Abstract
The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.
Highlights
The recent finding that p-nitrobenzofurazan (NBD)FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G
Hormone-sensitive lipase (HSL) in particular is thought to play an important role in the mobilization of fatty acids from the triacylglycerols (TAGs) stored in adipocytes, providing the main source of energy in mammals
Insulin acts as an antilipolytic hormone by phosphorylating and activating phosphodiesterase 3B, which hydrolyzes cAMP and reduces the hydrolysis of toward acylglycerol only moderately (TAG) [3]
Summary
Egg yolk phosphatidylcholine (PC), soybean phosphatidylinositol (PI), BSA, acetylcholinesterase, butyrylcholinesterase, pig liver esterase, 4-methylumbelliferyl butyrate, and 4-methylumbelliferyl palmitate were obtained from Sigma-Aldrich Fine Chemicals. 12-Aminododecanoic acid (compound 1) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (compound 2) were obtained from Fluka (Seelze, Germany). After being incubated for 5 min, the reaction mixture became clear and a solution of compound 2 (15 g, 75 mmol) in MeOH (300 ml) was added. To synthesize (S)-2,2-dimethyl[1,3]dioxolan-4-ylmethyl 12-(7nitrobenzo[1,2,5]oxadiazol-4-ylamino)dodecanoate (compound 5a), a solution of compound 1 (60 mg, 159 mol) in CH2Cl2 (2 ml) was treated with dicyclohexylcarbodiimide (160 mg, 770 mol) and stirred at 25ЊC for 30 min. The solvent was distilled off in vacuo, and the residue was purified by flash chromatography (2:1, 1:1 toluene-EtOAc). The resulting fat-free supernatant was mixed with 1 g of heparin-Sepharose (washed five times with 25 mM Tris-HCl, pH 7.4, and 150 mM NaCl), incubated at 4ЊC for 1 h (under head-to-end rotation of the vial), and centrifuged for 10 min at 1,000 g at 4ЊC. This procedure considerably decreases the 2-MAG lipase levels (during the acid precipitation step) and the removal of 70% of the LPL (during the heparin-Sepharose step) from the adipocyte extract, as shown by our experimental data (unpublished results)
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