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

Jenson Qi,Wensheng Lang,Edward Giardino,Gary W Caldwell,Charles Smith,Lisa K Minor,Andrew L Darrow,Gustaaf Willemsens,Katharina Dewaepenaert,Peter Roevens,Joannes T.M Linders,Yin Liang,Margery A Connelly

doi:10.1194/jlr.d008029

Jenson Qi, Wensheng Lang + Show 11 more

Open Access

https://doi.org/10.1194/jlr.d008029

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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 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.

Highlights

Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates
Because triolein is the mostabundant TG in mammals and oleoyl-CoA may be the most-physiologically relevant acyl donor for the DGAT reaction, stable isotope-labeled oleic acid ([13C18]oleic acid) was chosen to metabolically trace DAG and TG synthesis
Activity was determined in cell-free recombinant human diacylglycerol acyltransferase 1 (DGAT1), human DGAT2, human ACAT1, human ACAT2, microsomal triglyceride transfer protein (MTP), the peroxisome proliferator-activated receptor ␣ (PPAR␣), PPAR␥, stearoyl-CoA desaturase 1 (SCD-1), and apolipoprotein B secretion assays

Summary

Introduction

Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates. We describe a novel, high-throughput LC/ MS/MS-based cellular assay for detecting newly synthesized TG using endogenously expressing DGAT1 cells and a stable isotope-labeled oleic acid. The major species of stable isotope-labeled oleoyl-incorporated TG from plasma were detected by LC/MS/MS after a one-step sample extraction in high-throughput format.

Results

Conclusion