Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells
2003; Elsevier BV; Volume: 44; Issue: 2 Linguagem: Inglês
10.1194/jlr.m200335-jlr200
ISSN1539-7262
AutoresFred Y. Xu, William A. Taylor, Jeffrey A. Hurd, Grant M. Hatch,
Tópico(s)Adipose Tissue and Metabolism
ResumoWe examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine.The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL. We examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL. Cardiolipin (CL) is a major membrane phospholipid in most mammalian cells and is localized primarily to the inner mitochondrial membrane where it comprises approximately 20% of the mitochondrial phospholipid mass (1Hostetler K.Y. Polyglycerophospholipids: phosphatidylglycerol, diphosphatidylglycerol and bis(monoacylglycerol)phosphate.in: Hawthorne J.N. Ansell G.B. Phospholipids. Elsevier Press, Amsterdam1982: 215-261Google Scholar, 2Daum G. Lipids of mitochondria.Biochim. Biophys. Acta. 1985; 882: 1-42Google Scholar, 3Poorthuis B.J. Yazaki P.J. Hostetler K.Y. An improved two-dimensional thin layer chromatrography system for the separation of phosphatidylglycerol and its derivatives.J. Lipid Res. 1976; 17: 433-437Google Scholar). In addition, CL has been identified in the outer mitochondrial membrane (4Hovius R. Lambrechts H. Nicolay K. de Kruijff B. Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the outer mitochondrial membrane.Biochim. Biophys. Acta. 1990; 1021: 217-226Google Scholar, 5Hovius R. Thijssen J. van der Linden P. Nicolay K. de Kruijff B. Phospholipid asymmetry of the outer membrane of rat liver mitochondria. Evidence for the presence of cardiolipin on the outside of the outer membrane.FEBS Lett. 1993; 330: 71-76Google Scholar). CL was shown to be required for the reconstituted activity of a number of key mammalian mitochondrial enzymes involved in cellular energy metabolism (6Vik S.B. Georgevich G. Capaldi R.A. Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase.Proc. Natl. Acad. Sci. USA. 1981; 78: 1456-1460Google Scholar, 7Fiol C.J. Bieber L.L. Sigmoidal kinetics of purified beef heart mitochondrial carnitine palmitoyltransferase.J. Biol. Chem. 1984; 259: 13084-13088Google Scholar, 8Muller M. Moser R. Cheneval D. Carafoli E. Cardiolipin is the membrane receptor for creatine phosphokinase.J. Biol. Chem. 1985; 260: 3839-3843Google Scholar, 9Hutson S.M. Roten S. Kaplan R.S. Solubilization and reconstitution of the branched-chain-alpha-keto acid transporter from rat heart mitochondria.Proc. Natl. Acad. Sci. USA. 1990; 87: 1028-1031Google Scholar, 10Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. Purification and characterization of the reconstitutively active tricarboxylate transporter from rat liver mitochondria.J. Biol. Chem. 1990; 265: 13379-13385Google Scholar, 11Belezani Z.S. Janesik V. Role of cardiolipin in the function of the L-glycerol-3-phosphate dehydrogenase.Biochem. Biophys. Res. Commun. 1989; 159: 132-139Google Scholar, 12Kadenbach B. Mende P. Kolbe H.V. Stipani I. Palmieri F. The mitochondrial phosphate carrier has an essential requirement for cardiolipin.FEBS Lett. 1982; 139: 109-112Google Scholar, 13Hoffman B. Stockl A. Schlame M. Beyer K. Klingenberg M. The reconstituted ADP/ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine mutants.J. Biol. Chem. 1994; 269: 1940-1944Google Scholar, 14Eble K.S. Coleman W.B. Hantgan R.R. Cunningham C.C. Tightly associated cardiolipin in bovine heart mitochondrial ATP synthase as analysed by 31P nuclear magnetic resonance spectroscopy.J. Biol. Chem. 1990; 265: 19434-19440Google Scholar). More recently, peroxidation of CL in mitochondria of rat basophile leukemia cells resulted in dissociation of cytochrome c from mitochondrial inner membranes, an initial step in cytochrome c-mediated apoptosis (15Nomura K. Imai H. Koumura T. Kobayashi T. Nakagawa Y. Mitochondrial hydroperoxide glutathione peroxidase inhibits the release of cytochrome c from mitochondria by suppressing the peroxidation of cardiolipin in hypoglycemia-induced apoptosis.Biochem. J. 2000; 351: 183-193Google Scholar). In mammalian tissues, CL contains four fatty acid side chains occupied primarily by unsaturated fatty acids of 16–18 carbons in length (16Schlame M. Brody S. Hostetler K.Y. Mitochondrial cardiolipin in diverse eukaryotes: comparison of biosynthetic reactions and molecular acyl species.Eur. J. Biochem. 1993; 212: 727-735Google Scholar). The fatty acyl species appeared to be the important structural requirement for the high protein binding affinity of CL (17Schlame M. Hovath L. Vigh L. Relationship between lipid saturation and lipid-protein interaction in liver mitochondria modified by catalytic hydrogenation with reference to cardiolipin molecular species.Biochem. J. 1990; 265: 79-85Google Scholar). Dietary modification of the fatty acyl species composition of CL was shown to alter the oxygen consumption in cardiac mitochondria (18Yamaoka S. Urade R. Kido M. Cardiolipin molecular species in rat heart mitochondria are sensitive to essential fatty acid-deficient dietary lipids.J. Nutr. 1990; 120: 415-421Google Scholar, 19Yamaoka-Koseki S. Urade R. Kito M. Cardiolipins from rats fed different diets affect bovine heart cytochrome c oxidase activity.J. Nutr. 1991; 121: 956-958Google Scholar). The specific activity of cytochrome c oxidase reconstituted with CL varied markedly and significantly with different molecular species of the phospholipid. Thus, the appropriate fatty acyl composition of CL is important for optimum mitochondrial respiratory activity. CL de novo biosynthesis begins with the conversion of phosphatidic acid (PtdOH) to cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol (CDP-DG) catalyzed by PtdOH:CTP cytidylyltransferase (EC 2.7.7.41) (20Kiyasu J.Y. Pieriniger R.A. Paulus H. Kennedy E.P. The biosynthesis of phosphatidylglycerol.J. Biol. Chem. 1963; 238: 2293-2298Google Scholar). CDP-DG is then condensed with sn-glycerol-3-phosphate to form phosphatidylglycerol (PtdGro) phosphate catalyzed by PtdGro phosphate synthase (EC 2.7.8.5). The PtdGro phosphate is immediately converted to PtdGro by a highly active PtdGro phosphate phosphatase (EC 3.1.3.27). Finally, PtdGro is condensed with another molecule of CDP-DG to form CL catalyzed by CL synthase (21Hostetler K.Y. Van den Bosch H. Van Deenen L.L.M. Biosynthesis of cardiolipin in rat liver.Biochim. Biphys. Acta. 1971; 239: 113-119Google Scholar). The de novo CL biosynthetic enzymes lack fatty acyl species specificity (22Rustow B. Schlame M. Rabe H. Reichman G. Kunze D. Species pattern of phosphatidic acid, diacylglycerol, CDP-diacylglycerol and phosphatidylglycerol synthesized de novo in rat liver mitochondria.Biochim. Biophys. Acta. 1989; 1002: 261-263Google Scholar, 23Hostetler K.Y. Galesloot J.M. Boer P. van den Bosch H. Further studies on the formation of cardiolipin and phosphatidylglycerol in rat liver mitochondria: effect of divalent cations and the fatty acid composition of CDP-diglyceride.Biochim. Biophys. Acta. 1975; 380: 382-389Google Scholar). Thus, like other phospholipids, newly synthesized CL must be rapidly remodeled by a deacylation-reacylation pathway to obtain fatty acyl groups (24Schlame M. Rustow B. Lysocardiolipin formation and regulation in isolated rat liver mitochondria.Biochem. J. 1990; 272: 589-595Google Scholar). The enzyme responsible for CL deacylation in the mitochondria is phospholipase A2 (25Nachbaur J. Vignais P.M. Localization of phospholipase A2 in outer membrane of mitochondria.Biochem. Biophys. Res. Commun. 