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• W2096596761 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. 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. 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. Lipases (EC 3.1.1.3) play a key role in human lipid metabolism, as they degrade dietary as well as stored lipids and thus initiate and regulate the release of free fatty acids into the serum. Lipases are therefore promising targets for the development of drugs in the field of obesity, diabetes, and atherosclerosis. 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 (for review, see 1), providing the main source of energy in mammals. In vivo, HSL is activated by phosphorylation via cAMP-dependent kinase in response to various lipolytic hormones such as catecholamines. The phosphorylation of HSL leads to its translocation from the cytoplasm to the lipid droplet (2Clifford G.M. Kraemer F.B. Yeaman S.J. Vernon R.G. Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation during the lactation cycle of the rat.Metabolism. 2001; 50: 1264-1269Abstract Full Text PDF PubMed Scopus (16) Google Scholar). Insulin acts as an antilipolytic hormone by phosphorylating and activating phosphodiesterase 3B, which hydrolyzes cAMP and thus reduces the hydrolysis of TAG (3Degerman E. Landström T.R. Wijkander J. Holst L.S. Ahmad F. Belfrage P. Manganiello V.V. Phosphorylation and activation of hormone-sensitive adipocyte phosphodiesterase type 3B.Methods. 1998; 14: 43-53Crossref PubMed Scopus (67) Google Scholar). In addition to adipocytes, HSL is expressed in other tissues (4Kraemer F.B. Patel S. Saedi M.S. Sztalryd C. Detection of hormone-sensitive lipase in various tissues. I. Expression of an HSL/bacterial fusion protein and generation of anti-HSL antibodies.J. Lipid Res. 1993; 34: 663-671Abstract Full Text PDF PubMed Google Scholar), including skeletal muscle, heart, brain, pancreatic β cells, adrenal gland, ovaries, testes, and macrophages (1Holm C. Osterlund T. Laurell H. Contreras J.A. Molecular mechanisms regulating hormone-sensitive lipase and lipolysis.Annu. Rev. Nutr. 2000; 20: 365-393Crossref PubMed Scopus (342) Google Scholar). Because neutral lipases are water-soluble enzymes hydrolyzing insoluble long-chain TAG substrates and some phospholipids to a variable extent, the cleavage reaction has to occur at the lipid-water interface (5Bengtsson-Olivecrona G. Olivecrona T. Medical aspects of triglyceride lipases.in: Wooley P. Petersen S.B. Lipases. Cambridge University Press, Cambridge, UK1994: 315-336Google Scholar, 6Hide W.A. Chan L. Li W.H. Structure and evolution of the lipase superfamily.J. Lipid Res. 1992; 33: 167-178Abstract Full Text PDF PubMed Google Scholar, 7Verger R. 'Interfacial activation’ of lipases: facts and artifacts.Trends Biotechnol. 1997; 15: 32-38Abstract Full Text PDF Scopus (700) Google Scholar). The mechanisms involved in the enzymatic lipolysis depend strongly on the mode of organization of the lipid substrate in interfacial structures such as monolayers, micelles, liposomal dispersions, and oil-in-water emulsions. Lipases interact with these lipid complexes, or “supersubstrates,” via hydrophobic domains that are exposed upon contact as the result of a substrate-induced conformational change, which sometimes has been called “interfacial activation” (8Schmid R.D. Verger R. Lipases: interfacial enzymes with attractive applications.Angew. Chem. Int. Ed. Engl. 1998; 37: 1608-1633Crossref PubMed Google Scholar, 9Panaitov I. Verger R. Enzymatic reactions at interfaces: interfacial and temporal organization of enzymatic lipolysis.in: Baszkin A. Norde W. Physical Chemistry of Biological Interfaces. Marcel Dekker, Inc., New York2000: 359-400Google Scholar). The two-dimensional nature of this lipase reaction does not obey Michaelis-Menten kinetics and depends critically on the quality of the interface (7Verger R. 