Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. Hydrolysis of glycerolipids generates FFAs for further usage as a source of energy. Besides FFA production, lipolysis is also an important source of several metabolic signals. As an integral part of the glycerolipid/fatty acid cycle (1.Prentki M. Madiraju S.R. Glycerolipid metabolism and signaling in health and disease.Endocr. Rev. 2008; 29: 647-676Crossref PubMed Scopus (194) Google Scholar), lipolysis produces lipid signals that modify cellular functions such as glucose-stimulated insulin secretion (2.Prentki M. Matschinsky F.M. Madiraju S.R. Metabolic signaling in fuel-induced insulin secretion.Cell Metab. 2013; 18: 162-185Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar) and metabolic pathways and also alter transcription of various genes (1.Prentki M. Madiraju S.R. Glycerolipid metabolism and signaling in health and disease.Endocr. Rev. 2008; 29: 647-676Crossref PubMed Scopus (194) Google Scholar, 3.Young S.G. Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis.Genes Dev. 2013; 27: 459-484Crossref PubMed Scopus (243) Google Scholar). TG breakdown to glycerol and fatty acids is accomplished by the sequential action of adipose triglyceride lipase (ATGL), which hydrolyzes TG to 2,3- or 1,3-diacylglycerol (DAG), followed by hormone sensitive lipase (HSL)-mediated DAG hydrolysis to generate 1- or 2-monoacylglycerol (MAG) (1.Prentki M. Madiraju S.R. Glycerolipid metabolism and signaling in health and disease.Endocr. Rev. 2008; 29: 647-676Crossref PubMed Scopus (194) Google Scholar, 3.Young S.G. Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis.Genes Dev. 2013; 27: 459-484Crossref PubMed Scopus (243) Google Scholar). Finally MAG is hydrolyzed by either the classical MAG lipase (MAGL) or the recently described α/β-hydrolase domain 6 (ABHD6) to glycerol and FFA (4.Zhao S. Mugabo Y. Iglesias J. Xie L. Delghingaro-Augusto V. Lussier R. Peyot M.L. Joly E. Taib B. Davis M.A. et al.alpha/beta-Hydrolase domain-6-accessible monoacylglycerol controls glucose-stimulated insulin secretion.Cell Metab. 2014; 19: 993-1007Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 5.Navia-Paldanius D. Savinainen J.R. Laitinen J.T. Biochemical and pharmacological characterization of human alpha/beta-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12).J. Lipid Res. 2012; 53: 2413-2424Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 6.Blankman J.L. Simon G.M. Cravatt B.F. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.Chem. Biol. 2007; 14: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar). Receptor-mediated signaling at the plasma membrane leads to the phospholipase-C-dependent formation of 1,2-DAG, which is further hydrolyzed by sn-1-DAG lipases (DAGL) α or β (7.Bisogno T. Howell F. Williams G. Minassi A. Cascio M.G. Ligresti A. Matias I. Schiano-Moriello A. Paul P. Williams E.J. et al.Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain.J. Cell Biol. 2003; 163: 463-468Crossref PubMed Scopus (835) Google Scholar), to form mostly 2-MAG. Recent studies indicated the physiological importance of many of these lipases. Thus, ATGL has been implicated in lipid homeostasis in adipocytes, myocardium and skeletal muscle, cancer cachexia (1.Prentki M. Madiraju S.R. Glycerolipid metabolism and signaling in health and disease.Endocr. Rev. 2008; 29: 647-676Crossref PubMed Scopus (194) Google Scholar, 3.Young S.G. Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis.Genes Dev. 2013; 27: 459-484Crossref PubMed Scopus (243) Google Scholar, 8.Das S.K. Eder S. Schauer S. Diwoky C. Temmel H. Guertl B. Gorkiewicz G. Tamilarasan K.P. Kumari P. Trauner M. et al.Adipose triglyceride lipase contributes to cancer-associated cachexia.Science. 