1968; 33: 315-320Google Scholar, 26Severina E.P. Evtodienko I. Phospholipase A2 localization in mitochondria.Biokhimiia. 1981; 46: 1199-1201Google Scholar, 27Levrat C. Louisot P. Dual localization of the mitochondrial phospholipase A2: outer membrane contact sites and inner membrane.Biochem. Biophys. Res. Commun. 1992; 183: 719-724Google Scholar). We have characterized the activity of monolysocardiolipin acyltransferase (MLCL AT) specific for the acylation of MLCL to CL in the heart and mammalian tissues (28Ma B.J. Taylor W.A. Dolinsky V. Hatch G.M. Acylation of monolysocardiolipin in rat heart.J. Lipid Res. 1999; 40: 1837-1845Google Scholar). Recent studies support the hypothesis of functionally independent acyl-CoA pools within mammalian cells that may be channeled toward specific fates rather than being freely available for all possible enzymatic reactions (29Muoio D.M. Lewin T.M. Weidmar P. Coleman R.A. Acyl-CoA’s are functionally channelled in liver: Potential role of acyl-CoA synthetase.Am. J. Physiol. 2000; 279: E1366-E1373Google Scholar, 30Fulgencio J.P. Kohl C. Girard J. Pegorier J.P. Troglitazone inhibits fatty acid oxidation and esterification, and gluconeogenesis in isolated hepatocytes from starved rats.Diabetes. 1996; 45: 1556-1562Google Scholar, 31Coleman R.A. Lewin T.M. Muoio D.M. Physiological and nutritional regulation of enzymes of triacylglycerol synthesis.Annu. Rev. Nutr. 2000; 20: 77-103Google Scholar, 32Hatch G.M. Smith A.J. Xu F.Y. Hall A.M. Bernlohr D.A. FATP1 channels exogenous FA into 1,2,3-triacyl-sn-glycerol and down-regulates sphingomyelin and cholesterol metabolism in growing 293 cells.J. Lipid Res. 2002; 43 (In press)Google Scholar). Etomoxir is a member of the oxirane carboxylic acid carnitine palmitoyl transferase I inhibitors and has been suggested as a therapeutic agent for the treatment of heart failure (33Bartlett K. Turnball D.M. Sherratt H.S.A. Inhibition of hepatic and skeletal muscle carnitine palmitoyltransferase I by 2[5(4-chlorophenyl)pentyl]-oxirane-2-carboxyl-CoA.Biochem. Soc. Trans. 1984; 12: 688-689Google Scholar, 34Bristow M. Etomoxir: a new approach to treatment of heart failure.Lancet. 2000; 356: 1621-1622Google Scholar, 35Schmidt-Schweda S. Holubarsch C. First clinical trial with etomoxir in patients with chronic congestive heart failure.Clin. Sci. 2000; 99: 27-35Google Scholar). Acute etomoxir treatment irreversibly inhibits the activity of carnitine palmitoyltransferase I. As a result, fatty acid import into the mitochondria and β-oxidation is reduced, whereas cytosolic fatty acid accumulates and glucose oxidation is elevated. Prolonged incubation (24 h) with etomoxir produces diverse effects on the expression of several metabolic enzymes (36Hegardt F.G. Serra D. Asins G. Influence of etomoxir on the expression of several genes in liver, testes and heart.Gen. Pharmacol. 1995; 26: 897-904Google Scholar). It had been postulated that acute etomoxir treatment might stimulate de novo phospholipid biosynthesis in the mammalian heart (37Dhalla N.S. Elimban V. Rupp H. Paradoxical role of lipid metabolism in heart function and dysfunction.Mol. Cell. Biochem. 1992; 116: 3-9Google Scholar). However, this had never been demonstrated. In this study, we examined if acute etomoxir treatment of H9c2 cardiac myoblast cells indeed stimulated de novo CL and phospholipid biosynthesis, and if etomoxir mediated distinct metabolic channeling of fatty acid and glycerol into CL. Our results indicate that etomoxir treatment of H9c2 cardiac myoblast cells produces a diverse plethora of effects on overall de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir produces a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL in H9c2 cells. Rat heart H9c2 myoblastic cells were obtained from American Type Culture