'Interfacial activation’ of lipases: facts and artifacts.Trends Biotechnol. 1997; 15: 32-38Abstract Full Text PDF Scopus (700) Google Scholar, 8Schmid R.D. Verger R. Lipases: interfacial enzymes with attractive applications.Angew. Chem. Int. Ed. Engl. 1998; 37: 1608-1633Crossref PubMed Google Scholar, 9Panaitov I. Verger R. Enzymatic reactions at interfaces: interfacial and temporal organization of enzymatic lipolysis.in: Baszkin A. Norde W. Physical Chemistry of Biological Interfaces. Marcel Dekker, Inc., New York2000: 359-400Google Scholar). Obtaining accurate (i.e., substrate-specific) measurements of lipase activity as well as developing reliable lipase assay systems require taking these unique features into account. We published a critical review describing the various lipase detection and assay methods available (10Beisson F. Tiss A. Rivière C. Verger R. Methods for lipase detection and assay: a critical review.Eur. J. Lipid Sci. Technol. 2000; 1: 133-153Crossref Google Scholar). Generally speaking, these methods can be classified in two groups: chemical methods, in which the amount of substrate disappearing or the amount of product released is measured; and physical methods, which are based on the changes with time in a given physical property, such as the conductivity, turbidity, or interfacial tension during the lipolytic reaction (10Beisson F. Tiss A. Rivière C. Verger R. Methods for lipase detection and assay: a critical review.Eur. J. Lipid Sci. Technol. 2000; 1: 133-153Crossref Google Scholar). In addition, we have developed a continuous lipase assay using naturally occurring fluorescent TAG isolated from Parinari glaberrium (11Beisson F. Ferte N. Nari J. Noat G. Arondel V. Verger R. Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings.J. Lipid Res. 1999; 40: 2313-2321Abstract Full Text Full Text PDF PubMed Google Scholar). Synthetic octadeca-9,11, 13,15-tetronic-3-hydroxy-octadecyloxypropylester, a 1-acyl-2-alkyl glycerol from parinaric acid, is a diacylglycerol (DAG) analog that provides an efficient substrate for HSL. But its pronounced sensitivity to oxidation precludes its use under routine conditions (S. Petry, H. Jordan, H. Kleine, and N. Tennagels, unpublished results). An alternative ultraviolet spectrophotometric assay based on the use of TAG from Aleutris fordii seeds, which is less sensitive to oxidation, was recently introduced by our group (12Pencreac'h G. Graille J. Pina M. Verger R. An ultraviolet spectrophotometric assay for measuring lipase activity using long-chain triacyglycerols from Aleurites fordii seeds.Anal. Biochem. 2002; 303: 17-24Crossref PubMed Scopus (27) Google Scholar). Various fluorogenic substrates have been used to measure lipase activity (for review, see 13Hendrickson H.S. Fluorescence-based assays of lipases, phospholipases, and other lipolytic enzymes.Anal. Biochem. 1994; 219: 1-8Crossref PubMed Scopus (56) Google Scholar). It has been established that fluorophores such as BODIPY (14Meshulam T. Herscovitz H. Casavant D. Bernardo J. Roman R. Haugland R.P. Strohmeier G.S. Diamond R.D. Simons E.R.R. Flow cytometric kinetic measurements of neutrophil phospholipase A activation.J. Biol. Chem. 1992; 267: 21465-21470Abstract Full Text PDF PubMed Google Scholar), rhodamine (15Agmon V. Cherbu S. Dagan A. Grace M. Grabowski G.A. Gatt S.S. Synthesis and use of novel fluorescent glycosphingolipids for estimating beta-glucosidase activity in vitro in the absence of detergents and subtyping Gaucher disease variants following administration into intact cells.Biochim. Biophys. Acta. 1993; 1170: 72-79Crossref PubMed Scopus (13) Google Scholar), and pyrene (16Scholze H. Stutz H. Paltauf F. Hermetter A. Fluorescent inhibitors for the qualitative and quantitative analysis of lipolytic enzymes.Anal. Biochem. 