Disruption of Mitochondrial β-Oxidation of Unsaturated Fatty Acids in the 3,2-trans-Enoyl-CoA Isomerase-deficient Mouse
2002; Elsevier BV; Volume: 277; Issue: 22 Linguagem: Inglês
10.1074/jbc.m110993200
ISSN1083-351X
Autores Tópico(s)Metabolism and Genetic Disorders
ResumoCellular energy metabolism is largely sustained by mitochondrial β-oxidation of saturated and unsaturated fatty acids. To study the role of unsaturated fatty acids in cellular lipid and energy metabolism we generated a null allelic mouse, deficient in 3,2-trans-enoyl-CoA isomerase (ECI) (eci−/− mouse). ECI is the link in mitochondrial β-oxidation of unsaturated and saturated fatty acids and essential for the complete degradation and for maximal energy yield. Mitochondrial β-oxidation of unsaturated fatty acids is interrupted in eci−/−mice at the level of their respective 3-cis- or 3-trans-enoyl-CoA intermediates. Fasting eci−/− mice accumulate unsaturated fatty acyl groups in ester lipids and deposit large amounts of triglycerides in hepatocytes (steatosis). Gene expression studies revealed the induction of peroxisome proliferator-activated receptor activation in eci−/− mice together with peroxisomal β- and microsomal ω-oxidation enzymes. Combined peroxisomal β- and microsomal ω-oxidation of the 3-enoyl-CoA intermediates leads to a specific pattern of medium chain unsaturated dicarboxylic acids excreted in the urine in high concentration (dicarboxylic aciduria). The urinary dicarboxylate pattern is a reliable diagnostic marker of the ECI genetic defect. The eci−/− mouse might be a model of a yet undefined inborn mitochondrial β-oxidation disorder lacking the enzyme link that channels the intermediates of unsaturated fatty acids into the β-oxidation spiral of saturated fatty acids. Cellular energy metabolism is largely sustained by mitochondrial β-oxidation of saturated and unsaturated fatty acids. To study the role of unsaturated fatty acids in cellular lipid and energy metabolism we generated a null allelic mouse, deficient in 3,2-trans-enoyl-CoA isomerase (ECI) (eci−/− mouse). ECI is the link in mitochondrial β-oxidation of unsaturated and saturated fatty acids and essential for the complete degradation and for maximal energy yield. Mitochondrial β-oxidation of unsaturated fatty acids is interrupted in eci−/−mice at the level of their respective 3-cis- or 3-trans-enoyl-CoA intermediates. Fasting eci−/− mice accumulate unsaturated fatty acyl groups in ester lipids and deposit large amounts of triglycerides in hepatocytes (steatosis). Gene expression studies revealed the induction of peroxisome proliferator-activated receptor activation in eci−/− mice together with peroxisomal β- and microsomal ω-oxidation enzymes. Combined peroxisomal β- and microsomal ω-oxidation of the 3-enoyl-CoA intermediates leads to a specific pattern of medium chain unsaturated dicarboxylic acids excreted in the urine in high concentration (dicarboxylic aciduria). The urinary dicarboxylate pattern is a reliable diagnostic marker of the ECI genetic defect. The eci−/− mouse might be a model of a yet undefined inborn mitochondrial β-oxidation disorder lacking the enzyme link that channels the intermediates of unsaturated fatty acids into the β-oxidation spiral of saturated fatty acids. Long chain saturated and unsaturated (mono- and polyunsaturated) fatty acids comprising members of the ω-3 (α-linolenic), ω-6 (linoleic), and ω-9 (oleic acid) families occur almost equally as acyl groups of phospholipids and triglycerides. In phospholipids they are essential in the regulation of the fluidity of biological membranes. ω-3 and ω-6 polyunsaturated fatty acids are the precursors in eicosanoid synthesis (prostaglandins, prostacyclins, thromboxanes, and leukotrienes). As constituents of triglycerides, unsaturated fatty acids are a main energy source for muscle work.Following the classical pathway of mitochondrial β-oxidation of unsaturated fatty acids with cis double bonds at odd-numbered C atoms, e.g. of oleic acid (18:19), linoleic acid (18:19,12), and α-linolenic acid (18:19,12,15), yields 3-cis-enoyl-CoA-intermediates. They are isomerized by the mitochondrial 3,2-trans-enoyl-CoA isomerase (ECI) 1The abbreviations used are: ECI3,2-trans-enoyl-CoA isomerasepTFEperoxisomal trifunctional enzymePPARperoxisomal proliferator-activating receptorSCADshort chain acyl-CoA dehydrogenaseCYP 4A1cytochrome P450 IVA1RTreverse transcription1The abbreviations used are: ECI3,2-trans-enoyl-CoA isomerasepTFEperoxisomal trifunctional enzymePPARperoxisomal proliferator-activating receptorSCADshort chain acyl-CoA dehydrogenaseCYP 4A1cytochrome P450 IVA1RTreverse transcription (EC 5.3.3.8) to their respective 2-trans-enoyl-CoA isomers, common substrates of enoyl-CoA hydratase of the β-oxidation cycle of saturated fatty acyl-CoA esters (1Stoffel W. Ditzer R. Caesar H. Hoppe Seylers Z. Physiol. Chem. 1964; 339: 167-181Crossref PubMed Scopus (59) Google Scholar). cis double bonds at even C atoms yield 2-trans-4-cis-intermediates, which are reduced and isomerized by a mitochondrial NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34) to their respective 3-trans-intermediates (2Kunau W.H. Dommes P. Eur. J. Biochem. 