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Oleic acid is a potent inhibitor of fatty acid and cholesterol synthesis in C6 glioma cells

Open AccessPublished:June 13, 2007DOI:https://doi.org/10.1194/jlr.M700051-JLR200
      Glial cells play a pivotal role in brain fatty acid metabolism and membrane biogenesis. However, the potential regulation of lipogenesis and cholesterologenesis by fatty acids in glial cells has been barely investigated. Here, we show that physiologically relevant concentrations of various saturated, monounsaturated, and polyunsaturated fatty acids significantly reduce [1-14C]acetate incorporation into fatty acids and cholesterol in C6 cells. Oleic acid was the most effective at depressing lipogenesis and cholesterologenesis; a decreased label incorporation into cellular palmitic, stearic, and oleic acids was detected, suggesting that an enzymatic step(s) of de novo fatty acid biosynthesis was affected. To clarify this issue, the activities of acetyl-coenzyme A carboxylase (ACC) and FAS were determined with an in situ digitonin-permeabilized cell assay after incubation of C6 cells with fatty acids. ACC activity was strongly reduced (∼80%) by oleic acid, whereas no significant change in FAS activity was observed. Oleic acid also reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). The inhibition of ACC and HMGCR activities is corroborated by the decreases in ACC and HMGCR mRNA abundance and protein levels. The downregulation of ACC and HMGCR activities and expression by oleic acid could contribute to the reduced lipogenesis and cholesterologenesis.

      Abbreviations

      ACC
      acetyl-coenzyme A carboxylase
      HMGCR
      3-hydroxy-3-methylglutaryl coenzyme A reductase
      MTT
      3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
      After white adipose tissue, the brain is the organ with the highest lipid content of the body. The biosynthesis and deposition of lipids play an important role in maintaining brain structure and function, for example, during development-associated biogenesis of neural cell membranes. It is well established that alterations in lipid metabolism are the cause of or are associated with many neurological diseases (
      • Dexter D.T.
      • Carter C.J.
      • Wells F.R.
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      • Agid Y.
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      Basal lipid peroxidation in substantia nigra is increased in Parkinson's disease.
      ,
      • Van Geel B.M.
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      • Wanders R.J.A.
      • Barth P.G.
      X-linked adrenoleukodystrophy: clinical presentation, diagnosis, and therapy.
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      • Lukiw W.J.
      • Papolla M.
      • Pelaez R.Palacios
      • Bazan N.G.
      Alzheimer's disease—a dysfunction in cholesterol and lipid metabolism.
      ).
      Astrocytes, the major class of glial cells in the mammalian brain, play an active role in brain metabolism. These cells surround intraparenchymal blood capillaries so that they represent the first cellular barrier for nutrients and other substances entering the brain system. A metabolic coupling between astrocytes and neurons to maintain energy metabolism homeostasis has been described (
      • Bezzi P.
      • Volterra A.
      A neuron-glia signalling network in the active brain.
      ,
      • Hansson E.
      • Rönnbäck L.
      Glial neuronal signaling in the central nervous system.
      ). Metabolic regulation in the brain has been investigated extensively, and those studies focused mostly on carbohydrate and amino acid metabolism (for review, see Ref.
      • Tsacopoulos M.
      Metabolic signaling between neurons and glial cells: a short review.
      ). During neuronal activity, glucose taken up by astrocytes is converted into lactate, which is then released into the extracellular space to be used by neurons (
      • Tsacopoulos M.
      Metabolic signaling between neurons and glial cells: a short review.
      ). Regarding lipid metabolism, astroglial ketone body synthesis, showing characteristics strikingly similar to those of hepatic ketogenesis (
      • Guzmán M.
      • Blázquez C.
      Ketone body synthesis in the brain: possible neuroprotective effects.
      ), may represent an important pathway for brain energy production and/or biosynthetic processes. The involvement of fatty acids in cell death pathways, particularly in the context of lipid-mediated apoptotic signaling, has also been described (
      • Bazan Jr, N.G.
      Effects of ischemia and electroconvulsive shock on free fatty acid pool in the brain.
      ,
      • Zhu Y.
      • Schwarz S.
      • Ahlemeyer B.
      • Grzeschik S.
      • Klumpp S.
      • Krieglstein J.
      Oleic acid causes apoptosis and dephosphorylates Bad.
      ). It has been shown that exogenous fatty acids may influence the fatty acid composition of neuronal and glial membranes (
      • Bourre J.M.
      Biochemistry of brain lipids (especially fatty acids). In situ synthesis and exogenous origin during development. Various aspects of nutritional effects.
      ,
      • Rapoport S.I.
      In vivo fatty acid incorporation into brain phospholipids in relation to signal transduction and membrane remodeling.
      ,
      • Di Biase A.
      • Avellino C.
      • Pieroni F.
      • Quaresima T.
      • Grisolia A.
      • Cappa M.
      • Salvati S.
      Effects of exogenous hexacosanoic acid on biochemical myelin composition in weaning and post-weaning rats.
      ,
      • Horrocks L.A.
      • Farooqui A.A.
      Docosahexaenoic acid in the diet: its importance in maintenance and restoration of neural membrane function.
      ,
      • Contreras M.A.
      • Rapoport S.I.
      Recent studies on interactions between n-3 and n-6 polyunsaturated fatty acids in brain and other tissues.
      ), and these changes in turn can affect cellular metabolism and regulatory (
      • Rodríguez-Rodríguez R.A.
      • Tabernero A.
      • Velasco A.
      • Lavado E.M.
      • Medina J.M.
      The neurotrophic effect of oleic acid includes dentritic differentiation and the expression of the neuronal basic helix-loop-helix transcription factor NeuroD2.
      ) and inflammatory processes (
      • Farooqui A.A.
      • Horrocks L.A.
      • Farooqui T.
      Modulation of inflammation in brain: a matter of fat.
      ). Furthermore, in primary cultures of rat astroglia, it has been shown that the addition of oleic and linoleic acids to the medium reduces several aminopeptidase activities (
      • Ramírez-Expósito M.J.
      • García M.J.
      • Mayas M.D.
      • Ramírez M.
      • Martínez-Martos J.M.
      Effects of exogenous fatty acids and cholesterol on aminopeptidase activities in rat astroglia.
      ).
      Despite the great impact of lipid-metabolizing processes in brain development and homeostasis, the potential regulation of lipogenesis and cholesterologenesis by fatty acids has not been studied in glial cells. Moreover, to our knowledge, only a few studies (
      • Bourre J.M.
      Biochemistry of brain lipids (especially fatty acids). In situ synthesis and exogenous origin during development. Various aspects of nutritional effects.
      ,
      • Volpe J.J.
      • Marasa J.C.
      Regulation of palmitic acid synthesis in cultured glial cells: effects of lipid on fatty acid synthetase, acetyl-CoA carboxylase, fatty acid and cholesterol synthesis.
      ,
      • Volpe J.J.
      • Marasa J.C.
      Short term regulation of fatty acid synthesis in cultured glial and neuronal cells.
      ,
      • Volpe J.J.
      • Hennessy S.W.
      Cholesterol biosynthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured glial and neuronal cells. Regulation by lipoprotein and by certain free sterols.
      ) have been reported regarding lipid synthesis in brain cells. Therefore, the aim of this work was to study fatty acid and cholesterol biosynthesis and their regulation by different exogenous fatty acids in glial cells. For this purpose, we used the rat C6 glioma cell line, which expresses a large repertoire of astrocyte-expressing enzymatic activities (
      • Volpe J.J.
      • Fujimoto K.
      • Marasa J.C.
      • Agrawal H.C.
      Relation of C-6 glial cells in culture to myelin.
      ,
      • McMorris F.A.
      Norepinephrine induces glial-specific enzyme activity in cultured glioma cells.
      ) and exhibits a prevalent astrocyte-like phenotype when cultured in serum-rich medium (
      • Nave K.A.
      • Lemke G.
      Induction of the myelin proteolipid protein (PLP) gene in C6 glioblastoma cells: functional analysis of the PLP promoter.
      ). We found that oleic acid greatly inhibits fatty acid and cholesterol synthesis by a mechanism that involves, at least in part, the downregulation of either activity and the expression of acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR; EC 1.1.1.34), key regulatory enzymes of lipogenesis and cholesterologenesis, respectively.

