Uptake and utilization of lipids by Paramecium tetraurelia

Uptake rates of a variety of 14C-labeled fatty acids and complex lipids by Paramecium tetraurelia during 48 h of log-phase growth varied. Fatty acid uptake was maximal during lag phase of growth when phagosome (food vacuole) formation was minimal. Food vacuole formation was shown to be suppressed by the presence of exogenous lipids and by starvation. The rates of uptake of lipids were significantly greater than those of small organic compounds such as amino acids, cyclitols, fatty acid precursors and metabolic intermediates. Significant amounts of radioactivity from 14C-labeled fatty acids were metabolized to 14CO2. The uptake rates of different saturated, straight-chain fatty acids of even carbon numbers were different and were not correlated with chain length, results suggesting that the primary mechanism for uptake of these compounds was neither by bulk transport nor simple diffusion and that carrier-mediated processes could possibly be involved.     

Translocation of fatty acids across the basolateral rat liver plasma membrane is driven by an active potential-sensitive sodium-dependent transport system

In previous studies it was shown that hepatocellular uptake of fatty acids is mediated by a specific fatty acid binding membrane protein. To determine now directly the driving forces for their entry into hepatocytes, the uptake of a representative long chain fatty acid, [3H]oleate, by basolateral rat liver plasma membrane vesicles was examined. Influx of oleate was stimulated by increasing the Na+ concentration of the medium. In the presence of an inwardly directed Na+ gradient (NaSCN, NaNO3, NaCl) oleate was accumulated during the initial uptake phase (20 s) at a concentration of 1.4-1.9-fold that at equilibrium (overshoot). This activation of influx was not observed after replacement of Na+ by Li+, K+, or choline+. Na+-dependent oleate uptake was significantly stimulated by creation of a negative intravesicular potential, either by altering the accompanying anions or by valinomycin-induced K+ diffusion potentials, suggesting an electrogenic transport mechanism. Na+-dependent fatty acid uptake was temperature dependent, with maximal overshoots occurring at 37 degrees C, and revealed saturation kinetics with a Km of 83.1 nM and Vmax of 2.9 nmol X min-1 X mg protein-1. These studies demonstrate that the carrier-mediated hepatocellular uptake of fatty acids represents an active potential-sensitive Na+-fatty acid cotransport system.     

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.     

Fatty acid composition of cardiolipin from guinea pig Harderian gland

The fatty acid composition of cardiolipin from the Harderian gland of guinea pig was examined by gas chromatography and gas chromatography-mass spectrometry. At least 33 kinds of fatty acids were detected. Oleic acid was the most prominent component, accounting for 18.2 mol% of the total fatty acids. About 70.2 mol% of fatty acids had methyl branches. Ethyl branches were also detected (1.3 mol%). Straight chain saturated acids comprised only 10.3 mol%. On the other hand, linoleic, linolenic, and arachidonic acids were not found in this lipid. The 2-(2'-) acyl moieties contained larger amounts of oleic acid and smaller amounts of branched chain acids than the 1-(1'-)acyl moieties, but the saturated straight chain acids showed even distribution between the 1-(1'-) and 2-(2'-)positions. The fatty acids of cardiolipin from the liver of the same animal were also examined. Linoleic acid was the most abundant component (66.9 mol%), and saturated straight chain acids occupied 21.9 mol%. Branched chain acids were detected but comprised only 11.2 mol%.     

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.     

Fatty acid transport by the lipophilic bacterium Nocardia asteroides.

Hexadecanoate was translocated in Nocardia asteroides by a constitutive transport system(s), which transported short, medium, and long-chain fatty acids. Inhibition of hexadenocanoate transport by homologues suggested that at least two systems are present: one specific for short-chain fatty acids and the other specific for medium- and long-chain fatty acids. Saturation kinetics typical of a carrier-mediated transport system (Kt = 870 muM)were observed, and concentration of fatty acids against a gradient was achieved. Inhibitor studies indicated that free sulfhydryl groups, a functional respiratory chain, and energy are required for translocation. Efflux of [14C]hexadecanoate in the presence of excess unlabeled hexadecanoate or 2,4-dinitrophenol and the cytoplasmic localization of acyl-coenzyme A synthetase (acid:coenzyme A ligase [adenosine monophosphate]; EC 6.2.1.3) (Calmes and Deal, 1973) are consistent with the hypothesis that fatty acids are transported and released intracellularly as free fatty acids.     