2011; 333: 233-238Crossref PubMed Scopus (378) Google Scholar), and the regulation of insulin secretion in pancreatic β-cells (9.Peyot M.L. Guay C. Latour M.G. Lamontagne J. Lussier R. Pineda M. Ruderman N.B. Haemmerle G. Zechner R. Joly E. et al.Adipose triglyceride lipase is implicated in fuel- and non-fuel-stimulated insulin secretion.J. Biol. Chem. 2009; 284: 16848-16859Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). HSL was shown to be important in adipose lipid metabolism (10.Wu J.W. Wang S.P. Casavant S. Moreau A. Yang G.S. Mitchell G.A. Fasting energy homeostasis in mice with adipose deficiency of desnutrin/adipose triglyceride lipase.Endocrinology. 2012; 153: 2198-2207Crossref PubMed Scopus (65) Google Scholar) and in the regulation of glucose-stimulated insulin secretion (11.Peyot M.L. Nolan C.J. Soni K. Joly E. Lussier R. Corkey B.E. Wang S.P. Mitchell G.A. Prentki M. Hormone-sensitive lipase has a role in lipid signaling for insulin secretion but is nonessential for the incretin action of glucagon-like peptide 1.Diabetes. 2004; 53: 1733-1742Crossref PubMed Scopus (65) Google Scholar, 12.Fex M. Haemmerle G. Wierup N. Dekker-Nitert M. Rehn M. Ristow M. Zechner R. Sundler F. Holm C. Eliasson L. et al.A beta cell-specific knockout of hormone-sensitive lipase in mice results in hyperglycaemia and disruption of exocytosis.Diabetologia. 2009; 52: 271-280Crossref PubMed Scopus (40) Google Scholar). MAGL, which hydrolyzes the endocannabinoid 2-arachidonoylglycerol, has been implicated in the control of pain mechanisms (13.Ignatowska-Jankowska B.M. Ghosh S. Crowe M.S. Kinsey S.G. Niphakis M.J. Abdullah R.A. Tao Q. O'Neal S.T. Walentiny D.M. Wiley J.L. et al.In vivo characterization of the highly selective monoacylglycerol lipase inhibitor KML29: antinociceptive activity without cannabimimetic side effects.Br. J. Pharmacol. 2014; 171: 1392-1407Crossref PubMed Scopus (99) Google Scholar) and cancer cell proliferation (14.Nomura D.K. Lombardi D.P. Chang J.W. Niessen S. Ward A.M. Long J.Z. Hoover H.H. Cravatt B.F. Monoacylglycerol lipase exerts dual control over endocannabinoid and fatty acid pathways to support prostate cancer.Chem. Biol. 2011; 18: 846-856Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). DAGL enzymes have been found to be important in the production of 2-arachidonoylglycerol and in the control of appetite and pain (15.Reisenberg M. Singh P.K. Williams G. Doherty P. The diacylglycerol lipases: structure, regulation and roles in and beyond endocannabinoid signalling.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2012; 367: 3264-3275Crossref PubMed Scopus (102) Google Scholar) and also in the regulation of Ca2+ influx into cells (16.Liu L. Heneghan J.F. Michael G.J. Stanish L.F. Egertova M. Rittenhouse A.R. L- and N-current but not M-current inhibition by M1 muscarinic receptors requires DAG lipase activity.J. Cell. Physiol. 2008; 216: 91-100Crossref PubMed Scopus (14) Google Scholar). We recently showed that 1-MAG is a coupling factor linking glucose metabolism to insulin secretion in pancreatic β-cells and that ABHD6 specifically controls β-cell 1-MAG levels (4.Zhao S. Mugabo Y. Iglesias J. Xie L. Delghingaro-Augusto V. Lussier R. Peyot M.L. Joly E. Taib B. Davis M.A. et al.alpha/beta-Hydrolase domain-6-accessible monoacylglycerol controls glucose-stimulated insulin secretion.Cell Metab. 2014; 19: 993-1007Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Various compounds have been used to inhibit particular lipolytic enzymes, assuming that the inhibitor is specific. However, no systematic studies have been done comparing the specificity of many of these inhibitor compounds and whether their effectiveness differs among species. These issues make a significant impact on our understanding of lipid metabolism in different organisms and also on the discovery of drugs targeting these enzymes for a possible use against human diseases. It is necessary to have reliable assay procedures to measure the activity of an enzyme before embarking on identifying its physiological role and also for the development of drugs targeting the particular enzyme. In the present study, we describe reliable assays of lipolytic enzymes using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. Our findings reveal that some of