      MATERIALS AND METHODS

      Materials

      Rat C6 glioma cells were from the American Type Culture Collection. DMEM, FBS, penicillin/streptomycin, PBS, and pCR 2.1 TOPO vector were from Gibco-Invitrogen, Ltd. (Paisley, UK); [1-14C]acetate was from GE Healthcare (Little Chalfont, UK); [1-14C]acetyl-CoA, [3H]water, [3-14C]HMG-CoA, and [α-32P]UTP were from Perkin-Elmer (Boston, MA). Brij97 detergent, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), fatty acid sodium salts, and peroxidase-conjugated streptavidin were purchased from Sigma-Aldrich (St. Louis, MO). Primary antibodies for HMGCR, α-tubulin, and horseradish peroxidase-conjugated IgGs were from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents (from Sigma-Aldrich) were of analytical grade.

      Cell culture

      C6 cells were grown in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2. C6 cells were seeded at a density of 5 × 105 cells per 35 mm diameter Petri dishes; 24 h after plating, the medium was changed and, after another 24 h, sodium salts of different fatty acids at 99% of purity (C18:0, stearic acid; C18:1 cis, oleic acid, cis9-octadecaenoic acid; C18:1 trans, elaidic acid, trans9-octadecaenoic acid; C18:2, linoleic acid, all-cis9,12-octadecadienoic acid; C20:4, arachidonic acid, all-cis5,8,11,14-eicosatetraenoic acid; C20:5, all-cis5,8,11,14,17-eicosapentaenoic acid; C22:6, all-cis4,7,10,13,16,19-docosahexaenoic acid) were added to the serum-rich (10% FBS) medium, obtaining 100 μM final concentration, as reported by Salvati et al. (
      • Salvati S.
      • Natali F.
      • Attorri L.
      • Raggi C.
      • Biase A.Di
      • Sanchez M.
      Stimulation of myelin proteolipid protein gene expression by eicosapentaenoic acid in C6 glioma cells.
      ). Unless specified otherwise, cells were in contact with the exogenous fatty acid for a total period of 4 h. In each experiment and for each determination, control dishes without any fatty acid addition were used.

      MTT assay

      An MTT assay was used for the quantification of metabolically active, living cells. C6 cells were plated at a density of 0.5 × 104 cells/well in a 96-well culture dish. After 24 h, the serum-rich medium was refreshed, and after another 24 h, cells were incubated for 4 h with the indicated fatty acid sodium salts. Then, cell monolayers were incubated for 3 h with 1 mg/ml MTT. Mitochondria of living cells convert the yellow tetrazolium compound to its purple formazan derivative. After removal of the unconverted MTT, the formazan product was dissolved in isopropanol and the absorbance of formazan dye was measured at 450 nm. Viability was calculated as percentage of absorbance relative to control cells.

      Determination of the rate of fatty acid and cholesterol synthesis

      Lipogenic activity was determined by monitoring the incorporation of [1-14C]acetate (16 mM, 0.96 mCi/mol) or [3H]water (5 mCi/ml) into fatty acids and cholesterol essentially as reported (
      • Gnoni G.V.
      • Geelen M.J.H.
      • Bijleveld C.
      • Quagliariello E.
      • Van den Bergh S.G.
      Short-term stimulation of lipogenesis by triiodothyronine in maintenance cultures of rat hepatocytes.
      ). Labeled substrate was added 1 h before ending the experiment.
      To terminate the lipogenic assay, the medium was aspirated, cells were washed three times with ice-cold 0.14 M KCl to remove unreacted labeled substrate, and the reaction was stopped with 1.5 ml of 0.5 M NaOH. The cells were scraped off with a rubber policeman, transferred to a test tube, saving 100 μl for protein assay (
      • Lowry O.H.
      • Rosebrough N.J.
      • Farr A.L.
      • Randall R.J.
      Protein measurement with the Folin phenol reagent.
      ), and saponified with 4 ml of ethanol and 2 ml of double-distilled water for 90 min at 90°C. Unsaponifiable sterols and, after acidification with 1 ml of 7 M HCl, fatty acids were extracted with 3 × 5 ml of petroleum ether. The extracts were collected, dried under a stream of nitrogen, and counted for radioactivity.