Collection. [14C]glycerol-3-phosphate, [5-3H]CTP, [1,3-3H]glycerol, [1-14C]palmitic acid, [1-14C]oleic acid, [1-14C]oleoyl-CoA, [3H]serine, myo-[3H]inositol, [3H]ethanolamine, and [methyl-3H]choline were obtained from either Dupont, Mississauga, Ontario, or Amersham, Oakville, Ontario, Canada. [14C]PtdGro was synthesized from [14C]glycerol-3-phosphate as previously described (38Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells by cytidine-5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Google Scholar). Sodium etomoxir, 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate was obtained from Research Biochemicals Incorporated, Natick, MD. Etomoxiryl-CoA was synthesized as described (39Morillas M. Clotet J. Rubi B. Serra D. Arino J. Hegardt F.G. Asins G. Inhibition by extomoxir of rat liver carnitine octanoyltransferase is produced through the coordinate interaction with two histidine residues.Biochem. J. 2000; 351: 495-502Google Scholar). DMEM and fetal bovine serum were products of Canadian Life Technologies (GIBCO), Burlington, Onatrio, Canada. Lipid standards were obtained from Serdary Research Laboratories, Englewood Cliffs, NJ. MLCL was obtained from Avanti Polar Lipids, Alabaster, AL. Thin layer plates (silica gel G, 0.25 mm thickness) were obtained from Fisher Scientific, Winnipeg, Canada. Ecolite scintillant was obtained from ICN Biochemical, Montreal, Quebec, Canada. All other biochemicals were certified ACS grade or better and obtained from either Sigma Chemical Co., MO or Fisher Scientific. Rat heart H9c2 myoblastic cells were incubated in DMEM containing 10% fetal bovine serum until near confluence. In some experiments, cells were preincubated for 2 h with DMEM (serum-free) in the absence or presence of 1–80 μM etomoxir and then incubated for 2 h with 0.1 mM [1-14C]oleic acid (10 μCi/dish, bound to BSA in a 1:1 molar ratio). In other experiments, cells were preincubated for 2 h plus or minus 40 μM etomoxir and then incubated for 2 h with 0.1 μM or 0.1 mM [1,3-3H]glycerol (10 μCi/dish), 0.1 mM [1-14C]oleic acid (2 μCi/dish, bound to BSA in a 1:1 molar ratio), 0.1 mM [1-14C]palmitic acid (2 μCi/dish, bound to BSA in a 1:1 molar ratio), 28 μM [3H]ethanolamine (2 μCi/dish), 28 μM [methyl-3H]choline (2 μCi/dish), 0.4 mM [3H]serine (20 μCi/dish), or 40 μM myo-[3H]inositol (10 μCi/dish). The medium was removed and the cells washed twice with ice-cold saline and then harvested from the dish with 2 ml methanol-water (1:1, v/v) for lipid extraction. An aliquot of the homogenate was taken for the determination of total uptake of radioactivity into cells. Phospholipids were then isolated and radioactivity in these determined as previously described (38Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells by cytidine-5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Google Scholar). For enzyme studies, H9c2 cells were washed twice with ice-cold saline and harvested with 2 ml homogenization buffer (10 mM Tris-HCL, pH 7.4, 0.25 M sucrose). The cells were homogenized with 15 strokes of a Dounce A homogenizer. The homogenate was centrifuged at 1,000 g for 5 min. The resulting supernatant was centrifuged at 10,000 g for 15 min. The resulting pellet was resuspended in 1 ml of homogenizing buffer and designated the mitochondrial fraction. The resulting supernatant was centrifuged at 100,000 g for 60 min to obtain the cytosolic fraction. The resulting pellet from this centrifugation was resuspended in 1 ml of homogenizing buffer and designated the microsomal fraction. Glycerol kinase in cytosol was determined by measuring the conversion of [1,3-3H]glycerol to [1,3-3H]glycerol-3-phosphate as described (40Thorner J.W. Glycerol kinase.Methods Enzymol. 