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

Abstract

Carnitine palmitoyltransferase-1 (CPT-1) catalyzes the rate-limiting step of mitochondrial beta-oxidation of long chain fatty acids (LCFA), the most abundant fatty acids in mammalian membranes and in energy metabolism. Human deficiency of the muscle isoform CPT-1b is poorly understood. In the current study, embryos with a homozygous knockout of Cpt-1b were lost before embryonic day 9.5-11.5. Also, while there were normal percentages of CPT-1b+/- pups born from both male and female CPT-1b+/- mice crossed with wild-type mates, the number of CPT-1b+/- pups from CPT-1b+/- breeding pairs was under-represented (63% of the expected number). Northern blot analysis demonstrated approximately 50% Cpt-1b mRNA expression in brown adipose tissue (BAT), heart and skeletal muscles in the CPT-1b+/- male mice. Consistent with tissue-specific expression of Cpt-1b mRNA in muscle but not liver, CPT-1+/- mice had approximately 60% CPT-1 activity in skeletal muscle and no change in total liver CPT-1 activity. CPT-1b+/- mice had normal fasting blood glucose concentration. Consistent with expression of CPT-1b in BAT and muscle, approximately 7% CPT-1b+/- mice (n=30) developed fatal hypothermia following a 3h cold challenge, while none of the CPT-1b+/+ mice (n=30) did. With a prolonged cold challenge (6h), significantly more CPT-1b+/- mice developed fatal hypothermia (52% CPT-1b+/- mice vs. 21% CPT-1b+/+ mice), with increased frequency in females of both genotypes (67% female vs. 38% male CPT-1b+/- mice, and 33% female vs. 8% male CPT-1b+/+ mice). Therefore, lethality of homozygous CPT-1b deficiency in the mice is consistent with paucity of human cases.

Figure 1

Levels of Cpt-1a and Cpt-1b mRNA. A) Northern blot analysis of total RNA in 6- to 8- week-old non-fasted CPT-1b+/− male mice (born from a founder CPT-1b+/− male and a C56BL/6J female): Cpt-1a and Cpt-1b mRNA expression in heart (H), brown adipose tissue (BAT), and skeletal muscles including quadricep (Q) and gastrocnemus muscles (G). B) Densitometry quantification of Cpt-1b mRNA expression. The integrated density reading of each band of Cpt-1b mRNA were first standardized with the amount of total RNA shown in (A), and then the average levels of which in the CPT-1b+/+ hearts (n=3) were arbitrarily set to 1. Mean+/− SE, *p<0.05, **p<0.005.

Figure 2. Enzyme activity of CPT-1 in skeletal muscle (gastroc) and liver

Male CPT-1b+/− mice (n=5) had ∼60% and ∼92% CPT-1 activity in gastroc muscles and liver, respectively as compared to the male CPT-1b+/+ mice (n=4, **p<0.001). Results were also significantly different in female CPT-1b+/− mice (n=5) and CPT-1b+/+ mice (n=2, *p<0.05).

Figure 3. Survival curves of 6 hr cold challenge

A) Among male mice, there were 5 CPT-1b+/− mice and 1 CPT-1b+/+ mouse with fatal hypothermia; the survival rates at 6 hr for the CPT-1b+/− mice and the CPT-1b+/+ mice were 62% and 92%, respectively. Among the female mice, there were 8 CPT-1b+/− mice and 4 CPT-1b+/+ mice with fatal hypothermia; the survival rates at 6 hr for the CPT-1b+/− mice and the CPT-1b+/+ mice were 33% and 67%, respectively. In both sexes, there was no significant difference between survival of the CPT-1b+/− and CPT-1b+/+ groups (p=0.160 in male, p=0.198 in female). B) When combining the data from both sexes, there were 13 CPT-1b+/− mice and 4 CPT-1b+/+ mice that developed fatal hypothermia. The survival rates at 6h for the CPT-1b+/− mice and the CPT-1b+/+ were 48% and 79%, respectively (*p<0.05 by Kaplan-Meier survival analysis: Gehan-Breslow test).

Figure 4. Serum acylcarnitines

Following an 18 h fast, in CPT-1b+/− female mice (7−10 weeks old), there were small reductions in serum C₁₆ and C₁₈ acylcarnitine levels (n=6 for each genotype, mean+/−S.E, *p<0.05); however, the males (n=6 for each genotype, mean+/−S.E) showed no differences.

Figure 5. Blood glucose levels

In both sexes (6−8 weeks old), there was no significant difference in blood glucose concentrations following either an 18 h fast or a 6h cold challenge between the two genotypes. Also, blood glucose levels in all 4 groups increased significantly following a 3 h but not a 6h cold challenge (n=12−14 for each group, mean+/−S.E, *p<0.05, †p<0.005, ‡p<0.001). Moreover, following a 6h cold challenge in female CPT-1b+/− mice (n=13), blood glucose concentrations were significantly lower than that in CPT-1b+/+ controls (n=12, mean+/−S.E, *p<0.05).