1999; 276: 72-80Crossref PubMed Scopus (21) Google Scholar) incorporated into lipase substrates do not interfere with the cleavage of these substrates by lipolytic enzymes. In general, the chromophore should be as small as possible and should be hydrophobic to ensure optimum interactions with the lipase. In addition, the chromophore group should not interact with colored compounds and should be insensitive to oxidation. For these reasons, we selected the p-nitrobenzofurazan (NBD) moiety as a fluorescence label. For instance, the NBD group has been used previously as a fluorophore in discontinuous phospholipase A2 assays (17Dagan A. Yedgar S. A facile method for direct determination of phospholipase A2 activity in intact cells.Biochem. Int. 1987; 15: 801-808PubMed Google Scholar, 18Wittenauer L.A. Shirai K. Jackson R.L. Johnson J.D. Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase.Biochem. Biophys. Res. Commun. 1984; 118: 894-901Crossref PubMed Scopus (59) Google Scholar). We have also previously reported that NBD-FA is taken up by adipocytes and incorporated into acylglycerol in an insulin-sensitive manner and that NBD-FA is released from the NBD-FA-labeled acylglycerols upon challenging the adipocytes with catecholamines, which shows that NBD-modified fatty acid and lipid precursors/derivatives generally are accepted as substrates by lipid-handling enzymes (e.g., acyltransferases, HSL) (19Muller G. Jordan H. Petry S. Wetekam E.M. Schindler P. Analysis of lipid metabolism in adipocytes using a fluorescent fatty acid derivative. I. Insulin stimulation of lipogenesis.Biochim. Biophys. Acta. 1997; 1347: 23-39Crossref PubMed Scopus (14) Google Scholar). Starting with NBD-FA, we synthesized a water-insoluble lipase substrate, monoacylglycerol (NBD-MAG) in the form of mixed phospholipid liposomes, which constitutes a sensitive substrate for HSL as well as for other lipases tested. 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). Collagenase (type I, 250 U/mg; Worthington) was provided by Biochrom (Berlin, Germany); male Wistar rats (220–250 g, fed ad libitum) were delivered from the Aventis Pharma animal breeding station (Kastengrund, Germany); Si-60 silica gel plates were purchased from Merck (Darmstadt, Germany). Nonidet-P40 and protease inhibitor cocktails were from Roche Diagnostics. Heparin-Sepharose CL-6B was obtained from Pharmacia-Biotech (Freiburg, Germany). ProBond Nickel-Chelating Resin was from Invitrogen Life Technologies. Porcine colipase devoid of phospholipase contamination was purified by J. de Caro (Enzymology at Interfaces and Physiology of Lipolysis, Marseille, France). All other chemicals and solvents were of reagent or better quality and were obtained from local suppliers. For the synthesis of NBD-FA (compound 3), sodium methanolate solution (14.5 ml, 76 mmol) was added under stirring to a solution of compound 1 (18 g, 83.7 mmol) in methanol (MeOH; 300 ml). 