High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity
| Autor: | Yin Liang, Margery A. Connelly, Gary W. Caldwell, Jenson Qi, Lisa Minor, Peter Walter Maria Roevens, Joannes Theodorus Maria Linders, Andrew L. Darrow, Katharina DeWaepenaert, Wensheng Lang, Charles E. Smith, Edward C. Giardino, Gustaaf Henri Maria Willemsens |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Male
Very low-density lipoprotein Insecta [13C18]oleic acid high-content assay QD415-436 Lipoproteins VLDL Kidney Tandem mass spectrometry Biochemistry Rats Sprague-Dawley chemistry.chemical_compound Endocrinology Tandem Mass Spectrometry In vivo Methods Animals Humans Carbon Radioisotopes Diacylglycerol O-Acyltransferase triglyceride Cells Cultured Triglycerides Enzyme Assays Diacylglycerol kinase Triglyceride biology Chemistry Cellular Assay Cell Biology Enzyme assay Rats Oleic acid Liver Hepatocytes biology.protein lipids (amino acids peptides and proteins) glycerol-3-phosphate pathway liquid chromatography/tandem mass spectrometry Chromatography Liquid Oleic Acid |
| Zdroj: | Journal of Lipid Research, Vol 51, Iss 12, Pp 3559-3567 (2010) |
| ISSN: | 0022-2275 |
| DOI: | 10.1194/jlr.d008029 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|