title: The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2.
abstract: 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 (13)C(3)-D(5)-glycerol or (13)C(18)-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that (13)C(3)-D(5)-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, (13)C(18)-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D(5)-glycerol to mice and measured plasma levels of D(5)-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D(5)-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered (13)C(18)-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.
authors.affiliation: Cardiovascular and Metabolic Disease Research and Community of Research Excellence and Advanced Technology, Janssen Pharmaceutical Companies of Johnson and Johnson, Spring House, PA 19477, USA. jqi@its.jnj.com
authors.forename: Jenson
authors.initials: J
authors.lastname: Qi
authors.forename: Wensheng
authors.initials: W
authors.lastname: Lang
authors.forename: John G
authors.initials: JG
authors.lastname: Geisler
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Ioanna
authors.initials: I
authors.lastname: Petrounia
authors.forename: Selyna
authors.initials: S
authors.lastname: Mai
authors.forename: Charles
authors.initials: C
authors.lastname: Smith
authors.forename: Hossein
authors.initials: H
authors.lastname: Askari
authors.forename: Geoffrey T
authors.initials: GT
authors.lastname: Struble
authors.forename: Robyn
authors.initials: R
authors.lastname: Williams
authors.forename: Sanjay
authors.initials: S
authors.lastname: Bhanot
authors.forename: Brett P
authors.initials: BP
authors.lastname: Monia
authors.forename: Shariff
authors.initials: S
authors.lastname: Bayoumy
authors.forename: Eugene
authors.initials: E
authors.lastname: Grant
authors.forename: Gary W
authors.initials: GW
authors.lastname: Caldwell
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Todd
authors.forename: Yin
authors.initials: Y
authors.lastname: Liang
authors.forename: Micheal D
authors.initials: MD
authors.lastname: Gaul
authors.forename: Keith T
authors.initials: KT
authors.lastname: Demarest
authors.forename: Margery A
authors.initials: MA
authors.lastname: Connelly
mesh_terms: D000818:Animals; D051048:Diacylglycerol O-Acyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004951:Esterification / Q000187:drug effects; D005227:Fatty Acids / Q000096:biosynthesis / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D007553:Isotope Labeling; D008079:Lipoproteins, VLDL / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D019301:Oleic Acid / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics; D014280:Triglycerides / Q000096:biosynthesis
chemical_list: D004791:Enzyme Inhibitors; D005227:Fatty Acids; D008079:Lipoproteins, VLDL; D016376:Oligonucleotides, Antisense; D014280:Triglycerides; D019301:Oleic Acid; C494881:DGAT1 protein, human; C494885:DGAT2 protein, human; D051048:Diacylglycerol O-Acyltransferase; D005990:Glycerol