      Incorporation of radiolabeled acetate into lipid fractions

      Because newly synthesized labeled fatty acids are incorporated mainly into complex lipids, phospholipid analysis was carried out. Experimental conditions were the same as those reported for fatty acid and cholesterol synthesis assays. At the end of the incubation period, the reaction was blocked by washing the cells three times with ice-cold 0.14 M KCl and treated with 2 ml of KCl/CH3OH (1:2, v/v); total lipids were extracted according to Giudetti et al. (
      • Giudetti A.M.
      • Leo M.
      • Geelen M.J.
      • Gnoni G.V.
      Short-term stimulation of lipogenesis by 3,[email protected]@ce:[email protected]@[email protected]@/ce:[email protected]@-diiodothyronine in cultured rat hepatocytes.
      ).
      Phospholipids were resolved by TLC on silica gel plates using CHCl3/CH3OH/28% NH4OH (65:25:4) as a developing system (
      • Touchstone J.C.
      Thin-layer chromatographic procedures for lipid separation.
      ). Lipid spots were visualized by placing the plate in a tank saturated with iodine vapor. The areas corresponding to the individual lipid classes were marked and scraped individually into counting vials for radioactivity measurement.

      HPLC analysis of newly synthesized radiolabeled fatty acids

      Total extracted fatty acids were separated by HPLC using a modification of the method described by Mehta, Oeser, and Carlson (
      • Mehta A.
      • Oeser A.M.
      • Carlson M.G.
      Rapid quantitation of free fatty acids in human plasma by high-performance liquid chromatography.
      ). Briefly, fatty acid extract, obtained from six Petri dishes as described above, was resuspended in 100 μl of α-bromoacetophenone (15 mg/ml in acetone) and 100 μl of triethylamine (25 mg/ml in acetone). Samples were put into a boiling-water bath for 15 min and then cooled at room temperature. A total of 150 μl of acetic acid (10 mg/ml in acetone) was added to the sample, which was heated again for 5 min, dried under a stream of nitrogen, resuspended in 40 μl of acetonitrile, and centrifuged for a few seconds. For HPLC analysis, 20 μl of each sample was injected into a Beckman System Gold chromatograph equipped with a C18 ODS column (4.6 × 250 mm). The chromatographic system was programmed for elution using two mobile phases: solvent A, acetonitrile-water (4:1) and solvent B, acetonitrile. Solvent A ran for 45 min, and then solvent B ran for 15 min. The flow rate was 2 ml/min, and detection was at 242 nm. Eluted fractions, corresponding to the different fatty acids, were collected for radioactivity measurement.

      Assay of de novo fatty acid synthesis enzymatic activities

      A procedure that allows one to assay directly in situ the activities of the lipogenic enzymes, ACC and FAS (EC 2.3.1.85), was set up. To this end, after incubation with exogenous fatty acids, culture medium was removed and C6 glioma cells were permeabilized using 400 μl of assay mixture containing digitonin (400 μg/ml). The reaction mixture was prepared within 15 min before use by mixing a known amount of digitonin, dissolved in an EGTA stock solution by heating in a boiling-water bath, with the other components of the assay mixture. The assay mixture did not contain any exogenous fatty acid.
      Because ACC and FAS are cytosolic enzymes and they leak from permeabilized cells at the digitonin concentration used, cell permeabilization and ACC and FAS assays were carried out simultaneously (
      • Geelen M.J.H.
      The use of digitonin-permeabilized mammalian cells for measuring enzyme activities in the course of studies on lipid metabolism.
      ). ACC activity was determined as the incorporation of radiolabeled acetyl-CoA into fatty acids in a reaction coupled with that catalyzed by FAS, essentially as described by Bijleveld and Geelen (
      • Bijleveld C.
      • Geelen M.J.H.
      Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes.
      ) in isolated rat hepatocytes. This method avoids a number of interferences associated with the classical bicarbonate fixation assay of ACC activity (
      • Geelen M.J.H.
      The use of digitonin-permeabilized mammalian cells for measuring enzyme activities in the course of studies on lipid metabolism.
      ).
      The ACC assay reaction mixture contained 100 mM HEPES (pH 7.9), 4.2 mM MgCl2, 1 mM citrate, 5 mM EGTA, 20 mM KHCO3, 20.5 mM NaCl, 4 mM ATP, 1 mM NADPH, 0.44 mM dithioerythritol, 0.85% (w/v) BSA, 800 μg/ml digitonin, 0.12 mM [1-14C]acetyl-CoA (0.5 μCi/ml), 0.12 mM butyryl-CoA, and 3 mU of purified FAS (before use, FAS was preincubated for 30 min at room temperature with 12.5 mM dithioerythritol). FAS was purified from rat liver according to Linn (
      • Linn T.C.
      Purification and crystallization of rat liver fatty acid synthetase.
      ) and stored at −80°C. The assay mixture was diluted 1:1 in culture medium, and 400 μl of this solution was added to the plates that were incubated at 37°C for 8 min.
      FAS activity was assayed in permeabilized cells essentially as reported (
      • Geelen M.J.H.
      The use of digitonin-permeabilized mammalian cells for measuring enzyme activities in the course of studies on lipid metabolism.
      ). The incubation time was 10 min at 37°C. The lipogenic assays were stopped by the addition of 100 μl of 10 M NaOH.
      Thereafter, cells were scraped off with a rubber policeman and transferred to a test tube. Plates were washed twice with 450 μl of 0.5 M NaOH, and these washing solutions were collected into the same tubes. One drop of phenol red and 5 ml of CH3OH were added, and the samples were saponified by boiling for 45–60 min in capped tubes. After cooling and acidification with 200 μl of 12 M HCl, fatty acids were extracted three times with 4 ml of petroleum ether each time. The combined petroleum ether extracts were evaporated to dryness. Residua were dissolved in scintillation fluid and counted for radioactivity.
      The activities of ACC and FAS are expressed as nanomoles of [1-14C]acetyl-CoA incorporated into fatty acids per minute per milligram of protein.

