Oleic acid is a potent inhibitor of fatty acid and cholesterol synthesis in C6 glioma cells
2007; Elsevier BV; Volume: 48; Issue: 9 Linguagem: Inglês
10.1194/jlr.m700051-jlr200
ISSN1539-7262
AutoresFrancesco Natali, Luisa Siculella, Serafina Salvati, Gabriele V. Gnoni,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoGlial 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. 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. acetyl-coenzyme A carboxylase 3-hydroxy-3-methylglutaryl coenzyme A reductase 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 (1.Dexter D.T. Carter C.J. Wells F.R. Javoy-Agid F. Agid Y. Lees A. Jenner P. Marsden C.D. Basal lipid peroxidation in substantia nigra is increased in Parkinson's disease.J. Neurochem. 1989; 52: 381-389Crossref PubMed Scopus (1227) Google Scholar, 2.Van Geel B.M. Assies J. Wanders R.J.A. Barth P.G. X-linked adrenoleukodystrophy: clinical presentation, diagnosis, and therapy.J. Neurol. Neurosurg. Psychiatry. 1997; 63: 4-14Crossref PubMed Scopus (114) Google Scholar, 3.Lukiw W.J. Papolla M. Pelaez R.Palacios Bazan N.G. Alzheimer's disease—a dysfunction in cholesterol and lipid metabolism.Cell. Mol. Neurobiol. 2005; 25: 475-483Crossref PubMed Scopus (36) Google Scholar). 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 (4.Bezzi P. Volterra A. A neuron-glia signalling network in the active brain.Curr. Opin. Neurobiol. 2001; 11: 387-394Crossref PubMed Scopus (360) Google Scholar, 5.Hansson E. Rönnbäck L. Glial neuronal signaling in the central nervous system.FASEB J. 2003; 17: 341-348Crossref PubMed Scopus (252) Google Scholar). Metabolic regulation in the brain has been investigated extensively, and those studies focused mostly on carbohydrate and amino acid metabolism (for review, see Ref. 6.Tsacopoulos M. Metabolic signaling between neurons and glial cells: a short review.J. Physiol. (Paris). 2002; 96: 283-288Crossref PubMed Scopus (41) Google Scholar). 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 (6.Tsacopoulos M. Metabolic signaling between neurons and glial cells: a short review.J. Physiol. (Paris). 2002; 96: 283-288Crossref PubMed Scopus (41) Google Scholar). Regarding lipid metabolism, astroglial ketone body synthesis, showing characteristics strikingly similar to those of hepatic ketogenesis (7.Guzmán M. Blázquez C. Ketone body synthesis in the brain: possible neuroprotective effects.Prostaglandins Leukot. Essent. Fatty Acids. 2004; 70: 287-292Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), 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 (8.Bazan Jr, N.G. Effects of ischemia and electroconvulsive shock on free fatty acid pool in the brain.Biochim. Biophys. Acta. 1970; 218: 1-10Crossref PubMed Scopus (688) Google Scholar, 9.Zhu Y. Schwarz S. Ahlemeyer B. Grzeschik S. Klumpp S. Krieglstein J. Oleic acid causes apoptosis and dephosphorylates Bad.Neurochem. Int. 2005; 46: 127-135Crossref PubMed Scopus (59) Google Scholar). It has been shown that exogenous fatty acids may influence the fatty acid composition of neuronal and glial membranes (10.Bourre J.M. Biochemistry of brain lipids (especially fatty acids). In situ synthesis and exogenous origin during development. Various aspects of nutritional effects.Reprod. Nutr. Dev. 1982; 22: 179-191Crossref PubMed Scopus (5) Google Scholar, 11.Rapoport S.I. In vivo fatty acid incorporation into brain phospholipids in relation to signal transduction and membrane remodeling.Neurochem. Res. 1999; 24: 1403-1415Crossref PubMed Scopus (55) Google Scholar, 12.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.Neurochem. Res. 1997; 22: 327-331Crossref PubMed Scopus (9) Google Scholar, 13.Horrocks L.A. Farooqui A.A. Docosahexaenoic acid in the diet: its importance in maintenance and restoration of neural membrane function.Prostaglandins Leukot. Essent. Fatty Acids. 2004; 70: 361-372Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 14.Contreras M.A. Rapoport S.I. Recent studies on interactions between n-3 and n-6 polyunsaturated fatty acids in brain and other tissues.Curr. Opin. Lipidol. 2002; 13: 267-272Crossref PubMed Scopus (73) Google Scholar), and these changes in turn can affect cellular metabolism and regulatory (15.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.J. Neurochem. 2004; 88: 1041-1051Crossref PubMed Scopus (50) Google Scholar) and inflammatory processes (16.Farooqui A.A. Horrocks L.A. Farooqui T. Modulation of inflammation in brain: a matter of fat.J. Neurochem. 2007; 101: 577-599Crossref PubMed Scopus (336) Google Scholar). 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 (17.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.Cell Biochem. Funct. 2002; 20: 285-290Crossref PubMed Scopus (5) Google Scholar). 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 (10.Bourre J.M. Biochemistry of brain lipids (especially fatty acids). In situ synthesis and exogenous origin during development. Various aspects of nutritional effects.Reprod. Nutr. Dev. 1982; 22: 179-191Crossref PubMed Scopus (5) Google Scholar, 18.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.J. Neurochem. 1975; 25: 333-340Crossref PubMed Scopus (36) Google Scholar, 19.Volpe J.J. Marasa J.C. Short term regulation of fatty acid synthesis in cultured glial and neuronal cells.Brain Res. 1977; 120: 91-106Crossref Scopus (10) Google Scholar, 20.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.Biochim. Biophys. Acta. 1977; 486: 408-420Crossref PubMed Scopus (62) Google Scholar) 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 (21.Volpe J.J. Fujimoto K. Marasa J.C. Agrawal H.C. Relation of C-6 glial cells in culture to myelin.Biochem. J. 1975; 152: 701-703Crossref PubMed Scopus (68) Google Scholar, 22.McMorris F.A. Norepinephrine induces glial-specific enzyme activity in cultured glioma cells.Proc. Natl. Acad. Sci. USA. 1977; 74: 4501-4505Crossref PubMed Scopus (74) Google Scholar) and exhibits a prevalent astrocyte-like phenotype when cultured in serum-rich medium (23.Nave K.A. Lemke G. Induction of the myelin proteolipid protein (PLP) gene in C6 glioblastoma cells: functional analysis of the PLP promoter.J. Neurosci. 