Altered brain lipid composition in cyclooxygenase-2 knockout mouse
2007; Elsevier BV; Volume: 48; Issue: 4 Linguagem: Inglês
10.1194/jlr.m600400-jlr200
ISSN1539-7262
AutoresKaizong Ma, Robert Langenbach, Stanley I. Rapoport, Mireille Basselin,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoCyclooxygenase (COX)-2 plays an important role in brain arachidonic acid (20:4n-6) metabolism, and its expression is upregulated in animal models of neuroinflammation and excitotoxicity. Our hypothesis was that brain lipid composition would be altered in COX-2 knockout (COX-2−/−) compared with wild-type (COX-2+/+) mice, reflecting the important role of COX-2 in brain lipid metabolism. Concentrations of different lipids were measured in high-energy microwaved brain from COX-2−/− and COX-2+/+ mice. Compared with the COX-2+/+ mouse brain, the brain of the COX-2−/− mouse had a statistically significant 15% increase in phosphatidylserine (PtdSer) and significant 37, 27, and 32% reductions in triacylglycerol and cholesterol concentrations and in the cholesterol-to-phospholipid ratio, respectively. The normalized concentration of palmitic acid (16:0) was increased in PtdSer, as was the brain concentration of unesterified arachidic acid (20:0). A lifetime absence of COX-2 produces multiple changes in brain lipid composition. These changes may be related to reported changes in fatty acid kinetics and in resistance to neuroinflammation and excitotoxicity in the COX-2−/− mouse. Cyclooxygenase (COX)-2 plays an important role in brain arachidonic acid (20:4n-6) metabolism, and its expression is upregulated in animal models of neuroinflammation and excitotoxicity. Our hypothesis was that brain lipid composition would be altered in COX-2 knockout (COX-2−/−) compared with wild-type (COX-2+/+) mice, reflecting the important role of COX-2 in brain lipid metabolism. Concentrations of different lipids were measured in high-energy microwaved brain from COX-2−/− and COX-2+/+ mice. Compared with the COX-2+/+ mouse brain, the brain of the COX-2−/− mouse had a statistically significant 15% increase in phosphatidylserine (PtdSer) and significant 37, 27, and 32% reductions in triacylglycerol and cholesterol concentrations and in the cholesterol-to-phospholipid ratio, respectively. The normalized concentration of palmitic acid (16:0) was increased in PtdSer, as was the brain concentration of unesterified arachidic acid (20:0). A lifetime absence of COX-2 produces multiple changes in brain lipid composition. These changes may be related to reported changes in fatty acid kinetics and in resistance to neuroinflammation and excitotoxicity in the COX-2−/− mouse. Cyclooxygenase (COX)-2 catalyzes the conversion of the second messenger, arachidonic acid (AA; 20:4n-6), to prostaglandins and thromboxanes (1Needleman P. Turk J. Jakschik B.A. Morrison A.R. Lefkowith J.B. Arachidonic acid metabolism. Annu. Rev. Biochem. 1986; 55: 69-102Crossref PubMed Google Scholar, 2Otto J.C. Smith W.L. Prostaglandin endoperoxide synthases-1 and -2. J. Lipid Mediat. Cell Signal. 1995; 12: 139-156Crossref PubMed Scopus (180) Google Scholar). It participates in neuroreceptor-initiated signaling involving the activation of cytosolic phospholipase A2 (cPLA2), which releases AA from membrane phospholipids (3Kaufmann W.E. Worley P.F. Pegg J. Bremer M. Isakson P. COX-2, a synaptically induced enzyme, is expressed by excitatory neurons at postsynaptic sites in rat cerebral cortex. Proc. Natl. Acad. Sci. USA. 1996; 93: 2317-2321Google Scholar, 4Yamagata K. Andreasson K.I. Kaufmann W.E. Barnes C.A. Worley P.F. Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids. Neuron. 1993; 11: 371-386Google Scholar, 5Basselin M. Villacreses N.E. Langenbach R. Ma K. Bell J.M. Rapoport S.I. Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mice. J. Neurochem. 2006; 96: 669-679Google Scholar). Brain COX-2 expression is upregulated in animal models of excitotoxicity, neuroinflammation, and ischemia (6Mirjany M. Ho L. Pasinetti G.M. Role of cyclooxygenase-2 in neuronal cell cycle activity and glutamate-mediated excitotoxicity. J. Pharmacol. Exp. Ther. 2002; 301: 494-500Google Scholar, 7Kelley K.A. Ho L. Winger D. Freire-Moar J. Borelli C.B. Aisen P.S. Pasinetti G.M. Potentiation of excitotoxicity in transgenic mice overexpressing neuronal cyclooxygenase-2. Am. J. Pathol. 1999; 155: 995-1004Scopus (212) Google Scholar, 8Consilvio C. Vincent A.M. Feldman E.L. Neuroinflammation, COX-2, and ALS—a dual role? Exp. Neurol. 2004; 187: 1-10Google Scholar, 9Sasaki T. Kitagawa K. Sugiura S. Omura-Matsuoka E. Tanaka S. Yagita Y. Okano H. Matsumoto M. Hori M. Implication of cyclooxygenase-2 on enhanced proliferation of neural progenitor cells in the adult mouse hippocampus after ischemia. J. Neurosci. Res. 2003; 72: 461-471Google Scholar), and COX-2 has been considered a target of nonsteroidal anti-inflammatory drugs in the treatment of clinical neuroinflammation (10Minghetti L. Cyclooxygenase-2 (COX-2) in inflammatory and degenerative brain diseases. J. Neuropathol. Exp. Neurol. 2004; 63: 901-910Crossref PubMed Scopus (601) Google Scholar). COX-2 knockout (COX-2−/−) mice have been created to elucidate the roles of COX-2 in brain and other organs (11Morham S.G. Langenbach R. Loftin C.D. Tiano H.F. Vouloumanos N. Jennette J.C. Mahler J.F. Kluckman K.D. Ledford A. Lee C.A. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell. 1995; 83: 473-482Google Scholar). Compared with its wild-type (COX-2+/+) littermate, the brain of the COX-2−/− mouse shows altered neurotransmitter-initiated signaling involving AA and its metabolites and increased resistance to ischemia, stroke, and N-methyl-d-aspartate-induced excitotoxicity (5Basselin M. Villacreses N.E. Langenbach R. Ma K. Bell J.M. Rapoport S.I. Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mice. J. Neurochem. 2006; 96: 669-679Google Scholar, 9Sasaki T. Kitagawa K. Sugiura S. Omura-Matsuoka E. Tanaka S. Yagita Y. Okano H. Matsumoto M. Hori M. Implication of cyclooxygenase-2 on enhanced proliferation of neural progenitor cells in the adult mouse hippocampus after ischemia. J. Neurosci. Res. 2003; 72: 461-471Google Scholar, 12Iadecola C. Niwa K. Nogawa S. Zhao X. Nagayama M. Araki E. Morham S. Ross M.E. Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice. Proc. Natl. Acad. Sci. USA. 2001; 98: 1294-1299Google Scholar). It also demonstrates upregulated expression of a number of enzymes involved in AA metabolism (13Shimizu T. Wolfe L.S. Arachidonic acid cascade and signal transduction. J. Neurochem. 1990; 55: 1-15Google Scholar), including COX-1, cPLA2, and secretory phospholipase A2 (sPLA2), as well as a reduced concentration of prostaglandin E2, a COX-2-mediated product of AA (14Brock T.G. McNish R.W. Peters-Golden M. Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2. J. Biol. Chem. 1999; 274: 11660-11666Google Scholar, 15Bosetti F. Langenbach R. Weerasinghe G.R. Prostaglandin E2 and microsomal prostaglandin E synthase-2 expression are decreased in the cyclooxygenase-2-deficient mouse brain despite compensatory induction of cyclooxygenase-1 and Ca2+-dependent phospholipase A2. J. Neurochem. 