Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Insect tissues, not microorganisms, produce linoleic acid in the house cricket and the American cockroach

Biosynthesis of linoleic acid, 18:2 (n-6), was unambiguously demonstrated to occur in the cockroach, Periplaneta americana, and the cricket, Acheta domesticus. Axenic tissue from both of these insect species was demonstrated by radio-gas-liquid chromatography (radio-GLC) and radio-high-performance liquid chromatography (radio-HPLC) to incorporate [1-14C]acetate and [1-14C]oleate into this essential fatty acid.     

Long-chain fatty acid uptake by skeletal myocytes: a confocal laser scanning microscopy study

Studies of regulation of free fatty acid (FFA) utilization by skeletal muscles have focused on plasma FFA delivery and on intracellular factors affecting FFA metabolism. The present study was conducted to directly analyse the uptake process of fatty acids into single myocytes. Cells were isolated from the rat flexor digitorum brevis muscle. Confocal laser scanning microscopy was utilized to analyse the uptake of the fluorescent fatty acid derivative 12-NBD-stearate, which is not metabolized by muscle tissue. Uptake represented a saturable function of the unbound fatty acid concentration in the medium (K _m 366 ± 118 nM, V _max 2.1 ± 0.3 AU/s) and depended on the medium sodium concentration. Reduced buffer pH increased initial uptake rates, whereas lactate (10 mM) had no effect. Membrane hyper- and depolarization decreased uptake rates. This study demonstrates for the first time kinetic data from isolated myocytes with evidence for a carrier-mediated transport mechanism for long-chain fatty acids. Received 31 March 1998; accepted 8 May 1998     

Lipid accumulation by a cellulolytic strain of Aspergillus niger.

Lipid accumulation by a cellulolytic mold, Aspergillus niger, was studied. The amount of lipid accumulated ranged from 13.6-16.6% on various carbon sources, namely glucose, xylose, avicel (microcrystalline cellulose) and bagasse (a natural lignocellulosic substrate). Neutral lipids, phospholipids and glycolipids of the mycelia varied from 41.0-46.2%, 34.9-38.4% and 18.7-22.6% of total lipids, respectively. Unsaturated fatty acids comprised around 80% of total fatty materials with linoleic and oleic acid predominating. Of the four nitrogen sources tested, NH4Cl was the best source for lipid synthesis from cellulose (bagasse). Optimum temperature range for growth and lipid synthesis was 25-30 degrees C.     

Inhibition of high affinity uptake of GABA by branched fatty acids

Summary Several branched fatty acids including an antiepileptic agent nDPA were tested as potential inhibitors of high affinity uptake of GABA by brain slices and synaptosomes. Only three compounds (2-butyl-3-propylhexanoic acid, 5-propyloctanoic acid, 2-propylpenten-2-oic acid) were found to be relatively weak inhibitors of the uptake system. There was no correlation between anticonvulsant properties of the branched fatty acids and their potencies as inhibitors of high affinity uptake of GABA.     

Chemie und Stoffwechsel der Polyenfettsäuren

Summary The chemical structure of the polyenoic fatty acids occuring in organ phosphatides and in fish oils is reviewed. The double bonds of all these polyenoic acids are arranged in divinylmethane pattern. Except some of the C₁₆-polyenoic acids of fish oils, these polyenoic acids belong either to the oleic, linoleic or linolenic acid type and have chain lengths C₁₈, C₂₀, and C₂₂. Polyenoic acids of the oleic acid type are present only in small amounts in phosphatides of mammalian origin. Fish oils are lacking in these but predominantly contain polyenoic acids of the linolenic acid type.Metabolic studies have shown that polyenoic acids of linoleic acid type (e.g. arachidonic acid) originate from linoleic acid and those of the linolenic acid type (e.g. C₂₀-pentaenoic and C₂₂-hexaenoic acid) from linolenic acid-both supplemented exogenous-by extension of the carbon chain by acetate on the side of the carboxylic acid group and introduction of additional double bonds in the divinylmethane pattern directed toward the carboxylic acid group. There is some evidence, however, that a total synthesis of the polyenoic acids of the oleic acid type occurs in the animal body.The transformation of linolenic to C₂₂-hexaenoic acid and some intermediate reactans have been investigated more precisely by means of the tracer method. As far as the biosynthesis of the polyenoic fatty acids is concerned there are no fundamental differences in different vertebrates.