1978; 91: 533-544Crossref PubMed Scopus (105) Google Scholar, 3Luo M.J. Smeland T.E. Shoukry K. Schulz H. J. Biol. Chem. 1994; 269: 2384-2388Abstract Full Text PDF PubMed Google Scholar, 4Koivuranta K.T. Hakkola E.H. Hiltunen J.K. Biochem. J. 1994; 304: 787-789Crossref PubMed Scopus (31) Google Scholar). Hydration to thed(−)-3-hydroxy derivative followed by epimerization by 3-hydroxyacyl-CoA epimerase (EC 5.1.2.3) is apparently an alternative but minor pathway. Likewise, another alternative pathway has been proposed, according to which a cis-5 double bond when encountered in the β-oxidation of an odd-numbered double bond in unsaturated fatty acids is removed through an NADPH-dependent reduction of 5-enoyl-CoA, possibly mediated by a 5-enoyl-CoA reductase (5Tserng K., Y. Jin S., J. J. Biol. Chem. 1991; 266: 11614-11620Abstract Full Text PDF PubMed Google Scholar). The enzyme, however, has neither been characterized on the protein level, nor has it been cloned. Its contribution to the overall mitochondrial β-oxidation of unsaturated fatty acids awaits further clarification.According to the classical pathway of mitochondrial β-oxidation, all unsaturated fatty acids are channeled via their 3-cis- and/or 3-trans-enoyl-CoA isomers to their respective 2-trans-enoyl-CoA intermediates, which are regular intermediates of the β-oxidation spiral. Mitochondrial 3,2-trans-enoyl-CoA isomerase is the essential link between saturated and unsaturated β-oxidation. The peroxisomal trifunctional enzyme (pTFE) contains an isomerase subunit, which carries out the equivalent reaction when peroxisomal β-oxidation is challenged,e.g. by lipid-lowering drugs (6Novikov D.K. Koivuranta K.T. Helander H.M. Filppula S.A. Yagi A.I. Qin Y.M. Hiltunen K.J. Adv. Exp. Med. Biol. 1999; 466: 301-309Crossref PubMed Google Scholar).Mature ECI of mouse, rat, bovine, and man are 29-kDa soluble mitochondrial matrix proteins (7Stoffel W. Grol M. Hoppe Seylers Z. Physiol. Chem. 1978; 359: 1777-1782Crossref PubMed Scopus (28) Google Scholar, 8Euler-Bertram S. Stoffel W. Biol. Chem. Hoppe Seyler. 1990; 371: 603-610Crossref PubMed Scopus (16) Google Scholar). The cDNA-derived amino acid sequence of rodents encodes a 289-residue polypeptide (32 kDa) with a 28-residue N-terminal signal sequence, and that of human encodes a 302-residue ECI (33 kDa) with a 41-residue N-terminal signal sequence, which are processed to the 261-residue mature enzyme during mitochondrial import (9Muller-Newen G. Stoffel W. Biol. Chem. Hoppe Seyler. 1991; 372: 613-624Crossref PubMed Scopus (32) Google Scholar, 10Muller-Newen G. Janssen U. Stoffel W. Eur. J. Biochem. 1995; 228: 68-73Crossref PubMed Scopus (77) Google Scholar, 11Muller-Newen G. Stoffel W. Biochemistry. 1993; 32: 11405-11412Crossref PubMed Scopus (40) Google Scholar). The human eci locus has been assigned to chromosome 16p13.3 (12Janssen U. Fink T. Lichter P. Stoffel W. Genomics. 1994; 23: 223-228Crossref PubMed Scopus (22) Google Scholar) and the mouse gene characterized (13Stoffel W. Duker M. Hofmann K. FEBS Lett. 1993; 333: 119-122Crossref PubMed Scopus (11) Google Scholar).A growing number of inborn errors of mitochondrial β-oxidation enzymes form a new class of metabolic diseases following the first description of the carnitine-palmitoyl transferase deficiency (14Bennett M.J. Ann. Clin. Biochem. 1990; 27: 519-531Crossref PubMed Scopus (41) Google Scholar, 15Di Mauro S. Di Mauro P.M. Science. 1973; 182: 929-931Crossref PubMed Scopus (388) Google Scholar, 16Amendt B.A. Greene C. Sweetman L. Cloherty J. Shih V. Moon A. Teel L. Rhead W.J. J. Clin. Invest. 1987; 79: 1303-1309Crossref PubMed Scopus (142) Google Scholar, 17Wanders R.J. IJlst L. Poggi F. Bonnefont J.P. Munnich A. Brivet M. Rabier D. Saudubray J.M. Biochem. Biophys. Res. Commun. 1992; 188: 1139-1145Crossref PubMed Scopus (150) Google Scholar) and the molecular basis of several additional β-oxidation enzyme defects are awaiting clarification.Common to these genetic defects is an impaired utilization of fatty acids as primary energy source. Starvation or increased energy requirement, particularly of newborns and children, causes severe hypoketotic hyperglycemia, elevated cellular and serum fatty acid concentrations, and enhanced ω-oxidation with medium chain length dicarboxylic acids as end products that are excreted in the urine.We studied the function of mitochondrial β-oxidation of unsaturated fatty acids in cellular energy metabolism in a null allelic mouse model in which the key enzyme of mitochondrial β-oxidation of unsaturated fatty acids, ECI, has been disrupted by homologous recombination in mouse ES cells.The complete ablation of eci in the mouse severely perturbs the metabolism of unsaturated fatty acids, particularly on short interval starvation. The fatty acid pattern of complex phospholipids is strongly altered. Saturated fatty acids become substituted by unsaturated fatty acids, and triglycerides massively accumulate in hepatocytes (steatosis). The lack of 3,2-trans-enoyl-CoA isomerase interrupts β-oxidation at the level of their 3-cis- or 3-trans-enoyl-CoA intermediates. They are further processed to specific medium chain saturated and unsaturated dicarboxylic acid end products and excreted into the urine. The urinary dicarboxylate pattern may serve as an unambiguous and valuable diagnostic tool in the diagnosis of the ECI genetic defect.DISCUSSIONThis report describes the generation and characterization of the first mouse model that addresses the catabolism of unsaturated fatty acids. ECI is the enzyme that links unsaturated and saturated fatty acid mitochondrial β-oxidation essential for the complete degradation of unsaturated fatty acids for optimal energy yield. In the eci−/− mouse, mitochondrial unsaturated fatty acid β-oxidation is interrupted at the stage of their 3-cis- or 3-trans-enoyl-CoA intermediates.eci−/− mice show no obvious phenotypic differences under normal physiological conditions. However, like patients affected by mitochondrial β-oxidation defects of saturated fatty acids (15Di Mauro S. Di Mauro P.M. Science. 