1991; 11: 3060-3069Crossref PubMed Google Scholar). 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. 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. 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, cis-Δ9-octadecaenoic acid; C18:1 trans, elaidic acid, trans-Δ9-octadecaenoic acid; C18:2, linoleic acid, all-cis-Δ9,12-octadecadienoic acid; C20:4, arachidonic acid, all-cis-Δ5,8,11,14-eicosatetraenoic acid; C20:5, all-cis-Δ5,8,11,14,17-eicosapentaenoic acid; C22:6, all-cis-Δ4,7,10,13,16,19-docosahexaenoic acid) were added to the serum-rich (10% FBS) medium, obtaining 100 μM final concentration, as reported by Salvati et al. (24.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.Neurochem. Int. 2004; 44: 331-338Crossref PubMed Scopus (26) Google Scholar). 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. 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. 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 (25.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.Biochem. Biophys. Res. Commun. 1985; 128: 525-530Crossref PubMed Scopus (28) Google Scholar). 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 (26.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), 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. 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. (27.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.Endocrinology. 2005; 146: 3959-3966Crossref PubMed Scopus (25) Google Scholar). Phospholipids were resolved by TLC on silica gel plates using CHCl3/CH3OH/28% NH4OH (65:25:4) as a developing system (28.Touchstone J.C. Thin-layer chromatographic procedures for lipid separation.J. Chromatogr. B. 1995; 671: 169-195Crossref PubMed Scopus (130) Google Scholar). 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. Total extracted fatty acids were separated by HPLC using a modification of the method described by Mehta, Oeser, and Carlson (29.Mehta A. Oeser A.M. Carlson M.G. Rapid quantitation of free fatty acids in human plasma by high-performance liquid chromatography.J. Chromatogr. B Biomed. Sci. Appl. 1998; 719: 9-23Crossref PubMed Scopus (79) Google Scholar). 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. 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 (30.Geelen M.J.H. The use of digitonin-permeabilized mammalian cells for measuring enzyme activities in the course of studies on lipid metabolism.Anal. Biochem. 2005; 347: 1-9Crossref PubMed Scopus (34) Google Scholar). 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 (31.Bijleveld C. Geelen M.J.H. Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes.Biochim. Biophys. Acta. 1987; 918: 274-283Crossref PubMed Scopus (51) Google Scholar) in isolated rat hepatocytes. This method avoids a number of interferences associated with the classical bicarbonate fixation assay of ACC activity (30.Geelen M.J.H. The use of digitonin-permeabilized mammalian cells for measuring enzyme activities in the course of studies on lipid metabolism.Anal. Biochem. 2005; 347: 1-9Crossref PubMed Scopus (34) Google Scholar). 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 (32.Linn T.C. Purification and crystallization of rat liver fatty acid synthetase.Arch. Biochem. Biophys. 1981; 209: 613-619Crossref PubMed Scopus (88) Google Scholar) 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 (30.Geelen M.J.H. The use of digitonin-permeabilized mammalian cells for measuring enzyme activities in the course of studies on lipid metabolism.Anal. Biochem. 2005; 347: 1-9Crossref PubMed Scopus (34) Google Scholar). 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. The HMGCR activity assay was performed essentially as described by Volpe and Hennessy (20.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.Biochim. Biophys. Acta. 1977; 486: 408-420Crossref PubMed Scopus (62) Google Scholar). 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. Three fragments of ACC, FAS, and HMGCR cDNA were amplified by reverse transcriptase polymerase chain reaction, as reported by Siculella et al. (33.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.J. Lipid Res. 2004; 45: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), 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. Antisense RNAs were synthesized by an in vitro transcription reaction as reported (33.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.J. Lipid Res. 2004; 45: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Nuclear RNA (25 μg) isolated from ∼5 × 106 C6 cells, as described by Chomczynski and Sacchi (34.Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thioacetate-phenol-chloroform extraction.Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63191) Google Scholar), 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. 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 (35.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.J. Lipid Res. 2003; 44: 2135-2141Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). 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 (9.Zhu Y. Schwarz S. Ahlemeyer B. Grzeschik S. Klumpp S. Krieglstein J. Oleic acid causes apoptosis and dephosphorylates Bad.Neurochem. Int. 2005; 46: 127-135Crossref PubMed Scopus (59) Google Scholar). 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. 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. 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. 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 an
Referência(s)