The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and...

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Bibliographic Details
Published in:	Journal of lipid research Vol. 53; no. 6; pp. 1106 - 1116
Main Authors:	Qi, Jenson, Lang, Wensheng, Geisler, John G., Wang, Ping, Petrounia, Ioanna, Mai, Selyna, Smith, Charles, Askari, Hossein, Struble, Geoffrey T., Williams, Robyn, Bhanot, Sanjay, Monia, Brett P., Bayoumy, Shariff, Grant, Eugene, Caldwell, Gary W., Todd, Matthew J., Liang, Yin, Gaul, Micheal D., Demarest, Keith T., Connelly, Margery A.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-06-2012 The American Society for Biochemistry and Molecular Biology Elsevier
Subjects:	13C18-oleic acid 13C3-D5-glycerol Animals diacylglycerol acyltransferase Diacylglycerol O-Acyltransferase - antagonists & inhibitors Diacylglycerol O-Acyltransferase - genetics Diacylglycerol O-Acyltransferase - metabolism Enzyme Assays - methods Enzyme Inhibitors - pharmacology Esterification - drug effects Fatty Acids - biosynthesis Fatty Acids - metabolism Glycerol - metabolism Hep G2 Cells Humans Isotope Labeling Lipoproteins, VLDL - metabolism liquid chromatography tandem mass spectrometry Liver - enzymology Male Mice Oleic Acid - metabolism Oligonucleotides, Antisense - genetics triglyceride synthesis Triglycerides - biosynthesis very low density lipoprotein triglyceride triglyceride synthesis liquid chromatography tandem mass spectrometry very low density lipoprotein triglyceride diacylglycerol acyltransferase 13C18-oleic acid 13C3-D5-glycerol
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Summary:	Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with 13C3-D5-glycerol or 13C18-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that 13C3-D5-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, 13C18-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D5-glycerol to mice and measured plasma levels of D5-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D5-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered 13C18-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0022-2275 1539-7262
DOI:	10.1194/jlr.M020156