1973; 182: 929-931Crossref PubMed Scopus (388) Google Scholar), the eci−/−mouse develops pathological symptoms as soon as energy supply becomes dependent on mitochondrial fatty acids oxidation, e.g. in hypoglycemic state during fasting periods.In general two independent β-oxidation systems, a mitochondrial and a peroxisomal, are involved in the degradation of saturated and unsaturated fatty acids. Peroxisomes catalyze fatty acid oxidation in a reaction sequence similar to the mitochondrial β-oxidation spiral, although only through two to five cycles for fatty acyl chain shortening. Unlike in mitochondria the reduction equivalents released in the peroxisomal β-oxidation cannot be utilized by oxidative phosphorylation. Therefore, the energy production is only minor and contributes under prolonged fasting conditions to total cellular fatty acid oxidation no more than 20% (25Krahling J.B. Gee R. Murphy P.A. Kirk J.R. Tolbert N.E. Biochem. Biophys. Res. Commun. 1978; 82: 136-141Crossref PubMed Scopus (35) Google Scholar, 26Lazarow P.B. De Duve C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2043-2046Crossref PubMed Scopus (1172) Google Scholar). Therefore, ECI is essential for the energy supply by mitochondrial β-oxidation from unsaturated fatty acids. Their abundance defines them as essential metabolic fuel for the energy supply by mitochondrial β-oxidation. The important function of 3,2-trans-enoyl-CoA isomerase in the complete mitochondrial degradation of unsaturated fatty acids to acetyl-CoA is obvious.Mitochondrial ECI and the isomerase subunit of pTFE are equivalent in their catalytic function. The mouse and human ECI gene organization (11Muller-Newen G. Stoffel W. Biochemistry. 1993; 32: 11405-11412Crossref PubMed Scopus (40) Google Scholar, 12Janssen U. Fink T. Lichter P. Stoffel W. Genomics. 1994; 23: 223-228Crossref PubMed Scopus (22) Google Scholar) and of pTFE consist of seven exons. Exon I–V of ptfe encode the peroxisomal isomerase-hydratase activity with 25% identity to the heci (27Ishii N. Hijikata M. Osumi T. Hashimoto T. J. Biol. Chem. 1987; 262: 8144-8150Abstract Full Text PDF PubMed Google Scholar). However, the exon/intron positions of mitochondrial eci and peroxisomal rn tfe show no similarity, which indicates the independent evolutionary development of the mitochondrial and peroxisomal β-oxidation to acetyl-CoA.The important question arose of whether in the eci−/− mouse mutant peroxisomal β-oxidation can compensate for the deficiency of mitochondrial β-oxidation and carry out the complete degradation of the accumulating 3-cis- and 3-trans-isomeric intermediates of unsaturated fatty acid β-oxidation.Surprisingly, eci−/− mice are viable, fertile, and phenotypically indistinguishable from wt mice as long as they are unchallenged by metabolic stress. The genotypes of the siblings of heterozygous crosses (eci+/−mice) followed the Mendelian law and excluded an influence of ECI deficiency on embryonic development.Most human mitochondrial β-oxidation enzyme defects described thus far are involved in mitochondrial β-oxidation of saturated fatty acids. Affected individuals develop a pathological phenotype only when an enhanced energy requirement must be covered by mitochondrial fatty acid β-oxidation, e.g. during prolonged fasting (29Wood P.A. Amendt B.A. Rhead W.J. Millington D.S. Inoue F. Armstrong D. Pediatr. Res. 1989; 25: 38-43Crossref PubMed Scopus (82) Google Scholar).Our ECI-deficient mice show three characteristic features. 1) A massive accumulation of neutral lipids, mainly triglycerides, occurs in liver and kidney upon short fasting conditions (Fig. 3). 2) Starvation induces hormonal activation of adipose tissue triglyceride lipase, elevates free fatty acid concentration in serum, and enhances mitochondrial and extra mitochondrial fatty acid oxidation. The activation of the hormone-sensitive adipose tissue triglyceride lipase releases long chain unsaturated fatty acids selectively from fat cell triglycerides (28Raclot T. Groscolas R. J. Lipid Res. 1995; 36: 2164-2173Abstract Full Text PDF PubMed Google Scholar). Liver of eci−/− mice is supplied with these unsaturated long chain fatty acids. They are utilized for the synthesis of ester lipids (triglyceride and phospholipids). Because of their impaired mitochondrial β-oxidation, they are mainly stored as cytosolic triglycerides in hepatocytes, as documented in the TLC analysis of eci−/− liver and kidney lipids (Fig. 3).Triglyceride storage in the liver described here for the eci−/− mouse has also been observed in SCAD-deficient mice (29Wood P.A. Amendt B.A. Rhead W.J. Millington D.S. Inoue F. Armstrong D. Pediatr. Res. 1989; 25: 38-43Crossref PubMed Scopus (82) Google Scholar). Both the unchallenged eci−/− and the scad−/− mouse are clinically asymptomatic, different from the SCAD deficiency in human, characterized by severe clinical symptoms.3) PPAR isoforms are activated by free fatty acids and accumulating metabolites (30Tontonoz P., Hu, E. Spiegelman B.M. Curr. Opin. Genet. Dev. 1995; 5: 571-576Crossref PubMed Scopus (402) Google Scholar, 31Vidal-Puig A. Jimenez-Linan M. Lowell B.B. Hamann A., Hu, E. Spiegelman B. Flier J.S. Moller D.E. J. Clin. Invest. 