| High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity. | |
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MedLine Citation:
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PMID: 20805092 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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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Authors:
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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:
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Type: Evaluation Studies; Journal Article Date: 2010-08-28 |
Journal Detail:
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Title: Journal of lipid research Volume: 51 ISSN: 0022-2275 ISO Abbreviation: J. Lipid Res. Publication Date: 2010 Dec |
Date Detail:
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Created Date: 2010-11-10 Completed Date: 2011-02-22 Revised Date: 2011-12-21 |
Medline Journal Info:
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Nlm Unique ID: 0376606 Medline TA: J Lipid Res Country: United States |
Other Details:
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Languages: eng Pagination: 3559-67 Citation Subset: IM |
Affiliation:
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Johnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PA, USA. jqi@its.jnj.com |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Carbon Radioisotopes / diagnostic use, metabolism Cells, Cultured Chromatography, Liquid Diacylglycerol O-Acyltransferase / metabolism* Enzyme Assays / methods* Hepatocytes / cytology, enzymology Humans Insects / cytology, enzymology, virology Kidney / cytology, embryology, enzymology Lipoproteins, VLDL / chemistry, metabolism Liver / enzymology* Male Oleic Acid / diagnostic use, metabolism Rats Rats, Sprague-Dawley Tandem Mass Spectrometry Triglycerides / chemistry, metabolism |
| Chemical | |
Reg. No./Substance:
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0/Carbon Radioisotopes; 0/Lipoproteins, VLDL; 0/Triglycerides; 112-80-1/Oleic Acid; EC 2.3.1.20/Diacylglycerol O-Acyltransferase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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