2004; 91: 1389-1397Google Scholar). To further address the role of COX-2 in brain lipid metabolism, in this study we examined brain lipid composition in COX-2−/− and COX-2+/+ mice. We used high-energy microwaving to prepare the brain for analysis to prevent ischemia-induced changes in concentrations of brain unesterified fatty acids, acyl-CoAs, anandamide, and eicosanoids (16Bazinet R.P. Lee H.J. Felder C.C. Porter A.C. Rapoport S.I. Rosenberger T.A. Rapid high-energy microwave fixation is required to determine the anandamide (N-arachidonoylethanolamine) concentration of rat brain. Neurochem. Res. 2005; 30: 597-601Google Scholar, 17Deutsch J. Rapoport S.I. Purdon A.D. Relation between free fatty acid and acyl-CoA concentrations in rat brain following decapitation. Neurochem. Res. 1997; 22: 759-765Google Scholar, 18Cenedella R.J. Galli C. Paoletti R. Brain free fatty levels in rats sacrificed by decapitation versus focused microwave irradiation. Lipids. 1975; 10: 290-293Google Scholar, 19Bosisio E. Galli C. Galli G. Nicosia S. Spagnuolo C. Tosi L. Correlation between release of free arachidonic acid and prostaglandin formation in brain cortex and cerebellum. Prostaglandins. 1976; 11: 773-781Google Scholar, 20Poddubiuk Z.M. Blumberg J.B. Kopin I.J. Brain prostaglandin content in rats sacrificed by decapitation vs focused microwave irradiation. Experientia. 1982; 38: 987-988Google Scholar). We measured brain concentrations of unesterified fatty acids and "stable" lipids [phospholipids, total cholesterol (cholesterol plus cholesteryl ester), and triacylglycerol] as well as concentrations of fatty acids esterified in these stable lipids. 1,2-Diheptadecanoyl-sn-glycero-3-phosphocholine (di-17:0-PC) and heptadecanoic acid (17:0), used as internal standards, were obtained from Sigma-Aldrich (St. Louis, MO). Silica gel TLC plates were purchased from EMD Chemicals (Gibbstown, NJ). All solvents were HPLC grade and were purchased from Fisher Scientific (Fair Lawn, NJ) or EMD Chemicals. Nembutal® (50 mg/ml) was obtained from Abbott Laboratories (Chicago, IL). K603-100 kits were purchased from BioVision Research Products (Mountain View, CA), and TR0100 kits were obtained from Sigma-Aldrich. This study was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication 86-23) under a protocol approved by the Animal Care and Use Committee of the National Institute of Child Health and Human Development. Three month old male COX-2−/− and COX-2+/+ mice from a C57BL/6J×129/Ola genetic background (11Morham S.G. Langenbach R. Loftin C.D. Tiano H.F. Vouloumanos N. Jennette J.C. Mahler J.F. Kluckman K.D. Ledford A. Lee C.A. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell. 1995; 83: 473-482Google Scholar) (Taconic Farms, Germantown, NY) were provided free access to standard rodent chow (NIH-31; Zeigler, Gardners, PA) and water. They were euthanized by an overdose of Nembutal® (100 mg/kg, ip) and then subjected to head-focused microwave irradiation (5.5 kW, 1.2 s; Cober Electronics, Stamford, CT) to stop brain metabolism (16Bazinet R.P. Lee H.J. Felder C.C. Porter A.C. Rapoport S.I. Rosenberger T.A. Rapid high-energy microwave fixation is required to determine the anandamide (N-arachidonoylethanolamine) concentration of rat brain. Neurochem. Res. 2005; 30: 597-601Google Scholar, 17Deutsch J. Rapoport S.I. Purdon A.D. Relation between free fatty acid and acyl-CoA concentrations in rat brain following decapitation. Neurochem. Res. 1997; 22: 759-765Google Scholar, 18Cenedella R.J. Galli C. Paoletti R. Brain free fatty levels in rats sacrificed by decapitation versus focused microwave irradiation. Lipids. 