1996; 97: 2553-2561Crossref PubMed Scopus (576) Google Scholar). Upon starvation the eci−/− mouse rapidly develops a disrupted lipid metabolism. PPARα expression is up-regulated within the first 24 h, associated with a strongly enhanced expression of microsomal cytochrome P450 A1 and the peroxisomal β-oxidation enzymes, as documented here for the gene expression of CYP 4A1 and pTFE in the liver (Fig. 5).In eci−/− mice unsaturated fatty acid intermediates become substrates of these enzymes. As a consequence large amounts of saturated and unsaturated dicarboxylic acids are excreted in the urine, leading to a characteristic urinary dicarboxylate pattern in which hex-3-cis-ene-1,6-dioic acid is a dominant end product exceeding that of wild type control mice 30-fold. Because of the abundance of oleic acid among unsaturated fatty acids in phospholipids and triglycerides, ECI deficiency leads to a halt of the regular β-oxidation of oleic acid on the dodec-3-cis-enoyl-CoA level, which becomes the substrate of the P450-dependent microsomal ω-oxidation. Dodec-3- and 9-cis-ene-1,6-dioic acid is then activated by the microsomal dicarboxylyl-CoA synthase (32Vamecq J. de Hoffmann E. Van Hoof F. Biochem. J. 1985; 230: 683-693Crossref PubMed Scopus (65) Google Scholar) and further degraded either in mitochondria or in peroxisomes to hex-3-cis-ene-1,6-dicarboxylic acid. Octadi-2,5-cis-ene-1,8-dioic acid is another characteristic and abundant degradation product, which is derived from linoleic acid (18:29,12). Degradation of octadi-2,5-cis-ene-1,8-dioic acids via peroxisomal β-oxidation complex (hydratase to the d-3-hydroxy- and epimerase to the l-3-hydroxy-intermediate, dehydrogenase, and thiolase) might further increase the hex-3-cis-ene-1,6-dioic acid concentration in the urinary dicarboxylic acid pattern.All isomeric hexene, octene, octadiene, and decadiene dioic acids have been identified and characterized by gas liquid chromatography except the cis-/trans-isomerism of the latter. Triglyceride storage and dicarboxylic aciduria upon fasting as described above is a phenotypic sign, which is shared between the eci−/− mouse and the human inborn error SCAD deficiency. In SCAD deficiency starvation also causes a massive increase in urinary ethylmalonate and methylsuccinate as specific dicarboxylate metabolites, which results from ω-oxidation of incompletely β-oxidized saturated fatty acids.The eci−/− mouse model deficient in the 3,2-trans-enoyl-CoA isomerase, which links mitochondrial β-oxidation of saturated and unsaturated fatty acids, furthers our understanding of the role of unsaturated fatty acids in energy metabolism in response to normal and fasting conditions. The molecular pathology and the analysis of its phenotype has provided specific diagnostic tools. The eci−/− mouse mutant might prove to be a valuable mimicry of human ECI deficiency and facilitate the discovery of the equivalent human inherited disease in the pool of hitherto unknown mitochondrial fatty acid β-oxidation defects observed in new born and infants. The mouse model might also be useful in the development of dietary therapeutic strategies. Long chain saturated and unsaturated (mono- and polyunsaturated) fatty acids comprising members of the ω-3 (α-linolenic), ω-6 (linoleic), and ω-9 (oleic acid) families occur almost equally as acyl groups of phospholipids and triglycerides. In phospholipids they are essential in the regulation of the fluidity of biological membranes. ω-3 and ω-6 polyunsaturated fatty acids are the precursors in eicosanoid synthesis (prostaglandins, prostacyclins, thromboxanes, and leukotrienes). As constituents of triglycerides, unsaturated fatty acids are a main energy source for muscle work. Following the classical pathway of mitochondrial β-oxidation of unsaturated fatty acids with cis double bonds at odd-numbered C atoms, e.g. of oleic acid (18:19), linoleic acid (18:19,12), and α-linolenic acid (18:19,12,15), yields 3-cis-enoyl-CoA-intermediates. They are isomerized by the mitochondrial 3,2-trans-enoyl-CoA isomerase (ECI) 1The abbreviations used are: ECI3,2-trans-enoyl-CoA isomerasepTFEperoxisomal trifunctional enzymePPARperoxisomal proliferator-activating receptorSCADshort chain acyl-CoA dehydrogenaseCYP 4A1cytochrome P450 IVA1RTreverse transcription1The abbreviations used are: ECI3,2-trans-enoyl-CoA isomerasepTFEperoxisomal trifunctional enzymePPARperoxisomal proliferator-activating receptorSCADshort chain acyl-CoA dehydrogenaseCYP 4A1cytochrome P450 IVA1RTreverse transcription (EC 5.3.3.8) to their respective 2-trans-enoyl-CoA isomers, common substrates of enoyl-CoA hydratase of the β-oxidation cycle of saturated fatty acyl-CoA esters (1Stoffel W. Ditzer R. Caesar H. Hoppe Seylers Z. Physiol. Chem. 1964; 339: 167-181Crossref PubMed Scopus (59) Google Scholar). cis double bonds at even C atoms yield 2-trans-4-cis-intermediates, which are reduced and isomerized by a mitochondrial NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34) to their respective 3-trans-intermediates (2Kunau W.H. Dommes P. Eur. J. Biochem. 1978; 91: 533-544Crossref PubMed Scopus (105) Google Scholar, 3Luo M.J. Smeland T.E. Shoukry K. Schulz H. J. Biol. Chem. 1994; 269: 2384-2388Abstract Full Text PDF PubMed Google Scholar, 4Koivuranta K.T. Hakkola E.H. Hiltunen J.K. Biochem. J. 1994; 304: 787-789Crossref PubMed Scopus (31) Google Scholar). Hydration to thed(−)-3-hydroxy derivative followed by epimerization by 3-hydroxyacyl-CoA epimerase (EC 5.1.2.3) is apparently an alternative but minor pathway. Likewise, another alternative pathway has been proposed, according to which a cis-5 double bond when encountered in the β-oxidation of an odd-numbered double bond in unsaturated fatty acids is removed through an NADPH-dependent reduction of 5-enoyl-CoA, possibly mediated by a 5-enoyl-CoA reductase (5Tserng K., Y. Jin S., J. J. Biol. Chem. 1991; 266: 11614-11620Abstract Full Text PDF PubMed Google Scholar). The enzyme, however, has neither been characterized on the protein level, nor has it been cloned. Its contribution to the overall mitochondrial β-oxidation of unsaturated fatty acids awaits further clarification. 