoplasmic fatty acid-binding proteins.

Rat intestinal 15 kDa protein (I-15P) is a member of the family of cytoplasmic fatty acid-binding proteins. Using a specific antiserum against I-15P, we studied the tissue distribution and subcellular localization of this protein in the entire rat body. By immunoblot analysis of cytosolic proteins, I-15P was detected not only in the distal portion of small intestine but also in the ovary and adrenal gland. Immunohistochemically, I-15P was localized to the absorptive epithelial cells as well as a subpopulation of enterochromaffin cells in the intestine, the lutein cells in the ovary, and subpopulations of cortical cells in the adrenal gland. Furthermore, I-15P-like immunoreactivity was also demonstrated in the surface mucous cells of stomach and the granular convoluted tubule cells of submandibular gland. Immuno-electron microscopy showed that the immunoreactivity was confined to the cytoplasmic matrix region, except in the enterochromaffin cells and granular convoluted tubule cells, where it was localized in the secretory granules. The present findings suggest that I-15P plays a role in the cellular metabolism of steroids.

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-FABP c) 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-FABP c 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-FABP c tended to be lower in SHR than in WKY hearts (874 ± 53 μg/g and 955 ± 51 μg/g, respectively; 0.1< P<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-FABP c 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<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 μ M.

Cellular fatty acid-binding proteins: current concepts and future directions

At least three different proteins are implicated in the cellular transport of fatty acid moieties: a plasmalemmal membrane and a cytoplasmic fatty acid-binding protein (FABPPM and FABPC, respectively) and cytoplasmic acyl-CoA 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 are found in remarkable abundance in the cytoplasmic compartment of a variety of tissues. Although their mechanism of action is not yet fully elucidated, current knowledge suggests that the function of this set of proteins reaches beyond simply aiding cytoplasmic solubilization of hydrophobic ligands, but that they can be assigned several regulatory roles in cellular lipid homeostasis.

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

In order to develop specific antibodies against human heart cytoplasmic fatty acid-binding protein (H-FABPc), four oligo-peptides of 15-20 amino-acids each and corresponding with different antigenic parts of the human H-FABPc 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-FABPc. 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.

In situ labeling of the adipocyte lipid binding protein with 3-[125I]iodo-4-azido-N-hexadecylsalicylamide. Evidence for a role of fatty acid binding proteins in lipid uptake.

Differentiating 3T3-L1 cells have been used to investigate the process of fatty acid uptake, its cellular specificity, and the involvement of cytoplasmic carrier proteins. The profile of fatty acid uptake in both differentiated and undifferentiated cells was biphasic, consisting of an initial rapid phase (0-20 s) followed by a second slower phase (60-480 s). In both cell types the initial phase of fatty acid (FA) uptake was temperature-insensitive whereas the rate of uptake during the second phase decreased 4-fold when measurements were made at 4 degrees C. The rate of [9,10-3H]oleate uptake in 3T3-L1 adipocytes was 10-fold greater than in the fibroblastic precursor cells. The acquisition of a differentially expressed cytoplasmic fatty acid binding protein (adipocyte lipid binding protein (ALBP] occurs coincident with the increased ability of these cells to take up FAs. Uptake experiments with 3-[125I]iodo-4-azido-N-hexadecylsalicylamide demonstrated that this photoactivatable FA analogue accumulated intracellularly in a time-, temperature-, and cell-specific fashion. Moreover, when 3T3-L1 adipocytes were presented with 3-[125I]iodo-4-azido-N-hexadecylsalicylamide and then irradiated, a single cytoplasmic 15-kDa protein was labeled. The in situ-labeled 15-kDa protein was identified as ALBP by its ability to be immunoprecipitated with anti-ALBP antisera. Taken together these results indicate that fatty acids traverse the plasma membrane and are bound by ALBP in the cytoplasmic compartment. It is likely that lipid uptake in other cell systems, such as liver, heart, intestine, and nerve tissue, proceeds by a similar process and that this represents a general mechanism for cell-specific FA uptake and utilization.

Characteristics of fatty acid-binding proteins and their relation to mammary-derived growth inhibitor

Based on sequence relationships the cytoplasmic fatty acid-binding proteins (FABPs) of mammalian origin are divided into at least three distinct types, namely the hepatic-, intestinal- and cardiac-type. Highly conserved sequences of FABPs within the same type correlate with immunological crossreactivities. Isoforms of hepatic-type FABP are found in several mammalian species and for bovine liver FABP specific shifts in isoelectric points upon lipidation with fatty acids are observed. Isoforms of intestinal-type FABP are not known and the occurrence of cardiac-type isoforms so far is confined to bovine heart tissue. A bovine mammary-derived growth inhibitor (MDGI) is 95% homologous to the cardiac-type FABP from bovine heart. Dissociation constants of FABP/fatty acid complexes are in the range of 1 microM and 1:1 stoichiometries are usually found, but the neutral isoform of hepatic FABP from bovine liver binds 2 fatty acids. On subcellular levels hepatic- and cardiac-type FABPs are differently distributed. Though mainly cytosolic in either case, immunoelectron microscopy as well as a gelchromatographic immunofluorescence assay demonstrate the association of hepatic FABP in liver cells with microsomal and outer mitochondrial membranes and with nuclei, whereas in heart cells cardiac FABP is confined to mitochondrial matrix and nuclei. In mammary epithelial cells MDGI is associated with neither mitochondria nor endoplasmic reticulum, and is expressed in a strictly developmental-dependent spatial and temporal pattern. The specific role proposed for MDGI is to arrest growth of mammary epithelial cells when they become committed to differentiation in the mammary gland.

Self-aggregation, a new property of cardiac fatty acid-binding protein. Predictable influence on energy production in the heart.

The binding isotherm of 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxyloxazolidine to the 12,000 molecular weight cytoplasmic fatty acid-binding protein purified from pig's heart and as analyzed by electron spin resonance is shown to be sigmoidal and dependent on protein concentration. Its maximum binding capacity, Vmax, is 9.81 microM X g-1 when the protein concentration is 1.98 g . liter-1 and 14.33 microM . g-1 when the concentration is 0.198 g . liter-1. The molar ellipticity, theta, measured by circular dichroism at 225 nm in the protein range of 0.2-3 g . liter-1 is also shown to depend on concentration, with two maxima at 1.7 and 1.2 g . liter-1. The rotational correlation time, tau eff, of a spin-labeled N-(2,2,5,5-tetramethyl-3-carbonyl-pyrroline-1-oxyl)imidazole specifically bound to tyrosine residues of the protein surface, as analyzed by electron spin resonance, is decreasing as a function of protein concentration. To account for these three concentration-dependent variations, we suggest that the protein self-aggregates since these variations will not be found if the protein is monomeric. A very significant influence of this aggregation property of the protein on the activity of fatty acid-dependent membrane-bound enzymes is predicted by a model analysis and, thus, should be considered as a new parameter in the control of energy production in the heart.