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. 2006 May 30;103(22):8552-7.
doi: 10.1073/pnas.0603115103. Epub 2006 May 22.

Liver-specific deletion of acetyl-CoA carboxylase 1 reduces hepatic triglyceride accumulation without affecting glucose homeostasis

Jianqiang Mao  1 , Francesco J DeMayo, Huiguang Li, Lutfi Abu-Elheiga, Ziwei Gu, Tattym E Shaikenov, Parichher Kordari, Subrahmanyam S Chirala, William C Heird, Salih J Wakil
Affiliations

Liver-specific deletion of acetyl-CoA carboxylase 1 reduces hepatic triglyceride accumulation without affecting glucose homeostasis

Jianqiang Mao et al. Proc Natl Acad Sci U S A. .

Abstract

In animals, liver and white adipose are the main sites for the de novo fatty acid synthesis. Deletion of fatty acid synthase or acetyl-CoA carboxylase (ACC) 1 in mice resulted in embryonic lethality, indicating that the de novo fatty acid synthesis is essential for embryonic development. To understand the importance of de novo fatty acid synthesis and the role of ACC1-produced malonyl-CoA in adult mouse tissues, we generated liver-specific ACC1 knockout (LACC1KO) mice. LACC1KO mice have no obvious health problem under normal feeding conditions. Total ACC activity and malonyl-CoA levels were approximately 70-75% lower in liver of LACC1KO mice compared with that of the WT mice. In addition, the livers of LACC1KO mice accumulated 40-70% less triglycerides. Unexpectedly, when fed fat-free diet for 10 days, there was significant up-regulation of PPARgamma and several enzymes in the lipogenic pathway in the liver of LACC1KO mice compared with the WT mice. Despite the significant up-regulation of the lipogenic enzymes, including a >2-fold increase in fatty acid synthase mRNA, protein, and activity, there was significant decrease in the de novo fatty acid synthesis and triglyceride accumulation in the liver. However, there were no significant changes in blood glucose and fasting ketone body levels. Hence, reducing cytosolic malonyl-CoA and, therefore, the de novo fatty acid synthesis in the liver, does not affect fatty acid oxidation and glucose homeostasis under lipogenic conditions.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Generation of tissue-specific knockout mice. (A) The strategy to delete, in a tissue-specific manner, the biotin binding site (Met-Lys-Met) containing exon is shown. The targeting vector contained a floxed Neo gene inserted in intron 21 and a loxP site inserted in intron 22, and, in addition, the TK gene for negative selection of nonhomologous events. Homologous recombination in ES cells generated targeted allele (ACC1neo) allele. Chimeras generated by using the ES cells were bred with C57BL/6J mice to generate mice containing an ACC1neo allele. The generation of mice containing ACC1lox allele and tissue-specific deletion of exon 22 (ACC1del) are described in Materials and Methods. LoxP sites are indicated by arrowheads. Primers for PCR analysis are indicated by arrows. (B) A typical Southern blot analysis of ES cell DNA by using the probes indicated in A. (C) PCR-based genotyping of tail DNA to distinguish WT (lox−/−, lane 1), heterozygote (lox+/−, lane 2), and homozygote (lox+/+, lane 3) by using the primers a and b and c and d, indicated in A. (D) PCR-based genotyping of liver DNA by using the primers a and d. (E) RT-PCR analysis for liver mRNA by using primers derived from the exons 21 and 24. In D and E, + and − refer to the presence and absence of Cre.
Fig. 2.
Fig. 2.
Analysis of ACC1 expression in the WT and LACC1KO mice. The preparation of liver extracts, affinity purification of biotinylated proteins, and determination of ACC activities were performed as described in Materials and Methods. (A) Avidin-affinity purified proteins from liver homogenates were fractioned by 6% Tris-glycine SDS/PAGE. The gel was stained with Coomassie (Left, lanes 1–4). A similar gel was electroblotted to nitrocellulose sheet and probed with avidin–horseradish peroxidase (Right, lanes 7–10). The mutated ACC1 protein is 10 kDa less than the WT ACC1. Lanes 5 and 6 show the 250-kDa molecular mass standard. (B and C) ACC activities in the liver extracts and avidin-affinity purified proteins, respectively, of mice (n = 4–5) after 2 days of fasting, followed by 2 days of refeeding with fat-free diet. Open bars, WT; filled bars, LACC1KO. (D) Malonyl-CoA content in the livers from mice (n = 5) fed fat-free diet as described in B and C. (E) Liver histology of WT and LACC1KO of mice fed fat-free diet for 28 days. Liver sections were stained with Oil Red O as described in ref. .
Fig. 3.
Fig. 3.
De novo lipogenesis in the isolated primary hepatocytes. Hepatocytes isolated from the perfused livers of WT and LACC1KO mice were incubated with 14C-acetate for 2 h, and the radioactivity in the nonsaponifiable (cholesterol) and saponifiable (fatty acids) fractions was measured. Where indicated, insulin was added to the medium at 0.2 μM. ∗, P < 0.001 (LACC1KO vs. WT mice).
Fig. 4.
Fig. 4.
Analysis of gene expression in the livers of LACC1KO mice. (A) Relative mRNA levels of genes expressed in the livers of LACC1KO mice over that of the WT mice as determined by real-time PCR. ACL, ATP-citrate lyase; LCE, long-chain fatty acid elongase; SCD-1, stearoyl-CoA desaturase-1; HNF4α, hepatic nuclear factor 4α. (B) Coomassie staining of bands obtained by gel electrophoresis (4–12% NuPAGE gel with Mes buffer) of 30 μg of protein samples from livers of WT mice (lanes 1–3) and from livers of LACC1KO mice (lanes 4–6). (C) Western blot analysis by using anti-FAS antibody. Arrows indicate the position of FAS protein, and M denotes standard protein, 188 kDa.
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