ATP-Citrate Lyase Deficiency in the Mouse

Abstract

ATP-citrate lyase (Acly) is one of two cytosolic enzymes that synthesize acetyl-coenzyme A (CoA). Because acetyl-CoA is an essential building block for cholesterol and triglycerides, Acly has been considered a therapeutic target for hyperlipidemias and obesity. To define the phenotype of Acly-deficient mice, we created Acly knockout mice in which a ␤-galactosidase marker is expressed from Acly regulatory sequences. We also sought to define the cell type-specific expression patterns of Acly to further elucidate the in vivo roles of the enzyme. Homozygous Acly knockout mice died early in development. Heterozygous mice were healthy, fertile, and normolipidemic on both chow and high fat diets, despite expressing half-normal amounts of Acly mRNA and protein. Fibroblasts and hepatocytes from heterozygous Acly mice contained half-normal amounts of Acly mRNA and protein, but this did not perturb triglyceride and cholesterol synthesis or the expression of lipid biosynthetic genes regulated by sterol regulatory elementbinding proteins. The expression of acetyl-CoA synthetase 1, another cytosolic enzyme for producing acetylCoA, was not up-regulated. As judged by ␤-galactosidase staining, Acly was expressed ubiquitously but was expressed particularly highly in tissues with high levels of lipogenesis, such as in the livers of mice fed a highcarbohydrate diet. ␤-Galactosidase staining was intense in the developing brain, in keeping with the high levels of de novo lipogenesis of the tissue. In the adult brain, ␤-galactosidase staining was in general much lower, consistent with reduced levels of lipogenesis; however, ␤-galactosidase expression remained very high in cholinergic neurons, likely reflecting the importance of Acly in generating acetyl-CoA for acetylcholine synthesis. The Acly knockout allele is useful for identifying cell types with a high demand for acetyl-CoA synthesis. ATP-citrate lyase (Acly)1 is a cytosolic enzyme that catalyzes the formation of acetyl-coenzyme A (CoA) and oxaloacetate from citrate and CoA, with the hydrolysis of ATP to ADP and phosphate. Acetyl-CoA is the key building block for de novo lipogenesis. Acly is not, however, the sole source of acetyl-CoA in the cytosol. Another enzyme, acetyl-CoA synthetase 1 (Acs1), generates acetyl-CoA from acetate and CoA, with the hydrolysis of ATP to AMP and diphosphate (1). Whether Asc1 might be sufficient in the setting of a genetic deficiency in Acly is not known. The expression of both Acly and Acs1 is controlled by the sterol regulatory element-binding protein (SREBP)-1c, a transcriptional regulator of fatty acid synthesis (2, 3).An important role for Acly in lipid biosynthesis in the liver has been suggested by studies with the Acly inhibitor hydroxycitrate. In rats treated with hydroxycitrate, the rate of fatty acid and cholesterol synthesis in the liver fall (4). Those experiments and studies with other inhibitors (5-10) have suggested that Acly could represent a therapeutic target for elevated plasma lipid levels. As with other genes involved in lipid...

N-Acetylaspartate in the CNS: From neurodiagnostics to neurobiology

“…ATP citrate lyase is strongly expressed in the developing brain, and ATP citrate lyase-deficient mice die early in development, highlighting the essential nature of this reaction in many cell types, and the fact that ACS-1 can not compensate for the loss of the citrate lyase pathway (Beigneux et al, 2004). It appears likely that the brain uses both of these acetyl CoA synthesizing pathways together to meet the strong demand for lipid synthesis in the brain during postnatal myelination.…”

Agpat6 deficiency causes subdermal lipodystrophy and resistance to obesity

“…For b-galactosidase staining, tissues were fixed and stained as described previously (23). For adipose size measurement, randomly selected sections from three wild-type mice and three Agpat6 2/2 mice were used.…”

Agpat6—a novel lipid biosynthetic gene required for triacylglycerol production in mammary epithelium

“…A cDNA probe comprising sequences within the 3V untranslated region of Agpat6 (a region not conserved in Agpat8) was amplified by RT-PCR with 5V-GTGGCAGGACAAGGTCA-GAGCTACA-3V and 5V-TCCCTCCTGACTCACCAGTTCTTCC-3V. The lacZ probe was prepared as described previously (21). b-Actin AGPAT6 and mammary glandand 18S cDNA probes were used as controls for RNA integrity and loading.…”

“…[ 32 P]dCTP-labeled cDNA probes were prepared with Allin-One random prime labeling mixture (Sigma). Standard prehybridization, hybridization, and washing procedures were used (21).…”

“…For b-galactosidase staining, tissues were fixed and stained as described previously (21). For hematoxylin/eosin staining, mammary glands were fixed in 10% buffered formalin for 24 h, dehydrated in ethanol, transitioned into xylene, embedded in paraffin, sectioned (1 Am thick), and stained.…”