---

---

Nach einem Vortrag auf der gemeinsamen Tagung der deutschen, französischen und schweizerischen Biochemiker in Zürich vom 10.–12. Oktober 1960.     

Fatty acid profiles of major food sources of howler monkeys (Alouatta palliata) in the neotropics

Wild howler monkeys (Alouatta palliata) get most of their calories from carbohydrates (65%) and fats (18%) of native tropical plants, but little is known about their intake of individual fatty acids. The fatty acid composition of several natural food sources of howler monkeys collected in Panama was determined by gas-liquid chromatography. The predominant fatty acids were palmitic (30%), linoleic (23%), linoleic (23%), -linolenic (16%) and oleic (15%). Fatty acids with less than 16, and more than 18, carbon chains were uncommon (0–7%). Although total saturated fatty acids were high in some specific food sources (22–54% of total fatty acids and 8 energy %), most of the calories from fat in the animals' diets are derived from mono-and polyunsaturated fatty acids (9.75 energy %) All food sources had significant amounts of the -3 fatty acid, -linolenic acid (2.9 energy %). In terms of human diets, the howler monkey's fat consumption would not be considered atherogenic. Unless these animals show a particular adverse susceptibility to dietary fat, it is unlikely that their fat intake is the primary cause of the low, but significant, incidence of atherosclerosis that develops in these animals in the wild state.     

Cellular uptake of long-chain fatty acids: role of membrane-associated fatty-acid-binding/transport proteins

The critical importance of long-chain fatty acids in cellular homeostasis demands an efficient uptake system for these fatty acids and their metabolism in tissues. Increasing evidence suggests that the plasma-membrane-associated and cytoplasmic fatty-acid-binding proteins are involved in cellular fatty acid uptake, transport and metabolism in tissues. These binding proteins may also function in the fine tuning of cellular events by modulating the metabolism of long-chain fatty acids implicated in the regulation of cell growth and various cellular functions. Several membrane-associated fatty-acid-binding/transport proteins such as plasma membrane fatty-acid-binding protein (FABPpm, 43 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transporter protein (FATP, 63 kDa) have been identified. In the feto-placental unit, preferential transport of maternal plasma arachidonic and docosahexaenoic acids across the placenta is of critical importance for fetal growth and development. Our studies have shown that arachidonic and docosahexaenoic acids are preferentially taken up by placental trophoblasts for fetal transport. The existence of a fatty-acid-transport system comprising multiple membrane-binding proteins (FAT, FATP and FABPpm) in human placenta may be essential to facilitate the preferential transport of maternal plasma fatty acids in order to meet the requirements of the growing fetus. The preferential uptake of arachidonic and docosahexaenoic acids by the human placenta has the net effect of shunting these maternal plasma fatty acids towards the fetus. The roles of plasma membrane-associated binding/transport proteins (FABPpm, FAT and FATP) in tissue-specific fatty acid uptake and metabolism are discussed.     

High affinity binding of 5-hydroxytryptamine to some membrane preparations from cattle brain. Relationship to lysergic acid diethylamide (LSD)]

5 hydroxytryptamine binds to crude brain membrane preparations with two different affinities (KD = 1 to 2 X 10(-9) M for the highest, 1 to 2 X 10(-8) M for the lowest). LSD also binds with two affinities (KD = 3 to 4 X 10(-9) M and KD = 2 to 3 X 10(-8) M). Subcellular distribution of these sites shows that binding involves the two binding affinities in microsomal membranes but solely the high affinity binding sites are present in purified synaptosomal membranes. High affinity sites for 5 HT and for LSD are different as no direct competitive inhibition is observed in that case. On microsomal membranes, direct relationship occurs between low affinity binding for 5 HT and high affinity binding for LSD.     