the inhibitors available commercially as "specific" against a particular enzyme are in fact not specific and also that certain inhibitors discovered using rodent enzymes are ineffective against their human counterparts. Purified carboxylesterase 1 (CES1) and RHC80267 were obtained from Sigma (St. Louis, MO). Purified human HSL (hHSL), human MAGL (hMAGL), Cay10499, WWL70, JZL184, JZL195, fatty acid amide hydrolase (FAAH) inhibitor screening assay kit (#10005196) and 1-S-arachidonoylthioglycerol were purchased from Cayman Chemical Co. (Ann Arbor, MI). ThioGlo-1 was from Covalent Associates (Corvallis, OR). ATGL inhibitors ATGListatin (17.Mayer N. Schweiger M. Romauch M. Grabner G.F. Eichmann T.O. Fuchs E. Ivkovic J. Heier C. Mrak I. Lass A. et al.Development of small-molecule inhibitors targeting adipose triglyceride lipase.Nat. Chem. Biol. 2013; 9: 785-787Crossref PubMed Scopus (133) Google Scholar) and WWL64, HSL inhibitors WWL11 (18.Bachovchin D.A. Mohr J.T. Speers A.E. Wang C. Berlin J.M. Spicer T.P. Fernandez-Vega V. Chase P. Hodder P.S. Schurer S.C. et al.Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors.Proc. Natl. Acad. Sci. USA. 2011; 108: 6811-6816Crossref PubMed Scopus (91) Google Scholar) and Compound 13f (19.Ebdrup S. Refsgaard H.H. Fledelius C. Jacobsen P. Synthesis and structure-activity relationship for a novel class of potent and selective carbamate-based inhibitors of hormone selective lipase with acute in vivo antilipolytic effects.J. Med. Chem. 2007; 50: 5449-5456Crossref PubMed Scopus (36) Google Scholar), DAGL inhibitors KT109 and KT172, and ABHD6 inhibitor KT195 (20.Hsu K.L. Tsuboi K. Adibekian A. Pugh H. Masuda K. Cravatt B.F. DAGLbeta inhibition perturbs a lipid network involved in macrophage inflammatory responses.Nat. Chem. Biol. 2012; 8: 999-1007Crossref PubMed Scopus (162) Google Scholar) were synthesized according to the published procedures. All stock solutions of inhibitors were prepared in dimethylsulfoxide and diluted in assay medium at required concentrations just prior to assays. Plasmids for the expression of human ABHD6 (hABHD6) (pCMV6-AC; SC320252), human ATGL (hATGL) (pCMV6-XL5; SC107379), mouse ATGL (pCMV6; MC210442), hHSL (pCMV6-XL5; SC303624), mouse HSL (pCMV6; MC200824), and human DAGLα (hDAGLα) (pCMV6-XL5; SC101029) were obtained from OriGene (Rockville, MD). All other chemicals used were of the highest purity available. Fluorescence and absorbance were measured using the FLUOstar Optima reader (BMG, Ortenberg, Germany). Antibodies against hATGL and hHSL were from Cell Signaling, anti-DAGLα antibody was from AbNova, and anti-tubulin antibody was from Abcam. Anti-ABHD6 antibody was a gift from Dr. J. Mark Brown (Cleveland, OH). Secondary antibodies were from Santa Cruz and BioRad. HEK 293T cells were cultured in DMEM high glucose, 4 mM l-glutamine without sodium pyruvate (HyClone, Logan, UT), supplemented with 10% FBS (Gibco, Grand Island, NY). For lipase expression, 9 × 105 cells were cultured overnight in a 10 cm Petri dish and then transfected with 20 μg of lipase expression plasmids using 20 μl of lipofectamine 2000 (Invitrogen, Carlsbad, CA) in 3 ml of Opti-MEM media (Invitrogen) and 10 ml of above DMEM. After 24 h, transfection medium was replaced by DMEM. Cells were harvested 72 h posttransfection in the corresponding lysis buffers described below, for different lipases after two washes in PBS. Human and mouse ATGL were expressed separately in 293T cells. Cell extracts were prepared in 1 ml of buffer A (50 mM HEPES, pH 7.2, 100 mM NaCl, 5 mM CaCl2, 0.5 mM DTT, 2% DMSO, 0.1% Triton X-100), by three cycles of freeze (in liquid nitrogen)/thaw sonication at high power in a cup horn sonicator (Misonix Sonicator 3000; Qsonica LLC, CT) for 5 min, followed by centrifugation at 1,000 g for 5 min at 4°C, and the supernatant was used as the source of ATGL after measuring protein concentration. Aliquots containing 2–4 µg/µl of proteins were stored at −80°C. ATGL expression was verified by Western blotting using antibodies