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. The reaction mixture, which immediately became dark, was stirred for 18 h at 25°C. Methanolic HCl (1 M, 100 ml, 100 mmol) was then added and the solvent was distilled off in vacuo. The residue was taken up in MeOH and filtered through silica gel. The filtrate was concentrated to dryness, and after being solubilized it was purified by flash chromatography [1:1 toluene-ethyl acetate (EtOAc)]. Compound 3 was obtained in the form of a red solid (26.4 g, 93%). Relative mobility (Rf): 0.16 (1:1 toluene-EtOAc). 1H-NMR (250 MHz, CDCl3): δ 8.5 (d, 1 H, ArH), 6.35 (m, 1 H, NH), 6.18 (d, 1 H, ArH), 3.48 (dt, 2 H, CH2NH2), 2.36 (t, 2 H, CH2COOH), 1.9–1.2 (m, 18 H, 9 CH2). MS (electrospray ionization-MS): 379.2 (M+1). To synthesize 2,3-dihydroxypropyl 12-(7-nitrobenzo[1,2,3]oxadiazol-4-ylamino)dodecanoate (compound 4), a solution of compound 1 (12 g, 31.7 mmol) and 2,3-epoxypropanol (50 ml) in isopropanol (50 ml) was stirred at 50°C for 16 h. The solvent was distilled off in vacuo, and the residue was dried at 0.01 torr and then purified by flash chromatography (diisopropyl ether, ether, EtOAc). Compound 4 was obtained as a red oil (10.3 g, 71.8%). Rf: 0.18 (1:1 toluene-EtOAc); Rf: 0.5 (30:5:1 CH2Cl2-MeOH-NH3), which crystallized from EtOAc-diethyl ether. 1H-NMR (250 MHz, CDCl3): δ 8.5 (d, 1H, ArH), 6.35 (m, 1 H, NH), 6.18 (d, 1 H, ArH), 4.19 (dd, 2 H, H-1, H-1′), 3.94 (m, 1 H, H-2), 3.65 (dd, 2 H, H-3, H-3′), 3.48 (dt, 2 H, CH2NH2), 2.35 (t, 2 H, CH2COOH), 1.8 (m, 2 H, CH2), 1.6 (m, 2 H, CH2), 1.27–1.15 (m, 14 H, 7 CH2). MS (electrospray ionization-MS): 453.4 (M+1). To synthesize (S)-2,2-dimethyl[1,3]dioxolan-4-ylmethyl 12-(7-nitrobenzo[1,2,5]oxadiazol-4-ylamino)dodecanoate (compound 5a), a solution of compound 1 (60 mg, 159 μmol) in CH2Cl2 (2 ml) was treated with dicyclohexylcarbodiimide (160 mg, 770 μmol) and stirred at 25°C for 30 min. A solution of (R)-(2,2-dimethyl[1,3]dioxolan-4-yl)methanol (100 mg, 760 μmol) and dimethylaminopyridine (94 mg, 770 μmol) in CH2Cl2 (2 ml) was then added, and the mixture was stirred for 4 h at 25°C. The solvent was distilled off in vacuo, and the residue was then purified by flash chromatography (15:1 toluene-EtOAc). Compound 5a was obtained in the form of a yellow fluorescent oil (46 mg, 58%). Rf: 0.29 (4:1 toluene-EtOAc). 1H-NMR (CDCl3): δ 8.5 (d, 1 H, aromatic), 6.2 (m, 1 H, NH), 6.16 (d, 1 H, aromatic), 4.31 (m, 1 H), 4.1 (m, 3 H), 3.73 (dd, 1 H), 3.48 (dt, 2 H, CH2NH2), 2.35 (t, 2 H, CO-CH2), 2.0–1.2 (m, 18 H, 9 CH2), 1.42 (s, 3 H, CMe2), 1.37 (s, 3 H, CMe2). (R)-2,2-Dimethyl[1,3]dioxolan-4-ylmethyl 12-(7-nitrobenzo [1,2,5]oxadiazol-4-ylamino)dodecanoate (compound 5b) was synthesized as described for compound 5a, but starting with (S)-(2,2-dimethyl[1,3]dioxolan-4-yl)methanol and NBD-FA (compound 3). The protected compound 5b was obtained in the form of a yellow fluorescent oil (51.4 mg, 65%). Rf: 0.29 (4:1 toluene-EtOAc). 1H-NMR (CDCl3): δ 8.5 (d, 1 H, ArH), 6.2 (m, 1 H, NH), 6.16 (d, 1 H, ArH), 4.31 (m, 1 H), 4.1 (m, 3 H), 3.73 (dd, 1 H), 3.48 (dt, 2 H, CH2NH2), 2.32 (t, 2 H, CO-CH2), 2.0–1.2 (m, 18 H, 9 CH2), 1.42 (s, 3 H, CMe2), 1.37 (s, 3 H, CMe2). To synthesize (S)-2,3-dihydroxypropyl 12-(7-nitrobenzo[1,2,5] oxadiazol-4-ylamino)dodecanoate (compound 4a) and (R)-2,3-dihydroxypropyl 12-(7-nitrobenzo[1,2,5]oxadiazol-4-ylamino)dodecanoate (compound 4b), methanolic HCl (1 M, 200 μl) was added separately to a 13.9 mg (28.2 μmol) solution of compound 4a or a 17.8 mg (36.1 μmol) solution of compound 4b in 25 ml of MeOH. The mixture was stirred for 1.5 h at 25°C. The solvent was distilled off in vacuo, and the residue was purified by flash chromatography (2:1, 1:1 toluene-EtOAc). The fatty acylesters, compounds 4a and 4b, were obtained in yields of 10.5 mg (82%) and 9.3 mg (57%), respectively. 