Fatty acid uptake in diabetic rat adipocytes.

The effect of diabetic status and insulin on adipocyte plasma membrane properties and fatty acid uptake was examined. Studies with inhibitors and isolated adipocyte ghost plasma membranes indicated 9Z, 11E, 13E, 15Z-octatetraenoic acid (cis-parinaric acid) uptake was protein mediated. Cis-parinaric acid uptake was inhibited by trypsin treatment or incubation with phloretin, and competed with stearic acid. The initial rate, but not maximal uptake, of cis-parinaric acid uptake was enhanced two-fold in adipocytes from diabetic rats. Concomitantly, the structure and lipid composition of adipocyte ghost membranes was dramatically altered. However, the increased initial rate of cis-parinaric acid uptake in the diabetic adipocytes was not explained by membrane alterations or by a two-fold decrease in cytosolic adipocyte fatty acid binding protein (ALBP), unless ALBP stimulated fatty acid efflux. Thus, diabetic status dramatically altered adipocyte fatty acid uptake, plasma membrane structure, lipid composition, and cytosolic fatty acid binding protein.

Fibroblast membrane sterol kinetic domains: modulation by sterol carrier protein-2 and liver fatty acid binding protein.

The mechanism(s) of intracellular sterol trafficking among subcellular organelle membranes is not well understood. Relative contributions of vesicular, sterol carrier protein, and membrane sterol domain pathways are not resolved. A sterol kinetic assay was used to resolve multiple sterol domains in microsome (MICRO), mitochondria (MITO), and plasma (PM) membrane: exchangeable, 20-40% of total; non-exchangeable, 60-80% of total. Spontaneous sterol transfer between dissimilar donor and acceptor membranes was vectorial and depended both on acceptor and donor membrane properties. For example, sterol transfer from PM to MICRO or to MITO, or from MICRO to MITO was 3- to 5-fold slower as compared to sterol movement in the opposite direction. Sterol carrier protein-2 (SCP-2) stimulated sterol transfer in most donor/acceptor membrane combinations by decreasing exchange half-time but not domain size. SCP-2 enhanced sterol transfer selectively: PM-MICRO (12-fold); MITO-MITO, MICRO-MICRO, MICRO-PM (3-fold); PM-PM (1.4-fold); PM-MITO, MICRO-MITO (no effect). Thus, SCP-2-mediated sterol movement was vectorial and not necessarily down a membrane sterol concentration gradient. In contrast, liver fatty acid binding protein (L-FABP) revealed a modest (2-fold) stimulatory effect on sterol transfer only between PM-MITO and MICRO-MICRO. In conclusion, in vitro studies of sterol transfer among isolated subcellular membranes provided kinetic evidence for sterol domains in microsomes and mitochondria as well as plasma membranes. Furthermore, both spontaneous and protein-mediated sterol transfer appeared vectorial and selective in nature.

Spontaneous and protein-mediated sterol transfer between intracellular membranes.

Relatively little is known regarding intracellular cholesterol trafficking pathways. To resolve some of these potential pathways, spontaneous and protein-mediated sterol transfer was examined between different donor-acceptor membrane pairs in vitro using L-cell fibroblast plasma membrane (PM) and microsomal (MICRO) and mitochondrial (MITO) membranes. Several new exciting insights were provided. First, the initial rate of spontaneous molecular sterol transfer was more dependent on the type of acceptor than donor membrane, i.e. spontaneous intracellular sterol trafficking was vectorial. Therefore, the rate of sterol desorption from the donor membrane was not necessarily the rate-limiting step in molecular sterol transfer. Second, the rate of molecular sterol transfer was not obligatorily correlated with the direction of the cholesterol gradient. For example, although PM had a 3.2-fold higher cholesterol/phospholipid ratio than MITO, spontaneous sterol transfer was 4-5-fold faster up (MITO to PM) rather than down (PM to MITO) the concentration gradient. Third, sterol carrier protein-2 differentially stimulated the initial rate of sterol transfer for all donor-acceptor combinations, being most effective with PM donors: PM-MICRO, 27-fold; and PM-MITO, 12-fold. Sterol carrier protein-2 was less effective in enhancing sterol transfer in the reverse direction, i.e. MICRO-PM and MITO-PM (5- and 4-fold, respectively). Fourth, liver fatty acid-binding protein was limited in stimulating the initial rate of sterol transfer from PM to PM (1.5-fold), from PM to MITO (3-fold), and from MICRO to MITO (3-fold). In summary, these observations present important insights into potential sterol trafficking pathways between the major membrane components of the cell.

Liver fatty acid binding protein enhances sterol transfer by membrane interaction.

