Molecular characterization and tissue-specific expression of the acetyl-CoA carboxylase α gene from Grass carp, Ctenopharyngodon idella.

Acetyl-CoA carboxylase α (ACC1), the major regulatory enzyme of fatty acid biosynthesis, catalyzes the conversion of acetyl-CoA to malonyl-CoA. The full-length cDNA coding ACC1 isoform was cloned from liver of grass carp. The cDNA obtained was 7515bp with a 7173bp open reading frame encoding 2389 amino acids. The ACC1 protein has a calculated molecular weight of 269.2kDa and isoelectric point of 6.23. Tissue distribution of ACC1 mRNA in brain, mesenteric adipose, spleen, white muscle and liver of grass carp was analyzed by real-time PCR method using ß-actin as an internal control for cDNA normalization. The results showed that the expressions of ACC1 mRNA were detected in all examined tissues. Relative expression profile of ACC1 mRNA in liver normalized with ß-actin level was 15, 92, 135 and 165-fold compared with the level in brain, white muscle, mesenteric adipose and spleen, respectively. In addition, we present evidence for the presence of two isoforms of ACC1 (265.7kDa and 267.2kDa) in grass carp liver that differ from the 269.2kDa ACC1 by the absence of 34 and 15 amino acids. In conclusion, the liver is one of the main ACC1 producing tissues in grass carp and ACC1 gene was highly homologous to that of mammals.

Discovery of novel (4-piperidinyl)-piperazines as potent and orally active acetyl-CoA carboxylase 1/2 non-selective inhibitors: F-Boc and triF-Boc groups are acid-stable bioisosteres for the Boc group.

Novel (4-piperidinyl)-piperazine derivatives were synthesized and evaluated as ACC1/2 non-selective inhibitors. Optimization of the substituents on the nitrogen of the piperidine ring led to the identification of the fluorine substituted tert-butoxycarbonyl group. Advanced analog, 1,1,1-trifluoro-2-methylpropan-2-yl 4-{4-[(2-amino-6-methyl-1-benzothiophen-3-yl)carbonyl]piperazin-1-yl}piperidine-1-carboxylate (12c) showed potent inhibitory activities in enzyme-assay and cell-based assays. Compound 12c also exhibited reduction of hepatic de novo fatty acid synthesis in rats after oral administration.

Genes encoding the alpha-carboxyltransferase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution.

Heteromeric acetyl coenzyme A carboxylase (ACCase), a rate-limiting enzyme in fatty acid biosynthesis in dicots, is a multi-enzyme complex consisting of biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase (alpha-CT and beta-CT). In the present study, four genes encoding alpha-CT were cloned from Brassica napus, and two were cloned from each of the two parental species, B. rapa and B. oleracea. Comparative and cluster analyses indicated that these genes were divided into two major groups. The major divergence between group-1 and group-2 occurred in the second intron. Group-2 alpha-CT genes represented the ancestral form in the genus Brassica. The divergence of group-1 and group-2 genes occurred in their common ancestor 12.96-17.78 million years ago (MYA), soon after the divergence of Arabidopsis thaliana and Brassica (15-20 MYA). This time of divergence is identical to that reported for the paralogous subgenomes of diploid Brassica species (13-17 MYA). Real-time reverse transcription PCR revealed that the expression patterns of the two groups of genes were similar in different organs, except in leaves. To better understand the regulation and evolution of alpha-CT genes, promoter regions from two sets of orthologous gene copies from B. napus, B. rapa, and B. oleracea were cloned and compared. The function of the promoter of gene Bnalpha-CT-1-1 in group-1 and gene Bnalpha-CT-2-1 in group-2 was examined by assaying beta-glucuronidase activity in transgenic A. thaliana. Our results will be helpful in elucidating the evolution and regulation of ACCase in oilseed rape.

Acetyl-coenzyme A carboxylase alpha gene variations may be associated with the direct effects of some antipsychotics on triglyceride levels.

Acetyl-coenzyme A carboxylase alpha (ACACA) single-nucleotide polymorphism (SNP) (rs2229416) was significantly associated with hypertriglyceridemia, during exploration of antipsychotic direct effects on lipids. Neuropeptide Y (NPY) gene (rs1468271) and ACACB gene (rs2241220) SNPs were significantly associated with severe hypercholesterolemia. In the same sample (173 patients on olanzapine, quetiapine, chlorpromazine or mirtazapine [increasing the risk of hyperlipidemia] and 184 controls taking other antipsychotics), three (rs1266175, rs12453407 and rs9906543) of eight additional ACACA SNPs were significantly associated with hypertriglyceridemia in those taking drugs of interest, but not in controls. Five other ACACA SNPs, three additional NPY SNPs, and seven additional ACACB SNPs were not significant.

Chronic suppression of acetyl-CoA carboxylase 1 in beta-cells impairs insulin secretion via inhibition of glucose rather than lipid metabolism.

Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to beta-cells suppressed [(14)C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.