Metabolic tracing of monoacylglycerol acyltransferase-2 activity in vitro and in vivo

Anal Biochem. 2017 May 1:524:68-75. doi: 10.1016/j.ab.2016.09.017. Epub 2016 Sep 22.

Abstract

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/kg U13C-TG oil. The newly resynthesized plasma heavy TGs containing three 13C in the glycerol backbone and two U13C-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 and U13C-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.

Keywords: Fat absorption assay; LC/ESI/MS/MS; MGAT2; MGAT2 cell-based assay; Stable isotope incorporated TG.

MeSH terms

  • Animals
  • Chromatography, Liquid / methods
  • Enzyme Inhibitors / pharmacology
  • Glycerides / metabolism*
  • Glycerol / metabolism*
  • HEK293 Cells
  • Humans
  • Lipid Metabolism*
  • Mice
  • N-Acetylglucosaminyltransferases / metabolism*
  • Tandem Mass Spectrometry / methods*

Substances

  • Enzyme Inhibitors
  • Glycerides
  • N-Acetylglucosaminyltransferases
  • alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase
  • Glycerol