The effect of endogenous essential and nonessential fatty acids on the uptake and subsequent agonist-induced release of arachidonate

We have demonstrated that the uptake and agonist-induced release of a pulse of arachidonate are influenced by the size and composition of preexisting endogenous fatty acid pools. EFD-1 cells, an essential fatty acid-deficient mouse fibrosarcoma cell line, were incubated with radiolabeled (14C or 3H] arachidonate, linoleate, eicosapentaenoate (EPA), palmitate, or oleate in concentrations of 0-33 microM for 24 h. After 24 h, the cells were pulsed with 0.67 microM radiolabeled (3H or 14C, opposite first label) arachidonate for 15 min and then stimulated with 10 microM bradykinin for 4 min. Because EFD-1 cells contain no endogenous essential fatty acids, we were able to create essential fatty acid-repleted cells for which the specific activity of the newly constructed endogenous essential fatty acid pool was known. Loading the endogenous pool with the essential fatty acids arachidonate, eicosapentaenoate, or linoleate (15-20 nmol of fatty acid incorporated/10(6) cells) decreased the uptake of a pulse of arachidonate from 200 to 100 pmol/10(6) cells but had no effect on palmitate uptake. The percent of arachidonate incorporated during the pulse which was released upon agonist stimulation increased 2-fold (4-8%) as the endogenous pool of essential fatty acids was increased from 0 to 15-20 nmol/10(6) cells. This 8% release was at least 3-fold greater than the percent release from the various endogenous essential fatty acid pools. In contrast, loading the endogenous pool with the nonessential fatty acids oleate or palmitate to more than 2-3 times their preexisting cellular level had no effect on the uptake of an arachidonate pulse. Like the essential fatty acids, increasing endogenous oleate increased (by 2-fold) the percent release of arachidonate incorporated during the pulse, whereas endogenous palmitate had no effect on subsequent agonist-induced release from this arachidonate pool. These studies show that preexisting pools of essential and nonessential fatty acids exert different effects on the uptake and subsequent releasability of a pulse of arachidonate.     

FATP4 contributes as an enzyme to the basal and insulin-mediated fatty acid uptake of C₂C₁₂ muscle cells

The function of membrane proteins in long-chain fatty acid transport is controversial. The acyl-CoA synthetase fatty acid transport protein-4 (FATP4) has been suggested to facilitate fatty acid uptake indirectly by its enzymatic activity, or directly by transport across the plasma membrane. Here, we investigated the function of FATP4 in basal and insulin mediated fatty acid uptake in C(2)C(12) muscle cells, a model system relevant for fatty acid metabolism. Stable expression of exogenous FATP4 resulted in a twofold higher fatty acyl-CoA synthetase activity, and cellular uptake of oleate was enhanced similarly. Kinetic analysis demonstrated that FATP4 allowed the cells to reach apparent saturation of fatty acid uptake at a twofold higher level compared with control. Short-term treatment with insulin increased fatty acid uptake in line with previous reports. Surprisingly, insulin increased the acyl-CoA synthetase activity of C(2)C(12) cells within minutes. This effect was sensitive to inhibition of insulin signaling by wortmannin. Affinity purified FATP4 prepared from insulin-treated cells showed an enhanced enzyme activity, suggesting it constitutes a novel target of short-term metabolic regulation by insulin. This offers a new mechanistic explanation for the concomitantly observed enhanced fatty acid uptake. FATP4 was colocalized to the endoplasmic reticulum by double immunofluorescence and subcellular fractionation, clearly distinct from the plasma membrane. Importantly, neither differentiation into myotubes nor insulin treatment changed the localization of FATP4. We conclude that FATP4 functions by its intrinsic enzymatic activity. This is in line with the concept that intracellular metabolism plays a significant role in cellular fatty acid uptake.     