      HMGCR activity assay

      The HMGCR activity assay was performed essentially as described by Volpe and Hennessy (
      • Volpe J.J.
      • Hennessy S.W.
      Cholesterol biosynthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured glial and neuronal cells. Regulation by lipoprotein and by certain free sterols.
      ). Briefly, C6 cells were seeded at a density of 2 × 106 cells per 100 mm diameter Petri dish. At 24 h after plating, medium was changed and, after another 24 h, exogenous fatty acid sodium salt was added to the serum-rich (10% FBS) medium for 4 h. Afterward, the medium from each Petri dish was discarded and the cells were washed twice with 4 ml of ice-cold PBS. Cells were scraped with a rubber policeman into 1 ml of buffer containing 0.05 M Tris-HCl (pH 7.4) and 0.15 M NaCl. After centrifugation (900 g, 3 min, room temperature), the pellet was frozen once in liquid nitrogen and kept at −80°C until use.
      Cell extracts were prepared by dissolving the thawed pellet of C6 cells in 0.2 ml of buffer containing 50 mM K2HPO4 (pH 7.5), 5 mM DTT, 1 mM EDTA, and 0.25% Brij97. A total of 100–250 μg of protein from cell extract was preincubated for 10 min at 37°C in a total volume of 0.2 ml containing 0.1 M K2HPO4 (pH 7.5), 5 mM DTT, and 2.5 mM NADPH. The reaction was started by the addition of [3-14C]HMG-CoA (75 μM, 1.8 Ci/mol). After incubation at 37°C for 120 min, the reaction was stopped by the addition of 20 μl of 7 M HCl. Conversion to mevalonolactone was carried out with an additional 60 min incubation at 37°C, and the radioactive product was isolated by TLC using toluene-acetone (1:1) as the mobile phase. Silica spots were recovered and subjected to scintillation counting.

      Probe design for the RNase protection assay

      Three fragments of ACC, FAS, and HMGCR cDNA were amplified by reverse transcriptase polymerase chain reaction, as reported by Siculella et al. (
      • Siculella L.
      • Damiano F.
      • Sabetta S.
      • Gnoni G.V.
      n-6 PUFAs downregulate expression of the tricarboxylate carrier in rat liver by transcriptional and posttranscriptional mechanisms.
      ), using rat liver total RNA as the template and the following primers: F1, 5′-GTCATGCCTCCGAGAACC-3′, and R1, 5′-GCCAATCCACTCGAAGACC-3′ (National Center for Biotechnology Information accession number J03808), for the ACC probe; F2, 5′-TTGCCCGAGTCAGAGAACC-3′, and R2, 5′-CGTCCACAATAGCTTCATAGC-3′ (accession number M76767), for the FAS probe; and F3, 5′-CTCACAGGATGAAGTAAGGG-3′, and R3, 5′-CTGAGCTGCCAAATTGGACG-3′ (accession number NM_013134), for the HMGCR probe.
      The amplified products (180, 197, and 244 bp for the ACC, FAS, and HMGCR probes, respectively) were subcloned into pCR 2.1 TOPO vector, and their identities were verified by sequence analysis. After linearization, the recombinant plasmids were used in the in vitro transcription reactions.

      RNase protection assay

      Antisense RNAs were synthesized by an in vitro transcription reaction as reported (
      • Siculella L.
      • Damiano F.
      • Sabetta S.
      • Gnoni G.V.
      n-6 PUFAs downregulate expression of the tricarboxylate carrier in rat liver by transcriptional and posttranscriptional mechanisms.
      ). Nuclear RNA (25 μg) isolated from ∼5 × 106 C6 cells, as described by Chomczynski and Sacchi (
      • Chomczynski P.
      • Sacchi N.
      Single-step method of RNA isolation by acid guanidinium thioacetate-phenol-chloroform extraction.
      ), was hybridized with 2 × 105 cpm of 32P-labeled specific antisense probe in 20 μl of hybridization reaction at 50°C for 16 h. For the normalization, a β-actin antisense 32P-labeled RNA probe was added in each hybridization reaction. Probes were also hybridized with 10 μg of yeast RNA used as a control to test the RNase activity (data not shown). After digestion with RNase A/T1, the protected fragments were separated onto a 6% denaturing polyacrylamide gel. Gels were dried and exposed for radiography, and the intensity of the bands was evaluated by densitometry with Molecular Analyst software.

      Western blot analysis

      Cells grown in six-well dishes were treated with C18:1 cis as indicated above and lysed with a pH 7.5 buffer containing 50 mM HEPES, 250 mM mannitol, 10 mM citrate, 4 mM MgCl2, 20 mM Tris-HCl, 500 mM NaCl, 0.5 μM PMSF, 0.05% Tween 20, 0.5% β-mercaptoethanol, and protease inhibitors. The extracts were heat-denatured for 5 min, and samples containing an equal amount of total protein (25 μg) were loaded on 7% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane (
      • Giudetti A.M.
      • Sabetta S.
      • Summa R.di
      • Leo M.
      • Damiano F.
      • Siculella L.
      • Gnoni G.V.
      Differential effects of coconut oil- and fish oil-enriched diets on tricarboxylate carrier in rat liver mitochondria.
      ). To detect biotinylated ACC, the blot was incubated with peroxidase-conjugated streptavidin at a dilution of 1:4,000 at room temperature for 2 h. To detect HMCGR, the blot was first incubated with HMGCR antibody (dilution, 1:400) for 1 h at room temperature and then for 1 h with donkey anti-goat horseradish peroxidase-conjugated IgG (dilution, 1:5,000). Signals were detected by enhanced chemiluminescence. For signal normalization, α-tubulin detection was used (
      • Zhu Y.
      • Schwarz S.
      • Ahlemeyer B.
      • Grzeschik S.
      • Klumpp S.
      • Krieglstein J.
      Oleic acid causes apoptosis and dephosphorylates Bad.
      ).

      Statistical analysis

      Results shown represent means ± SD of the number of experiments indicated in every case. In each experiment, determinations were carried out in triplicate. Statistical analysis was performed with Student's t-test. Differences were considered statistically significant at P < 0.05.