3,2-trans-enoyl-CoA isomerase peroxisomal trifunctional enzyme peroxisomal proliferator-activating receptor short chain acyl-CoA dehydrogenase cytochrome P450 IVA1 reverse transcription 3,2-trans-enoyl-CoA isomerase peroxisomal trifunctional enzyme peroxisomal proliferator-activating receptor short chain acyl-CoA dehydrogenase cytochrome P450 IVA1 reverse transcription According to the classical pathway of mitochondrial β-oxidation, all unsaturated fatty acids are channeled via their 3-cis- and/or 3-trans-enoyl-CoA isomers to their respective 2-trans-enoyl-CoA intermediates, which are regular intermediates of the β-oxidation spiral. Mitochondrial 3,2-trans-enoyl-CoA isomerase is the essential link between saturated and unsaturated β-oxidation. The peroxisomal trifunctional enzyme (pTFE) contains an isomerase subunit, which carries out the equivalent reaction when peroxisomal β-oxidation is challenged,e.g. by lipid-lowering drugs (6Novikov D.K. Koivuranta K.T. Helander H.M. Filppula S.A. Yagi A.I. Qin Y.M. Hiltunen K.J. Adv. Exp. Med. Biol. 1999; 466: 301-309Crossref PubMed Google Scholar). Mature ECI of mouse, rat, bovine, and man are 29-kDa soluble mitochondrial matrix proteins (7Stoffel W. Grol M. Hoppe Seylers Z. Physiol. Chem. 1978; 359: 1777-1782Crossref PubMed Scopus (28) Google Scholar, 8Euler-Bertram S. Stoffel W. Biol. Chem. Hoppe Seyler. 1990; 371: 603-610Crossref PubMed Scopus (16) Google Scholar). The cDNA-derived amino acid sequence of rodents encodes a 289-residue polypeptide (32 kDa) with a 28-residue N-terminal signal sequence, and that of human encodes a 302-residue ECI (33 kDa) with a 41-residue N-terminal signal sequence, which are processed to the 261-residue mature enzyme during mitochondrial import (9Muller-Newen G. Stoffel W. Biol. Chem. Hoppe Seyler. 1991; 372: 613-624Crossref PubMed Scopus (32) Google Scholar, 10Muller-Newen G. Janssen U. Stoffel W. Eur. J. Biochem. 1995; 228: 68-73Crossref PubMed Scopus (77) Google Scholar, 11Muller-Newen G. Stoffel W. Biochemistry. 1993; 32: 11405-11412Crossref PubMed Scopus (40) Google Scholar). The human eci locus has been assigned to chromosome 16p13.3 (12Janssen U. Fink T. Lichter P. Stoffel W. Genomics. 1994; 23: 223-228Crossref PubMed Scopus (22) Google Scholar) and the mouse gene characterized (13Stoffel W. Duker M. Hofmann K. FEBS Lett. 1993; 333: 119-122Crossref PubMed Scopus (11) Google Scholar). A growing number of inborn errors of mitochondrial β-oxidation enzymes form a new class of metabolic diseases following the first description of the carnitine-palmitoyl transferase deficiency (14Bennett M.J. Ann. Clin. Biochem. 1990; 27: 519-531Crossref PubMed Scopus (41) Google Scholar, 15Di Mauro S. Di Mauro P.M. Science. 1973; 182: 929-931Crossref PubMed Scopus (388) Google Scholar, 16Amendt B.A. Greene C. Sweetman L. Cloherty J. Shih V. Moon A. Teel L. Rhead W.J. J. Clin. Invest. 1987; 79: 1303-1309Crossref PubMed Scopus (142) Google Scholar, 17Wanders R.J. IJlst L. Poggi F. Bonnefont J.P. Munnich A. Brivet M. Rabier D. Saudubray J.M. Biochem. Biophys. Res. Commun. 1992; 188: 1139-1145Crossref PubMed Scopus (150) Google Scholar) and the molecular basis of several additional β-oxidation enzyme defects are awaiting clarification. Common to these genetic defects is an impaired utilization of fatty acids as primary energy source. Starvation or increased energy requirement, particularly of newborns and children, causes severe hypoketotic hyperglycemia, elevated cellular and serum fatty acid concentrations, and enhanced ω-oxidation with medium chain length dicarboxylic acids as end products that are excreted in the urine. We studied the function of mitochondrial β-oxidation of unsaturated fatty acids in cellular energy metabolism in a null allelic mouse model in which the key enzyme of mitochondrial β-oxidation of unsaturated fatty acids, ECI, has been disrupted by homologous recombination in mouse ES cells. The complete ablation of eci in the mouse severely perturbs the metabolism of unsaturated fatty acids, particularly on short interval starvation. The fatty acid pattern of complex phospholipids is strongly altered. Saturated fatty acids become substituted by unsaturated fatty acids, and triglycerides massively accumulate in hepatocytes (steatosis). The lack of 3,2-trans-enoyl-CoA isomerase interrupts β-oxidation at the level of their 3-cis- or 3-trans-enoyl-CoA intermediates. They are further processed to specific medium chain saturated and unsaturated dicarboxylic acid end products and excreted into the urine. The urinary dicarboxylate pattern may serve as an unambiguous and valuable diagnostic tool in the diagnosis of the ECI genetic defect. DISCUSSIONThis report describes the generation and characterization of the first mouse model that addresses the catabolism of unsaturated fatty acids. ECI is the enzyme that links unsaturated and saturated fatty acid mitochondrial β-oxidation essential for the complete degradation of unsaturated fatty acids for optimal energy yield. In the eci−/− mouse, mitochondrial unsaturated fatty acid β-oxidation is interrupted at the stage of their 3-cis- or 3-trans-enoyl-CoA intermediates.eci−/− mice show no obvious phenotypic differences under normal physiological conditions. However, like patients affected by mitochondrial β-oxidation defects of saturated fatty acids (15Di Mauro S. Di Mauro P.M. Science. 