Ultrastructural studies of the intracellular translocation of endocytosed alpha-foetoprotein (AFP) by cytochemistry and of the uptake of 3H-arachidonic acid bound to AFP by autoradiography in rat rhabdomyosarcoma cells

A covalent conjugate of alpha-foetoprotein (AFP) and horseradish peroxidase (HRP) has been used to follow, at the ultrastructural level, the pathway of AFP uptake and translocation in a rat rhabdomyosarcoma cell line. The cells were incubated for several times at 4 degrees C and/or 37 degrees C, and fixed. AFP-HRP was found to enter the cells via coated pits and receptosomes and to move to tubular elements of the trans-reticular portion of the Golgi. Some observations suggest that AFP can be recycled back to the cell surface. On the other hand, the cells were incubated with a noncovalent conjugate of AFP and 3H-arachidonic acid [3H-(20:4)], and the uptake of the fatty acid molecules studied by ultrastructural autoradiography. The cytoplasmic labeling, very low after an incubation in the presence of [3H-(20:4)]-AFP for 2 hours at 4 degrees C, increased rapidly after transfer of the cells for 5 minutes to 37 degrees C. These observations support the hypothesis that AFP plays a role in the intracellular delivery of polyunsaturated fatty acids.     

Characteristics of lysophosphatidylcholine in its inhibition of taurine uptake by human intestinal Caco-2 cells

The characteristics of lysophosphatidylcholine (LPC) in its inhibition of the taurine uptake by human intestinal Caco-2 cells were investigated. By treating the cells with 200 microM of LPC, the taurine uptake was rapidly decreased by approximately 60%. This decrease was accompanied by an increase in the Km value for the uptake. A rapid uptake of LPC itself by the cells was also observed. The inhibitory activity of LPC was specific to the uptake of taurine and certain amino acids, while the uptake of glucose, glutamic acid and peptide (glycylglutamine) was not affected by LPC. The activity was dependent on the structure of a polar head and the bound fatty acid. The phosphorylcholine residue was likely to have played an important role, and surface active LPC with fatty acids of C14 or longer was highly inhibitory. These results suggest that the interaction of LPC with the taurine transporter in the intestinal cell membrane was the cause of the reduced taurine uptake.     

Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1

Fatty acid transport protein 1 (FATP1) is an approximately 63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16-24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the Km values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.     

Alanine transport by Chinese hamster ovary cells with altered phospholipid acyl chain composition

The Na+-dependent transport of alanine has been examined in Chinese hamster ovary (CHO) cells as a function of the fatty acid composition of their membrane lipids. Significant changes in the fatty acid composition of the CHO cell phospholipids were achieved by supplementation of the growth medium with specific saturated (palmitate) or monoenoic (oleate) free fatty acids. Arrhenius plots of the temperature-dependent uptake of alanine were constructed for cells of altered fatty acid composition. Alanine uptake was characterized by a single discontinuity in the Arrhenius plot. The temperature of this break was observed to be dependent upon the fatty acid composition of the cell phospholipids, ranging from 16 degrees C for cells enriched with oleate to 32 degrees C for cells enriched in palmitate. Calculation of the Km value for the uptake process showed no significant change with temperature or fatty acid supplementation. Correlations are made between the physical state of the membrane lipids and the temperature-dependence for alanine transport. The results are discussed in terms of membrane fatty acid composition, ordered in equilibrium fluid phase transitions and amino acid transport.     

Hepatocellular uptake of aflatoxin B1 by non-ionic diffusion. Inhibition of bile acid transport by interference with membrane lipids

Aflatoxin B1 permeates isolated rat hepatocytes by non-ionic diffusion. Its uptake is neither saturable nor influenced by metabolic energy and not inhibited by treatment of cells with proteases. The initial rate of aflatoxin B1 uptake measured at 7 degrees C is between 40 and 50% compared to that at 37 degrees C. However, after an incubation period of 7 minutes identical equilibrium uptake is reached at both temperatures. The apparent activation energies, calculated for aflatoxin B1 uptake by Arrhenius diagrams ranged between 1.69 and 4.5 kcal/mol. A Q10 value of 1.34 was calculated for a temperature interval of 7-17 degrees C but decreased to 1.05 for the interval of 27-37 degrees C. Liposomes or lipoproteins added to the cell suspension inhibited the aflatoxin B1 uptake into hepatocytes. Liposomes mainly composed of unsaturated fatty acids bind twice as much aflatoxin B1 as those composed of saturated ones, indicating that the lipophilicity of the mycotoxin is crucial in the determination of its uptake into liver cells. At concentrations above 5 micrograms/ml, aflatoxin B1 inhibited the carrier-mediated uptake of cholic acid and of phalloidin into hepatocytes. This effect was reversible and abolished by washing the cells after preincubation with aflatoxin. In concentrations below 5 micrograms/ml the uptake of phallotoxin and cholic acid was however stimulated by 15-25%. These results indicate, that a carrier-mediated uptake into hepatocytes via the multispecific bile salt transporter is not responsible for the organoselective clearance of aflatoxins by the liver. On the other hand, the cholestatic effect of aflatoxin B1 results at least partially from the inhibition of the multispecific bile acid transport system. This inhibition may arise from affinity of aflatoxins to lipid domains of the cell membrane.     

