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 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.
Qi
Jenson
J
Johnson and Johnson Pharmaceutical Research and Development, LLC, Spring House, PA, USA. jqi@its.jnj.com
Lang
Wensheng
W
Giardino
Edward
E
Caldwell
Gary W
GW
Smith
Charles
C
Minor
Lisa K
LK
Darrow
Andrew L
AL
Willemsens
Gustaaf
G
Dewaepenaert
Katharina
K
Roevens
Peter
P
Linders
Joannes T M
JT
Liang
Yin
Y
Connelly
Margery A
MA
D000818:Animals; D002250:Carbon Radioisotopes / Q000378:metabolism; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D051048:Diacylglycerol O-Acyltransferase / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000201:enzymology / Q000821:virology; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000201:enzymology; D008079:Lipoproteins, VLDL / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D019301:Oleic Acid / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry; D014280:Triglycerides / Q000737:chemistry / Q000378:metabolism
D002250:Carbon Radioisotopes; D008079:Lipoproteins, VLDL; D014280:Triglycerides; D019301:Oleic Acid; D051048:Diacylglycerol O-Acyltransferase
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