Thermodynamics of Fatty Acid Transfer

There is continuing controversy about the mechanism for transfer of fatty acids (FA) between plasma and the interior of cells and vice versa. One view is that this is a spontaneous process. The generally accepted view is that each step of the process is facilitated by a specialized protein. Whether uptake is spontaneous or facilitated, the components of the uptake system, e.g., albumin, water, FA, plasma membrane, and putative transport proteins of the plasma membrane, must behave according to the rules of the physical chemistry of the system. We review these features to illustrate the constraints they impose on the design of experiments to adduce the mechanism of uptake. Analysis of the literature in the context of the physical chemistry of the uptake system indicates that arguments for a facilitated mechanism of uptake for FA are not supported by any data extant. By contrast, comparison of the rates for individual steps of the pathway traversed by FA moving from albumin to the inside of a cell (or vesicles of a model system) with rates of uptake of FA of tissues in the steady state shows that the rates of the former are sufficient to account for the rate of the latter. Received: 18 January 2000/Revised: 17 April 2000     

Transmembrane transport of fatty acids in the heart

Summary Although fatty acid uptake by the myocardium is rapid and efficient, the mechanism of their transmembrane transport has been unclear. Fatty acids are presented to the plasma membrane of cardiomyocytes as albumin complexes within the plasma. Since albumin is not taken up by the cells, it was postulated that specific high affinity binding sites at the sarcolemma may mediate the dissociation of fatty acids from the albumin molecules, before they are transported into the cells. In studies with a representative long-chain fatty acid, oleate, it was in fact shown that fatty acids bind with high affinity to isolated plasma membranes of rat heart myocytes revealing a K_D of 42 nM. Moreover, a specific membrane fatty acid-binding protein (MFABP) was isolated from these membranes. It had a molecular weight of 40 kD, an isoelectric point of 9.0, and lacked carbohydrate or lipid components. Binding to a specific membrane protein might represent the first step of a carrier mediated uptake process. Therefore, the uptake kinetics of oleate by isolated rat heart myocytes was determined under conditions where only cellular influx and not metabolism occurred. Uptake revealed saturation kinetics and was temperature dependent which were considered as specific criteria for a facilitated transport mechanism. For evaluation whether uptake is mediated by MFABP, the effect of a monospecific antibody to this protein on cellular influx of oleate was examined. Inhibition of uptake of fatty acids but not of glucose by the antibody to MFABP indicated the physiologic significance of this protein as transmembrane carrier in the cellular uptake process of fatty acids. Such a transporter might represent an important site for the metabolic regulation of fatty acid influx into the myocardium.     

Cellular fatty acid uptake is acutely regulated by membrane-associated fatty acid-binding proteins

Cellular long-chain fatty acid uptake is believed to occur largely by protein-mediated transmembrane transport of fatty acids, and also by passive diffusional uptake. It is postulated that the membrane proteins function in trapping of fatty acids from extracellular sources, whereafter their transmembrane translocation occurs by passive diffusion through the lipid bilayer. The key membrane-associated proteins involved are plasma membrane fatty acid-binding protein (FABP(pm)) and fatty acid translocase (FAT/CD36). Their plasma membrane contents are positively correlated with rates of fatty acid uptake. In studies with heart and skeletal muscle we observed that FAT/CD36 is regulated acutely, in that both contraction and insulin can translocate FAT/CD36 from an intracellular depot to the sarcolemma, thereby increasing the rate of fatty acid uptake. In addition, from studies with obese Zucker rats, an established rodent model of obesity and insulin resistance, evidence has been obtained that in heart, muscle and adipose tissue FAT/CD36 is permanently relocated from an intracellular pool to the plasma membrane, resulting in increased fatty acid uptake rates in this condition. These combined observations indicate that protein-mediated fatty acid uptake is a key step in cellular fatty acid utilization, and suggest that malfunctioning of the uptake process could be a critical factor in the pathogenesis of insulin resistance.     