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.     

Metabolism of saturated and polyunsaturated very-long-chain fatty acids in fibroblasts from patients with defects in peroxisomal beta-oxidation.

The metabolism of [1-14C]lignoceric acid (C24:0) and [1-14C]tetracosatetraenoic acid (C24:4, n-6) was studied in normal skin fibroblast cultures and in cultures from patients with defects in peroxisomal beta-oxidation (but normal peroxisomal numbers). Cells from X-linked adrenoleukodystrophy (ALD) patients with a presumed defect in a peroxisomal acyl-CoA synthetase, specific for fatty acids of carbon chain lengths greater than 22 (very-long-chain fatty acids; VLCFA), showed a relatively normal production of radiolabelled CO2 and water-soluble metabolites from [1-14C]C24:0. However, the products of synthesis from acetate de novo (released by beta-oxidation), i.e. C16 and C18 fatty acids, were decreased, and carbon chain elongation of the fatty acid was increased. In contrast, cell lines from two patients with an unidentified lesion in peroxisomal beta-oxidation (peroxisomal disease, PD) showed a marked deficiency in CO2 and water-soluble metabolite production, a decreased synthesis of C16 and C18 fatty acids and an increase in carbon chain elongation. The relatively normal beta-oxidation activity of ALD cells appears to be related to low uptake of substrate, as a defect in beta-oxidation is apparent when measurements are performed on cell suspensions under high uptake conditions. Oxidation of [1-14C]C24:4 was relatively normal in ALD cells and in the cells from one PD patient but abnormal in those from the other. Our data suggest that, despite the deficiency in VLCFA CoA synthetase, ALD cells retain a near normal ability to oxidize both saturated and polyunsaturated VLCFA under some culture conditions. However, acetate released by beta-oxidation of the saturated VLCFA and, to a much lesser degree, the polyunsaturated VLCFA, appears to be used preferentially for the production of CO2 and water-soluble products, and acetate availability for fatty acid synthesis in other subcellular compartments is markedly decreased. It is likely that the increased carbon chain elongation of the saturated VLCFA which is also observed reflects the increased availability of substrate (C24:0) and/or an increase in microsomal elongation activity in ALD cells.     

Induction of an endothermic transition in the Arrhenius plot of fatty acid uptake by lipid-depleted ascites tumor cells

Cultured ascites tumor cells and their lipid-depleted variants containing 35-40% less membrane phospholipid and cholesterol were used to study uptake and metabolism of fatty acids complexed to albumin. Uptake of stearate and oleate at 37 degrees C was considerably higher in the lipid-depleted cells, but no significant difference in the affinity constants for stearate uptake of 3.70 microM for the lipid-depleted and 2.50 microM for the control cells was observed. Similar rates of uptake of both cultures were observed at lower temperatures up to 30 degrees C. The drastic increase in stearate uptake above 30 degrees C resulted in an endothermic transition in the Arrhenius plot with an activation energy of 20.8 kJ/mol versus 6.5 kJ/mol for the control cells. Uptake of stearate and oleate of the control cells was only slightly reduced by metabolic inhibitors, which was similar to stearic acid transport in the lipid-depleted variants. However, oleate uptake was substantially decreased in these variants. Incorporated stearate was esterified to about 50% in both cultures, and oleate between 85 and 90%. Mainly triacylglycerols and phospholipids with phosphatidylcholine (41%) and phosphatidylethanolamine (35%) as major polar lipid components, and also lower acylglycerols and cholesterol were found to be labeled. Under lipid-depleted conditions, a pronounced increase in the relative proportion of oleate incorporation into triacylglycerols was determined. It is suggested that fatty acid uptake is controlled by the number of active sites of the putative transport protein, which increases upon lipid depletion as shown from the V values. This increase may result from the segregation of membrane-bound proteins into domains (Haeffner et al. (1986) Cell Mol. Biol. 32, 359-368), which are known to be formed as a consequence of lipid phase separation in the lipid-depleted cells.     