Characterization of tryptophan transport in human placental brush-border membrane vesicles.

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.     

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.     

Adipose differentiation related protein (ADRP) expressed in transfected COS-7 cells selectively stimulates long chain fatty acid uptake.

Adipose differentiation related protein (ADRP) is a 50-kDa novel protein cloned from a mouse 1246 adipocyte cDNA library, rapidly induced during adipocyte differentiation. We have examined ADRP function, and we show here that ADRP facilitates fatty acid uptake in COS cells transfected with ADRP cDNA. We demonstrate that uptake of long chain fatty acids was significantly stimulated in a time-dependent fashion in ADRP-expressing COS-7 cells compared with empty vector-transfected control cells. Oleic acid uptake velocity increased significantly in a dose-dependent manner in ADRP-expressing COS-7 cells compared with control cells. The transport Km was 0.051 microM, and Vmax was 57.97 pmol/10(5) cells/min in ADRP-expressing cells, and Km was 0.093 microM and Vmax was 20.13 pmol/10(5) cells/min in control cells. The oleate uptake measured at 4 degrees C was only 10% that at 37 degrees C. ADRP also stimulated uptake of palmitate and arachidonate but had no effect on uptake of medium chain fatty acid such as octanoic acid and glucose. These data suggest that ADRP specifically enhances uptake of long chain fatty acids by increasing the initial rate of uptake and provide novel information about ADRP function as a saturable transport component for long chain fatty acids.     

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.     

Fatty acid transport proteins

PURPOSE OF REVIEW: Fatty acid transport proteins are a family of proteins involved in fatty acid uptake and activation. This review summarizes recent progress in elucidating the function of fatty acid transport proteins. RECENT FINDINGS: Recent experiments clearly establish FATP1 as a regulated fatty acid transporter in both adipose tissue and muscle with important roles in energy homeostasis, thermogenesis and insulin resistance. Knockout of FATP5 in mice show it to be a bifunctional protein required for both hepatic fatty acid uptake and bile acid reconjugation. The most striking phenotype of FATP4 deletion is a defect in skin homeostasis, which may be due to its very long chain acyl-coenzyme A synthetase activity. Fatty acid transport proteins are increasingly being recognized as multifunctional proteins that can mediate the uptake of fatty acids as well as catalyze the formation of coenzyme A derivatives using long-chain and very-long chain fatty acids, bile acids and bile acid precursors as substrates. SUMMARY: Modulation of fatty acid transport protein function can result in altered energy homeostasis and insulin sensitivity, defective skin homeostasis, and altered bile acid metabolism. Both fatty acid uptake and enzymatic activity of fatty acid transport proteins likely contribute to these phenotypes. Future studies are needed to better understand the molecular mechanism of fatty acid transport protein function and the physiological role of FATP2, FATP3, and FATP6.     

Fatty acid composition of lipids which copurify with band 3

In a previous study (L. R. Maneri and P. S. Low (1988) J. Biol. Chem. 263, 16170-16178) we determined that the anion transport protein, band 3, was significantly stabilized by lipids containing saturated and/or long chain fatty acids. To determine whether this thermodynamic preference is reflected in the composition of lipids tightly associating with the anion transporter in vivo, we have analyzed the fatty acid content of phospholipids co-isolating with the purified integral domain of band 3. Our data demonstrate that although stearic acid comprises only 14% of the bulk lipid fatty acids of the red cell membrane, it constitutes -68% of the fatty acids of lipids co-isolating with band 3. Certain other long chain fatty acids were also enriched in the adherent lipids. These results suggest that the fatty acids which most effectively stabilize band 3 also have the highest affinity for the transport protein.     