against human (Cell Signaling #2138) or mouse ATGL (Cell Signaling #2439). ATGL activity was assayed using EnzChek lipase substrate (Life Technologies) (21.Basu D. Manjur J. Jin W. Determination of lipoprotein lipase activity using a novel fluorescent lipase assay.J. Lipid Res. 2011; 52: 826-832Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Control cell extracts prepared from empty vector-transfected cells were used to ascertain that the lipase activity measured is due to the overexpressed ATGL. Assays were performed in 96-well opaque black plates (Corning #3915) containing 30 μg of ATGL cell extract in 90 μl of buffer A, to which 5 μl of test inhibitor (diluted in 30% DMSO to appropriate working stock concentrations to achieve different inhibitor concentrations) was added to give the required final concentration. After 30 min preincubation at room temperature with 700 rpm orbital shaking, 5 µl of 20 μM EnzChek lipase substrate working solution was added to each well to a final concentration of 1 µM to start the reaction at 37°C. EnzChek lipase substrate stock solution (1 mM) was prepared in DMSO and then diluted 1:50 in buffer A, just before use. Final concentration of DMSO was kept at 5% (v/v) in all the wells. Fluorescence (excitation 485 nm; emission 510 nm) was recorded every 30 s for 60 to 90 min with 2 s of shaking preceding each reading. ATGL activity was calculated by subtracting background activity (no enzyme added), using the linear portion of the velocity curve, after the first 15 min of the reaction. Endogenous ATGL activity in extracts prepared from non- or empty vector-transfected cells was found to be low and did not contribute significantly to the activity measured with ATGL enzyme extracts. Human and mouse HSL proteins were expressed separately in 293T cells. Cell extracts were prepared in PBS as described above for ATGL. Human and mouse HSL expression was verified using HSL antibody (Cell Signaling #4107) in Western blots. Alternatively, we also tested commercially available purified hHSL for some assays. The assay conditions were similar to those described below for ABHD6, except that potassium phosphate buffer was adjusted to pH 7.0 and 1 µg cell extract/well was used. Because HSL is capable of hydrolyzing 1-MAG, we used 1-S-arachidonoylthioglycerol as the substrate and measured the release of thioglycerol at 37°C using ThioGlo-1 (see ABHD6 Assay below for more details). DAGLα could not be expressed using the plasmid pCMV6-XL5-hDAGLα from OriGene in 293T cells. The DAGLα cDNA harbors relatively long 5′ and 3′ untranslated regions (UTRs; 104 and 2,570 bp, respectively). In addition, the 5′ UTR has several out of frame ATG start sites. We observed a similar expression problem when using adenoviral vector with the same DAGLα cDNA. We realized that the 5′ UTR interfered with protein expression. Thus, we have used a PCR-based strategy to remove both 5′ and 3′ UTRs, keeping only the coding sequence and the natural ATG start site. The coding sequence (3,129 bp long) from the pCMV6-XL5-hDAGLα was amplified by PCR using DAGL forward primer 5′ CAT CTA GAG CCA TGC TGC CCG GGA TCG TGG T and the reverse primer 5′ CAC TCG AGC TAG CGT GCT GAG ATG ACC A. The amplified product was purified and subcloned into pIRES2-EGFP (enhanced green fluorescence protein) plasmid (Clontech). DNA sequencing was performed to confirm in frame ligation of the construct. The plasmid pIRES2-DAGLα thus generated was used to transfect 293T cells, and cell extracts were prepared in 300 µl of 0.25 M sucrose, 20 mM HEPES, pH 7.0 (buffer B). To this end, cells were lysed by three rapid cycles of freeze/thaw/sonication 2 min (10 s on/off cycles) at high power in ice-cold water in a cup horn sonicator, and the extract was centrifuged at 51,000 g for 30 min at 4°C. The membrane pellet was suspended in 200 µl buffer B by sonication, and the protein content was measured. Aliquots containing 2–5 µg/µl of protein were stored at −80°C. DAGLα expression was verified using hDAGLα antibody (Abnova #PAB11515) in Western blots. DAGLα activity was assayed by following the hydrolysis of p-nitrophenylbutyrate (pNPB), as described earlier (22.Pedicord D.L. Flynn M.J. Fanslau C. Miranda M. Hunihan L. Robertson B.J. Pearce B.C. Yu X.C. Westphal R.S. Blat Y. Molecular characterization and identification of surrogate substrates for diacylglycerol lipase alpha.Biochem. Biophys. Res. Commun. 