Significance of non-esterified fatty acids in iron uptake by intestinal brush-border membrane vesicles

Iron uptake from Fe/ascorbate by mouse brush-border membrane vesicles is not greatly inhibited by prior treatment with a variety of protein-modification reagents or heat. Non-esterified fatty acid levels in mouse proximal small intestine brush-border membrane vesicles show a close positive correlation with initial Fe uptake rates. Loading of rabbit duodenal brush-border membrane vesicles with oleic acid increases Fe uptake. Depletion of mouse brush-border membrane vesicle fatty acids by incubation with bovine serum albumin reduces Fe uptake. Iron uptake by vesicles from Fe/ascorbate is enhanced in an O2-free atmosphere. Iron uptake from Fe/ascorbate and Fe3+-nitrilotriacetate (Fe3+-NTA) were closely correlated. Incorporation of oleic acid into phosphatidylcholine/cholesterol (4:1) liposomes leads to greatly increased permeability to Yb3+, Tb3+, Fe2+/Fe3+ and Co2+. Ca2+ and Mg2+ are also transported by oleic acid-containing liposomes, but at much lower rates than transition and lanthanide metal ions. Fe3+ transport by various non-esterified fatty acids was highest with unsaturated acids. The maximal transport rate by saturated fatty acids was noted with chain length C14-16. It is suggested that Fe transport can be mediated by formation of Fe3+ (fatty acid)3 complexes.     

Cellular uptake and intracellular trafficking of long chain fatty acids.

While aspects of cellular fatty acid uptake have been studied as early as 50 years ago, recent developments in this rapidly evolving field have yielded new functional insights on the individual mechanistic steps in this process. The extremely low aqueous solubility of long chain fatty acids (LCFA) together with the very high affinity of serum albumin and cytoplasmic fatty acid binding proteins for LCFA have challenged the limits of technology in resolving the individual steps of this process. To date no single mechanism alone accounts for regulation of cellular LCFA uptake. Key regulatory points in cellular uptake of LCFA include: the aqueous solubility of the LCFA; the driving force(s) for LCFA entry into the cell membrane; the relative roles of diffusional and protein mediated LCFA translocation across the plasma membrane; cytoplasmic LCFA binding protein-mediated uptake and/or intracellular diffusion; the activity of LCFA-CoA synthetase; and cytoplasmic protein mediated targeting of LCFA or LCFA-CoAs toward specific metabolic pathways. The emerging picture is that the cell has multiple, overlapping mechanisms that assure adequate uptake and directed intracellular movement of LCFA required for maintenance of physiological functions. The upcoming challenge is to take advantage of new advances in this field to elucidate the differential interactions between these pathways in intact cells and in tissues.     

Selective inhibition of long-chain fatty acid uptake in short-term cultured rat hepatocytes by an antibody to the rat liver plasma membrane fatty acid-binding protein

Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein.     

Utilization of free fatty acids complexed to human plasma lipoproteins by mammalian cell suspensions

The purpose of this study was to determine whether lipoprotein-bound free fatty acid could be utilized by isolated mammalian cells. Ehrlich ascites tumor cells were incubated in vitro with radioactive free fatty acids that were bound to human plasma lipoproteins. Under these conditions, lipoprotein-bound free fatty acids were readily taken up by the cells. After 2 min of incubation with free fatty acids bound to low density lipoproteins, most of the radioactivity that was associated with the cells was in the form of free fatty acids. As the incubation continued, increasing amounts of radioactivity were incorporated into CO(2) and cell lipids, particularly phospholipids. Most of the free fatty acid uptake was the result of fatty acid transfer from low density lipoproteins to the cell, not from irreversible incorporation of the intact free fatty acid-low density lipoprotein complex. Fatty acid uptake increased as the ratio of free fatty acid to low density lipoprotein was raised. When albumin was added to the medium, free fatty acid uptake decreased. A large percentage of the newly incorporated cellular radioactivity was released into the medium if the cells were exposed subsequently to a solution containing albumin. Most of the released radioactivity was in the form of free fatty acid. The results with this experimental model suggest that lipoprotein-bound free fatty acid, like albumin-bound free fatty acid, is readily available for uptake by isolated cells. The mechanism of free fatty acid utilization by the Ehrlich cell is similar when either low density lipoprotein or serum albumin serves as the fatty acid carrier.     