Spectrum of effects of dietary long-chain fatty acids on rat intestinal glucose and lipid uptake

Isocaloric modification in the ratio of dietary polyunsaturated-to-saturated fatty acids influences intestinal uptake of actively and passively transported nutrients. This study was undertaken to determine which dietary fatty acid was responsible for these alterations in absorption. Adult female rats were fed isocaloric semisynthetic diets high in palmitic and stearic acids (SFA), oleic acid (OA), linoleic acid (LA), or linolenic acid (LNA). An in vitro technique was used to measure the uptake of varying concentrations of glucose as well as a series of fatty acids and cholesterol. Jejunal uptake of 40 mM glucose was highest in rats fed SFA and lowest in those fed LA; ileal glucose uptake was similar in OA, LA, and LNA, but was lowest in SFA. Jejunal uptake of medium-chain fatty acids (8:0-12:0) was higher in OA than in other diet groups; ileal uptake of medium-chain fatty acids was unaffected by diet. Jejunal and ileal uptake of 18:2 was higher in LNA than in SFA or OA; the uptake of the other long-chain saturated or unsaturated fatty acids was unchanged by diet. The ileal but not the jejunal uptake of cholesterol was increased in LA as compared with SFA or OA, and reduced in LNA as compared with LA. These transport changes were not explained by differences in the animals' food consumption, body weight gain, intestinal mass, or mucosal surface area. We postulate that these diet-induced transport alterations may be mediated via changes in brush border membrane phospholipid fatty acyl composition. Thus, intestinal transport of nutrients may be varied by isocaloric changes in the dietary content of individual fatty acids.     

Regulation of transmembrane ion transport by reaction products of phospholipase A2. II. Effects of arachidonic acid and other fatty acids on mitochondrial Ca2+ transport

The effects of arachidonic acid and other fatty acids on mitochondrial Ca2+ transport were studied. Cis-unsaturated fatty acids generally strongly inhibited mitochondrial Ca2+ uptake, induced a net Ca2+ efflux, and thereby increased the extramitochondrial Ca2+ concentration, whereas trans-unsaturated fatty acids were ineffective. Saturated fatty acids exhibited slight activity at chain lengths from C(10) to C(14) only. The structure-activity relationship and the inability of some of the effective fatty acids such as palmitoleic and myristoleic acid to be metabolized to eicosanoids suggest that Ca2+ release was induced by the fatty acids themselves and resulted from changes in the mitochondrial membrane bilayer structure. There was a correlation between Ca2+-releasing potency and reduction of mitochondrial membrane potential, which is the main driving force for mitochondrial Ca2+ uptake. There were, however, considerable differences compared with the effects of lysophospholipids on the membrane potential. The mechanism of action of fatty acids may be that of a fluidizing effect on the hydrophobic core of the membrane, thereby modulating the activity of integral membrane proteins of the respiratory chain.     

Fatty Acid Transport and Utilization for the Developing Brain

Abstract: To determine the transport and utilization of dietary saturated, monounsaturated, and n-6 and n-3 polyunsaturated fatty acids for the developing brain and other organs, artificially reared rat pups were fed a rat milk substitute containing the perdeuterated (each 97 atom% deuterium) fatty acids, i.e., palmitic, stearic, oleic, linoleic, and linolenic, from day 7 after birth to day 14 as previously described. Fatty acids in lipid extracts of the liver, lung, kidney, and brain were analyzed by gas chromatography-mass spectrometry to determine their content of each of the deuterated fatty acids. The uptake and metabolism of perdeuterated fatty acid lead to the appearance of three distinct groups of isotopomers: the intact perdeuterated, the newly synthesized (with recycled deuterium), and the natural unlabeled fatty acid. The quantification of these isotopomers permits the estimation of uptake and de novo synthesis of these fatty acids. Intact perdeuterated palmitic, stearic, and oleic acids from the diet were found in liver, lung, and kidney, but not in brain. By contrast, perdeuterated linoleic acid was found in all these organs. Isotopomers of fatty acid from de novo synthesis were observed in palmitic, oleic, and stearic acids in all tissues. The highest enrichment of isotopomers with recycled deuterium was found in the brain. The data indicate that, during the brain growth spurt and the prelude to myelination, the major saturated and monounsaturated fatty acids in brain lipids are exclusively produced locally by de novo biosynthesis. Consequently, the n-6 and n-3 polyunsaturated fatty acids must be transported and delivered to the brain by highly specific mechanisms.     