1H-NMR (CDCl3) data and mass spectra were identical to those obtained for compound 4. The NBD-MAG enantiomers were synthesized starting with d- and l-1,2-O-isopropylideneglycerol, which was esterified with NBD-FA (1Holm C. Osterlund T. Laurell H. Contreras J.A. Molecular mechanisms regulating hormone-sensitive lipase and lipolysis.Annu. Rev. Nutr. 2000; 20: 365-393Crossref PubMed Scopus (342) Google Scholar) by dicyclohexylcarbodiimide activation. The protective group was removed using 1 N methanolic HCl. (R)-(2,2-Dimethyl [1,3]dioxolan-4-yl)methanol (5a) was the starting material for synthesizing 12-(7-nitrobenzofurazan-4-ylamino)-dodecanoic acid (S)-2,3-dihydroxy-propyl ester (4a), and (S)-(2,2-dimethyl[1,3]dioxolan-4-yl)methanol (5b) was the starting material for the corresponding R-enantiomer 12-(7-nitrobenzofurazan-4-ylamino)-dodecanoic acid (R)-2,3-dihydroxy-propyl ester (4b). The recombinant human HSL was expressed and purified from insect cells as described by Ben Ali et al. (20Ben Ali Y. Chahinian H. Petry S. Muller G. Carriere F. Verger R. Abousalham A.A. Might the kinetic behavior of hormone-sensitive lipase reflect the absence of the lid domain?.Biochemistry. 2004; 43: 9298-9306Crossref PubMed Scopus (33) Google Scholar). The recombinant human pancreatic lipase (HPL) was expressed and purified from insect cells as described by Thirstrup et al. (21Thirstrup K. Carrière F. Hjorth S. Rasmussen P.B. Wöldike H. Nielsen P.F. Thim L.L. One-step purification and characterization of human pancreatic lipase expressed in insect cells.FEBS Lett. 1993; 327: 79-84Crossref PubMed Scopus (65) Google Scholar). Purified Thermomyces lanuginosus lipase was a generous gift from S. Patkar (Novo Nordisk). Lipoprotein lipase (affinity purified) from bovine milk, defatted BSA (fraction V), and phospholipase A2 from human pancreas were purchased from Sigma (Deisenhofen, Germany). Adipocytes were isolated from epididymal fat pads of Wistar rats by performing digestion with collagenase and subsequent separation steps from undigested tissue using a nylon web, using previously published procedures (19Muller G. Jordan H. Petry S. Wetekam E.M. Schindler P. Analysis of lipid metabolism in adipocytes using a fluorescent fatty acid derivative. I. Insulin stimulation of lipogenesis.Biochim. Biophys. Acta. 1997; 1347: 23-39Crossref PubMed Scopus (14) Google Scholar, 22Müller G. Ertl J. Gerl M. Preibisch G. Leptin impairs metabolic actions of insulin in isolated rat adipocytes.J. Biol. Chem. 1997; 272: 10585-10593Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). Cells obtained from 10 rats were washed three times with 50 ml each of homogenization buffer (25 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, and 10 μg/ml each of leupeptin, antipain, and pepstatin) by flotation (500 g, 2 min, 25°C, swing-out rotor), suspended in 10 ml of homogenization buffer, and then homogenized by performing 10 strokes at 1,500 rpm in a loose-fitting Teflon-in-glass homogenizer (15°C). The homogenate was centrifuged for 10 min at 3,000 g at 4°C. The infranatant below the fat layer was aspirated and recentrifuged. This procedure was repeated three times to completely remove the residual lipid left at the top after the centrifugation. The final infranatant was centrifuged for 45 min at 48,000 g at 4°C. 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 4°C for 1 h (under head-to-end rotation of the vial), and then centrifuged for 10 min at 1,000 g at 4°C. The supernatant was adjusted to pH 5.2 and incubated for 30 min at 4°C. The precipitates were collected by centrifugation (25,000 g, 10 min, 4°C), suspended in 2.5 ml of 20 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1 mM DTT, 70 mM NaCl, 13% sucrose, and 10 μg/ml each of leupeptin, antipain, and pepstatin, and finally dialyzed (20 h, 4°C) against 3 × 500 ml of 25 mM Tris-HCl, pH 7.4, 50% glycerol, 1 mM EDTA, 1 mM DTT, and 10 μg/ml each of leupeptin, antipain, and pepstatin. Adipocyte extract was frozen in liquid N2 and stored at −70°C for up to 4 weeks. 