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Figure I. CPT 1 inhibition induces the expression of P PA Rut target genes involved in hepatic and cardiac fatty acid oxidation. Three-month-old male C57BL/6 x SJL/J mice were given daily intraperitoneal injections of vehicle (V) or cto-moxir (Eto, 50pg/g body mass) for 5 days. A representative autoradiognim is shown. "P-labeled cDNA probes used for the hybridizations encoded mouse acyl-CoA oxidase (ACO), rat cytochrome P450 4A1 (CYP 4A1) and P450 4A3 (CYP 4A3), mouse medium-chain acyl-CoA dehydrogenase (MCAD) and 18s ribosomal RNA (18s). CYP 4A1 and CYP 4A3 signals were only detected in liver. The signals on the Northern blot autoradi-ograms were quantified by laser densitometric analysis within the linear range of film sensitivity. Values were normalized to the signals obtained with 18s ribosomal cDNA probe. The bars represent the mean ± SE from at least 6 animals in each group normalized {= 1.0) to the results obtained with 5 days of vehicle administration. The asteriks denote a statistically significant difference (*p < 0.05; **p < 0.01; ***p < 0.001) compared with the value obtained with vehicle alone using analysis of variance and Fisher's test.

PPARa target genes encoding hepatic cytochrome P450 oxidation enzymes (CYP 4A1 and CYP 4A3) and the expression of the gene encoding medium-chain acyl-CoA dehydrogenase (MCAD), which catalyses a rate-limiting step in the mitochondrial oxidation of medium-chain fatty acyl-thioesters produced by peroxisomal ß-oxidation of LCFA. Thus, inhibition of mitochondrial long-chain fatty acid import induced the expression of PPARa target genes encoding cellular fatty acid oxidation enzymes. All of the mice tolerated the metabolic perturbation induced by etomoxir.

The CPT I inhibition experiments were repeated with mice homozygous for a targeted disruption of the PPARa gene (PPARa -/-). Western-blot analysis showed that

Table 1. CPT I inhibition results in gender-influenced death in PPARa -/- mice. The mice received a single daily intraperitoneal injection of vehicle (Veh, sterile water) or etomoxir (Eto, 50 (xg/g body mass) for 5 consecutive days. Other abbreviations are M: male, F: female.
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