1975; 10: 290-293Google Scholar, 19Bosisio E. Galli C. Galli G. Nicosia S. Spagnuolo C. Tosi L. Correlation between release of free arachidonic acid and prostaglandin formation in brain cortex and cerebellum. Prostaglandins. 1976; 11: 773-781Google Scholar, 20Poddubiuk Z.M. Blumberg J.B. Kopin I.J. Brain prostaglandin content in rats sacrificed by decapitation vs focused microwave irradiation. Experientia. 1982; 38: 987-988Google Scholar). The brain was removed, frozen on dry ice, and stored at −80°C until analyzed. Total lipids from frozen microwaved whole brain were extracted using a partition system of chloroform-methanol-0.5 M KCl (2:1:0.75, v/v) (21Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 1957; 226: 497-509Google Scholar). The lipids were separated by TLC on silica gel-60 plates. Phospholipid classes were separated using the solvent system chloroform-methanol-glacial acetic acid-water (60:50:1:4, v/v). This method separates ethanolamine glycerophospholipid (EtnGpl), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), choline glycerophospholipid (ChoGpl), and sphingomyelin (CerPCho). Cholesteryl esters, triacylglycerol, and unesterified fatty acids were separated using the solvent system heptane-diethylether-glacial acetic acid (60:40:3, v/v) (22Skipski V.P. Good J.J. Barclay M. Reggio R.B. Quantitative analysis of simple lipid classes by thin-layer chromatography. Biochim. Biophys. Acta. 1968; 152: 10-19Google Scholar). Each band was scraped from the TLC plate and converted to fatty acid methyl esters (FAMEs) with 1% H2SO4 in anhydrous methanol for 3 h at 70°C (23Makrides M. Neumann M.A. Byard R.W. Simmer K. Gibson R.A. Fatty acid composition of brain, retina, and erythrocytes in breast- and formula-fed infants. Am. J. Clin. Nutr. 1994; 60: 189-194Google Scholar). Individual FAMEs were separated by gas-liquid chromatography, and fatty acid concentrations were calculated by proportional comparison of gas-liquid chromatography peak areas with the areas of 17:0 and di-17:0-PC internal standards (24DeMar Jr., J.C. Ma K. Bell J.M. Rapoport S.I. Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids. J. Neurochem. 2004; 91: 1125-1137Google Scholar). Phospholipid content was determined by assaying the lipid phosphorus content of the TLC scrapings (25Rouser G. Siakotos A.N. Fleischer S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids. 1966; 1: 85-86Google Scholar). Briefly, phospholipid bands were scraped into a glass tube, 0.5 ml of water and 0.65 ml of perchloric acid (70%) were added, and the contents were digested at 180°C for 1 h. Samples were cooled to room temperature, 0.5 ml of ascorbic acid (10%, w/v), 0.5 ml of ammonium molybdate (2.5%, w/v), and 3.0 ml of water were added, and the reaction mixture was mixed. It then was boiled for 5 min and cooled, and the absorbance was measured at 797 nm. Phospholipid concentrations were determined using standard curves. Total cholesterol (cholesterol plus cholesteryl ester) and triacylglycerol concentrations were determined with the commercial kits K603-100 (Biovision) and TR0100 (Sigma-Aldrich), respectively. Statistical analysis was performed by unpaired, two-tailed t-tests using GraphPad Prism version 4.0b for Macintosh (GraphPad Software, San Diego, CA; www.graphpad.com). Results are expressed as means ± SD (n = 8). Significance was taken as P ⩽ 0.05. There was no statistically significant difference in mean brain weight between COX-2−/− and COX-2+/+ mice, 0.42 ± 0.05 and 0.39 ± 0.05 g, respectively (n = 8). Although not part of this study, we reported previously that there was no statistically significant difference in the mean concentrations of plasma palmitic (16:0), palmitoleic (16:1n-7), stearic (18:0), oleic (18:1n-9), linoleic (18:2n-6), α-linolenic (18:3n-3), arachidonic (20:4n-6), or docosahexaenoic (22:6n-3) acid between unanesthetized COX-2−/− and COX-2+/+ mice (5Basselin M. Villacreses N.E. Langenbach R. Ma K. Bell J.M. Rapoport S.I. Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mice. J. Neurochem. 2006; 96: 669-679Google Scholar). The brain concentration of total phospholipid (nmol/g wet weight) did not differ significantly between COX-2−/− and COX-2+/+ mice (Table 1 ). Of individual phospholipids measured, the brain concentration of PtdSer was increased significantly by 15% in the COX-2−/− mouse, whereas concentrations of EtnGpl, ChoGpl, PtdIns, and CerPCho did not differ significantly between groups (Table 1). Mean brain concentrations of triacylglycerol and cholesterol were decreased significantly by 37% and 27%, respectively (Table 1), and the cholesterol-to-phospholipid ratio was reduced by 32%.TABLE 1Brain lipid concentrations in COX-2+/+ and COX-2−/− miceLipidCOX-2+/+COX-2−/−μmol/g brainEtnGpl24.3 ± 2.426.6 ± 3.6PtdIns2.4 ± 0.42.2 ± 0.4PtdSer9.4 ± 0.410.8 ± 1.2aP < 0.01 versus COX-2+/+ mean.ChoGpl23.4 ± 3.324.1 ± 3.2CerPCho4.3 ± 1.04.5 ± 0.8Total phospholipid63.9 ± 7.668.9 ± 5.4Triglyceride0.51 ± 0.170.32 ± 0.12bP < 0.05 versus COX-2+/+ mean.Total cholesterol51.8 ± 8.138.0 ± 11.8bP < 0.05 versus COX-2+/+ mean.(cholesterol plus cholesteryl ester)Total cholesterol/total phospholipid0.810 ± 0.0950.551 ± 0.125cP < 0.001 versus COX-2+/+ mean.CerPCho, sphingomyelin; ChoGpl, choline glycerophospholipid; COX, cyclooxygenase; EtnGpl, ethanolamine glycerophospholipid; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine. Values shown are means ± SD (n = 8).a P < 0.01 versus COX-2+/+ mean.b P < 0.05 versus COX-2+/+ mean.c P < 0.001 versus COX-2+/+ mean. Open table in a new tab CerPCho, sphingomyelin; ChoGpl, choline glycerophospholipid; COX, cyclooxygenase; EtnGpl, ethanolamine glycerophospholipid; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine. Values shown are means ± SD (n = 8). Of the unesterified fatty acids examined (Table 2 ), only the brain concentration of arachidic acid (20:0) was altered significantly (+58%) in COX-2−/− compared with COX-2+/+ mice.TABLE 2Esterified fatty acid concentrations in brain lipids of COX-2+/+ and COX-2−/− miceFatty AcidCOX-2+/+COX-2−/−COX-2+/+COX-2−/−Cholesteryl EsterTriacylglycerol16:040.0 ± 15.423.6 ± 11.4aP < 0.05 versus COX-2+/+ mean.234 ± 88149 ± 31aP < 0.05 versus COX-2+/+ mean.16:1n-7ndnd17.0 ± 8.12.6 ± 1.3bP < 0.001 versus COX-2+/+ mean.18:054.1 ± 21.060.2 ± 37.1146 ± 16131 ± 1718:1n-931.1 ± 13.526.5 ± 11.2203 ± 9888 ± 21cP < 0.01 versus COX-2+/+ mean.18:2n-617.2 ± 7.79.9 ± 6.0173 ± 9556 ± 28cP < 0.01 versus COX-2+/+ mean.18:3n-3ndndndnd20:0ndnd8.0 ± 5.03.7 ± 1.2aP < 0.05 versus COX-2+/+ mean.20:4n-619.7 ± 8.016.8 ± 7.026.0 ± 9.018.3 ± 6.5aP < 0.05 versus COX-2+/+ mean.22:6n-329.5 ± 14.926.1 ± 9.183 ± 3557 ± 18Unesterified FattyAcid Phospholipid16:032.5 ± 6.027.3 ± 10.024,052 ± 3,55123,394 ± 171716:1ndnd460 ± 120395 ± 14618:051.5 ± 4.052.8 ± 14.824,632 ± 2,78225,143 ± 2,46118:1n-916.7 ± 4.813.4 ± 7.122,220 ± 5,07721,342 ± 1,63818:2n-615.5 ± 8.918.8 ± 11.1712 ± 112810 ± 11118:3n-3ndndndnd20:00.98 ± 0.371.55 ± 0.42aP < 0.05 versus COX-2+/+ mean.481 ± 112496 ± 10520:4n-65.1 ± 2.23.6 ± 2.39,718 ± 1,0609,611 ± 94022:6n-38.4 ± 4.67.2 ± 4.318,425 ± 2,40617,711 ± 1,456All values shown are means ± SD (nmol/g brain; n = 8). nd, not detected.a P < 0.05 versus COX-2+/+ mean.b P < 0.001 versus COX-2+/+ mean.c P < 0.01 versus