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Journal of Lipid Research, Vol. 46, 603-614, March 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Sensitive assay for hormone-sensitive lipase using NBD-labeled monoacylglycerol to detect low activities in rat adipocytes

Stefan Petry^1,2,*, Yassine Ben Ali^2,, Henri Chahinian, Holger Jordan^*, Horst Kleine^*, Günter Müller^*, Frédéric Carrière and Abdelkarim Abousalham^1,

^* Aventis Pharma Germany
Enzymology at Interfaces and Physiology of Lipolysis, Unité Propre de Recherche 9025, Centre National de la Recherche Scientifique 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France

¹ To whom correspondence should be addressed. e-mail: abousal{at}ibsm.cnrs-mrs.fr (A.A.); stefan.petry{at}aventis.com (S.P.)

in re-

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. Müller, 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.

Abbreviations: DAG, diacylglycerol; DOG, 1,2-dioleoylglycerol; EtOAc, ethyl acetate; HPL, human pancreatic lipase; HSL, hormone-sensitive lipase; MAG, monoacylglycerol; MeOH, methanol; NBD, p-nitrobenzofurazan; OD, optical density; PC, phosphatidylcholine; PI, phosphatidylinositol; R_f, relative mobility; TAG, triacylglycerol; TOG, trioleoylglycerol

Supplementary key words p-nitrobenzofurazan • liposomes • NBD-fatty acid

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