High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity

Abstract

that they are a major source of energy. TGs are synthesized from glycerol and three FA molecules; each FA is attached via an ester bond to hydroxyl groups of the glycerol backbone. Like many neutral lipids, TGs contain FA molecules with varying chain lengths; the most common are 16, 18, or 20 carbons. The two major biosynthetic pathways of TG are the glycerol-3-phosphate pathway, which exists primarily in liver and adipose tissues, and the monoacylglycerol pathway, which exists predominately in the intestine. The fi nal step of the glycerol-3-phosphate biosynthetic pathway can be catalyzed by either diacylglycerol acyltransferase 1 (DGAT1) or DGAT2 ( 1, 2 ). Although DGAT1 and DGAT2 both convert diacylglycerol (DAG) to TG, they do not share similarity in either their nucleotide or amino acid sequences.It has been reported that knockout mice lacking DGAT1 (DGAT1 Ϫ / Ϫ ) do not display obvious changes in TG metabolism in the liver ( 3 ). In contrast, knockout mice lacking DGAT2 (DGAT2 Ϫ / Ϫ ) display severely reduced TG content in the liver ( 4 ). Furthermore, studies have shown that suppression of DGAT2 with antisense oligonucleotides reduced hepatic TG content in rodents ( 5, 6 ) producing reversed diet-induced hepatic steatosis and insulin resistance in rats ( 5 ). These results suggest that DGAT1 and DGAT2 function differently in TG biosynthesis. The fi nding that multiple enzymes catalyze the synthesis of TG from DAG presents an opportunity to modulate one catalytic mechanism of this biochemical reaction. This type of modulation may produce therapeutic results without the potential adverse side effects that might occur if TG synthesis were completely inhibited in all tissues. By specifically inhibiting the activity of DGAT1 or DGAT2, compounds that inhibit the conversion of DAG to TG will be useful in lowering absorption and circulating concentrations of TG.Abstract 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 DGATexpressing cells with stable isotope-labeled [

The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2

“…Recombinant human DGAT1-or DGAT2-expressing Sf9 membranes were produced in a baculovirus expression system as previously described ( 11 ). DGAT activity was assayed in a solution containing 100 mM Tris-HCl (pH 7.5), 5 g/ml DGAT1-or DGAT2-expressing membranes, 0.25 M sucrose, 15 mM MgCl 2 , 1 mM EDTA, 0.1% BSA, 50 M oleoyl-CoA, and 50 M 1,2-dicapryl-sn -glycerol diluted to 1% acetone, in a total reaction volume of 100 l for 40 min.…”

Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models

“…Fortunately, advances in mass spectrometry and other areas now permit high-throughput, high-resolution measurements. For example, we, and others recently demonstrated the ability to measure the incorporation of [ 13 C 18 ]oleate into TG following a single bolus administration of tracer ( 5,12 ). We demonstrated the utilization of that approach in quantifying TG synthesis in vivo.…”

The use of stable-isotopically labeled oleic acid to interrogate lipid assembly in vivo: assessing pharmacological effects in preclinical species

“…Table 2 reports data for the isotopomer profi le for two triglycerides, 54:3 and 54:4, as well as the labeling of their respective precursor pools following oral administration of [ 13 C 18 ] oleic acid in various formulations to mice that had been fasted for 16 h prior to testing. When the tracer fatty acid was mixed in a vehicle containing endogenous 12 C lipid (olive oil or heavy cream), the labeled substrate was largely incorporated as a single equivalent into newly synthesized triglyceride. Conversely, when the tracer was administered in aqueous TPGS, the predominant triglyceride detected in plasma was the fully labeled (M 3 ) triolein; the response for the M 3 isotopomer exceeded the response for the endogenous 12 C form of TG 54:3 under these conditions.…”

“…When the tracer fatty acid was mixed in a vehicle containing endogenous 12 C lipid (olive oil or heavy cream), the labeled substrate was largely incorporated as a single equivalent into newly synthesized triglyceride. Conversely, when the tracer was administered in aqueous TPGS, the predominant triglyceride detected in plasma was the fully labeled (M 3 ) triolein; the response for the M 3 isotopomer exceeded the response for the endogenous 12 C form of TG 54:3 under these conditions. In this case, the precursor pool effectively consists of the [ 13 C 18 ] oleic acid tracer and whatever lipid can be derived from intracellular stores or the systemic circulation.…”

“…The ability of LC-MS to separate different classes of lipids, such as triglycerides, phosphatidylcholines, and cholesteryl esters, in a single run is another signifi cant advantage and allows the disposition of the tracer between different lipid pools to be easily explored. A recent publication by Qi et al ( 12 ) describes the utility of an LC-MS-based approach to monitoring the effects of pharmacological inhibitors of DGAT1 on di-and triglyceride assembly from stable-isotopically labeled oleic acid in vitro and in vivo. Although the authors demonstrated the capability of the LC-MS method to measure multiple labeled forms of di-and triglyceride simultaneously, little detail on the mass isotopomer distribution profi le was presented, and no analysis of precursor pool labeling was described.…”