Disruption of Mitochondrial β-Oxidation of Unsaturated Fatty Acids in the 3,2-trans-Enoyl-CoA Isomerase-deficient Mouse

Uwe Janssen,Wilhelm Stoffel

doi:10.1074/jbc.m110993200

Abstract

Cellular energy metabolism is largely sustained by mitochondrial beta-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 beta-oxidation of unsaturated and saturated fatty acids and essential for the complete degradation and for maximal energy yield. Mitochondrial beta-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 beta- and microsomal omega-oxidation enzymes. Combined peroxisomal beta- and microsomal omega-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 beta-oxidation disorder lacking the enzyme link that channels the intermediates of unsaturated fatty acids into the beta-oxidation spiral of saturated fatty acids.

Highlights

Long chain saturated and unsaturated fatty acids comprising members of the ␻-3 (␣-linolenic), ␻-6, and ␻-9 families occur almost as acyl groups of phospholipids and triglycerides
Another alternative pathway has been proposed, according to which a cis-5 double bond when encountered in the ␤-oxidation of an odd-numbered double bond in unsaturated fatty acids is removed through an NADPH-dependent reduction of 5-enoylCoA, possibly mediated by a 5-enoyl-CoA reductase [5]
We studied the function of mitochondrial ␤-oxidation of unsaturated fatty acids in cellular energy metabolism in a null allelic mouse model in which the key enzyme of mitochondrial ␤-oxidation of unsaturated fatty acids, enoyl-CoA isomerase (ECI), has been disrupted by homologous recombination in mouse ES cells

Summary

EXPERIMENTAL PROCEDURES

Construction of Replacement Vector—An 8-kb BamHI fragment encoding exons III–VII from the mouse genomic phage clone ␭-iso-8 described previously [13] was cloned into the BamHI site of pBluescript SKϩ. The neo resistance gene, isolated as a XhoI/BamHI fragment from the vector pMC1 neo poly(A) (Stratagene), was inserted by blunt end ligation into the SmaI site of exon IV (Fig. 1). The 9.2-kb insert was isolated as a BamHI fragment and cloned into the BamHI site of the pIC19R-MC-I TK vector [18] at the 3Ј end of the thymidine kinase box. Genomic DNA of ES cells and tail biopsies was isolated as described previously. A temperature gradient program was run between 100 and 240 °C; 10 °C/min; 15 min at 240 °C

RESULTS

DISCUSSION

ND ND