Diacylglycerol

Acyl-CoAs ([1-¹⁴ C]-labeled an unlabeled) used for enzymatic assays were synthesized according to the method developed by Sánchez et al. [47 (link)]. [1-¹⁴C]-fatty acids and triolein [carboxyl-¹⁴C] were purchased from Perkin Elmer or American Radiochemicals. Labeled diacylglycerol was derived through partial lipase treatment of triolein [carboxyl-¹⁴C] with Rhizopus arrhizus lipase (Sigma-Aldrich). The lipase products were separated on TLC plates (silica gel 60; Sigma-Aldrich). The DAG was eluted from the silica with methanol: chloroform (2:1, v/v), extracted with chloroform by Bligh and Dyer method (1959). Non-radioactive fatty acids and diacylglycerol were ordered from Larodan or Sigma-Aldrich. Internal standard of heptadecanoic acid methyl ester, coenzyme A and TAG standard for thin-layer chromatography were purchased also from Sigma-Aldrich.

The following lipid standards used in this study were purchased from Larodan Fine Chemicals AB:TG (11:1-11:1-11:1), diacylglycerol (DG) (8:0-8:0), monoacylglycerol (MG) (15:1), and cholesteryl ester (CE) (10:0). Additional lipid standards were purchased from Avanti Polar Lipids, Inc., including phophatidylcholine (PC) (10:0-10:0), phosphatidylethanolamine (PE) (10:0-10:0), phosphoserine (PS) (10:0-10:0), phosphatidylglycerol (10:0-10:0), phosphatidylinositol (8:0-8:0), phosphatidic acid (PA) (10:0-10:0), lysophosphatidylcholine (LPC) (13:0), lysophosphatidylethanolamine (LPE) (14:0), lysophosphoserine (LPS) (17:1), lysophosphatidylglycerol (14:0), lysophosphatidylinositol (13:0), lysophosphatidic acid (LPA) (14:0), sphingomyeline (SM) (d18:1-12:0), and ceramide (Cer) (d18:1-12:0). Each lipid standard was dissolved in methanol or chloroform (in the case of CE), stored at −20 °C, and diluted to 1 µg/mL for extraction.