High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity |
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Authors: | Jenson Qi Wensheng Lang Edward Giardino Gary W Caldwell Charles Smith Lisa K Minor Andrew L Darrow Gustaaf Willemsens Katharina DeWaepenaert Peter Roevens Joannes T M Linders Yin Liang Margery A Connelly |
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Institution: | *Johnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PA;†Johnson and Johnson Pharmaceutical Research and Development, LLC, and Beerse, Belgium |
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Abstract: | 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. |
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Keywords: | [13C18]oleic acid triglyceride glycerol-3-phosphate pathway liquid chromatography/tandem mass spectrometry high-content assay |
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