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). To prepare the NBD-MAG substrate, 41.5 μl of a PC solution (6 mg/ml in chloroform), 83.5 μl of a PI solution (6 mg/ml in chloroform), and 100 μl of a NBD-MAG solution (10 mg/ml in chloroform) were added to plastic scintillation vials and dried over a stream of N2. This NBD-MAG substrate incorporated into phospholipid liposomes can be stored ready for use for up to 3 days at 4°C without any significant loss of cleavage efficiency. The compound itself is crystalline and can be stored for longer than 1 year without any detectable degradation (purity after 2 years is >95%). The dried lipids were then resuspended in 20 ml of 25 mM Tris-HCl buffer, pH 7.4, and 150 mM NaCl and then subjected to an ultrasonic treatment in an ice bath using a Branson Sonifier (type II, standard microtip; 2 × 1 min at setting 2 followed by 2 × 1 min at setting 4 with 1 min intervals). During the ultrasonic treatment, the substrate suspension shifted from yellow (maximum absorbance at 481 nm) to pink (maximum absorbance at 550 nm) (Fig. 1). This suspension was used after a period of 15 min (minimum) to 2 h (maximum). To start the assay procedure, 180 μl of NBD-MAG substrate solution were warmed to 30°C and supplemented with either 30 μl of an adipocyte extract (appropriately diluted with 25 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.1% BSA) or 20 μl of recombinant human HSL in the wells of 96-well microtiter plates. The optical density (OD) at 481 nm was recorded continuously at regular intervals (from 1 to 30 min) using a microplate scanning spectrophotometer (PowerWave; Bio-Tek Instruments). Buffer alone was used in the control experiments. Alternatively, the products generated in the reaction mixture were analyzed by TLC. For this purpose, 200 μl of the reaction mixture was transferred into 2 ml reaction vials and supplemented with 1.3 ml of MeOH-chloroform-heptane (10:9:7, v/v/v) and then with 0.4 ml of 0.1 M HCl. After intense vortexing, phase separation was initiated by centrifugation (800 g, 20 min, 25°C), and 200 μl aliquots of the lower organic phase were removed, dried under a vacuum (SpeedVac evaporator), and suspended in 50 μl of tetrahydrofuran. Five to 10 μl samples were separated by performing TLC on silica gel Si-60 plates using diethylether-petrol ether-acetic acid (78:22:1, v/v/v) as the elution solvent system. In a pure lane, authentic NBD-FA was run and used as a marker. In some experiments, the amount of NBD-FA acid released was assessed by fluorescence imaging using a PhosphorImager (Molecular Dynamics; Storm 860 and ImageQuant software) with an excitation wavelength of 460 nm and an emission wavelength of 540–550 nm. The specific activity of recombinant human HSL was calculated from the steady-state reaction rate (ΔOD/min) using a molar extinction coefficient of 6,700 M−1 at pH 6.0 (see Results and Discussion). The specific activity was expressed in international units per milligram of purified lipase. One international unit corresponds to 1 μmol of fatty acid released per minute under the assay conditions. The OD at time 0 (ODt = 0) can be expressed using the following formula: ODt=0=εs[S]0(Eq. 1) where εs is the substrate (NBD-MAG) molar extinction coefficient and [S]0 is