Effect of membrane fatty acid composition on radiosensitivity of V79 Chinese hamster cells

Exponentially growing V79-379A Chinese hamster fibroblasts were transferred to low-lipid medium enriched with a single fatty acid of the C18 series. After 24 h at 37 degrees C, the fatty acid composition was determined by gas chromatography of the corresponding methyl esters for the total lipid extracts of the cells and for the nuclear membrane fraction. Radiation survival curves, based upon a clonogenic assay, were obtained by irradiation with low dose-rate 60Co gamma rays at either 4 degrees C or room temperature. We observe no effect of fatty acid upon radiosensitivity of these cells at either temperature, in confirmation of published reports with other mammalian cell lines.     

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.     

Characteristics of Lysophosphatidylcholine in Its Inhibition of Taurine Uptake by Human Intestinal Caco-2 Cells

The characteristics of lysophosphatidylcholine (LPC) in its inhibition of the taurine uptake by human intestinal Caco-2 cells were investigated. By treating the cells with 200 μM of LPC, the taurine uptake was rapidly decreased by approximately 60%. This decrease was accompanied by an increase in the K _m value for the uptake. A rapid uptake of LPC itself by the cells was also observed. The inhibitory activity of LPC was specific to the uptake of taurine and certain amino acids, while the uptake of glucose, glutamic acid and peptide (glycylglutamine) was not affected by LPC. The activity was dependent on the structure of a polar head and the bound fatty acid. The phosphorylcholine residue was likely to have played an important role, and surface active LPC with fatty acids of C14 or longer was highly inhibitory. These results suggest that the interaction of LPC with the taurine transporter in the intestinal cell membrane was the cause of the reduced taurine uptake.     

Constitutive uptake and degradation of fatty acids by Yersinia pestis.

Yersinia pestis was found to utilize palmitic acid as a primary carbon and energy source. No inhibition of growth by palmitic acid was observed. Comparison of palmitic acid uptake by cells pregrown either with or without palmitic acid demonstrated that fatty acid uptake was constitutive. High basal levels of two enzymes of beta-oxidation, beta-hydroxyacyl-coenzyme A dehydrogenase and thiolase, and the two enzymes of the glyoxylate shunt, isocitrate lyase and malate synthase, were found in cells grown in defined medium with glucose. Elevated levels of all four enzymes were found when cells were grown with acetate as a primary carbon and energy source, and even higher levels were observed when palmitic acid was provided as a primary carbon and energy source. High-pressure liquid chromatography was used to demonstrate that, in the presence of glucose, uniformly labeled [14C]palmitic acid was converted to intermediates of the tricarboxylic acid cycle and glyoxylate shunt. Pregrowth with palmitic acid was not required for this conversion. Strains lacking the 6- or the 47-megadalton plasmid did not take up [3H]palmitic acid but did possess levels of enzyme activity comparable to those observed in the wild-type strain.     

Bioenergetic Responses of Synechocystis 6803 Fatty Acid Desaturase Mutants at Low Temperatures