Uptake of fatty acids by the yeasts, Saccharomyces uvarum and Saccharomycopsis lipolytica

Yeast cells take up exogenous fatty acids with subsequent rapid incorporation into glycerolipids. beta-Oxidation does not occur in Saccharomyces uvarum and is observed in Saccharomycopsis lipolytica only 2-5 min after addition of radioactively labeled fatty acid. Rates of fatty acid uptake are linear up to 30 s with S. lipolytica and up to 2 min with S. uvarum. The uptake kinetics are consistent with a dual mode of transport, comprising a saturable component with KT values in the range 10(-5)-10(-6) M, and apparently simple diffusion that predominates at high substrate concentrations. Kinetics of fatty acid permeation are independent of metabolic energy and membrane potential. At least two fatty acid carrier systems exist in both S. lipolytica and S. uvarum, one being specific for fatty acids with 12 and 14 C atoms, respectively, the other for C16 and C18 saturated or unsaturated fatty acids. Octanoic acid and decanoic acid are not taken up by S. lipolytica. Internalization of lauric acid and oleic acid by S. lipolytica cells is preceded by a rapid (less than 5 s) initial uptake which most likely represents irreversible adsorption. This phenomenon was not observed with heat-inactivated S. lipolytica cells or with viable S. uvarum. In azide-poisoned cells of S. lipolytica an up to 20-fold accumulation of unesterified fatty acid was observed within 30 s after the addition of substrate.     

Inhibition of in vitro cholesterol synthesis by fatty acids.

Inhibitory effect of 44 species of fatty acids on cholesterol synthesis has been examined with a rat liver enzyme system. In the case of saturated fatty acids, the inhibitory activity increased with chain length to a maximum at 11 to 14 carbons, after which activity decreased rapidly. The inhibition increased with the degree of unsaturation of fatty acids. Introduction of a hydroxy group at the alpha-position of fatty acids abolished the inhibition, while the inhibition was enhanced by the presence of a hydroxy group located in an intermediate position of the chain. Branched chain fatty acids having a methyl group at the terminal showed much higher activity than the corresponding saturated straight chain fatty acids with the same number of carbons. With respect to the mechanism for inhibition, tridecanoate was found to inhibit acetoacetyl-CoA thiolase specifically without affecting the other reaction steps in the cholesterol synthetic pathway. The highly unsaturated fatty acids, arachidonate and linoleate, were specific inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA synthase. On the other hand, ricinoleate (hydroxy acid) and phytanate (branched-chain acid) diminished the conversion of mevalonate to sterols by inhibiting a step or steps between squalene and lanosterol.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

MODULATION OF FATTY ACIDS IN THE MEMBRANES OF ANACYSTIS NIDULANS (CYANOBACTERIA): INCORPORATION OF ODD-NUMBERED CARBON FATTY ACIDS1

Anacystis nidulans Richt., a unicellular cyanobacterium, can incorporate exogenously supplied fatty acids, including odd-numbered carbon fatty acid (OFAs), into the acylglycerols of cell membranes. Data are presented for the uptake of undecanoic acid (11:0) into cells of A. nidulans, the subsequent elongation up to C17, and incorporation of OFA into the four major membrane acylglycerols. The incorporation of OFAs was followed by desaturation of part of the saturated fatty acid to monoenoic fatty acid. Positional analyses of the double bonds of these manoenoic fatty acids suggest that there is one desaturase that inserts a Δ⁹ bond in both odd- and even-numbered fatty acids of varying chain length. Our data also suggest that there is no positional specificity for chain length on the glycerol backbone by the acyltransferases.     

Inhibition of pyruvate transport by fatty acids in isolated cells from rat small intestine.

1. A technique is described for the rapid separation of intestinal epithelial cells from the incubation medium by passage through a silicon-oil layer and collection in acid, in which their soluble constituents are released. 2. The inhibition by fatty acids of pyruvate oxidation is further studied. Measurement of pyruvate transport in epithelial cells at 0 degree C showed that short- and medium-chain fatty acids as well as ricinoleate inhibit this transport. Propionate inhibits pyruvate transport by another mechanism than octanoate. 3. Differences between pyruvate propionate and octanoate transport across the epithelial cell membrane were obtained in efflux studies. These studies revealed that acetate, propionate, butyrate and high concentrations of bicarbonate readily stimulate the efflux of pyruvate, probably by anionic counter-transport. No effects were seen with octanoate and hexanoate. The data obtained in these efflux studies suggest that lipophilicity and the pKa values of the monocarboxylic acids determine the contribution of non-ionic diffusion to overall transport. 4. Saturation kinetics, competitive inhibition by short-chain fatty acids and counter-transport suggest a carrier-mediated transport of pyruvate.