1975; 42: 148-156Google Scholar). Mitochondrial PtdOH:CTP cytidylyltransferase activity was determined by measuring the conversion of [5-3H]CTP and PtdOH to CDP-[3H]DG as described (38Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells by cytidine-5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Google Scholar). Mitochondrial PtdGro phosphate synthase and PtdGro phosphate phosphatase combined activities were determined by measuring the conversion of [14C]glycerol-3-phosphate and CDP-DG to [14C]PtdGro as described (38Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells by cytidine-5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Google Scholar). Mitochondrial CL synthase activity was determined by measuring the conversion of [14C]PtdGro and CDP-DG to [14C]CL as described (38Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells by cytidine-5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Google Scholar). Mitochondrial phospholipase A2 (PLA2) was determined by measuring the conversion of [14C]PtdGro to lyso[14C]PtdGro as described (41Hatch G.M. Cao S.G. Angel A. Decrease in cardiac phosphatidylglycerol in streptozotocin-induced diabetic rats does not affect cardiolipin biosynthesis: evidence for distinct pools of phosphatidylglycerol in the heart.Biochem. J. 1995; 306: 759-764Google Scholar). Mitochondrial MLCL AT activity was determined by measuring the conversion of [1-14C]oleoyl-CoA and MLCL to [14C]CL as described (28Ma B.J. Taylor W.A. Dolinsky V. Hatch G.M. Acylation of monolysocardiolipin in rat heart.J. Lipid Res. 1999; 40: 1837-1845Google Scholar). Mitochondrial and microsomal sn-glycerol-3-phosphate acyltransferase (GPAT) activities were determined as described (42Haldar D. Vancura A. Glycerophosphate acyltransferase from liver.Methods Enzymol. 1992; 209: 64-72Google Scholar). Microsomal 1,2-diacyl-sn-glycerol acyltransferase (DGAT) activity was determined as described (43Coleman R.A. Diacylglycerol acyltyransferase and monoacylglycerol acyltransferase from liver and intestine.Methods Enzymol. 1992; 209: 98-102Google Scholar). Total cell and membrane phosphatidate phosphohydrolase (PAP) activity was determined as described (44Gomez-Munoz A. Hatch G.M. Martin A. Jamal Z. Vance D.E. Brindley D.N. Effects of okadaic acid on the activities of two distinct phosphatidate phosphohydrolases in rat hepatocytes.FEBS Lett. 1992; 301: 101-106Google Scholar). In some experiments microsomal DGAT and cytosolic glycerol kinase activities were determined in the absence or presence of 30 μM etomoxiryl-CoA. The fatty acid composition of CL in H9c2 cells was determined as described (45Tardi P.J. Mukherjee J.J. Choy P.C. The quantitation of long chain acyl-CoA in mammalian tissue.Lipids. 1992; 27: 65-67Google Scholar). Phospholipid phosphorus was determined as described (46Rouser G. Fleischer S. Yamamoto A. Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots.Lipids. 1970; 5: 494-496Google Scholar). Protein was determined as described (47Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar), and Student’s t-test was used for statistical analysis. The level of significance was defined as P < 0.05. Fatty acid import into mitochondria and its subsequent β-oxidation is mediated by carnitine palmitoyltransferase I and II (48Kerner J. Hoppel C. Fatty acid import into mitochondria.Biochim. Biophys. Acta. 2000; 1486: 1-17Google Scholar). Etomoxir, 2-[6-(4-chlorophenoxy)hexyl] oxirane-2-carboxylate, is a compound known to inhibit β-oxidation in mitochondria by irreversibly inhibiting carnitine palmitoyltransferase I activity (33Bartlett K. Turnball D.M. Sherratt H.S.A. Inhibition of hepatic and skeletal muscle carnitine palmitoyltransferase I by 2[5(4-chlorophenyl)pentyl]-oxirane-2-carboxyl-CoA.Biochem. Soc. Trans. 