Fatty acid uptake and metabolism in a human intestinal cell line (Caco-2): comparison of apical and basolateral incubation

Free fatty acids can enter the enterocyte via the apical or basolateral plasma membrane. We have used the Caco-2 intestinal cell line to examine the polarity of free fatty acid uptake and metabolism in the enterocyte. Differentiated Caco-2 cells form polarized monolayers with tight junctions, and express the small intestine-specific enzymes sucrase and alkaline phosphatase. Cells were grown on permeable polycarbonate Transwell filters, thus allowing separate access to the apical and basolateral compartments. Total uptake of [3H]palmitate bound to bovine serum albumin (palmitate-BSA 4:1) was twofold higher (P less than 0.05 or less) at the apical surface than at the basolateral surface. The relative apical and basolateral membrane surface areas of the Caco-2 cells, as measured by partition of the fluorophore trimethylammonium-diphenylhexatriene TMA-DPH), was found to be 1:3. Thus, apical fatty acid uptake was sixfold higher than basolateral uptake per unit surface area. Analysis of metabolites after incubation with submicellar concentrations of [3H]palmitate showed that the triacylglycerol to phospholipid (TG:PL) ratio was higher for fatty acid added to the apical as compared to the basolateral compartment (20% at 60 min, P less than 0.025). Little fatty acid oxidation was observed. Preincubation with albumin-bound palmitate, alone or with monoolein, increased the incorporation of both apical and basolateral free fatty acids into TG. The results suggest that the net uptake of long-chain free fatty acids across the apical plasma membrane is greater than uptake across the basolateral membrane. In addition, a small increase in the TG:PL ratio for apically, compared to basolaterally, added free fatty acids suggests that polarity of metabolism occurs to a limited extent in Caco-2 enterocytes.     

Saturation kinetics of palmitate uptake in perfused skeletal muscle

We investigated the kinetics of palmitate uptake in a physiological skeletal muscle preparation by using the isolated perfused rat hindquarter. When plotted against the unbound plasma palmitate concentration, palmitate uptake displayed a simple Michaelis-Menten relation with a calculated Vmax and Km of 16.3 nmol.min-1.g-1 and 0.06 mumol.l-1, respectively. These results show that, as in isolated cell systems, uptake of free fatty acids in perfused skeletal muscle follows saturation kinetics consistent with carrier-mediated membrane transport of free fatty acids.     

Effect of supplementation with exogenous fatty acid on the biological properties of a fatty acid requiring auxotroph ofSalmonella typhimurium

The effects of changes in fatty acid composition of the cell membrane on different biological functions ofSalmonella typhimurium have been studied with the help of a temperature sensitive fatty acid auxotroph which cannot synthesise unsaturated fatty acids at high temperature. On being shifted to nonpermissive temperature the cells continue growing for another one and half to two generations. The rates of protein and DNA syntheses run parallel to the growth rate but the rate of RNA synthesis is reduced. Further, there is a gradual reduction in the rate of transport of exogenous uridine and thymidine into the soluble pool. The transport process can be restored by supplementing the growth medium with cis-unsaturated fatty acids but not trans-unsaturated ones although the growth of the cells is resumed by supplementation with eithercis or trans-unsaturated fatty acids. However, supplementation withtrans, trans-unsaturated fatty acids leads to only partial recovery of the transport process. The rate of oxygen uptake is also affected in cells grown in the presence of thetrans-unsaturated fatty acids, elaidic acid and palmitelaidic acid. Analysis of cells grown under different fatty acid supplementation indicate that fatty acid composition of the cell membrane, especially the ratio of unsaturated to saturated fatty acids varies with temperature shift and supplementation of the growth media with fatty acids.     