Functions of fatty acid binding proteins

Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14-15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABPpm). These proteins have not been fully characterized to date, but strong evidence suggest that they function in the transport of long-chain fatty acids across the plasma membrane.     

The egg-lipid composition of the ‘living fossil’ reptile tuatara (Sphenodon punctatus)

Summary The lipid composition of two tuatara eggs was examined. The eggs contained triacylglycerol (80%) and phospholipid (12%) as their major lipid fractions. Fatty acid analyses of the individual lipid classes indicated the presence of essential fatty acids, linoleic and arachidonic acids. The quantity of such acids in the egg yolk lipids would suggest they are factors for survival as illustrated in other species.     

The effect of endotoxin on membrane fatty acid composition in BCG-sensitized mice

The effects of endotoxin on mouse liver phospholipid fatty acid composition have been investigated. Administration of endotoxin from Salmonella abortus equi led to a decrease in the polyunsaturated fatty acid content of livers from mice sensitized with Bacille Calmette Guérin (BCG). The content of arachidonic acid fell significantly in both the phosphatidylcholine and phosphatidylinositol fractions whereas in the phosphatidylethanolamine fraction the linoleic acid content was significantly reduced. The polyunsaturated fatty acids were replaced by increased amounts of oleic acid and palmitic acid, leading to a reduction in the polyunsaturated to saturated fatty acid ratio.     

Antiproliferative effect of polyunsaturated fatty acids and interleukin-2 on normal and abnormal human lymphocytes

The polyunsaturated fatty acids (PUFAs), linoleic acid (LA), alpha linolenic acid (ALA), gamma linolenic acid (GLA), arachidonic acid (AA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), showed inhibition of growth of both normal and abnormal (Molt-4) human lymphocytes, and inhibition was concentration-dependent. Interestingly, the production of the lymphokine Interleukin-2 (IL-2) was elevated in Molt-4 cells, but it was reduced in the normal human lymphocytes. Addition of GLA or IL-2 or a combination of both showed enhancement of SO ₂ ^– and of lipid peroxidation levels, which were significantly higher in Molt-4 cells than in the normal lymphocytes. Reduction of protein concentration was also observed in both types of cells during this treatment. The data showed that the antiproliferative effects of GLA and IL-2 may partly be exerted through the elevated production of superoxide free radicals and peroxidatin products. This is a novel finding and therefore, further exploitation of combinations of PUFAs and IL-2 may be a possible way of combating cancer cell growth.     

Modulation of carcinoembryonic antigen release by HT-29 colon carcinoma line in the presence of different agents

In this study we followed the effects of various differentiating agents on the expression of carcinoembryonic antigen (CEA) released into the medium by a colon carcinoma cell line HT-29. Butyric acid 1 mM markedly increased the level of CEA (12-fold in comparison to control levels). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) 50 ng/ml and 5-azacytidine 4 x 10(-6) M increased the amount of CEA, 2- and 1.5-fold respectively. On the other hand retinoic acid 10(-5) M, N methyl-formamide 1% and N,N hexamethylene bisacetamide 2.5 mM decreased CEA 2-, 4- and 3-fold respectively. Our results emphasize that various differentiating agents affect CEA levels differently. Thus changes in CEA levels appear not to be reliable as a marker of a more differentiated phenotype.     

The effect of endotoxin on membrane fatty acid composition in BCG-sensitized mice

Summary The effects of endotoxin on mouse liver phospholipid fatty acid composition have been investigated. Administration of endotoxin fromSalmonella abortus equi led to a decrease in the polyunsaturated fatty acid content of livers from mice sensitized with Bacille Calmette Guérin (GCG). The content of arachidonic acid fell significantly in both the phosphatidylcholine and phosphatidylinositol fractions whereas in the phosphatidylethanolamine fraction the linoleic acid content was significantly reduced. The polyunsaturated fatty acids were replaced by increased amounts of oleic acid and palmitic acid, leading to a reduction in the polyunsaturated to saturated fatty acid ratio.     