      RESULTS

      Effect of exogenously added fatty acids on fatty acids and cholesterol synthesis

      After plating, exogenous fatty acids (100 μM) were added to C6 glioma cells and the cultures were incubated, unless specified otherwise, for 4 h. MTT test (Fig. 1), morphological observation, protein assay, and Trypan Blue exclusion showed that treated cells had the same viability as control cells during the experimental period, thus excluding a nonspecific toxic cellular effect of the added fatty acids.
      Figure thumbnail gr1
      Fig. 1.Effects of exogenous fatty acids on C6 cell viability. C6 cells were incubated for 4 h with 100 μM of different exogenous fatty acids in serum-rich medium. Cell viability was estimated by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Values, expressed as percentage of control, are means ± SD of five experiments.
      Radiolabeled acetate was added 1 h before ending the experiment, and its incorporation into fatty acids and cholesterol was monitored. Because acetyl-CoA is a precursor for both fatty acid and cholesterol synthesis, the capability of C6 glioma cells to incorporate acetate into these lipid fractions was measured. In the absence of exogenous fatty acids, a significant activity of cholesterologenesis (1.01 ± 0.04 nmol [1-14C]acetate incorporated/h/mg protein) and fatty acid biosynthesis (8.36 ± 0.43 nmol [1-14C]acetate incorporated/h/mg protein) was observed. After fatty acid addition to the cells, a general decrease in [1-14C]acetate incorporation into total fatty acids as well as into cholesterol was detected (Fig. 2). Both C18:1 cis and trans isomers showed the greatest inhibitory effect (80%) of [1-14C]acetate incorporation into fatty acids, whereas cholesterol synthesis was inhibited by ∼60%, mainly by the C18:1 cis isomer. A smaller reduction of labeled acetate incorporation into both fatty acids and cholesterol was observed by incubating C6 cells with 100 μM PUFAs (i.e., fatty acids from C18:2 to C22:6). The saturated fatty acid C18:0 showed a reducing effect more pronounced on fatty acid synthesis than on cholesterologenesis. As the C18:1 cis isomer showed the greatest inhibitory effect, only this fatty acid was tested in the next experiments.
      Figure thumbnail gr2
      Fig. 2.Effects of exogenous fatty acids on cholesterol and fatty acid synthesis in C6 glioma cells. After an initial 48 h plating period, C6 glioma cells, growing in serum-rich medium, were incubated for 4 h with 100 μM of different exogenous fatty acids. At 3 h, labeled acetate was added and its incorporation into cholesterol and fatty acids was followed. Values, expressed as percentage of control (no addition), are means ± SD of six independent experiments. In each experiment, determinations were carried out in triplicate. Control rates of cholesterol and fatty acid synthesis were 1.01 ± 0.04 and 8.36 ± 0.44 nmol [1-14C]acetate incorporated/h/mg protein, respectively.
      The effect exerted by C18:1 cis was dose-dependent (Fig. 3). A gradual decrease of lipogenic activity was found. The reduction of fatty acid and cholesterol synthesis was already evident at 25 μM C18:1 cis. At this concentration, labeled acetate incorporation into cholesterol and total fatty acids was reduced by ∼15% and 55%, respectively. Maximum inhibitory effect was observed at 100 μM C18:1 cis, at which cholesterol and fatty acid biosynthesis were reduced by ∼60% and 80%, respectively. No significant changes were observed at higher C18:1 cis concentrations.
      Figure thumbnail gr3
      Fig. 3.Dose-dependent effects of C18:1 cis on cholesterol and fatty acid synthesis. C6 cells were incubated with increasing oleic acid concentrations for 4 h in serum-rich medium. After 3 h, labeled acetate was added, and 1 h later its incorporation into cholesterol and fatty acids was stopped. Values, expressed as percentage of control, are means ± SD of six experiments. For control rates of cholesterol and fatty acid synthesis, see the legend to .
      Similar behavior was shown when [3H]water, instead of labeled acetate, was used as an independent index of lipogenic activity (
      • Jungas R.L.
      Fatty acid synthesis in adipose tissue incubated in tritiated water.
      ) (Fig. 4). At 100 μM C18:1 cis concentration, tritium incorporation into cholesterol was reduced by ∼55%, whereas label incorporation into fatty acids was inhibited by ∼70%.
      Figure thumbnail gr4
      Fig. 4.Effects of C18:1 cis on 3H incorporation into cholesterol and fatty acids in C6 cells. After an initial 48 h plating period, C6 glioma cells, growing in serum-rich medium, were incubated for 4 h with 100 μM C18:1 cis fatty acids. At 3 h, [3H]water was added and its incorporation into cholesterol and fatty acids was followed. Values, expressed as percentage of control (no addition), are means ± SD of three independent experiments. In each experiment, determinations were carried out in triplicate. Control rates of cholesterol and fatty acid synthesis were 3.29 ± 0.30 and 9.78 ± 0.78 nmol incorporated/h/mg protein, respectively.
      The time-dependent effect reported in Fig. 5 shows that 100 μM oleic acid addition inhibited lipogenesis already after 1 h of C6 cell incubation, reducing the incorporation of labeled acetate into fatty acids and into cholesterol by ∼60% and 30%, respectively, compared with the control. Maximum reduction of fatty acid (∼75%) and cholesterol (∼50%) synthesis was observed at 4 h of C18:1 cis addition. Incubation for longer than 4 h did not produce any further decrease.
      Figure thumbnail gr5
      Fig. 5.Time-dependent effects of C18:1 cis on cholesterol and fatty acid synthesis. [1-14C]acetate was added at 0, 1, 2, 3, 7, or 11 h, and 1 h later the lipogenic assay was terminated. Cells, growing in serum-rich medium, were in contact with 100 μM C18:1 cis for the indicated times. Rates of cholesterol and fatty acid synthesis in the presence of C18:1 cis are expressed as percentage of control. Each result is the mean of six experiments ± SD. For control rates of cholesterol and fatty acid synthesis, see the legend to .

      Effect of C18:1 cis on radiolabeled acetate incorporation into phospholipids

      The effect of C18:1 cis addition to C6 cells on [1-14C]acetate incorporation into phospholipid fractions and neutral lipids was tested (Fig. 6). A general decrease of labeled precursor incorporation into all phospholipids, and in particular into phosphatidylcholine, the most abundant phospholipid in C6 glioma cells, was observed. Interestingly, no significant change in the incorporation of labeled acetate into neutral lipids was detected after C18:1 cis addition to the cells.
      Figure thumbnail gr6
      Fig. 6.Effects of C18:1 cis on [1-14C]acetate incorporation into various lipid fractions in C6 cells. C6 cells, growing in serum-rich medium, were incubated with 100 μM oleic acid for a total period of 4 h, and labeled acetate was added 1 h before ending the incubation. Then, cells were washed and total lipids were extracted. Phospholipids were resolved by TLC, and the radioactivity associated with the different lipid fractions was counted. Values are means ± SD of five experiments.

