Metabolic tracing of monoacylglycerol acyltransferase-2 activity in vitro and in vivo.
Monoacylglycerol acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DAG) from free fatty acids (FFA) and sn-monoacylglycerol (MG), the two major hydrolysis products of dietary fat. To demonstrate MGAT2-mediated cellular activity of triglyceride (TG) synthesis, we utilized 1-oleoyl-glycerol-d5 as a substrate to trace MGAT2-driven 1-oleoyl-glycerol-d5 incorporation into TG in HEK293 cells stably expressing human MGAT2. The oleoyl-glycerol-d5 incorporated major TG species were then quantified by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) in a 96-well format. Conventional MGAT2 target-engagement in vivo assays measure the elevation of total plasma TG by orally dosing a bolus of TG oil. We developed a novel LC/ESI/MS/MS-based fat absorption assay to assess the ability of MGAT2 inhibitors to inhibit fat absorption in CD1 mice by a meal tolerance test consisting of a mixture of liquid Boost plus®and 0.59 g/kgU13C-TG oil. The newly resynthesized plasma heavy TGs containing three13C in the glycerol backbone and twoU13C-acyl-chains, which represented the digested, absorbed and resynthesized TGs, were then quantitated by LC/ESI/MS/MS. With this assay, we identified a potent MGAT2 inhibitor that blocked MGAT2-mediated activity in vitro and in vivo. The use of 1-oleoyl-glycerol-d5 andU13C-TG oil followed by LC/ESI/MS/MS detection of stable-isotopic labeled DAG, TG, or glycerol provides a wide range of applications to study pathophysiological regulation of the monoacylglycerol pathway and MGAT2 activity.
Cardiovascular and Metabolic Disease Research, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA. Electronic address: jqi@its.jnj.com.
Jenson
J
Qi
Discovery Sciences, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Wensheng
W
Lang
Cardiovascular and Metabolic Disease Research, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Margery A
MA
Connelly
Cardiovascular and Metabolic Disease Research, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Fuyong
F
Du
Cardiovascular and Metabolic Disease Research, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Yin
Y
Liang
Discovery Sciences, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Gary W
GW
Caldwell
Cardiovascular and Metabolic Disease Research, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Tonya
T
Martin
Cardiovascular and Metabolic Disease Research, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Michael K
MK
Hansen
Cardiovascular and Metabolic Disease Research, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Gee-Hong
GH
Kuo
Cardiovascular and Metabolic Disease Research, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Michael D
MD
Gaul
Cardiovascular and Metabolic Disease Research, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Alessandro
A
Pocai
Cardiovascular and Metabolic Disease Research, Janssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477-0776, USA.
Seunghun
S
Lee
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005989:Glycerides / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D050356:Lipid Metabolism; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004791:Enzyme Inhibitors; D005989:Glycerides; D017351:N-Acetylglucosaminyltransferases; C063313:alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase; D005990:Glycerol
Fat absorption assay; LC/ESI/MS/MS; MGAT2; MGAT2 cell-based assay; Stable isotope incorporated TG