Cell-based assay of MGAT2-driven diacylglycerol synthesis for profiling inhibitors: use of a stable isotope-labeled substrate and high-resolution LC/MS

摘要

To demonstrate monoacylglycerol acyltransferase 2 (MGAT2)-mediated enzyme activity in a cellular context, cells of the murine secretin tumor cell-1 line of enteroendocrine origin were used to construct human MGAT2-expressing recombinant cell lines. Low throughput and utilization of radiolabeled substrate in a traditional TLC technique were circumvented by development of a high-resolution LC/MS platform. Monitoring incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) allowed selective tracing of the cellular DAG synthesis activity. This assay format dramatically reduced background interference and increased the sensitivity and the signal window compared with the TLC method. Using this assay, several MGAT2 inhibitors from different chemotypes were characterized. The described cell-based assay adds a new methodology for the development and evaluation of MGAT2 inhibitors for the treatment of obesity and type 2 diabetes. To demonstrate monoacylglycerol acyltransferase 2 (MGAT2)-mediated enzyme activity in a cellular context, cells of the murine secretin tumor cell-1 line of enteroendocrine origin were used to construct human MGAT2-expressing recombinant cell lines. Low throughput and utilization of radiolabeled substrate in a traditional TLC technique were circumvented by development of a high-resolution LC/MS platform. Monitoring incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) allowed selective tracing of the cellular DAG synthesis activity. This assay format dramatically reduced background interference and increased the sensitivity and the signal window compared with the TLC method. Using this assay, several MGAT2 inhibitors from different chemotypes were characterized. The described cell-based assay adds a new methodology for the development and evaluation of MGAT2 inhibitors for the treatment of obesity and type 2 diabetes. Monoacylglycerol acyltransferase-2 (MGAT2) is a membrane-bound lipid acyltransferase that is an emerging molecular target for the treatment of obesity and type 2 diabetes (1Yen C.L. Cheong M.L. Grueter C. Zhou P. Moriwaki J. Wong J.S. Hubbard B. Marmor S. Farese Jr., R.V. Deficiency of the intestinal enzyme acyl CoA:monoacylglycerol acyltransferase-2 protects mice from metabolic disorders induced by high-fat feeding.Nat. Med. 2009; 15: 442-446Crossref PubMed Scopus (135) Google Scholar). MGAT2 is highly and selectively expressed in the small intestine, where it exerts an important role in the monoacylglycerol pathway of triacylglycerol (TAG) synthesis for the absorption of dietary fat (2Yen C.L. Farese Jr., R.V. MGAT2, a monoacylglycerol acyltransferase expressed in the small intestine.J. Biol. Chem. 2003; 278: 18532-18537Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 3Cao J. Lockwood J. Burn P. Shi Y. Cloning and functional characterization of a mouse intestinal acyl-CoA:monoacylglycerol acyltransferase, MGAT2.J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). When dietary fat is ingested, pancreatic lipase digests TAG into FFAs and monoacylglycerol, which are absorbed by intestinal epithelial enterocytes. Once inside enterocytes, FFAs and monoacylglycerol are used as building blocks to resynthesize TAG through two sequential acylation steps. The first acylation step is performed by MGAT, followed by diacylglycerol acyltransferase (DGAT) enzyme reactions. TAGs are then incorporated into chylomicrons and secreted into lymph to be used as an energy supply for the body (4Shi Y. Cheng D. Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E10-E18Crossref PubMed Scopus (158) Google Scholar). MGAT2 KO mice exhibit a multitude of healthy metabolic phenotypes relative to WT controls, including resistance to high-fat diet-induced obesity, improvement in insulin sensitivity, and decre