Cellular fatty acid uptake: the contribution of metabolism

PURPOSE OF REVIEW: The aim of this review is to highlight the importance of fatty acid metabolism as a major determinant in fatty acid uptake. In particular, we emphasize how the activation, intracellular transport and downstream metabolism of fatty acids influence their uptake into cells. RECENT FINDINGS: Studies examining fatty acid entry into cells have focused primarily on the roles of plasma membrane proteins or the question of passive diffusion. Recent studies, however, strongly suggest that a driving force governing fatty acid uptake is the metabolic demand for fatty acids. Both gain and loss-of-function experiments indicate that fatty acid uptake can be modulated by activation at both the plasma membrane and internal sites, by intracellular fatty acid binding proteins, and by enzymes in synthetic or degradative metabolic pathways. Although the mechanism is not known, it appears that converting fatty acids to acyl-CoAs and downstream metabolic intermediates increases cellular fatty acid uptake, probably by limiting efflux. SUMMARY: Altered fatty acid metabolism and the accumulation of triacylglycerol and lipid metabolites has been strongly associated with insulin resistance and diabetes, but we do not fully understand how the entry of fatty acids into cells is regulated. Future studies of cellular fatty acid uptake should consider the influence of fatty acid metabolism and the possible interactions between fatty acid metabolism or metabolites and fatty acid transport proteins.     

Translocation of oleic acid across the erythrocyte membrane. Evidence for a fast process

To clarify divergent views concerning the mechanism of fatty acid translocation across biomembranes this issue was now investigated in human erythrocytes. Translocation rates of exogenously inserted radioactive oleic acid across the membrane of native cells were derived from the time-dependent increase of the fraction of radioactivity becoming non-extractable by albumin. No accumulation of non-extractable unesterified oleic acid occurred. The rate of transfer was markedly suppressed by SH-reagents and by ATP-depletion. The suppression, however, resulted from a mere decrease of incorporation of oleic acid into phospholipids and was not accompanied by an increase of non-extractable unesterified oleic acid. These findings were reconcilable with the concept of a slow, possibly carrier-mediated fatty acid transfer as well as a very fast presumably, diffusional process not resolvable by the albumin extraction procedure. This ambiguity was resolved by using resealed ghosts, which are unable to incorporate oleic acid into phospholipids. In such ghosts all of the oleic acid inserted into the membrane remains extractable by albumin even after prolonged incubation. On the other hand, ghosts containing albumin accumulated non-extractable oleic acid. The rate of accumulation was beyond the time resolution of the albumin extraction procedure at 4 degrees C. Oleic acid uptake into albumin-containing ghosts became kinetically resolvable when the fatty acid was added as a complex with albumin. Correspondingly, time-resolvable release of oleic acid, originally complexed to internal albumin, into an albumin-containing medium was demonstrated at 4 degrees C. Rate and extent of these redistributions of oleic acid were dependent on the concentrations of internal and external albumin. This indicates limitation by the dissociation of oleic acid from albumin and not its translocation across the membrane. Translocation of oleic acid, which is probably a simple diffusive flip-flop process, must therefore occur with a half-time of less than 15 s. These findings raise doubts on the physiological role of presently discussed concepts of a carrier-mediated translocation of fatty acids across plasma membranes.     