1984; 12: 688-689Google Scholar). It was postulated that etomoxir stimulated phospholipid biosynthesis in mammalian heart (37Dhalla N.S. Elimban V. Rupp H. Paradoxical role of lipid metabolism in heart function and dysfunction.Mol. Cell. Biochem. 1992; 116: 3-9Google Scholar). However, the effect of etomoxir on the biosynthesis of CL, a phospholipid exclusively found in mitochondria, had not been investigated. We chose H9c2 cardiac myoblast cells since it is a cell line derived from embryonic rat heart and these cells readily take up radiolabeled glycerol and fatty acids and incorporate them into CL (38Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells by cytidine-5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Google Scholar). H9c2 cells were preincubated with 1–80 μM etomoxir for 2 h and incubated with 0.1 mM [1-14C]oleic acid bound to albumin (1:1 molar ratio) for 2 h, and radioactivity incorporated into CL determined. These concentrations of etomoxir have been used in various cell culture models to inhibit carnitine palmitoyltransferase I activity (49Spurway T.D. Sherratt H.S.A. Pogson C.I. Agius L. The flux control coefficient of carnitine palmitoyltransferase I on palmitate beta-oxidation in rat hepatocyte cultures.Biochem. J. 1997; 323: 119-122Google Scholar, 50Cabrero A. Alegret M. Sanchez R. Adzet T. Laguna J.C. Vazquez M. Etomoxir, sodium-2-[6-(4-chlorophenoxy)hexyl]- oxirane-2-carboxylate upregulates uncoupling protein-3 mRNA levels in primary culture of rat preadipocytes.Biochem. Biophys. Res. Commun. 1999; 263: 87-93Google Scholar). Oleic acid (C18:1) was chosen for these experiments since this is the major unsaturated fatty acid found in CL in H9c2 cells (Table 1). The 2 h treatment was chosen to avoid the complicated effects of altered gene expression seen during prolonged incubation (24 h) with this compound (36Hegardt F.G. Serra D. Asins G. Influence of etomoxir on the expression of several genes in liver, testes and heart.Gen. Pharmacol. 1995; 26: 897-904Google Scholar). In H9c2 cells, CL was comprised mainly of myristic (C14:0), palmitic (C16:0), and oleic (C18:1) acids. Incubation of H9c2 cells with etomoxir resulted in a concentration-dependent reduction in [1-14C]oleic acid incorporation into CL (Fig. 1). For subsequent studies, we chose to use 40 μM etomoxir since the reduction in [1-14C]oleic acid incorporation into CL was maximum at this concentration. The presence of 40 μM etomoxir in the medium of H9c2 cells did not alter the percent fatty acid composition of CL (Table 1). We next examined incorporation of [1-14C]oleic acid into lipid metabolites of the CDP-DG pathway. Incubation of cells with 40 μM etomoxir caused a 41% (P < 0.05) and 31% (P < 0.05) reduction in [1-14C]oleic acid incorporated into CL and PtdGro, respectively, compared with controls (Table 2). In contrast, radioactivity incorporated into PtdOH was unaltered. Radioactivity incorporated into CDP-DG was low, twice background (data not shown). Total radioactivity associated with the cells was unaltered by etomoxir treatment. Thus, etomoxir treatment reduced [1-14C]oleic acid incorporation into PtdGro and CL.TABLE 1The fatty acid composition of cardiolipin in H9c2 cells treated with etomoxir% Fatty AcidControl+ EtomoxirMyristate (C14:0)3.64.2Palmitate (C16:0)49.147.2Oleate (C18:1)37.338.2All others10.010.4H9c2 cells were incubated for 2 h in the absence or presence of 40 μM etomoxir. Cardiolipin (CL) was isolated and the percent fatty acid composition of CL determined. Results represent the mean of two experiments. Open table in a new tab TABLE 2Incorporation of [1-14C]oleic acid into PtdOH, PtdGro, and CL in H9c2 cells treated with etomoxirControl+ Etomoxirdpm × 103/mg proteinCL1.7 ± 0.11.0 ± 0.1aP < 0.05.PtdGro3.2 ± 0.32.2 ± 0.2aP < 0.05.PtdOH0.2 ± 0.10.2 ± 0.1Total cellular incorporation1370.1 ± 120.21420.7 ± 100.2H9c2 cells were preincubated for 2 h in the absence or presence of 40 μM etomoxir, incubated with 0.1 mM [1-14C]oleic acid (2 μCi/dish), and the radioactivity incorporated into cells, phosphatidic acid (PtdOH), phosphatidylglycerol (PtdGro), and CL determined. Results represent the mean ± SD of six experiments.a P < 0.05. Open table in a new tab H9c2 cells were incubated for 2 h in the absence or presence of 40 μM etomoxir. Cardiolipin (CL) was isolated and the percent fatty acid composition of CL determined. Results represent the mean of two experiments. H9c2 cells were preincubated for 2 h in the absence or presence of 40 μM etomoxir, incubated with 0.1 mM [1-14C]oleic acid (2 μCi/dish), and the radioactivity incorporated into cells, phosphatidic acid (PtdOH), phosphatidylglycerol (PtdGro), and CL determined. Results represent the mean ± SD of six experiments. The mechanism for the reduction in [1-14C]oleic acid incorporation into PtdGro and CL was examined. The phospholipid phosphorus concentration of these cells was 124 ± 14 nmol/mg protein and was unaltered by pretreatment with etomoxir for 2 h. The percent phospholipid phosphorus of CL in these cells was 4.2% and was unaltered by pretreatment with etomoxir for 2 h. Since [1-14C]oleic acid incorporated into PtdOH was unaltered, we examined if the reduction in [1-14C]oleic acid incorporated into PtdGro and CL was due to a reduction in the activities of the enzymes of the CDP-DG pathway of CL biosynthesis. Cells were incubated for 2 h in the absence or presence of 40 μM etomoxir. The cells were homogenized, mitochondrial fractions prepared, and the activities of the biosynthetic enzymes determined. As seen in Table 3, etomoxir did not affect mitochondrial PtdOH:CTP cytidylyltransferase, PtdGro phosphate synthase/phospha-tase, or CL synthase activities. Thus, the reduction in [1-14C]oleic acid incorporation into PtdGro and CL was not due to alterations in the activities of the enzymes of de novo CL biosynthesis.TABLE 3PtdOH:CTP cytidylyltransferase, PtdGro phosphate synthase/phosphatase, CL synthase, phospholipase A2 (PLA2), monolysocardiolipin (MLCL) acyltransferase (AT), sn-glycerol-3-phosphate acyltransferase (GPAT), 1,2-diacyl-sn-glycerol acyltransferase (DGAT), phosphatidate phosphohydrolase (PAP), and glycerol kinase activities in H9c2 cells treated with etomoxirControl+ Etomoxirpmol/min.mg proteinPtdOH:CTPcytidylyltransferase2.5 ± 0.42.4 ± 0.2PtdGro phosphatesynthase/phosphatase52.3 ± 7.246.4 ± 5.2CL synthase4.1 ± 1.04.2 ± 1.2PLA2154.4 ± 17.2161.4 ± 20.3MLCL AT268.4 ± 16.3260.2 ± 22.4GPATmitochondrial27.3 ± 4.326.5 ± 2.4microsomal31.8 ± 2.635.9 ± 3.6DGAT106 ± 1520 ± 5aP < 0.05.Glycerol kinase0.9 ± 0.11.2 ± 0.2aP < 0.05.Percent of Total Activity on MembranesPAP20 ± 227 ± 3aP < 0.05.H9c2 cells were incubated for 2 h in the absence of presence of 40 μM etomoxir. Mitochondrial, microsomal, and cytosolic fractions were prepared and PtdOH:CTP cytidylyltransferase, PtdGro phosphate synthase/phosphatase, CL synthase, MLCL AT, PLA2, GPAT, DGAT, PAP, and glycerol kinase activities determined. The results represent the mean ± SD of five separate experiments.a P < 0.05. Open table in a new tab H9c2 cells were incubated for 2 h in the absence of presence of 40 μM etomoxir. Mitochondrial, microsomal, and cytosolic fractions were prepared and PtdOH:CTP cytidylyltransferase, PtdGro phosphate synthase/phosphatase, CL synthase, MLCL AT, PLA2, GPAT, DGAT, PAP, and glycerol kinase activities determined. The results represent the mean ± SD of five separate experiments. Unsaturated fatty acids enter into phospholipids mainly by deacylation-reacylation pathways (51Akesson B. Initial esterification and co
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