      Analysis of newly synthesized radiolabeled fatty acids

      To investigate the effect of C18:1 cis addition to C6 cells on the individual fatty acids synthesized from labeled acetate, an HPLC analysis of a total fatty acid extract was carried out. Moreover, in these experiments, a comparison was also made with a saturated fatty acid (C18:0) or a PUFA (C20:4) added to the cells.
      The results in Fig. 7 show that in the control dishes, palmitic acid (C16:0) was the most prominent newly synthesized fatty acid; a noticeable label incorporation into stearic acid and oleic acid was also observed. A relatively small incorporation of label into PUFAs was found, in accordance with the fact that these fatty acids are hardly present in C6 cells (
      • Robert J.
      • Montaudon D.
      • Hugues P.
      Incorporation and metabolism of exogenous fatty acids by cultured normal and tumoral glial cells.
      ). However, exogenous C18:1 cis showed the greatest inhibitory effect on [1-14C]acetate incorporation into individual fatty acids. The decrease of radiolabel incorporation was particularly evident with regard to C16:0, which in the cell is the main product of de novo fatty acid biosynthesis, as well as C18:0 and C18:1 cis.
      Figure thumbnail gr7
      Fig. 7.Effects of different exogenous fatty acids on [1-14C]acetate incorporation into individual fatty acids. The effects of 100 μM C18:0, C18:1 cis, and C20:4 on the incorporation of labeled acetate into different fatty acids were assayed. The radiolabeled neosynthesized fatty acids were separated by HPLC. Eluted fractions, corresponding to the different fatty acids, were collected for radioactivity measurement. Values are means ± SD of six experiments.

      ACC, FAS, and HMGCR activity modulation by saturated, monounsaturated, and polyunsaturated fatty acids

      On the basis of the data reported in Fig. 7, the enzymatic activities of de novo fatty acid synthesis (i.e., ACC and FAS) were investigated with an in situ assay using digitonin-permeabilized C6 cells. This tool offers the advantage of a rapid measurement of intracellular enzyme activities in a more or less natural environment, thus reducing the necessity of preparing cellular fractions for enzyme assays (
      • Geelen M.J.H.
      The use of digitonin-permeabilized mammalian cells for measuring enzyme activities in the course of studies on lipid metabolism.
      ). In a first series of experiments (data not shown), the optimal concentration of digitonin and the time of exposure to digitonin necessary to permeabilize the C6 plasma membrane, without affecting subcellular organelle integrity, were determined. HMGCR activity was investigated by following labeled mevalonolactone synthesis (
      • Volpe J.J.
      • Hennessy S.W.
      Cholesterol biosynthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured glial and neuronal cells. Regulation by lipoprotein and by certain free sterols.
      ).
      The comparative effects of 100 μM C18:0, C18:1 cis, or C20:4 on ACC, FAS, and HMGCR activities were then investigated in C6 glioma cells. FAS activity was not affected by any fatty acid addition, whereas ACC activity was reduced (∼50%) by C20:4 and (∼80%) by C18:0 and C18:1 cis addition to the cells (Fig. 8A). Moreover, stearic and arachidonic acids similarly decreased HMGCR activity (∼20%), whereas oleic acid showed the strongest inhibitory effect on this enzyme (∼45%) (Fig. 8A).
      Figure thumbnail gr8
      Fig. 8.Effects of saturated, monounsaturated, and polyunsaturated fatty acids on acetyl-coenzyme A carboxylase (ACC), FAS, and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) activities. Acetyl-CoA carboxylase and fatty acid synthase activities were tested with an in situ assay in digitonin-permeabilized C6 cells. HMGCR activity was studied by following labeled mevalonolactone synthesis. A: C6 cells were incubated with 100 μM C18:0, C18:1 cis, and C20:4 for a total period of 4 h in serum-rich medium. After the incubation with exogenous fatty acids, culture medium was removed and, for ACC and FAS assay, C6 glioma cells were permeabilized using 400 μl of assay mixture containing digitonin (400 μg/ml) and no exogenous fatty acid. Values, expressed as percentage of the control C6 cells, are means ± SD of five experiments. In each experiment, determinations were carried out in triplicate. B: C6 cells, growing in serum-rich medium, were incubated with either 25 or 100 μM oleic acid for a total period of 4 h, as above. Values, expressed as percentage of control, are means ± SD of four independent experiments. Control specific activities were as follows: for ACC, 0.152 ± 0.005 nmol [1-14C]acetyl-CoA incorporated/min/mg protein; for FAS, 0.041 ± 0.002 nmol [1-14C]acetyl-CoA incorporated/min/mg protein; for HMGCR, 38.6 ± 2.4 pmol mevalonolactone formed/min/mg protein.
      To deepen the oleic acid effect, we next tested two oleic acid concentrations on ACC, FAS, and HMGCR activities. We found that the addition of either 25 or 100 μM C18:1 cis to C6 cells for 4 h decreased ACC activity by ∼40% and 70%, respectively (Fig. 8B). Again, FAS activity was not affected by any tested C18:1 cis concentration. Finally, in good agreement with the data reported in Figs. 2 and 3 on [1-14C]acetate incorporation into cholesterol, we found that the addition of either 25 or 100 μM C18:1 cis to C6 cells decreased HMGCR activity by ∼20% and 45%, respectively.