Fatty acid uptake by human hepatoma cell lines represents a carrier-mediated uptake process

Cellular influx kinetics of a representative long chain fatty acid, [3H]oleate, were examined in monolayer cultures of three different human hepatoma cell lines (Hep G2; PLC/PRF 5; Mz-Hep-1). The cultures were incubated with 173 microM [3H]oleate in the presence of various concentrations of albumin which served to modulate the unbound oleate concentration in the medium. For all [3H]oleate-albumin complexes incubated, it was shown that cellular uptake of [3H]oleate over the initial 30 s incubation period was maximal, linear and independent of intracellular fatty acid metabolism, representing cellular influx. With increasing unbound oleate concentrations in the medium cellular influx by all three cell lines revealed similar saturation kinetics with Km values of 112.6 +/- 14.5 nM and Vmax values of 7.19 +/- 0.32 nmol.min-1 per mg cell protein. When these hepatoma cell lines were pretreated with the IgG fraction of a monospecific antibody to the rat liver membrane fatty acid binding protein (MFABP), initial uptake of [3H]oleate was selectively inhibited compared to controls pretreated with the IgG fraction of the preimmune serum. Furthermore, immunoblot analysis with the monospecific antibody to the rat MFABP revealed reactivity with a single 40 kDa protein in the homogenates of all three cell lines. These data suggest that uptake of fatty acids by human hepatoma cells may be mediated by a specific membrane fatty acid binding protein.     

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

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose level is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigatedin 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 accomodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtainedin 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.Abbreviations FABP fatty acid-binding protein - L-FABP liver fatty acid-binding protein - I-FABP intestinal fatty acid-binding protein - H-FABP heart fatty acid-binding protein - A-FABP adipocyte fatty acid-binding protein - PMFABP plasma membrane fatty acid-binding protein - SCP-2 sterol carrier protein-2 - Dehydroergosterol (DHE) d-5,7,9(11),22-ergostatetraene-3b-ol - cis-parinaric acid-9Z, 11E, 13E, 15Z-octatetraenoic acid - trans parinaric acid, 9E, 11E, 13E, 14E-octatetraenoic acid - BSA bovine serum albumin - KRH Krebs-Ringer-Henseleit buffer     

Mechanism for binding of fatty acids to hepatocyte plasma membranes

The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.     

Rapid flip-flop of oleic acid across the plasma membrane of adipocytes

Nonesterified long-chain fatty acids may enter cells by free diffusion or by membrane protein transporters. A requirement for proteins to transport fatty acids across the plasma membrane would imply low partitioning of fatty acids into the membrane lipids, and/or a slower rate of diffusion (flip-flop) through the lipid domains compared to the rates of intracellular metabolism of fatty acids. We used both vesicles of the plasma membrane of adipocytes and intact adipocytes to study transmembrane fluxes of externally added oleic acid at concentrations below its solubility limit at pH 7.4. Binding of oleic acid to the plasma membrane was determined by measuring the fluorescent fatty acid-binding protein ADIFAB added to the external medium. Changes in internal pH caused by flip-flop and metabolism were measured by trapping a fluorescent pH indicator in the cells. The metabolic end products of oleic acid were evaluated over the time interval required for the return of intracellular pH to its initial value. The primary findings were that (i) oleic acid rapidly binds with high avidity in the lipid domains of the plasma membrane with an apparent partition coefficient similar to that of protein-free phospholipid bilayers; (ii) oleic acid rapidly crosses the plasma membrane by the flip-flop mechanism (both events occur within 5 s); and (iii) the kinetics of esterification of oleic acid closely follow the time dependence of the recovery of intracellular pH. Any postulated transport mechanism for facilitating translocation of fatty acid across the plasma membrane of adipocytes, including a protein transporter, would have to compete with the highly effective flip-flop mechanism.     

Selective uptake from LDL is stimulated by unsaturated fatty acids and modulated by cholesterol content in the plasma membrane: role of plasma membrane composition in regulating non-SR-BI-mediated selective lipid transfer