Fatty acid composition of the membrane lipids in the mesophilic cyanobacterium Synechocystis sp. PCC 6803 was altered in earlier work by targeted mutagenesis of genes for fatty acid desaturases. In this work, cells of several mutant strains, depleted in the unsaturated fatty acids in membrane lipids, were grown at 34 degrees C. Spheroplasts (permeabilized cells) were prepared by lysozyme digestion of the cell wall followed by gentle osmotic shock. The bioenergetic parameters ATP formation, electron transport, and H+ uptake were measured at various temperatures. All three bioenergetic parameters for spheroplasts from wild-type cells (which had abundant polyunsaturated fatty acids) were active down to the lowest temperatures used (1 degrees - 2 degrees C). In two strains, which lacked the capacity to desaturate fatty acids at the A 12 position and at the A 12 and A6 positions (designated as desA- and desA-/desD-, respectively), the spheroplasts lost the capacity to form ATP (measured as phenazine methosulfate cyclic phosphorylation) at about 5 degrees C but retained electron transport (water oxidation-dependent ferricyanide reduction) and H+ uptake linked to phenazine methosulfate cyclic electron transport. It appears that the absence of the unsaturation of fatty acids in the A 12 and A6 positions blocks the ability of the photosynthetic membranes to couple a bioenergetically competent proton-motive force to the ATP formation mechanism at temperatures below 5 degrees C. It remains to be determined whether the loss of ATP formation in the mutant strains is the failure of available protons to properly flow into the CF0CF1-ATP synthase or a failure in the CF1 part of the complex in coupling the dissipative H+ flow to the enzyme mechanism of the synthase.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Effect of the amount of glucose uptake on the rates of the pentose phosphate cycle and of fatty acid synthesis

In order to study the quantitative relationship between fatty acid synthesis and pentose phosphate-cycle activity under different hormonal and dietary conditions affecting the extent of glucose uptake, cells isolated from rat epididymal adipose tissue were incubated in bicarbonate buffer containing [U-(14)C]-, [1-(14)C]- or [6-(14)C]-glucose. From the amount of glucose taken up, the production of lactate and pyruvate, and the incorporation of (14)C from differently labelled [(14)C]glucose into CO(2), fatty acids and glyceride glycerol, the rates of glucose metabolism via different pathways and the extent of lipogenesis under various experimental conditions were determined. The contribution of the pentose phosphate-cycle to glucose metabolism under normal conditions was calculated to be 8%. Starvation and re-feeding, and the presence of insulin, caused an enhancement of glucose uptake, pentose phosphate-cycle activity and fatty acid synthesis. Plots of both pentose phosphate-cycle activity and fatty acid synthesis versus glucose uptake revealed that the extent of glucose uptake, over a wide range, determines the rates of fatty acid synthesis and glucose metabolism via the pentose phosphate cycle. A balance of formation and production of nicotinamide nucleotides in the cytoplasm was established. The total amount of cytoplasmic NADH and NADPH formed was only in slight excess over the hydrogen equivalents required for the synthesis of fatty acids, glyceride glycerol and lactate. Except in cells from starved animals, the pentose phosphate cycle was found to provide only about 60% of the NADPH required for fatty acid synthesis. The results are discussed with respect to an overall control of the different metabolic and biosynthetic reactions in the fat-cells by the amount of glucose transported into the cell.     

Effect of apoE on triglyceride emulsion interaction with hepatocyte and hepatoma G2 cells

The uptake and internalization of a triglyceride emulsion by rat hepatocytes in culture less than 24 hr was either inhibited or uninfluenced by apoE. ApoE significantly increased the uptake of these emulsions in later cultures. Specific low density lipoprotein (LDL) binding was similar for hepatocyte monolayers prior to and after 24 hr. Rat hepatocytes in culture for 2 days, which were treated with collagenase, detached and then replated within 1 hr and were apoE-responsive in 2 hr. Heparin inhibited the apoE stimulation in both hepatocytes and hepatoma monolayers. Heparin wash of hepatocytes or hepatoma cells incubated with apoE-[14C]triolein emulsions at 4 degrees C resulted in a considerable loss in radiolabeled cell lipid. A similar wash after 37 degrees C incubations produced little loss suggesting internalization. Hepatocytes had lower affinity but similar apoE-emulsion binding capacity compared to hepatoma cells. Triolein emulsions with apoE were significantly more rapidly metabolized by the hepatocyte than unsupplemented emulsions. The apoE-mediated hepatocyte lipid uptake was inhibited by apoC proteins. High molar ratios of free fatty acid/albumin also suppressed hepatocyte apoE-mediated lipid uptake. Both rat high density lipoprotein (HDL) and LDL inhibited with a potency directly related to their content of apoE. Human LDL and HDL without apoE also inhibited the interaction with less potency than the rat lipoproteins. Human HDL inhibition was diminished after removal of apoC proteins.     

Cholesterol is required for the fusion of single unilamellar vesicles with Mycoplasma capricolum.