Expression, isolation and characterization of a mutated human plasminogen kringle 3 with a functional lysine binding site

Each kringle of human plasminogen (HPg) except kringle 3 (K3) exhibits affinity for omega-aminocarboxylic acids. Assuming that the K3 domain contains a preformed but nonfunctional lysine binding site (LBS), Lys311 was altered by site-directed mutagenesis into Asp311 in accordance with the consensus sequence of the LBS. Cys297 involved in the interkringle disulfide bridge was mutated into Ser297 to minimize dimerization and aggregation. The mutated K3 TYQ[K3HPg/C297S/K311D]DS (r-K3mut) was expressed in Escherichia coli, isolated on an Ni2(+)-nitrilotriacetic acid-agarose column, refolded and purified on a lysine Bio-Gel column. Fluorescence titration indicates affinity of r-K3mut for omega-aminocarboxylic acids with the following association constants (Kass, mM-1): 5-aminopentanoic acid: 1.3; 6-aminohexanoic acid: 4.2; 7-aminoheptanoic acid: 0.5; trans-(aminomethyl)cyclohexanecarboxylic acid: 12.7; p-benzylaminesulfonic acid: 11.8. r-K3mut exhibits an affinity similar to native and mutated (R220G, E221D) K2. The results indicate the presence of a preformed but nonfunctional LBS in native K3 of HPg. We were able to demonstrate for the first time that an appropriate mutation in the LBS of a kringle produced a weak but distinct affinity for omega-aminocarboxylic acids.     

Inhibition by tetanus and botulinum A toxin of the release of [3H]noradrenaline and [3H]GABA from rat brain homogenate

Rat brain homogenate was preloaded with [3H]noradrenaline or [3H]GABA and stimulated with high K+. Tetanus toxin and botulinum A neurotoxin partially prevent the evoked [3H]noradrenaline release in the same range of toxin concentrations starting below 10(-10) M. In contrast, release of gamma-amino butyric acid (GABA) is much more sensitive to tetanus than to botulinum A toxin.     

Oxidative photochemical decarboxylation of zaragozic acid A

A unique decomposition reaction of the novel squalene synthase inhibitors called zaragozic acids has been studied. Under very mild conditions, e.g. by merely exposing their solutions to air and visible light at ambient temperature, these compounds, characterized by the 2,8-dioxabicyclo[3.2.1]octane-4,6,7-trihydroxy-3,4,5-tricarboxylic acid core, rapidly decompose. As relatively stable intermediates in the cascade of decomposition, the biologically active 2,8-dioxabicyclo[3.2.1]octane-6,7-dihydroxy-4-keto-5-caroxylic acid (or 3,4-decarboxy-4-dehydro) derivatives of these compounds have been isolated in ca. 20% yield. Derivatization on the highly reactive 4-carbonyl group yields stable derivatives, several of which are potent inhibitors of squalene synthase. Further decomposition results in the elimination of C3 and C4 atoms and the carboxylic acid on C5, the oxidation of C5 to carboxylic acid and the liberation of the oxo group on C1. Specific results obtained with zaragozic acid A, a key representative of the family of these potent cholesterol-lowering agents, are presented in this study.     

Measurement of cyclic AMP in the islets of Langerhans of newborn rats. Effect of amino acids]

Radioimmunoassay of cyclic AMP (cAMP) in the islets of Langerhans from 48-64 h old Rats was performed after succinylation of the samples. cAMP was detected at 0.03 nM. The cAMP content of islets increases when L-arginine, L-lysine and L alanine are added together in the incubation medium at a concentration of 5-10 mM each. When phosphodiesterase is inhibited by theophylline the three amino acids considerably increase the cAMP content of islets. Thus an increase in cAMP content of the islets was observed with a concentration of amino acids which is efficient in stimulating the insulin and glucagon secretion.     

Functions of fatty acid binding proteins

Summary Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14–15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABP_pm). These proteins have not been fully characterized to date, but strong evidence suggests that they function in the transport of long-chain fatty acids across the plasma membrane.