High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity

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 [C-13(18)]oleic acid. The [C-13(18)]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 [C-13(18)] oleic acid to rats. The [C-13(18)]oleic acid-incorporated VLDL-TG was detected after one-step extraction without conventional separation of TG and recovery by derivatizing [C-13(18)]oleic acid for detection. Using potent and selective DGAT1 inhibitors as pharmacological tools, we measured changes in [C-13(18)]oleoyl-incorporated TG and DAG and demonstrated that DGAT1 inhibition significantly reduced [C-13(18)]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.-Qi, J., W. Lang, E. Giardino, G. W. Caldwell, C. Smith, L. K. Minor, A. L. Darrow, G. Willemsens, K. DeWaepenaert, P. Roevens, J. T. M. Linders, Y. Liang, and M. A. Connelly. High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity. J. Lipid Res. 2010. 51: 3559-3567.