Among the large family of fatty acid binding proteins, the liver L-FABP is unique in that it not only binds fatty acids but also interacts with sterols to enhance sterol transfer between membranes. Nevertheless, the mechanism whereby L-FABP potentiates intermembrane sterol transfer is unknown. Both fluorescence and dialysis data indicate L-FABP mediated sterol transfer between L-cell fibroblast plasma membranes occurs by a direct membrane effect: First, dansylated-L-FABP (DNS-L-FABP) is bound to L-cell fibroblast plasma membranes as indicated by increased DNS-L-FABP steady state polarization and phase resolved limiting anisotropy. Second, coumarin-L-FABP (CPM-L-FABP) fluorescence lifetimes were significantly increased upon interaction with plasma membranes. Third, dialysis studies with 3H-cholesterol loaded plasma membranes showed that L-FABP added to the donor compartment of the dialysis cell stimulated 3H-cholesterol transfer whether or not the dialysis membrane was permeable to L-FABP. However, L-FABP mediated intermembrane sterol transfer did require a sterol binding site on L-FABP. Chemically blocking the ligand binding site also inhibited L-FABP activity in intermembrane sterol transfer. Finally, L-FABP did not act either as an aqueous carrier or in membrane fusion. The fact that L-FABP interacted with plasma membrane vesicles and required a sterol binding site was consistent with a mode of action whereby L-FABP binds to the membrane prior to releasing sterol from the bilayer.

Cholesterol interaction with recombinant human sterol carrier protein-2.

The interaction of human recombinant sterol carrier protein-2 (SCP-2) with sterols was examined. Two independent ligand binding methods, Lipidex 1000 binding of [3H]cholesterol and a fluorescent dehydroergosterol binding assay, were used to determine the affinity of SCP-2 for sterols. Binding analysis indicated SCP-2 bound [3H]cholesterol and dehydroergosterol with a Kd of 0.3 and 1.7 microM, respectively, and suggested the presence of a single binding site. Phase fluorometry and circular dichroism were used to characterize the SCP-2 sterol binding site. Alterations in dehydroergosterol lifetime, SCP-2 tryptophan lifetime, and SCP-2 tryptophan quenching by acrylamide upon cholesterol binding demonstrated a shielding of the SCP-2 tryptophan from the aqueous solvent by bound sterol. Differential polarized phase fluorometry revealed decreased SCP-2 tryptophan rotational correlation time upon cholesterol binding. Circular dichroism of SCP-2 indicated that cholesterol elicited a small decrease in SCP-2 alpha helical content. The data suggest that SCP-2 binds sterols with affinity consistent with a lipid transfer protein that may act either as an aqueous carrier or at a membrane surface to enhance sterol desorption.

Sterol carrier protein-2 stimulates intermembrane sterol transfer by direct membrane interaction.

It is unclear how the cytosolic sterol carrier protein-2 (SCP-2) binds sterols and enhances sterol transfer between membranes. Therefore, human recombinant SCP-2 was used in conjunction with phase fluorometry, dialysis, and chemical labeling techniques to show if a direct membrane effect accounted for this activity. SCP-2 directly interacted with L-cell fibroblast plasma membrane vesicles as determined by increased fluorescence anisotropy of coumarin-labeled protein (CPM-SCP-2). Furthermore, a new fluorescence lifetime component due to plasma membrane-bound CPM-SCP-2 was observed. Dialysis studies with 3H- cholesterol loaded plasma membranes indicated that SCP-2, added to the donor compartment, stimulated sterol transfer whether or not the dialysis membrane was permeable to SCP-2. Nevertheless, ligand-binding experiments indicated that chemically blocking the SCP-2 sterol binding site inhibited the ability of SCP-2 to enhance sterol transfer between plasma membrane vesicles. SCP-2 did not stimulate plasma membrane fusion. Addition of SCP-2 to plasma membranes increased the anisotropy plasma membrane proteins covalently reacted with CPM, but not that of lipids labeled with the fatty acid analogue octadecyl rhodamine B. In conclusion, the data are consistent with SCP-2 stimulating intermembrane sterol transfer by direct interaction with sterol in the membrane and enhancing its desorption from the membrane.

Cholesterol domains in biological membranes.

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intraplasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50-60% of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains alter transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca(2+)-ATPase and Na+, K(+)-ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.

Binding of fluorescent and spin-labeled C-terminal hirudin analogs to thrombin.

Synthetic peptides based on the sequence of the negatively charged carboxyl tail of hirudin exhibit anticoagulant activity. Several antithrombin agents are being developed by chemical and structural optimization of these "hirupeptides". The present work demonstrates the design and use of novel spin-labeled and fluorescent-labeled C-terminal hirudin analogs to study the interactions of these antithrombin agents with thrombin in solution. Three labeled hirulabels were synthesized based upon the amino acid sequence of the antithrombin agent MDL 28050, X-NH-(CH2)7-CO-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Cha-D-Glu-OH, where X = anthraniloyl, 1,5-dansyl, or 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl. The modifications did not significantly alter the potency of these inhibitors which showed Ki values of 100 nM. Their interactions with human and bovine thrombin were studied by ESR and fluorescence techniques. The spin-labeled hirupeptide was able to discern subtle differences in binding to human versus bovine thrombin. The 8-aminooctanoic acid spacer arm placed the nitroxide moieties near the active site, near regions of the autolysis loops which differentiates between human alpha- and gamma-thrombin. It was also able to discern paramagnetic quenching and fluorescence energy transfer interactions, respectively, between covalently attached spin labels and fluorescent probes at the active site Ser 195 and the fluorophore on the hirupeptide.