COX-2+/+ mean. Open table in a new tab All values shown are means ± SD (nmol/g brain; n = 8). nd, not detected. The concentration of esterified palmitic acid (16:0) in cholesteryl ester (nmol/g wet weight brain) was significantly less (−40%) in COX-2−/− than in COX-2+/+ mice, whereas concentrations of other fatty acids were unchanged. Similarly, calculated concentrations of esterified palmitic (16:0), palmitoleic (16:1n-7), oleic (18:1n-9), linoleic (18:2n-6), arachidic (20:0), and arachidonic (20:4n-6) acids were significantly less in brain triacylglycerol (Table 2). In the COX-2+/+ brain, ChoGpl contained mainly saturated palmitate and stearate, monounsaturated oleic acid, polyunsaturated AA, and docosahexaenoic acid. PtdIns was rich in stearate and AA, whereas PtdSer was enriched in stearate, oleate, and docosahexaenoate (Table 3 ). CerPCho was rich in stearate, lignoceric acid (24:0), and nervonic acid (24:1n-9). Although the esterified fatty acid concentration in total phospholipid did not differ significantly between groups (Table 2), esterified 16:0, 18:1n-9, and 20:4n-6 concentrations (nmol/g wet weight brain) were increased in PtdSer.TABLE 3Esterified fatty acid concentrations in brain phospholipid classes of COX-2+/+ and COX-2−/− miceFatty AcidCOX-2+/+COX-2−/−COX-2+/+COX-2−/−EtnGplPtdIns16:02,309 ± 3352,427 ± 420285 ± 118268 ± 4716:1123 ± 46134 ± 41ndnd18:09,816 ± 1,5319,883 ± 1,4072,323 ± 5422,211 ± 47318:1n-96,674 ± 7937,193 ± 1,576767 ± 127705 ± 22918:2n-6199 ± 78260 ± 7232 ± 2127 ± 1018:3n-3ndndndnd20:0238 ± 94231 ± 5972 ± 5540 ± 2020:4n-64,972 ± 8405,005 ± 5982,125 ± 7932,021 ± 61922:6n-310,681 ± 1,80010,622 ± 1,570280 ± 135301 ± 107PtdSerChoGpl16:0159 ± 58286 ± 76aP < 0.01 versus COX-2+/+ mean.18,990 ± 3,41020,749 ± 4,39416:1ndndndnd18:06,812 ± 1,3747,282 ± 9178,041 ± 1,2848,078 ± 1,36918:1n-92,604 ± 2303,390 ± 357bP < 0.001 versus COX-2+/+ mean.12,537 ± 2,58210,931 ± 1,68018:2n-649 ± 3352 ± 34386 ± 106384 ± 6618:3n-3ndndndnd20:096 ± 32105 ± 22147 ± 45128 ± 2920:4n-6323 ± 60404 ± 77cP < 0.05 versus COX-2+/+ mean.2,488 ± 5412,317 ± 32822:6n-34,100 ± 8654,503 ± 1,2642,641 ± 5812,481 ± 343CerPCho16:0125.2 ± 71.9100.0 ± 54.416:12.6 ± 1.03.8 ± 3.018:03,522 ± 4293,108 ± 58018:1n-9107 ± 7577.8 ± 30.418:2n-610.5 ± 1.816.6 ± 8.218:3n-3ndnd20:0160 ± 50141 ± 3920:4n-6181 ± 129130 ± 5524:0243 ± 201191 ± 9224:1n-9651 ± 189656 ± 27422:6n-330.4 ± 19.720.4 ± 11.2All values shown are means ± SD (nmol/g brain; n = 8). nd, not detected.a P < 0.01 versus COX-2+/+ mean.b P < 0.001 versus COX-2+/+ mean.c P < 0.05 versus COX-2+/+ mean. Open table in a new tab All values shown are means ± SD (nmol/g brain; n = 8). nd, not detected. When esterified fatty acid concentrations in the stable lipids were normalized to the concentration of the stable lipid in which they were found and calculated (nmol fatty acid/μmol lipid) (Table 4 ), the only significant effect of the COX-2−/− condition was a 62% increase of esterified palmitate (16:0) in PtdSer.TABLE 4Brain fatty acid concentration per μmol of phospholipid class, triacylglycerol, or cholesteryl ester class in COX-2+/+ and COX-2−/− miceFatty AcidCOX-2+/+COX-2−/−COX-2+/+COX-2−/−EtnGplPtdIns16:00.0997 ± 0.01870.0922 ± 0.01830.1172 ± 0.05040.1246 ± 0.036016:10.0051 ± 0.00230.0050 ± 0.0015ndnd18:00.4073 ± 0.08020.3738 ± 0.04980.9878 ± 0.33061.0222 ± 0.311818:1n-90.2769 ± 0.04830.2726 ± 0.06100.3248 ± 0.09440.3282 ± 0.135418:2n-60.0083 ± 0.00380.0098 ± 0.00270.0160 ± 0.00980.0180 ± 0.013018:3n-3ndndndnd20:00.0096 ± 0.00370.0088 ± 0.00230.0304 ± 0.00240.0260 ± 0.006220:4n-60.2058 ± 0.03900.1898 ± 0.02520.9100 ± 0.41640.9472 ± 0.391422:6n-30.4423 ± 0.08460.4031 ± 0.06710.1034 ± 0.03380.1418 ± 0.0624PtdSerChoGpl16:00.0170 ± 0.00700.0276 ± 0.0109aP < 0.05 versus COX-2+/+ mean.0.8600 ± 0.22300.8828 ± 0.171116:1ndndndnd18:00.6061 ± 0.24790.7001 ± 0.23000.3627 ± 0.08130.3443 ± 0.055418:1n-90.2636 ± 0.02290.3257 ± 0.09960.5604 ± 0.11500.4658 ± 0.064818:2n-60.0053 ± 0.00390.0049 ± 0.00360.0176 ± 0.00570.0164 ± 0.002318:3n-3ndndndnd20:00.0101 ± 0.00320.0102 ± 0.00410.0067 ± 0.00210.0055 ± 0.002020:4n-60.0343 ± 0.00620.0385 ± 0.01290.1127 ± 0.03280.0991 ± 0.015722:6n-30.4355 ± 0.08970.4330 ± 0.19010.1187 ± 0.03400.1066 ± 0.0204Triacylglycerol16:00.4937 ± 0.23950.4730 ± 0.217016:10.0467 ± 0.04000.0523 ± 0.032118:00.2973 ± 0.08510.3675 ± 0.109218:1n-90.4323 ± 0.23340.3392 ± 0.216618:2n-60.3685 ± 0.21450.2710 ± 0.148418:3n-3ndnd20:00.0298 ± 0.01300.0457 ± 0.019320:4n-60.0559 ± 0.02780.0619 ± 0.026122:6n-30.2816 ± 0.14310.1964 ± 0.0724All values shown are means ± SD (nmol/μmol; n = 8). nd, not detected.a P < 0.05 versus COX-2+/+ mean. Open table in a new tab All values shown are means ± SD (nmol/μmol; n = 8). nd, not detected. Brain lipid composition was altered in several ways in the COX-2−/− compared with the COX-2+/+ mouse. There was a statistically significant 15% increase in the brain concentration of PtdSer and 37% and 27% decreased concentrations of triacylglycerol and cholesterol, respectively. Esterified fatty acid concentrations (nmol/g wet weight brain) generally reflected the concentrations of the stable lipids in which they were found, with the exception of palmitate (16:0), which was disproportionately increased in PtdSer. In addition, unesterified arachidic acid (20:0) was increased by 58%. In another study, we reported no difference in plasma concentrations of unesterified fatty acids between COX-2−/− and COX-2+/+ mice (5Basselin M. Villacreses N.E. Langenbach R. Ma K. Bell J.M. Rapoport S.I. Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mice. J. Neurochem. 2006; 96: 669-679Google Scholar). Along with evidence that the activities of several enzymes in the AA cascade, cPLA2, sPLA2, and COX-1 (13Shimizu T. Wolfe L.S. Arachidonic acid cascade and signal transduction. J. Neurochem. 1990; 55: 1-15Google Scholar), are increased in the brain of the COX-2−/− mouse (15Bosetti F. Langenbach R. Weerasinghe G.R. Prostaglandin E2 and microsomal prostaglandin E synthase-2 expression are decreased in the cyclooxygenase-2-deficient mouse brain despite compensatory induction of cyclooxygenase-1 and Ca2+-dependent phospholipase A2. J. Neurochem. 2004; 91: 1389-1397Google Scholar) and that neuroreceptor-initiated signaling involving AA and its metabolites is altered (5Basselin M. Villacreses N.E. Langenbach R. Ma K. Bell J.M. Rapoport S.I. Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mice. J. Neurochem. 2006; 96: 669-679Google Scholar), our findings indicate that a lifelong absence of COX-2 produces important changes in brain lipid composition and AA metabolism. Our measured brain stable lipid and esterified fatty acid concentrations in the COX-2+/+ mice are comparable to previously published values for nonmicrowaved (% fatty acid composition) and microwaved (nmol/g wet weight brain) mouse brain (26Rosenberger T.A. Villacreses N.E. Contreras M.A. Bonventre J.V. Rapoport S.I. Brain lipid metabolism in the cPLA2 knockout mouse. J. Lipid Res. 2003; 44: 109-117Scopus (72) Google Scholar, 27Lomnitski L. Oron L. Sklan D. Michaelson D.M. Distinct alterations in phospholipid metabolism in brains of apolipoprotein E-deficient mice. J. Neurosci. Res. 1999; 58: 586-592Google Scholar, 28Nakashima