Homozygous carnitine palmitoyltransferase 1a (liver isoform) deficiency is lethal in the mouse

Abstract

To better understand carnitine palmitoyltransferase 1a (liver isoform, gene=Cpt-1a, protein=CPT-1a) deficiency in human disease, we developed a gene knockout mouse model. We used a replacement gene targeting strategy in ES cells that resulted in the deletion of exons 11-18, thus producing a null allele. Homozygous deficient mice (CPT-1a -/-) were not viable. There were no CPT-1a -/- pups, embryos or fetuses detected from day 10 of gestation to term. FISH analysis demonstrated targeting vector recombination at the expected single locus on chromosome 19. The inheritance pattern from heterozygous matings was skewed in both C57BL/6NTac, 129S6/SvEvTac (B6;129 mixed) and 129S6/SvEvTac (129 coisogenic) genetic backgrounds biased toward CPT-1a +/- mice (>80%). There was no sex preference with regard to germ-line transmission of the mutant allele. CPT-1a +/- mice had decreased Cpt-1a mRNA expression in liver, heart, brain, testis, kidney, and white fat. This resulted in 54.7% CPT-1 activity in liver from CPT-1a +/- males but no significant difference in females as compared to CPT-1a +/+ controls. CPT-1a +/- mice showed no fatty change in liver and were cold tolerant. Fasting free fatty acid concentrations were significantly elevated, while blood glucose concentrations were significantly lower in 6-week-old CPT-1a +/- mice compared to controls. Although the homozygous mutants were not viable, we did find some aspects of haploinsufficiency in the CPT-1a +/- mutants, which will make them an important mouse model for studying the role of CPT-1a in human disease.

Homozygous carnitine palmitoyltransferase 1b (muscle isoform) deficiency is lethal in the mouse

“…Also, in contrast to the up-regulation of Cpt-1b mRNA expression in livers of CPT-1a+/− mice [10], there was no compensatory up-regulation of Cpt-1a mRNA in heart and BAT of CPT-1b+/− mice. Therefore, ∼50% of the wild-type levels of Cpt-1b mRNA, as well as ∼60% of the CPT-1 enzyme activities in CPT-1b predominant tissues were sufficient for a normal gross appearance, histology, and general physiologic functions in the CPT-1b+/− mice.…”

“…This is because deficiency in FAO not only reduces fat-derived energy supply but also leads to accumulation of fatty acid substrates or metabolites in the blood and other extra-cellular fluid compartment, in cytoplasm, and in mitochondria. Because CPT-1 is the rate-limiting enzyme for mitochondrial LCFA β-oxidation important in gametogenesis and embryogenesis [29][30][31], it is not surprising that, among available mouse models with enzyme deficiencies of FAO, a severe phenotype, or homozygous lethality is found in mice deficient in either CPT-1a [10] or CPT-1b (this study), in contrast to those with homozygous deficiency of any one of the four fatty acyl-CoA dehydrogenases (ACADs), which include very-long-, long-, medium-, and short-chain acyl-CoA dehydrogenases (VLCAD [32]; LCAD [33], MCAD [34]; and SCAD [35,36]). Also, among the four mouse models with deficiency in one of the ACADs, LCAD−/ − mice have the most severe phenotype including fatty liver, fasting and cold intolerance and gestational loss [33].…”

“…Interestingly, some animal models of FAO enzyme deficiency are associated with skewed patterns of inheritance [10]. Whereas LCAD+/− genotype is under-represented in offspring [31], probably due to frequent gestational loss, as similar to the LCAD−/− pups, it is not clear why both the VLCAD+/− [32] and CPT-1a+/− [10] genotypes are over-represented.…”

“…Wild-type 129/S6 mice are completely "cold tolerant", with less than 3 °C drop following a 3 hr cold-challenge [10]. In the present study, 65% and 30% of the CPT-1b+/+ male (n=17) and female (n=10) mice, respectively, on a mixed genetic background (third generation offspring from a B6J×129X1/SvJ chimera backcrossed to B6J females) were "cold tolerant" (as defined above), suggesting that the B6J genetic background of CPT-1b+/+ mice might predispose to increased susceptibility to cold.…”

“…CPT-1a, or the liver isoform, is expressed in liver, kidney, white adipose tissue (WAT, in male but not female mice [9,10]), testis, ovary, pancreatic islet, lung, spleen, brain, intestine [8,9], and at much lower levels in heart [10]. In contrast, the muscle isoform, or CPT-1b, is expressed in brown adipose tissue (BAT), heart, skeletal muscle, testis, and WAT (in male rats [11,12], in female but not male mice, and in humans [9]).…”