      Effects of C18:1 cis on mRNA abundance and protein levels of ACC, FAS, and HMGCR

      To investigate the molecular mechanism responsible for the regulation of lipogenic activity by C18:1 cis, the levels of mRNA encoding for ACC and FAS were measured by RNase protection assay. Using ACC and HMGCR probes, we found that the amount of protected ACC and HMGCR RNAs was decreased by ∼55% and 35%, respectively, in C6 cells upon 100 μM C18:1 cis treatment (Fig. 9A). The reduction of both ACC and HMGCR mRNA abundances was already significant at 25 μM C18:1 cis. By contrast, no significant change in the abundance of FAS mRNA was observed. The amount of β-actin mRNA, used for normalization, was unmodified.
      Figure thumbnail gr9
      Fig. 9.Effects of C18:1 cis on ACC, FAS, and HMGCR mRNA accumulation and protein content in C6 glioma cells. C6 cells were incubated with either 25 or 100 μM oleic acid for a total period of 4 h in serum-rich medium. A: Approximately 25 μg of nuclear RNA from ∼5 × 106 C6 cells was analyzed by RNase Protection Assay and hybridized with 32P-labeled specific antisense probes for ACC, FAS, and HMGCR. For normalization, a β-actin antisense 32P-labeled riboprobe was added in each hybridization reaction. The protected fragments were separated onto a denaturing polyacrylamide gel. The radiolabeled gel was dried and exposed to X-ray film, and the intensity of the resulting bands was evaluated by densitometry with Molecular Analyst software. B: ACC from control C6 cells or from C18:1 cis-treated C6 cells was immunodecorated with peroxidase-conjugated streptavidin and revealed by enhanced chemiluminescence. HMGCR was immunodecorated with specific primary antibody and horseradish peroxidase-conjugated IgG and revealed by enhanced chemiluminescence. The contents of ACC, HMGCR, and α-tubulin, used as a control, were quantified by photodensitometric analysis. Control, control cells; C18:1 25 μM, C6 cells incubated for 4 h with 25 μM C18:1 cis; C18:1 100 μM, C6 cells incubated for 4 h with 100 μM C18:1 cis. For treatment with 25 or 100 μM C18:1 cis versus control, *** P < 0.001; for treatment with 100 μM C18:1 cis versus 25 μM C18:1 cis, ° P < 0.01, °° P < 0.001.
      Furthermore, after 4 h of 100 μM C18:1 cis supplementation, the ACC and HMGCR protein contents, quantified by Western blot analysis, decreased (∼50% and 40%, respectively) in treated cells compared with controls (Fig. 9B). Significant reductions of ACC and HMGCR protein levels were observed also at 25 μM C18:1 cis.

