Development of a novel LC/MS method to quantitate cellular stearoyl-CoA desaturase activity
Development of a novel LC/MS method to quantitate cellular stearoyl-CoA desaturase activity
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DOI：
10.1016/j.aca.2008.05.012
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发表时间：
2008-10-03
影响因子：
6.2
通讯作者：
Bhat, B. Ganesh
中科院分区：
文献类型：
--
作者：
Dillon, Roslyn;Greig, Michael J.;Bhat, B. Ganesh
硬脂酰辅酶A去饱和酶1(SCD1)是催化硬脂酰辅酶A和棕榈酰辅酶A从头合成油酸和棕榈酸的限速步骤。这些产品是膜磷脂、甘油三酯、胆固醇酯中含量最丰富的单不饱和脂肪酸。有报道显示，与野生型相比，具有SCD1基因靶向突变(SCD1-/-)的小鼠的葡萄糖耐量和胰岛素敏感性有所改善，这表明SCD1可能成为糖尿病和相关代谢性疾病的治疗靶点。SCD1活性的测定在技术上具有挑战性，传统的基于细胞的SCD1测定方法劳动强度大，吞吐量低。在这里，我们描述了一种新的基于细胞的中通量LC/MS分析方法，用于测定细胞的SCD1活性，便于筛选潜在的SCD1抑制剂化合物。融合的HepG2细胞生长在24孔板中，与载体或抑制剂孵育，然后与氚标记的饱和脂肪酸底物孵育。提取细胞总脂，用液-质联用法测定硬脂酸转化为油酸的转化率。Sterculate是一种已知的SCD1抑制剂，在本实验中以剂量依赖的方式抑制该酶的活性，计算的EC50为247 NM，这里描述的中通量方法是朝着寻找治疗糖尿病和相关代谢性疾病的SCD1抑制剂迈出的重要一步。(C)2008爱思唯尔B.V.保留所有权利。
Stearoyl-CoA desaturase 1 (SCD1) is an enzyme that catalyzes the rate-limiting step in de novo synthesis of monounsaturated fatty acids-mainly oleate and palmitoleate from stearoyl-CoA and palmitoyl-Co A, respectively. These products are the most abundant monounsaturated fatty acids in membrane phospholipids, triglycerides, cholesterol esters. Reports on mice with a targeted disruption of SCD1 gene (SCD1-/-) exhibit improved glucose tolerance and insulin sensitivity compared to wild-type suggesting SCD1 could be a therapeutic target for diabetes and related metabolic diseases. Measurement of SCD1 activity is technically challenging and traditional cell-based SCD1 assay procedure is labor intensive with low throughput. We describe here a novel medium-throughput LC/MS cell-based assay for determining cellular SCD1 activity, facilitating screening of potential SCD1 inhibitor compounds. Confluent HepG2 cells were grown in 24-well plates and incubated with vehicle or an inhibitor followed by incubation with deuterium labeled saturated fatty acid substrates. Total cell lipids were extracted and the conversion of stearate to oleate was measured by liquid chromatography-mass spectrometry. Sterculate, a known inhibitor of SCD1, inhibited the enzyme activity in a dose dependent manner in this assay with a calculated EC50 of 247 nM, The medium-throughput method described here is an important step towards identifying an inhibitor of SCD1 to treat diabetes and related metabolic diseases. (C) 2008 Elsevier B.V. All rights reserved.