1973; 182: 929-931Crossref PubMed Scopus (388) Google Scholar), the eci−/−mouse develops pathological symptoms as soon as energy supply becomes dependent on mitochondrial fatty acids oxidation, e.g. in hypoglycemic state during fasting periods.In general two independent β-oxidation systems, a mitochondrial and a peroxisomal, are involved in the degradation of saturated and unsaturated fatty acids. Peroxisomes catalyze fatty acid oxidation in a reaction sequence similar to the mitochondrial β-oxidation spiral, although only through two to five cycles for fatty acyl chain shortening. Unlike in mitochondria the reduction equivalents released in the peroxisomal β-oxidation cannot be utilized by oxidative phosphorylation. Therefore, the energy production is only minor and contributes under prolonged fasting conditions to total cellular fatty acid oxidation no more than 20% (25Krahling J.B. Gee R. Murphy P.A. Kirk J.R. Tolbert N.E. Biochem. Biophys. Res. Commun. 1978; 82: 136-141Crossref PubMed Scopus (35) Google Scholar, 26Lazarow P.B. De Duve C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2043-2046Crossref PubMed Scopus (1172) Google Scholar). Therefore, ECI is essential for the energy supply by mitochondrial β-oxidation from unsaturated fatty acids. Their abundance defines them as essential metabolic fuel for the energy supply by mitochondrial β-oxidation. The important function of 3,2-trans-enoyl-CoA isomerase in the complete mitochondrial degradation of unsaturated fatty acids to acetyl-CoA is obvious.Mitochondrial ECI and the isomerase subunit of pTFE are equivalent in their catalytic function. The mouse and human ECI gene organization (11Muller-Newen G. Stoffel W. Biochemistry. 1993; 32: 11405-11412Crossref PubMed Scopus (40) Google Scholar, 12Janssen U. Fink T. Lichter P. Stoffel W. Genomics. 1994; 23: 223-228Crossref PubMed Scopus (22) Google Scholar) and of pTFE consist of seven exons. Exon I–V of ptfe encode the peroxisomal isomerase-hydratase activity with 25% identity to the heci (27Ishii N. Hijikata M. Osumi T. Hashimoto T. J. Biol. Chem. 1987; 262: 8144-8150Abstract Full Text PDF PubMed Google Scholar). However, the exon/intron positions of mitochondrial eci and peroxisomal rn tfe show no similarity, which indicates the independent evolutionary development of the mitochondrial and peroxisomal β-oxidation to acetyl-CoA.The important question arose of whether in the eci−/− mouse mutant peroxisomal β-oxidation can compensate for the deficiency of mitochondrial β-oxidation and carry out the complete degradation of the accumulating 3-cis- and 3-trans-isomeric intermediates of unsaturated fatty acid β-oxidation.Surprisingly, eci−/− mice are viable, fertile, and phenotypically indistinguishable from wt mice as long as they are unchallenged by metabolic stress. The genotypes of the siblings of heterozygous crosses (eci+/−mice) followed the Mendelian law and excluded an influence of ECI deficiency on embryonic development.Most human mitochondrial β-oxidation enzyme defects described thus far are involved in mitochondrial β-oxidation of saturated fatty acids. Affected individuals develop a pathological phenotype only when an enhanced energy requirement must be covered by mitochondrial fatty acid β-oxidation, e.g. during prolonged fasting (29Wood P.A. Amendt B.A. Rhead W.J. Millington D.S. Inoue F. Armstrong D. Pediatr. Res. 1989; 25: 38-43Crossref PubMed Scopus (82) Google Scholar).Our ECI-deficient mice show three characteristic features. 1) A massive accumulation of neutral lipids, mainly triglycerides, occurs in liver and kidney upon short fasting conditions (Fig. 3). 2) Starvation induces hormonal activation of adipose tissue triglyceride lipase, elevates free fatty acid concentration in serum, and enhances mitochondrial and extra mitochondrial fatty acid oxidation. The activation of the hormone-sensitive adipose tissue triglyceride lipase releases long chain unsaturated fatty acids selectively from fat cell triglycerides (28Raclot T. Groscolas R. J. Lipid Res. 1995; 36: 2164-2173Abstract Full Text PDF PubMed Google Scholar). Liver of eci−/− mice is supplied with these unsaturated long chain fatty acids. They are utilized for the synthesis of ester lipids (triglyceride and phospholipids). Because of their impaired mitochondrial β-oxidation, they are mainly stored as cytosolic triglycerides in hepatocytes, as documented in the TLC analysis of eci−/− liver and kidney lipids (Fig. 3).Triglyceride storage in the liver described here for the eci−/− mouse has also been observed in SCAD-deficient mice (29Wood P.A. Amendt B.A. Rhead W.J. Millington D.S. Inoue F. Armstrong D. Pediatr. Res. 1989; 25: 38-43Crossref PubMed Scopus (82) Google Scholar). Both the unchallenged eci−/− and the scad−/− mouse are clinically asymptomatic, different from the SCAD deficiency in human, characterized by severe clinical symptoms.3) PPAR isoforms are activated by free fatty acids and accumulating metabolites (30Tontonoz P., Hu, E. Spiegelman B.M. Curr. Opin. Genet. Dev. 1995; 5: 571-576Crossref PubMed Scopus (402) Google Scholar, 31Vidal-Puig A. Jimenez-Linan M. Lowell B.B. Hamann A., Hu, E. Spiegelman B. Flier J.S. Moller D.E. J. Clin. Invest. 1996; 97: 2553-2561Crossref PubMed Scopus (576) Google Scholar). Upon starvation the eci−/− mouse rapidly develops a disrupted lipid metabolism. PPARα expression is up-regulated within the first 24 h, associated with a strongly enhanced expression of microsomal cytochrome P450 A1 and the peroxisomal β-oxidation enzymes, as documented here for the gene expression of CYP 4A1 and pTFE in the liver (Fig. 5).In eci−/− mice unsaturated fatty acid intermediates become substrates of these enzymes. As a consequence large amounts of saturated and unsaturated dicarboxylic acids are excreted in the urine, leading to a characteristic urinary dicarboxylate pattern in which hex-3-cis-ene-1,6-dioic acid is a dominant end product exceeding that of wild type control mice 30-fold. Because of the abundance of oleic acid among unsaturated fatty acids in phospholipids and triglycerides, ECI