Recombinant liver fatty acid binding protein interacts with fatty acyl-coenzyme A.

Rat liver fatty acid binding protein (L-FABP) and rat intestine fatty acid binding protein (I-FABP) are homologous proteins which are both found in intestinal epithelial cells. It was once well accepted that liver fatty acid binding protein bound fatty acyl-CoAs, but the recent finding of a novel acyl-CoA binding protein (ACBP) in preparations of L-FABP has challenged the role of FABPs in acyl-CoA metabolism. Prior to the discovery of ACBP, L-FABP preparations from liver were shown to modulate the rate of fatty acyl-CoA synthesis (Burrier et al., 1987) and their conversion to phospholipids (Bordewick et al., 1989). Studies using FABPs free of ACBP are needed to determine the role of I-FABP and L-FABP in fatty acyl-CoA metabolism. In this study, highly pure recombinant L-FABP and I-FABP were used first to establish binding to fatty acyl-CoAs and then to examine the effects of these FABPs on microsomal phosphatidic acid synthesis. The standard Lipidex-1000 binding assay using [14C]oleoyl-CoA and a new fluorescence binding assay using the fluorescent fatty acyl-CoA cis-parinaroyl-CoA were used to determine binding. The results of these assays indicate that L-FABP binds fatty acyl-CoAs at two sites with a high-affinity Kd = 3-14 microM. These binding assays showed that I-FABP has a much lower affinity for fatty acyl-CoAs than does L-FABP. Furthermore, in vitro only L-FABP significantly increases the rate of incorporation of oleoyl-CoA into lysophosphatidic acid and phosphatidic acid.

Mechanistic studies of sterol carrier protein-2 effects on L-cell fibroblast plasma membrane sterol domains.

The factors which regulate intermembrane sterol domains and exchange in biomembranes are not well understood. A new fluorescent sterol exchange assay allowed correlation of changes in polarization to sterol transfer. Analysis of spontaneous sterol exchange between L-cell plasma membranes indicated two exchangeable and one very slowly or nonexchangeable sterol domain. The exchangeable domains exhibited half-times of 23 and 140 min with fractional contributions of 5 and 30%, respectively. Sterol carrier protein-2 (SCP-2) enhanced sterol exchange between L-cell plasma membranes and altered sterol domain size in a concentration dependent manner. Previous model membrane studies indicate that SCP-2 alters sterol domains and exchange through interaction with anionic phospholipids. In contrast to these observations, the ionic shielding agents KCl, low pH, or neomycin were either totally or partially ineffective inhibitors of SCP-2 action in L-cell plasma membrane exchanges. Thus the mechanism of SCP-2 in sterol transfer appears to be less charge dependent in L-cell plasma membranes than in model membranes. The cholesterol lowering drug probucol was also capable of altering the sterol exchange kinetics.

Expression of rat L-FABP in mouse fibroblasts: role in fat absorption.

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose levels is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigated in vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accommodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtained in vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.

Expression of liver fatty acid binding protein alters plasma membrane lipid composition and structure in transfected L-cell fibroblasts.

Liver fatty acid binding protein, L-FABP, is an abundant protein that binds fatty acids in vitro. The effects of L-FABP on plasma membrane lipid composition, distribution, and physical structure were determined in intact L-cell fibroblasts transfected with cDNA encoding L-FABP. L-FABP expression altered plasma membrane phospholipids by decreasing both phosphatidylethanolamine and esterified oleic acid content, and increasing sphingomyelin. L-FABP also binds sterols and stimulates sterol uptake and esterification. The fluorescent sterol dehydroergosterol was used to examine sterol distribution in the transfected cell plasma membrane. Dehydroergosterol codistributed equally with the cholesterol in both the bulk membrane and the individual bilayer leaflets. The sterol/phospholipid ratio was decreased in the inner leaflet due to sterol depletion. Concomitantly, intermembrane sterol transfer from the rapidly exchangeable lateral sterol domains as measured by exchange of dehydroergosterol, was reduced. The fluidity of the plasma membrane was measured with the fluorescent molecule diphenylhexatriene by multifrequency (1-250 MHz) phase and modulation fluorometry. Both the bulk plasma membrane and the plasma membrane outer leaflet lipids were fluidized in transfected cells. These alterations of plasma membrane structure and composition are consistent with a role for L-FABP in regulating intracellular sterol and fatty acid distribution and thereby membrane lipid domain structure.