Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation

Abstract

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/- mice yielded an abnormally low number of LCAD +/- and -/- offspring, indicating frequent gestational loss. LCAD -/- mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD -/- males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD -/- mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.

Figure 1

Targeting of the mouse Acadl gene. (A) Schematic diagram of the targeting vector, pAcadl^tm1Uab, the endogenous Acadl locus, and the targeted Acadl^tm1Uab locus. Gap repair of the deleted region (an 821-bp NheI deletion of exon 3 and flanking intron sequence) in the targeting vector occurs on homologous recombination and was the basis for genotype screening by Southern blot analysis of EcoRI-digested DNA. E, EcoRI; H, HindIII; N, NotI; Nh, NheI. (B) Representative Southern blot from Acadl-targeted mice showing three genotypes. EcoRI-digested genomic DNA was probed with a 304-bp Acadl genomic fragment containing exon 3 and flanking intron sequence. Banding patterns are as follows: Acadl normal controls (+/+), −11 kb; heterozygous (+/−), −15 kb, 11 kb, and 8 kb; and homozygous mutant (−/−), −15 kb and 8 kb.

Figure 2

LCAD immunoblot. Immunoblot of liver mitochondrial proteins (50 μg) isolated from normal control (+/+), heterozygous mutant (+/−), and homozygous mutant (−/−) mice and probed with a rat anti-LCAD antibody. No detectable LCAD protein was observed in samples from the homozygous mutant mice.

Figure 3

Histopathology on LCAD +/+ (normal control) and LCAD −/− (deficient mice). (A) +/+ and (B) −/−: Oil-Red-O staining of frozen liver sections from 4- to 5-wk-old mice after an 18- to 20-hr fast. Abundant large lipid droplets present in the LCAD-deficient mice compared with minimal lipid accumulation in normal control. (C) +/+ and (D) −/−: Oil-Red-O staining of frozen hearts from 14- to 16-wk-old mice after an 18- to 20-hr fast. Note lipid accumulation in all cardiomyocytes of the LCAD-deficient mouse as compared with the LCAD-normal control. (E) +/+ and (F) −/−: Hematoxylin/eosin-stained heart sections from 16-wk-old, nonfasted male mice. Note prominent myocardial degeneration and fibrosis (arrows) present in the LCAD −/− mice (F). (A and B, ×25, C and D, ×40, and E and F, ×10.)

Figure 4

Butyl-ester acylcarnitine profiles of bile specimens obtained by using precursor-ion scanning (parent of m/z 85) electrospray/tandem mass spectrometry. Mice were sacrificed after overnight fast (18–20 hr), and bile specimens were collected as spots on filter paper. (Top) LCAD +/+ (normal control), no dilution. (Bottom) LCAD −/− (LCAD-deficient), 1:10 (vol/vol) dilution. Internal standards: m/z 263, d3-acetylcarnitine; m/z 347, d3-octanoylcarnitine; and m/z 459, d3-palmitoylcarnitine. Peak identification: m/z 260, acetylcarnitine (C₂); m/z 372, decanoylcarnitine (C₁₀); m/z 400, dodecanoylcarnitine (C₁₂); m/z 424, tetradecadienoylcarnitine (C_14:2); m/z 426, tetradecenoylcarnitine (C_14:1); m/z 456, palmitoylcarnitine (C₁₆); m/z 480, linoleylcarnitine (C_18:2); m/z 482, oleylcarnitine (C_18:1).