deficiency leads to a halt of the regular β-oxidation of oleic acid on the dodec-3-cis-enoyl-CoA level, which becomes the substrate of the P450-dependent microsomal ω-oxidation. Dodec-3- and 9-cis-ene-1,6-dioic acid is then activated by the microsomal dicarboxylyl-CoA synthase (32Vamecq J. de Hoffmann E. Van Hoof F. Biochem. J. 1985; 230: 683-693Crossref PubMed Scopus (65) Google Scholar) and further degraded either in mitochondria or in peroxisomes to hex-3-cis-ene-1,6-dicarboxylic acid. Octadi-2,5-cis-ene-1,8-dioic acid is another characteristic and abundant degradation product, which is derived from linoleic acid (18:29,12). Degradation of octadi-2,5-cis-ene-1,8-dioic acids via peroxisomal β-oxidation complex (hydratase to the d-3-hydroxy- and epimerase to the l-3-hydroxy-intermediate, dehydrogenase, and thiolase) might further increase the hex-3-cis-ene-1,6-dioic acid concentration in the urinary dicarboxylic acid pattern.All isomeric hexene, octene, octadiene, and decadiene dioic acids have been identified and characterized by gas liquid chromatography except the cis-/trans-isomerism of the latter. Triglyceride storage and dicarboxylic aciduria upon fasting as described above is a phenotypic sign, which is shared between the eci−/− mouse and the human inborn error SCAD deficiency. In SCAD deficiency starvation also causes a massive increase in urinary ethylmalonate and methylsuccinate as specific dicarboxylate metabolites, which results from ω-oxidation of incompletely β-oxidized saturated fatty acids.The eci−/− mouse model deficient in the 3,2-trans-enoyl-CoA isomerase, which links mitochondrial β-oxidation of saturated and unsaturated fatty acids, furthers our understanding of the role of unsaturated fatty acids in energy metabolism in response to normal and fasting conditions. The molecular pathology and the analysis of its phenotype has provided specific diagnostic tools. The eci−/− mouse mutant might prove to be a valuable mimicry of human ECI deficiency and facilitate the discovery of the equivalent human inherited disease in the pool of hitherto unknown mitochondrial fatty acid β-oxidation defects observed in new born and infants. The mouse model might also be useful in the development of dietary therapeutic strategies. This report describes the generation and characterization of the first mouse model that addresses the catabolism of unsaturated fatty acids. ECI is the enzyme that links unsaturated and saturated fatty acid mitochondrial β-oxidation essential for the complete degradation of unsaturated fatty acids for optimal energy yield. In the eci−/− mouse, mitochondrial unsaturated fatty acid β-oxidation is interrupted at the stage of their 3-cis- or 3-trans-enoyl-CoA intermediates.eci−/− mice show no obvious phenotypic differences under normal physiological conditions. However, like patients affected by mitochondrial β-oxidation defects of saturated fatty acids (15Di Mauro S. Di Mauro P.M. Science. 1973; 182: 929-931Crossref PubMed Scopus (388) Google Scholar), the eci−/−mouse develops pathological symptoms as soon as energy supply becomes dependent on mitochondrial fatty acids oxidation, e.g. in hypoglycemic state during fasting periods. In general two independent β-oxidation systems, a mitochondrial and a peroxisomal, are involved in the degradation of saturated and unsaturated fatty acids. Peroxisomes catalyze fatty acid oxidation in a reaction sequence similar to the mitochondrial β-oxidation spiral, although only through two to five cycles for fatty acyl chain shortening. Unlike in mitochondria the reduction equivalents released in the peroxisomal β-oxidation cannot be utilized by oxidative phosphorylation. Therefore, the energy production is only minor and contributes under prolonged fasting conditions to total cellular fatty acid oxidation no more than 20% (25Krahling J.B. Gee R. Murphy P.A. Kirk J.R. Tolbert N.E. Biochem. Biophys. Res. Commun. 1978; 82: 136-141Crossref PubMed Scopus (35) Google Scholar, 26Lazarow P.B. De Duve C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2043-2046Crossref PubMed Scopus (1172) Google Scholar). Therefore, ECI is essential for the energy supply by mitochondrial β-oxidation from unsaturated fatty acids. Their abundance defines them as essential metabolic fuel for the energy supply by mitochondrial β-oxidation. The important function of 3,2-trans-enoyl-CoA isomerase in the complete mitochondrial degradation of unsaturated fatty acids to acetyl-CoA is obvious. Mitochondrial ECI and the isomerase subunit of pTFE are equivalent in their catalytic function. The mouse and human ECI gene organization (11Muller-Newen G. Stoffel W. Biochemistry. 1993; 32: 11405-11412Crossref PubMed Scopus (40) Google Scholar, 12Janssen U. Fink T. Lichter P. Stoffel W. Genomics. 1994; 23: 223-228Crossref PubMed Scopus (22) Google Scholar) and of pTFE consist of seven exons. Exon I–V of ptfe encode the peroxisomal isomerase-hydratase activity with 25% identity to the heci (27Ishii N. Hijikata M. Osumi T. Hashimoto T. J. Biol. Chem. 1987; 262: 8144-8150Abstract Full Text PDF PubMed Google Scholar). However, the exon/intron positions of mitochondrial eci and peroxisomal rn tfe show no similarity, which indicates the independent evolutionary development of the mitochondrial and peroxisomal β-oxidation to acetyl-CoA. The important question arose of whether in the eci−/− mouse mutant peroxisomal β-oxidation can compensate for the deficiency of mitochondrial β-oxidation and carry out the complete degradation of the accumulating 3-cis- and 3-trans-isomeric intermediates of unsaturated fatty acid β-oxidation. Surprisingly, eci−/− mice are viable, fertile, and phenotypically indistinguishable from wt mice as long as they are unchallenged by metabolic stress. The genotypes of the siblings of heterozygous crosses (eci+/−mice) followed the Mendelian law and excluded an influence of ECI deficiency on embryonic development. Most human mitochondrial β-oxidation enzyme defects described thus far are involved in mitochondrial β-oxidation of saturated fatty acids. Affected individuals develop a pathological phenotype only when an enhanced energy requirement must be covered by mitochondrial fatty acid β-oxidation, e.g. during prolonged fasting (29Wood P.A. Amendt B.A. Rhead W.J. Millington D.S. Inoue F. Armstrong D. Pediatr. Res. 1989; 25: 38-43Crossref PubMed Scopus (82) Google Scholar). Our ECI-deficient mice show three characteristic features. 