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  • 标题:High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity
  • 本地全文:下载
  • 作者:Jenson Qi ; Wensheng Lang ; Edward Giardino
  • 期刊名称:JLR Papers In Press
  • 印刷版ISSN:0022-2275
  • 电子版ISSN:1539-7262
  • 出版年度:2010
  • 卷号:51
  • 期号:12
  • 页码:3559-3567
  • DOI:10.1194/jlr.D008029
  • 语种:English
  • 出版社:American Society for Biochemistry and Molecular Biology
  • 摘要:Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates. In the liver, the production of VLDL permits the delivery of hydrophobic TG from the liver to peripheral tissues for energy metabolism. We describe here a novel high-content, high-throughput LC/MS/MS-based cellular assay for determining DGAT activity. We treated endogenous DGAT-expressing cells with stable isotope-labeled [13C18]oleic acid. The [13C18]oleoyl-incorporated TG and DAG lipid species were profiled. The TG synthesis pathway assay was optimized to a one-step extraction, followed by LC/MS/MS quantification. Further, we report a novel LC/MS/MS method for tracing hepatic TG synthesis and VLDL-TG secretion in vivo by administering [13C18]oleic acid to rats. The [13C18]oleic acid-incorporated VLDL-TG was detected after one-step extraction without conventional separation of TG and recovery by derivatizing [13C18]oleic acid for detection. Using potent and selective DGAT1 inhibitors as pharmacological tools, we measured changes in [13C18]oleoyl-incorporated TG and DAG and demonstrated that DGAT1 inhibition significantly reduced [13C18]oleoyl-incorporated VLDL-TG. This DGAT1-selective assay will enable researchers to discern differences between the roles of DGAT1 and DGAT2 in TG synthesis in vitro and in vivo.
  • 关键词:[13C18]oleic acid ; triglyceride ; glycerol-3-phosphate pathway ; liquid chromatography/tandem mass spectrometry ; high-content assay
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