Inherited enzyme deficiencies of mitochondrial fatty acid β-oxidation are diseases with variable presentations from asymptomatic existence to life-threatening acute disease episodes. 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
], 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
]). 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
]. Moreover, no live mice with a double homozygous LCAD(−/−)/VLCAD(−/−) genotype were detected from double heterozygous matings [32
]. A potential explanation for embryonic lethality of CPT-1b−/− mice is that CPT-1b activity might be required during mouse pre-implantation development, because Cpt-1b
mRNA is first and temporally detected at the 2-cell stage and reappears at the morula and blastocyst stage [30
]. The importance of LCFA oxidation at this stage is also supported by our previous findings that the LCAD−/− genotype is associated with frequent gestational loss at the morula-to-blastocyst conversion [31
In humans, loss of function mutations have been identified in genes encoding SCAD (ACADS
]), MCAD (ACADM
].), VLCAD (ACADVL
]), and CPT-1A [40
], but not CPT-1B or LCAD [41
]. While homozygous CPT-1a deficiency is rare and potentially fatal in humans [40
], heterozygous CPT-1a deficiency has not been reported with abnormalities. Similarly, the scarcity of reported CPT-1b deficient human cases is possibly due to embryonic lethality, as implied by our current mouse study. Also, heterozygous CPT-1b deficiency in humans would likely be free of symptoms, unless metabolically challenged with extreme exercise or offending drugs.
In our study, northern blot analysis suggested no up-regulation of Cpt-1b
mRNA expression from the only functional copy of Cpt-1b
gene in the CPT-1b+/− mice. 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.
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. In the current study, the mutant Cpt-1b
(-) allele was under-represented during germ-line transmission in heterozygous matings.
mRNA was found to be expressed at a very low level in wild-type placenta, as compare to Cpt-1a
mRNA, we concluded that CPT-1a is the major isoform expressed. Furthermore, it is unlikely that CPT-1b deficiency is an important factor in the lethality of CPT-1b−/− embryos. The timing of Cpt-1b
mRNA expression suggests that it is involved in spermatogenesis [8
]; while CPT-1a is the sole isoform detected in immature testis, adult testis is much richer in Cpt-1b
mRNA, which predominates in spermatids [9
], and in meiotic and post-meiotic germ cells [8
]. However, given the equal percentage of CPT-1b+/+ and CPT-1b+/− offspring from breeders consisting of either a CPT-1b+/− male mice with a B6/J female or a CPT-1b+/− female and a 129S6 male, it is unlikely that the percentage of sperm and oocyte with the mutant allele of Cpt-1b
is reduced. Also, gestational loss of CPT-1b+/− pups is not a likely explanation. That is, the CPT-1b+/− breeders produced CPT-1b+/− pups and CPT-1b+/+ pups at a 1:1 ratio and that led us to postulate that perhaps the CPT-1b (+) sperm survive predominately before fertilization in heterozygous mating pairs. This is another possible explanation of the absence of CPT-1b−/− fetuses at embryonic day 9.5−11.5. Further study is required to investigate the mechanisms for under-representation of CPT-1b+/− pups from heterozygous mating.
Mice with impaired FAO often have reduced cold tolerance. Exposure to cold is detected by the brain, leading to activation of the sympathetic nervous system, which heavily innervates thermogenic targets such as BAT and skeletal muscle. Fatal hypothermia is 100% in homozygous knockout mice of VLCAD, LCAD, MCAD, and SCAD, whereas “single heterozygous” enzyme-deficient mice are usually cold-tolerant [42
]. There was ~33% fatal hypothermia in double heterozygous with VLCAD/LCAD, LCAD/SCAD, VLCAD/SCAD combinations on a mixed genetic background [42
]. In the current study, it took an extended challenge (4 − 6 hrs), yet significantly more CPT-1b+/− mice developed fatal hypothermia following a 6-hr cold challenge (). This and the finding that 6−7% CPT-1b+/− mice verses none of the control mice had fatal hypothermia following a 3-hr cold challenge are consistent with tissue-specific expression of this enzyme in BAT and skeletal muscle, which mediates nonshivering and shivering thermogenesis, respectively. Also, it is interesting that there was a trend for an increase in frequency of fatal hypothermia in female verses male mice with both CPT-1b+/+ and CPT-1b+/− genotypes. While we cannot rule out the lower body weight of female littermates might contribute to increased susceptibility to cold, as compared to the males, we found no correlation between lower body weight and fatal hypothermia within each genotype and each sex (data not shown). In fact, those that developed fatal hypothermia tended to be slightly heavier. Moreover, the additive effects of being female with Cpt-1b
+/− genotype is consistent with cold susceptibility as an autosomal dominant trait of the mutant Cpt-1b
gene with variable penetrance.
Glucose metabolism can be altered in some mouse models deficient in a FAO enzyme. For example, CPT-1a+/− mice have lower fasting blood glucose and higher fasting free fatty acid in plasma [10
]. In the current study, young CPT-1b+/− mice at 6−9 weeks of age had normal indexes of glucose homeostasis: random and fasting blood glucose and fasting insulin. Also, while it has not been observed previously in mice of other genetic backgrounds such as the 129S6 mice (data not shown), blood glucose concentrations significantly increased following a 3-hr cold challenge in both Cpt-1b
genotypes and sexes (). Cold challenge increases FAO, which might inhibit glucose utilization in these mice. Therefore, simply reducing Cpt-1b
mRNA levels by half is not sufficient to alter glucose metabolism enough to cause any changes in plasma insulin and blood glucose levels.
In conclusion, we have developed a mouse model of CPT-1b deficiency that resulted in no homozygous fetuses found at any stages examined. We did find a normal mode of allelic transmission from the CPT-1b+/− male and female mice in breeding pairs with wild-type mates. In contrast, the number of CPT-1b+/− pups produced from CPT-1b+/− breeding pairs was under-represented. Heterozygous CPT-1b mice had virtually a normal phenotype, including normal body weight, normal fasting blood glucose levels. Also, increased blood glucose following a 3-hr cold challenge might reflect decreased glucose utilization during cold challenge. Moreover, CPT-1b+/− mice tended to have decreased cold tolerance as compared to CPT-1b+/+ mice at >3 hrs of cold challenge, although the difference was not significant. Following an extended time of cold challenge, there was a significant increase in the percentage of CPT-1b+/− mice developing lethal hypothermia compared to CPT-1b+/+ mice. Thus since there have been few human patients described with CPT-1b deficiency, this may also represent a high rate of gestational lethality in humans.