S. Nagata K. Banno Y. Sakiyama T. Kitagawa T. Miyawaki S. Nozawa Y. A mouse model for Niemann-Pick disease: phospholipid class and fatty acid composition of various tissues. J. Lipid Res. 1984; 25: 219-227Google Scholar), whereas our unesterified fatty acid concentrations are similar to reported values in the microwaved mouse brain (26Rosenberger T.A. Villacreses N.E. Contreras M.A. Bonventre J.V. Rapoport S.I. Brain lipid metabolism in the cPLA2 knockout mouse. J. Lipid Res. 2003; 44: 109-117Scopus (72) Google Scholar, 29Rosenberger T.A. Hovda J.T. Peters J.M. Targeted disruption of peroxisomal proliferator-activated receptor β (δ) results in distinct gender differences in mouse brain phospholipid and esterified FA levels. Lipids. 2002; 37: 495-500Google Scholar). Concerning the sum of the FAMEs derived from fatty acids for glycerophospholipids in COX-2+/+ and COX-2−/− mice (Table 4), we obtained values of 2.04 and 1.92 for ChoGpl, 2.5 and 2.6 for PtdIns, 1.46 and 1.36 for EtnGpl, and 1.37 and 1.54 for PtdSer. These values are in the expected ranges, in that FAMEs derived from plasmanyl and plasmenyl components of EtnGpl and ChoGpl were not included in the FAME tables (30Rosenberger T.A. Oki J. Purdon A.D. Rapoport S.I. Murphy E.J. Rapid synthesis and turnover of brain microsomal ether phospholipids in the adult rat. J. Lipid Res. 2002; 43: 59-68Abstract Full Text Full Text PDF PubMed Google Scholar); nor were FAMEs of other fatty acids that we did not list (31Demar J.C., Jr. Ma K. Chang L. Bell J.M. Rapoport S.I. Alpha-linolenic acid does not contribute appreciably to docosahexaenoic acid within brain phospholipids of adult rats fed a diet enriched in docosahexaenoic acid. J. Neurochem. 2005; 94: 1063-1076Google Scholar). For triacylglycerol, the ratio was much <3, likely because our concentration estimate, with its large standard deviation, was too high (0.51 μmol/g compared with 0.12–0.3 μmol/g in the literature) (32Golovko M.Y. Hovda J.T. Cai Z.J. Craigen W.J. Murphy E.J. Tissue-dependent alterations in lipid mass in mice lacking glycerol kinase. Lipids. 2005; 40: 287-293Google Scholar). Although abnormal behavior has not been noted in the COX-2−/− mouse (33Ballou L.R. Botting R.M. Goorha S. Zhang J. Vane J.R. Nociception in cyclooxygenase isozyme-deficient mice. Proc. Natl. Acad. Sci. USA. 2000; 97: 10272-10276Crossref PubMed Scopus (201) Google Scholar), its brain compared with that of the COX-2+/+ mouse shows increased resistance to injury produced by ischemia or kainate or N-methyl-d-aspartic acid microinjection (5Basselin M. Villacreses N.E. Langenbach R. Ma K. Bell J.M. Rapoport S.I. Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mice. J. Neurochem. 2006; 96: 669-679Google Scholar, 9Sasaki T. Kitagawa K. Sugiura S. Omura-Matsuoka E. Tanaka S. Yagita Y. Okano H. Matsumoto M. Hori M. Implication of cyclooxygenase-2 on enhanced proliferation of neural progenitor cells in the adult mouse hippocampus after ischemia. J. Neurosci. Res. 2003; 72: 461-471Google Scholar, 12Iadecola C. Niwa K. Nogawa S. Zhao X. Nagayama M. Araki E. Morham S. Ross M.E. Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice. Proc. Natl. Acad. Sci. USA. 2001; 98: 1294-1299Google Scholar, 34Takemiya T. Maehara M. Matsumura K. Yasuda S. Sugiura H. Yamagata K. Prostaglandin E2 produced by late induced COX-2 stimulates hippocampal neuron loss after seizure in the CA3 region. Neurosci. Res. 2006; 56: 103-110Google Scholar). Some of the abnormalities in lipid composition that we report in this study may contribute to thi
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