Small unilamellar vesicles (SUV) were prepared from the total lipid extract of Mycoplasma capricolum. The SUV were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C, and in the presence of 5% polyethylene glycol, an increase in the R18 fluorescence with time was observed when the R18-labeled SUV were introduced to a native M. capricolum cell suspension. The fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes of M. capricolum, was interpreted as a result of lipid mixing during fusion between the SUV and the mycoplasma cells. The presence of cholesterol in the SUV was found to be obligatory to allow SUV-mycoplasma fusion to occur. Adaptation of M. capricolum cells to grow in a medium containing low cholesterol concentration provided cells in which the unesterified cholesterol content was as low as 17 micrograms/mg cell protein. The fusion activity of the adapted cells was very low or nonexistent. Nonetheless, when an early exponential phase culture of the adapted cells was transferred to a cholesterol-rich medium, the cells accumulated cholesterol and regained their fusogenic activity. The cholesterol requirement for fusion in the target mycoplasma membrane was met by a variety of planar sterols having a free beta-hydroxyl group, but differing in the aliphatic side chain, e.g., beta-sitosterol or ergosterol, even though these sterols, having a bulky side chain, are preferentially localized in the outer leaflet of the lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Does an increase in membrane unsaturated fatty acids account for Tween 80 stimulation of glucosyltransferase secretion by Streptococcus salivarius?

When Streptococcus salivarius was grown in batch culture in the presence of various Tween detergents, the fatty acid moiety of the detergent was incorporated into the lipids of its membrane. Tween 80 (containing primarily oleic acid) markedly stimulated the production of extracellular glucosyltransferase and also increased the degree of unsaturation of the membrane lipid fatty acids. The possibility that an increase in membrane unsaturated fatty acids promoted extracellular glucosyltransferase production was examined by growing cells at different temperatures in the presence or absence of Tween 80. The membrane lipids of cells grown at 30 degrees C, 37 degrees C and 40 degrees C without Tween 80 exhibited unsaturated/saturated fatty acid ratios of 2.06, 1.01 and 0.87 respectively. A significant increase in the production of extracellular glucosyltransferase was observed at 30 degrees C compared to cells grown at 40 degrees C. However, cells produced much more exoenzyme at all temperatures when grown with Tween 80. The results indicated that an increase in the unsaturated fatty acid content of the membrane lipids was not by itself sufficient to account for the stimulation of extracellular glucosyltransferase production by Tween 80, but that the surfactant also had to be present.     

Insulin and leptin do not affect fatty acid uptake and metabolism in human placental choriocarcinoma (BeWo) cells

Placental transport of long chain polyunsaturated fatty acids is important for fetal growth and development. In order to examine the effects of leptin and insulin on fatty acid uptake by the placenta, placental choriocarcinoma (BeWo) cells were used. BeWo cells were incubated for 5h at 37 degrees C in the absence or presence of different concentrations of insulin (0.6, 60, and 100 ng) or leptin (10 ng) with 200 microM of various radiolabeled fatty acids (docosahexaenoic acid, arachidonic acid, eicosapentaenoic acid, and oleic acid, mixed with 1:1 bovine serum albumin (fat free). After incubation, the uptake and distribution of these fatty acids into different cellular lipid fractions were determined. The uptakes of oleic, eicosapentaenoic, arachidonic, and docosahexaenoic acids were 15.36+/-4.1, 19.95+/-3.6, 28.56+/-8.1, and 62.25+/-9.5 nmol/mg of protein, respectively, in BeWo cells. Incubation of these cells with insulin (0.6 or 60 ng/ml) or leptin (10 ng/ml) did not significantly alter uptake of any of these fatty acids (P>0.5). Insulin or leptin also did not affect beta oxidation of fatty acids in these cells. In contrast, leptin (10 ng/ml) and insulin (0.60 ng/ml)) stimulated the uptake of oleic acid (7.4+/-2.3 nmol/mg protein) in human adipose cells, SGBS cells by 1.28- and 2.48-fold (P<0.05), respectively. The distribution of fatty acids in different cellular lipid fractions was also not affected by these hormones. Our data indicate that unlike adipose tissue, fatty acid uptake and metabolism in placental trophoblasts is not regulated by insulin or leptin.