Massive Production of Farnesol-Derived Dicarboxylic Acids in Mice Treated with the Squalene Synthase Inhibitor Zaragozic Acid A

doi:10.1006/abbi.1998.0704

Archives of Biochemistry and Biophysics

Volume 355, Issue 1, 1 July 1998, Pages 84-92

https://doi.org/10.1006/abbi.1998.0704 Get rights and content

Abstract

The zaragozic acids are potent inhibitors of squalene synthase.In vivostudies in mice confirmed our earlier observations that inhibition of squalene synthase by zaragozic acid A was accompanied by an increase in the incorporation of label from [³H]mevalonate into farnesyl-diphosphate (FPP)-derived isoprenoic acids (J. D. Bergstromet al.,1993,Proc. Natl. Acad. Sci. USA90, 80–84). Farnesyl-diphosphate-derived metabolites appear transiently in the liver. We were unable to detect any farnesol formation in the zaragozic acid-treated animals which indicates that FPP is readily converted to farnesoic acid and dicarboxylic acids in the liver. These metabolites were found to be produced only in the liver and not in the kidney.trans-3,7-Dimethyl-2-octaen-1,8-dioic acid and 3,7-dimethyloctan-1,8-dioic acid were identified as the major end products of farnesyl-diphosphate metabolism in the urine of mice treated with zaragozic acid A. Quantitative analysis of these FPP-derived dicarboxylic acids by gas–liquid chromatography revealed that approximately 11 mg of total dicarboxylic acids is excreted per day into the urine of a mouse after 3 days of treatment with zaragozic acid A.

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Mendelian disorders of cholesterol biosynthesis typically result in multi-system clinical phenotypes, underlining the importance of cholesterol in embryogenesis and development. FDFT1 encodes for an evolutionarily conserved enzyme, squalene synthase (SS, farnesyl-pyrophosphate farnesyl-transferase 1), which catalyzes the first committed step in cholesterol biosynthesis. We report three individuals with profound developmental delay, brain abnormalities, 2-3 syndactyly of the toes, and facial dysmorphisms, resembling Smith-Lemli-Opitz syndrome, the most common cholesterol biogenesis defect. The metabolite profile in plasma and urine suggested that their defect was at the level of squalene synthase. Whole-exome sequencing was used to identify recessive disease-causing variants in FDFT1. Functional characterization of one variant demonstrated a partial splicing defect and altered promoter and/or enhancer activity, reflecting essential mechanisms for regulating cholesterol biosynthesis/uptake in steady state.
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Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.
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Recent guidelines in North America and Europe recommend lowering low density lipoprotein associated cholesterol (LDLC) to achieve optimal coronary heart disease risk reduction. Statins have been the therapy of choice and proven successful and relatively safe. However, we are now facing new challenges and it appears that additional or alternative drugs are urgently needed. This boosts research in the field, reopening old cases like other inhibitors of cholesterol synthesis or making attractive tools from the latest technologies like gene silencing by anti-sense oligonucleotides.
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Many pathways can be considered to lower LDLC, but the road has been paved with disappointments and difficulties. With new targets identified and diversification of the drugs, a new era for better LDLC management is plausible.
Functional characterization of the atypical integral membrane lipid phosphatase PDP1/PPAPDC2 identifies a pathway for interconversion of isoprenols and isoprenoid phosphates in mammalian cells
2010, Journal of Biological Chemistry
Citation Excerpt :
PDP1/PPPAPDC2 may also be a determinant of the efficacy and toxicity of clinically important mevalonate pathway inhibitors. In particular, squalene synthase inhibitors are a novel class of cholesterol-lowering agents, but their usefulness is limited by toxicity associated with the accumulation of isoprenols that are formed by the dephosphorylation of isoprenoid diphosphates (46). Finally, direct quantitation of polyisoprenoid diphosphates in cells and tissues has been challenging.
The polyisoprenoid diphosphates farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are intermediates in the synthesis of cholesterol and related sterols by the mevalonate pathway and precursors for the addition of isoprenyl anchors to many membrane proteins. We developed tandem mass spectrometry assays to evaluate polyisoprenoid diphosphate phosphatase activity of an unusual integral membrane lipid enzyme: type 1 polyisoprenoid diphosphate phosphatase encoded by the PPAPDC2 gene (PDP1/PPAPDC2). In vitro, recombinant PDP1/PPAPDC2 preferentially hydrolyzed polyisoprenoid diphosphates, including FPP and GGPP over a variety of glycerol- and sphingo-phospholipid substrates. Overexpression of mammalian PDP1/PPAPDC2 in budding yeast depletes cellular pools of FPP leading to growth defects and sterol auxotrophy. In mammalian cells, PDP1/PPAPDC2 localizes to the endoplasmic reticulum and nuclear envelope and, unlike the structurally related lipid phosphate phosphatases, is predicted to be oriented with key residues of its catalytic domain facing the cytoplasmic face of the membrane. Studies using synthetic isoprenols with chemical properties that facilitate detection by mass spectrometry identify a pathway for interconversion of isoprenols and isoprenoid diphosphates in intact mammalian cells and demonstrate a role for PDP1/PPAPDC2 in this process. Overexpression of PDP1/PPAPDC2 in mammalian cells substantially decreases protein isoprenylation and results in defects in cell growth and cytoskeletal organization that are associated with dysregulation of Rho family GTPases. Taken together, these results focus attention on integral membrane lipid phosphatases as regulators of isoprenoid phosphate metabolism and suggest that PDP1/PPAPDC2 is a functional isoprenoid diphosphate phosphatase.
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Regular ArticleMassive Production of Farnesol-Derived Dicarboxylic Acids in Mice Treated with the Squalene Synthase Inhibitor Zaragozic Acid A

Abstract

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Lipid Res.

J. Lipid Res.

Anal. Biochem.

J. Lipid Res.

Biochem. Biophys. Acta.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Regular Article
Massive Production of Farnesol-Derived Dicarboxylic Acids in Mice Treated with the Squalene Synthase Inhibitor Zaragozic Acid A