the concentration of NBD-MAG at time 0. The OD measured at time t (ODt) was determined using the following formula: ODt=εs[S]t+εp[P]t(Eq. 2) where εp is the molar extinction coefficient of the product (NBD-FA), [S]t is the concentration of NBD-MAG at time t, and [P]t is the concentration of the reaction product (NBD-FA) at time t. [P]t and [S]t, therefore, could be expressed as Xpt [S]0 and (1 − Xpt) [S]0, respectively, where Xpt is the reaction progress coefficient (0 < Xpt < 1). The variation of the OD at 481 nm (ΔOD481 nm) is given by the following equation: ΔOD481 nm = (Eq. 1) − (Eq. 2) = (εp − εs) [S] 0 Xpt(Eq. 3) εp and εs were determined using the linear part of the Beer-Lambert law in a 3 ml quartz cuvette containing 0.16 mM pure NBD-FA and after being incorporated under the same experimental lipase assay conditions into PC and PI liposomes, respectively. The absorbance spectra were recorded at wavelengths between 280 and 680 nm (Fig. 1) using a Uvikon 860 spectrophotometer (Kontron Instruments). Because the length of the optical path of the quartz cuvette (1 cm) and that of the microtiter plates (∼0.6 cm) were different, a correction factor of 1.66 was applied when using the microtiter plate. HSL activity was measured at pH values ranging from 3.0 to 9.0. The buffers used were 50 mM sodium acetate (pH 4.0–6.0), 50 mM Tris (pH 6.0–8.0), and 50 mM glycine (pH 8.0–10.0). Protein concentration was determined using the bicinchoninic acid method (Pierce, Rockford, IL) with BSA as the standard. Previous studies (1Holm C. Osterlund T. Laurell H. Contreras J.A. Molecular mechanisms regulating hormone-sensitive lipase and lipolysis.Annu. Rev. Nutr. 2000; 20: 365-393Crossref PubMed Scopus (342) Google Scholar, 5Bengtsson-Olivecrona G. Olivecrona T. Medical aspects of triglyceride lipases.in: Wooley P. Petersen S.B. Lipases. Cambridge University Press, Cambridge, UK1994: 315-336Google Scholar, 6Hide W.A. Chan L. Li W.H. Structure and evolution of the lipase superfamily.J. Lipid Res. 1992; 33: 167-178Abstract Full Text PDF PubMed Google Scholar, 23Santamarina-Fojo S. Haudenschild C. Amar M. The role of hepatic lipase in lipoprotein metabolism and atherosclerosis.Curr. Opin. Lipidol. 1998; 9: 211-219Crossref PubMed Scopus (211) Google Scholar, 24Goldberg I.J. Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis.J. Lipid Res. 1996; 37: 693-707Abstract Full Text PDF PubMed Google Scholar, 25Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J.J. A novel endothelial-derived lipase that modulates HDL metabolism.Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (412) Google Scholar, 26Hirata K. Dichek H.L. Cioffi J.A. Choi S.Y. Leeper N.J. Quintana L. Kronmal G.S. Cooper A.D. Quertermous T.T. Cloning of a unique lipase from endothelial cells extends the lipase gene family.J. Biol. 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Chem. 1981; 256: 6311-6320Abstract Full Text PDF PubMed Google Scholar) have shown that HSL, LPL, pancreatic lipase, endothelial lipase, and hepatic lipase all hydrolyze long-chain TAG, 1,2/1,3-DAG, and 1/3-MAG, although with varying efficiencies. By contrast, 2-MAG is assumed to be cleaved specifically by a 2-MAG lipase (31Tornqvist H. Nilsson-Ehle P. Belfrage P. Enzymes catalyzing the hydrolysis of long-chain monoacyglycerols in rat adipose tissue.Biochim. Biophys. Acta. 1978; 530: 474-486Crossref PubMed Scopus (34) Google Scholar) but may also rapidly isomerize to 1/3-MAG, thus leading to the complete degradation of TAG into fatty acids and glycerol by the above mentioned lipases. The po" @default.
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