      DISCUSSION

      Studies from a number of research groups have established that variations in dietary fatty acid levels are able to change the pattern of fatty acyl moieties as well as the cholesterol content of neuronal and glial membranes. These changes, in turn, can influence cellular metabolism and regulatory processes (
      • Murphy M.G.
      Effects of exogenous linoleic acid on fatty acid composition, receptor-mediated cAMP formation and transport functions in rat astrocytes in primary culture.
      ,
      • Marteinsdottir I.
      • Horrobin D.F.
      • Stenfors C.
      • Theodorsson E.
      • Mathé. A.A.
      Changes in dietary fatty acids alter phospholipid fatty acid composition in selected regions of rat brain.
      ,
      • Fernstrom J.D.
      Effects of dietary polyunsaturated fatty acids on neuronal function.
      ). It has also been reported that cultured astrocytes take up exogenous linoleic acid (C18:2) and incorporate its metabolites into phospholipids, modifying their membrane fatty acid composition and certain specific cell properties (
      • Murphy M.G.
      Effects of exogenous linoleic acid on fatty acid composition, receptor-mediated cAMP formation and transport functions in rat astrocytes in primary culture.
      ). These biochemical changes can be accompanied by alterations in the physical state of the membrane, for example in membrane fluidity (
      • Marteinsdottir I.
      • Horrobin D.F.
      • Stenfors C.
      • Theodorsson E.
      • Mathé. A.A.
      Changes in dietary fatty acids alter phospholipid fatty acid composition in selected regions of rat brain.
      ). Moreover, the addition of C18:1 cis and C18:2 to cultured astrocytes decreases several aminopeptidase activities (
      • Ramírez-Expósito M.J.
      • García M.J.
      • Mayas M.D.
      • Ramírez M.
      • Martínez-Martos J.M.
      Effects of exogenous fatty acids and cholesterol on aminopeptidase activities in rat astroglia.
      ). Furthermore, it has been shown that C18:1 cis addition determines a dose-dependent inhibition of GAP junction permeability in cultured rat astrocytes. The authors suggested that C18:1 cis may play an important role in the transduction pathway leading to the inhibition of intracellular communication (
      • Lavado E.
      • Sánchez-Abarca L.I.
      • Tabernero A.
      • Bolaños J.P.
      • Medina J.M.
      Oleic acid inhibits GAP junction permeability and increases glucose uptake in cultured rat astrocytes.
      ).
      However, despite the crucial role of fatty acids in brain function and metabolism, little is known about the effect of exogenous fatty acids on lipogenesis in brain cells (
      • Volpe J.J.
      • Marasa J.C.
      Regulation of palmitic acid synthesis in cultured glial cells: effects of lipid on fatty acid synthetase, acetyl-CoA carboxylase, fatty acid and cholesterol synthesis.
      ,
      • Volpe J.J.
      • Marasa J.C.
      Short term regulation of fatty acid synthesis in cultured glial and neuronal cells.
      ). Therefore, the two major aims in this study were as follows: i) to determine the capability of glioma cells to synthesize different lipid fractions, starting from labeled acetate; and ii) to investigate the effect of exogenous fatty acids on fatty acid and cholesterol biosynthesis in these cells. We show that both fatty acid and cholesterol biosynthesis are rather active in cultured glioma cells when [1-14C]acetate is used as the common precursor for both metabolic pathways. In fact, in control cells (i.e., in the absence of any addition to the culture medium), specific activities of 8.36 ± 0.43 nmol [1-14C]acetate incorporated into fatty acids/h/mg protein and 1.01 ± 0.04 nmol [1-14C]acetate incorporated into cholesterol/h/mg protein were found. It must be emphasized that these values are much higher than those observed previously, under similar experimental conditions, in cultured hepatocytes with regard to both fatty acid synthesis and cholesterogenesis (
      • Gnoni G.V.
      • Geelen M.J.H.
      • Bijleveld C.
      • Quagliariello E.
      • Van den Bergh S.G.
      Short-term stimulation of lipogenesis by triiodothyronine in maintenance cultures of rat hepatocytes.
      ,
      • Gnoni G.V.
      • Landriscina C.
      • Quagliariello E.
      Thyroid hormone stimulation of lipogenesis in isolated rat hepatocytes.
      ). Our findings on lipid biosynthesis add further support to previous studies showing very active de novo fatty acid and cholesterol synthesis in human malignant glial cells compared with their normal counterparts (
      • Fumagalli R.
      • Grossi E.
      • Paoletti P.
      • Paoletti R.
      Studies on lipids in brain tumors. Occurrence and significance of sterol precursor of cholesterol in human brain tumors.
      ,
      • Prasanna P.
      • Thibault A.
      • Liu L.
      • Samid D.
      Lipid metabolism as a target for brain cancer therapy: synergistic activity of lovastatin and sodium phenylacetate against human glioma cells.
      ,
      • Brusselmans K.
      • Schrijver E.De
      • Verhoeven G.
      • Swinnen J.V.
      RNA interference-mediated silencing of the acetyl-CoA-carboxylase-α gene induces growth inhibition and apoptosis of prostate cancer cells.
      ).
      A general decrease of fatty acid synthesis by exogenous fatty acids was observed; the reduction was particularly pronounced when the C18:1 cis or C18:1 trans isomer was added to the culture medium. Overall, the reduction of cholesterogenesis by exogenous fatty acids was often less pronounced, compared with fatty acid synthesis, especially in the case of C18:1 trans. It might be argued that the observed reduction of label incorporation into fatty acids and cholesterol after fatty acid addition could be attributable to the precursor-dilution effect by exogenous fatty acids. β-Oxidation of fatty acids and consequently the generation of acetyl-CoA might dilute the [14C]acetyl-CoA pool derived from [14C]acetate and may lead to a lower apparent synthesis rate of 14C-labeled fatty acids and [14C]cholesterol. To circumvent this problem, experiments were carried out using [3H]water incorporation into lipids as an independent index of lipogenic activity (
      • Jungas R.L.
      Fatty acid synthesis in adipose tissue incubated in tritiated water.
      ). The rate of tritium incorporation into fatty acids and cholesterol was reduced by exogenous fatty acids in a manner similar to that measured by the incorporation of [1-14C]acetate. Thus, the occurrence of a dilution effect is unlikely.
      Of importance, the reduction of fatty acid synthesis by C18:1 cis addition to C6 glioma cells reached a maximum (80%) at 4 h, but it was already evident within 1 h of incubation, thus indicating that short-term regulation may cooperate with long-term mechanisms in the reduction of lipogenesis by exogenous fatty acids in C6 cells (
      • Volpe J.J.
      • Marasa J.C.
      Regulation of palmitic acid synthesis in cultured glial cells: effects of lipid on fatty acid synthetase, acetyl-CoA carboxylase, fatty acid and cholesterol synthesis.
      ).
      De novo fatty acid synthesis is catalyzed by two enzymatic systems functioning in sequence: ACC (generally considered the rate-determining step of this metabolic pathway), which leads to the production of malonyl-CoA, and FAS, leading to the formation of the end product C16:0. Interestingly, the results in Fig. 8A showing the inhibition of ACC activity by exogenous fatty acids are in good agreement with those reported in Fig. 2 regarding total fatty acid synthesis. Therefore, the global inhibitory effect we observed most likely can be ascribed to the rapid inhibition of ACC activity (in which allosteric regulation and phosphorylation-dephosphorylation of the enzyme could participate) and the downregulation of ACC expression exerted by C18:1 cis treatment. Volpe and Marasa (
      • Volpe J.J.
      • Fujimoto K.
      • Marasa J.C.
      • Agrawal H.C.
      Relation of C-6 glial cells in culture to myelin.
      ) also found that in C6 cells, ACC activity was downregulated (within seconds to minutes) by exogenous long-chain acyl-CoAs, whereas no significant change of FAS activity was observed. However, unlike us, they did not find any changes in either RNA and protein synthesis involved in the mechanism of ACC regulation. The very short incubation time they used to investigate the molecular mechanism underlying ACC regulation may explain this discrepancy.
      Lastly, because ACC is not required for cholesterol synthesis, the possibility that supplemented fatty acids could impair some enzymatic step(s) directly connected with this pathway, such as HMGCR, was considered. To this end, HMGCR activity, as well as the corresponding mRNA and protein levels, were investigated. Our data suggest that the inhibition of cholesterol biosynthesis in C18:1 cis-treated cells might be ascribed, at least in part, to a reduced activity and expression of HMGCR. Therefore, these findings could have potential implications in some brain dysfunction in which alteration of lipid metabolism occurs. Several investigations have shown an abnormally active synthesis of cholesterol from acetate in malignant glial cells compared with their normal counterparts (
      • Fumagalli R.
      • Grossi E.
      • Paoletti P.
      • Paoletti R.
      Studies on lipids in brain tumors. Occurrence and significance of sterol precursor of cholesterol in human brain tumors.
      ,
      • Prasanna P.
      • Thibault A.
      • Liu L.
      • Samid D.
      Lipid metabolism as a target for brain cancer therapy: synergistic activity of lovastatin and sodium phenylacetate against human glioma cells.
      ). The inhibition of HMGCR activity and expression by exogenous fatty acids reported in this study adds further support to previous findings (
      • Sykes J.E.
      • Lopes-Cardozo M.
      Effect of exogenous fatty acids on lipid synthesis, marker-enzymes, and development of glial cells maintained in serum-free culture.
      ,
      • Leonardi F.
      • Attorri L.
      • Benedetto R.Di
      • Biase A.Di
      • Sanchez M.
      • Nardini M.
      • Salvati S.
      Effect of arachidonic, eicosapentaenoic and docosahexaenoic acids on the oxidative status of C6 glioma cells.
      ), indicating that fatty acids exogenously added to C6 cells may represent specific means of controlling gliomatous growth.
      Overall, this study clearly shows rather active lipogenic and cholesterologenic activities of C6 glioma cells and a downregulation of fatty acid and cholesterol synthesis by exogenous fatty acids, mainly C18:1 cis. The reduced ACC and HMGCR expression may be considered important factors in this regulation. Experiments are in progress in our laboratory to ascertain the possible level of this regulation (i.e., transcriptional and/or posttranscriptional).

      Acknowledgments

      The authors thank Dr. Math J. H. Geelen for discussions and critical reading of the manuscript.

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