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 metabolis...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2010-12-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520313687 |
| _version_ | 1830487014081822720 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Jenson Qi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T18:54:08Z |
| format | Article |
| id | doaj.art-adcb468b74c344d7843f3780adbc81d2 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-21T18:54:08Z |
| publishDate | 2010-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-adcb468b74c344d7843f3780adbc81d22022-12-21T18:53:40ZengElsevierJournal of Lipid Research0022-22752010-12-01511235593567High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activityJenson Qi0Wensheng Lang1Edward Giardino2Gary W. Caldwell3Charles Smith4Lisa K. Minor5Andrew L. Darrow6Gustaaf Willemsens7Katharina DeWaepenaert8Peter Roevens9Joannes T.M. Linders10Yin Liang11Margery A. Connelly12To whom correspondence should be addressed:. jqi@its.jnj.com; Johnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PAJohnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PAJohnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PAJohnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PAJohnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PAJohnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PAJohnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PAJohnson and Johnson Pharmaceutical Research and Development, LLC and Beerse, BelgiumJohnson and Johnson Pharmaceutical Research and Development, LLC and Beerse, BelgiumJohnson and Johnson Pharmaceutical Research and Development, LLC and Beerse, BelgiumJohnson and Johnson Pharmaceutical Research and Development, LLC and Beerse, BelgiumJohnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PAJohnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PAAcyl-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.http://www.sciencedirect.com/science/article/pii/S0022227520313687[13C18]oleic acidtriglycerideglycerol-3-phosphate pathwayliquid chromatography/tandem mass spectrometryhigh-content assay |
| spellingShingle | 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 High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity Journal of Lipid Research [13C18]oleic acid triglyceride glycerol-3-phosphate pathway liquid chromatography/tandem mass spectrometry high-content assay |
| title | High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity |
| title_full | High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity |
| title_fullStr | High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity |
| title_full_unstemmed | High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity |
| title_short | High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity |
| title_sort | high content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity |
| topic | [13C18]oleic acid triglyceride glycerol-3-phosphate pathway liquid chromatography/tandem mass spectrometry high-content assay |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520313687 |
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