Document Detail

High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity.
MedLine Citation:
PMID:  20805092     Owner:  NLM     Status:  MEDLINE    
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.
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
Publication Detail:
Type:  Evaluation Studies; Journal Article     Date:  2010-08-28
Journal Detail:
Title:  Journal of lipid research     Volume:  51     ISSN:  0022-2275     ISO Abbreviation:  J. Lipid Res.     Publication Date:  2010 Dec 
Date Detail:
Created Date:  2010-11-10     Completed Date:  2011-02-22     Revised Date:  2013-05-28    
Medline Journal Info:
Nlm Unique ID:  0376606     Medline TA:  J Lipid Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3559-67     Citation Subset:  IM    
Johnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PA, USA.
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MeSH Terms
Carbon Radioisotopes / diagnostic use,  metabolism
Cells, Cultured
Chromatography, Liquid
Diacylglycerol O-Acyltransferase / metabolism*
Enzyme Assays / methods*
Hepatocytes / cytology,  enzymology
Insects / cytology,  enzymology,  virology
Kidney / cytology,  embryology,  enzymology
Lipoproteins, VLDL / chemistry,  metabolism
Liver / enzymology*
Oleic Acid / diagnostic use,  metabolism
Rats, Sprague-Dawley
Tandem Mass Spectrometry
Triglycerides / chemistry,  metabolism
Reg. No./Substance:
0/Carbon Radioisotopes; 0/Lipoproteins, VLDL; 0/Triglycerides; 112-80-1/Oleic Acid; EC O-Acyltransferase

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