Mutant mice lacking acetyl-CoA carboxylase 1 are embryonically lethal

Abstract

Acetyl-CoA carboxylases (ACC1 and ACC2) catalyze the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate metabolite that plays a pivotal role in the regulation of fatty acid metabolism. We previously reported that ACC2 null mice are viable, and that ACC2 plays an important role in the regulation of fatty acid oxidation through the inhibition of carnitine palmitoyltransferase I, a mitochondrial component of the fatty-acyl shuttle system. Herein, we used gene targeting to knock out the ACC1 gene. The heterozygous mutant mice (Acc1(+/-)) had normal fertility and lifespans and maintained a similar body weight to that of their wild-type cohorts. The mRNA level of ACC1 in the tissues of Acc1(+/-) mice was half that of the wild type; however, the protein level of ACC1 and the total malonyl-CoA level were similar. In addition, there was no difference in the acetate incorporation into fatty acids nor in the fatty acid oxidation between the hepatocytes of Acc1(+/-) mice and those of the wild type. In contrast to Acc2(-/-) mice, Acc1(-/-) mice were not detected after mating. Timed pregnancies of heterozygotes revealed that Acc(-/-) embryos are already undeveloped at embryonic day (E)7.5, they die by E8.5, and are completely resorbed at E11.5. Our previous results of the ACC2 knockout mice and current studies of ACC1 knockout mice further confirm our hypotheses that malonyl-CoA exists in two independent pools, and that ACC1 and ACC2 have distinct roles in fatty acid metabolism.

Fig. 1.

Targeted mutation of the Acc1 locus. (A) Strategy used to create the targeted mutation. The exon (dark box) that contains two of the exons upstream the biotin-binding motif (Met-Lys-Met) was replaced with a hypoxanthine phosphoribosyltransferase (HPRT) expression cassette. The 3′ and 5′ probes used for Southern blot analysis are indicated. (B) A typical pattern observed in genotyping by Southern blot analyses of genomic DNA extracted from mouse tails. The DNA was digested with SphI in duplicate. The blots were probed with the 5′ and 3′ probes indicated in A. The presence of only wild-type (+/+) and heterozygous (+/–) genotypes indicated that no homozygous (–/–) mice were born.

Fig. 2.

Developmental abnormalities in E9.5 and E7.5 of ACC1 mutant embryos. Whole embryos at the late head-fold stage (E8.5) and E6.5 were extracted from the uterus of a heterozygous (+/–) female mouse after crossbreeding with a heterozygous male (+/–) and were mounted for morphological study. (A) A wild-type or heterozygous embryo at E8.5. (B) A degenerating Acc1 mutant embryo recovered by dissecting the ectoplacental cone shown in C. (D) A normal, developed embryo at E7.5 compared to a smaller and unorganized embryo (E). The embryos in B and E account for ≈25% of all of the offspring of heterozygous female mice crossbred with heterozygous males.

Fig. 3.

Northern and Western blot analyses of total RNA from white adipose tissue and perfused hepatocytes of wild-type and heterozygous mutant Acc1^+/–mice. Total RNA from white adipose (A) and hepatocytes (B) of wild-type and mutant Acc^+/– mice were subjected to 1% agarose gel in the presence of formalin. The fractionated RNA was transferred to Hybond N (Amersham Pharmacia Biotech). The filters were hybridized with ³²P-labeled cDNA of a 362-bp mouse cDNA PCR fragment. To show equal loading, the gel were stained with ethedium bromide to visualize the 28S and 18S rRNA. (C and D) Western blot of liver crude extracts (25 μg) probed with avidin peroxidase and antiphospho-ACC (Ser-79) respectively; lanes: 1, wild type; 2, heterozygous (+/–); M, myosin.

Fig. 4.

Malonyl-CoA levels and fatty acid oxidation synthesis in hepatocytes of Acc^+/– and wild-type mice. (A) The levels of malonyl-CoA in liver extracts of wild-type (wt) and heterozygous (het) mice were determined by the incorporation of [³H]acetyl-CoA into palmitate in the presence of NADPH and highly purified chicken fatty acid synthase. The [³H]palmitic acid synthesized was extracted with petroleum ether, and the radioactivity was measured. The mice were either fed a normal chow (F) or were fasted for 48 h, followed by feeding (RF) a fat-free/high-carbohydrate diet for another 48 h before they were killed. (B) Hepatocytes prepared from perfused livers (2 × 10⁵ cells) were cultured in polylysine-coated bottles with shaking in Krebs-Ringer bicarbonate buffer. Fatty acid oxidation was determined by measuring the ¹⁴CO₂ generated from the oxidation of [1-¹⁴C] palmitate and trapped with benzethonium solution in the center wells. (C) [³H]acetyl-CoA incorporation into total lipids in perfused hepatocytes.

Fig. 5.

Body weight of male and female Acc^+/– heterozygous and wild-type mice. Ten- to 12-week-old female (A) and male (B) mice were fed normal diets, and their weights were determined weekly for 24 weeks. The data are shown as means ± SD (n = 5).

Fig. 6.

A schematic model shows that ACC1 and ACC2 are compartmentalized and produce two independent malonyl-CoA Pools. Shown are the biochemical pathways for generating acetyl-CoA and malonyl-CoA, the precursors for de novo fatty acid synthesis and regulators for fatty acid oxidation.