1) A massive accumulation of neutral lipids, mainly triglycerides, occurs in liver and kidney upon short fasting conditions (Fig. 3). 2) Starvation induces hormonal activation of adipose tissue triglyceride lipase, elevates free fatty acid concentration in serum, and enhances mitochondrial and extra mitochondrial fatty acid oxidation. The activation of the hormone-sensitive adipose tissue triglyceride lipase releases long chain unsaturated fatty acids selectively from fat cell triglycerides (28Raclot T. Groscolas R. J. Lipid Res. 1995; 36: 2164-2173Abstract Full Text PDF PubMed Google Scholar). Liver of eci−/− mice is supplied with these unsaturated long chain fatty acids. They are utilized for the synthesis of ester lipids (triglyceride and phospholipids). Because of their impaired mitochondrial β-oxidation, they are mainly stored as cytosolic triglycerides in hepatocytes, as documented in the TLC analysis of eci−/− liver and kidney lipids (Fig. 3). Triglyceride storage in the liver described here for the eci−/− mouse has also been observed in SCAD-deficient mice (29Wood P.A. Amendt B.A. Rhead W.J. Millington D.S. Inoue F. Armstrong D. Pediatr. Res. 1989; 25: 38-43Crossref PubMed Scopus (82) Google Scholar). Both the unchallenged eci−/− and the scad−/− mouse are clinically asymptomatic, different from the SCAD deficiency in human, characterized by severe clinical symptoms. 3) PPAR isoforms are activated by free fatty acids and accumulating metabolites (30Tontonoz P., Hu, E. Spiegelman B.M. Curr. Opin. Genet. Dev. 1995; 5: 571-576Crossref PubMed Scopus (402) Google Scholar, 31Vidal-Puig A. Jimenez-Linan M. Lowell B.B. Hamann A., Hu, E. Spiegelman B. Flier J.S. Moller D.E. J. Clin. Invest. 1996; 97: 2553-2561Crossref PubMed Scopus (576) Google Scholar). Upon starvation the eci−/− mouse rapidly develops a disrupted lipid metabolism. PPARα expression is up-regulated within the first 24 h, associated with a strongly enhanced expression of microsomal cytochrome P450 A1 and the peroxisomal β-oxidation enzymes, as documented here for the gene expression of CYP 4A1 and pTFE in the liver (Fig. 5). In eci−/− mice unsaturated fatty acid intermediates become substrates of these enzymes. As a consequence large amounts of saturated and unsaturated dicarboxylic acids are excreted in the urine, leading to a characteristic urinary dicarboxylate pattern in which hex-3-cis-ene-1,6-dioic acid is a dominant end product exceeding that of wild type control mice 30-fold. Because of the abundance of oleic acid among unsaturated fatty acids in phospholipids and triglycerides, ECI deficiency leads to a halt of the regular β-oxidation of oleic acid on the dodec-3-cis-enoyl-CoA level, which becomes the substrate of the P450-dependent microsomal ω-oxidation. Dodec-3- and 9-cis-ene-1,6-dioic acid is then activated by the microsomal dicarboxylyl-CoA synthase (32Vamecq J. de Hoffmann E. Van Hoof F. Biochem. J. 1985; 230: 683-693Crossref PubMed Scopus (65) Google Scholar) and further degraded either in mitochondria or in peroxisomes to hex-3-cis-ene-1,6-dicarboxylic acid. Octadi-2,5-cis-ene-1,8-dioic acid is another characteristic and abundant degradation product, which is derived from linoleic acid (18:29,12). Degradation of octadi-2,5-cis-ene-1,8-dioic acids via peroxisomal β-oxidation complex (hydratase to the d-3-hydroxy- and epimerase to the l-3-hydroxy-intermediate, dehydrogenase, and thiolase) might further increase the hex-3-cis-ene-1,6-dioic acid concentration in the urinary dicarboxylic acid pattern. All isomeric hexene, octene, octadiene, and decadiene dioic acids have been identified and characterized by gas liquid chromatography except the cis-/trans-isomerism of the latter. Triglyceride storage and dicarboxylic aciduria upon fasting as described above is a phenotypic sign, which is shared between the eci−/− mouse and the human inborn error SCAD deficiency. In SCAD deficiency starvation also causes a massive increase in urinary ethylmalonate and methylsuccinate as specific dicarboxylate metabolites, which results from ω-oxidation of incompletely β-oxidized saturated fatty acids. The eci−/− mouse model deficient in the 3,2-trans-enoyl-CoA isomerase, which links mitochondrial β-oxidation of saturated and unsaturated fatty acids, furthers our understanding of the role of unsaturated fatty acids in energy metabolism in response to normal and fasting conditions. The molecular pathology and the analysis of its phenotype has provided specific diagnostic tools. The eci−/− mouse mutant might prove to be a valuable mimicry of human ECI deficiency and facilitate the discovery of the equivalent human inherited disease in the pool of hitherto unknown mitochondrial fatty acid β-oxidation defects observed in new born and infants. The mouse model might also be useful in the development of dietary therapeutic strategies.
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