We previously reported that unsaturated fatty acids stimulated low-density lipoprotein (LDL) particle uptake in J774 macrophages by increasing LDL receptor activity. Since free fatty acids (FFA) also change plasma membrane properties, a putative cholesteryl ester (CE) acceptor for selective uptake (SU), we questioned the ability of FFA to modulate SU from LDL. Using [(3)H]cholesteryl ether/(125)I-LDL to trace CE core and whole particle uptake, we found that oleic acid and eicosapentaenoic acid, but not saturated stearic acid, increased SU by 30% over control levels. An ACAT inhibitor, Dup128, abolished FFA effects on SU, indicating that increased SU by FFA was secondary to changes in cell-free cholesterol (FC). Consistent with these observations, ACAT inhibition increased cell FC and reduced LDL SU by half. The important role of plasma membrane composition was further demonstrated in that beta-cyclodextrin- (beta-CD-) mediated FC removal from the plasma membrane increased SU from LDL and was further stimulated by U18666A, a compound that inhibits FC transport between lysosomes and the plasma membrane. In contrast, cholesterol-saturated beta-CD markedly reduced LDL SU. In contrast to LDL SU, oleic acid, ACAT inhibition, U18666A, or beta-CD had no effects on HDL SU. Moreover, HDL SU was inhibited by antimouse SR-BI antibody by more than 50% but had little effect on LDL SU. In C57BL/6 mice fed a high fat diet, plasma FFA levels increased, and SU accounted for an almost 4-fold increased proportion of total cholesterol delivery to the arterial wall. Taken together, these data suggest that LDL SU is mediated by pathways independent of SR-BI and is influenced by plasma membrane FC content. Moreover, in conditions where elevated plasma FFA occur, SU from LDL can be an important mechanism for cholesterol delivery in vivo.     

Calcium modulates fatty acid dynamics in rat liver plasma membranes

Modulation of free fatty acid binding in isolated rat liver plasma membranes was evaluated using the fluorescent fatty acids trans-parinaric and cis-parinaric acid as analogues for saturated and unsaturated fatty acids, respectively. Binding of trans-parinarate but not cis-parinarate was inhibited by physiological levels of Ca2+. The effect was reversed by addition of excess EGTA. Calcium decreased the aqueous to lipid partition coefficient, Kp, of trans-parinaric acid for liver plasma membranes while increasing the Kp for cis-parinaric acid. In addition, Ca2+ also altered the fluorescence lifetime, the quantum yield, and the relative partitioning of trans-parinaric and cis-parinaric acid into fluid and solid phases. Calcium and EGTA did not affect the binding of 1,6-diphenyl-1,3,5-hexatriene. The effect of Ca2+ on the liver plasma membrane structure was to increase the rigidity of the membrane, primarily the solid domain. The fluorescence polarization of trans-parinarate, cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene at 24 degrees C in liver plasma membranes in the absence of Ca2+ was 0.295 +/- 0.008, 0.253 +/- 0.007, and 0.284 +/- 0.005, respectively. Calcium (2.4 mM) increased the polarization of these probe molecules in liver plasma membranes by 8-10%. EGTA (3.4 mM) reversed or abolished the increase in polarization. Thus, the fluorescent fatty acids trans-parinarate and cis-parinarate may be used to monitor fatty acid binding by isolated membranes, to evaluate factors such as Ca2+ which modulate fatty acid binding, and to investigate the microenvironment in which the fatty acids residue. The data suggest that Ca2+ may be an important regulator of fatty acid uptake by the liver plasma membrane, and thereby interact with intermediary metabolism of lipids at a step not involving lipolytic or synthetic enzymes.     

Alteration of the fatty acid composition of membrane phospholipids in mouse lymphoid cells

A simple method is described for introducing exogenous fatty acids into the membrane phospholipids of the murine leukemia cell EL-4, and into the membrane phospholipids of resting mouse lymphocytes. The method involves culturing of the cells with free or methylated fatty acids at concentrations up to 50 microgram/ml. The presence of serum in the culture medium does not interfere with fatty acid uptake, but does increase the growth rate and viability of the cells. Membrane lipid composition returns to normal after the cells are grown in medium without exogenous fatty acid. Fractionation of the cell membranes confirmed that exogenous fatty acids were incorporated into the phospholipids of the plasma membrane.