2011; 411: 809-814Crossref PubMed Scopus (20) Google Scholar). Endogenous DAGLα activity in extracts prepared from pIRES2-EGFP-transfected cells was found to be low and did not contribute significantly to the activity measured with DAGLα enzyme extracts. The assay system premix in a 96-well black plate with clear bottom (PerkinElmer Viewplate-96 F TC), in a volume of 90 μl per well, contained 0.25 M sucrose, 50 mM HEPES, pH 7.3 (buffer C), and 10 μg DAGLα enzyme (cell membranes) extract to which 5 μl of test inhibitor (diluted in 30% DMSO) was added. After 10 min preincubation at room temperature with 700 rpm orbital mixing and 20 min incubation at 37°C with mild shaking, 5 µl of freshly prepared 7.88 mM pNPB (in 70% DMSO in buffer C) was added to each well to start the reaction. The plates were shaken rapidly, and change in absorbance at 405 nm was measured every 30 s for 45 min. DAGLα activity was calculated by subtracting background activity (wells without enzyme extract) using the linear portion of the velocity curve. The final concentration of DMSO was adjusted to 5% in all wells. Activity was also measured using membrane extracts prepared from EGFP-expressing cells and was found to be negligible. DAGLα was also assayed using EnzChek lipase substrate described above for the ATGL assay. The assay system in a 96-well black plate was similar to the one used with pNPB above. However, reactions were started with 5 µl of 100 μM EnzChek lipase substrate solution to a final concentration of 5 µM. In parallel wells, CaCl2 was added at 5 mM final concentration. Enzyme was preincubated with 6 μM KT109 DAGL inhibitor where indicated, prior to substrate addition. Fluorescence was recorded as described for the ATGL assay above. hMAGL was assayed using purified recombinant enzyme (Cayman Chemical). The assay conditions were similar to those described below for ABHD6 with potassium phosphate buffer adjusted to pH 7.4 and an enzyme dilution of 1:7,500 in potassium phosphate buffer. Final concentration of MAGL enzyme protein in the assay was 0.025 ng per well. 1-S-arachidonoylthioglycerol was used as the substrate, and the release of thioglycerol at 37°C was measured with ThioGlo-1 (see below). hABHD6 was expressed in 293T cells by transfecting with pCMV6-AC-hABHD6 plasmid. Cell extracts were prepared in PBS as described above for ATGL. ABHD6 expression was verified using ABHD6 antibody (kindly provided by Dr. J. Mark Brown, Cleveland, OH) in Western blots. Aliquots containing 4 µg/µl of protein were stored at −80°C. ABHD6 assay was based on the hydrolysis of 1-S-arachidonoylthioglycerol to release 1-thioglycerol, which spontaneously reacts with ThioGlo-1 to form a fluorescent adduct (23.Storey B.T. Alvarez J.G. Thompson K.A. Human sperm glutathione reductase activity in situ reveals limitation in the glutathione antioxidant defense system due to supply of NADPH.Mol. Reprod. Dev. 1998; 49: 400-407Crossref PubMed Scopus (64) Google Scholar). Assays were performed in 96-well opaque black plates containing 90 µl of 50 mM potassium phosphate buffer, pH 7.2 and 5 µl of test inhibitor in 30% DMSO. The plates were then preincubated for 30 min at room temperature, after which 5 µl of premixed 100 µM 1-S-arachidonolythioglycerol and 0.26 mM ThioGlo-1 in 20% DMSO was added to all but the gain and background wells. These wells received the ThioGlo-1 only. The plates were immediately placed in the plate reader at 37°C and shaken for 10 s, and fluorescence was continuously recorded at excitation 380 nm and emission 510 nm for 45 min at 30 s intervals. Sensitivity of the detection was adjusted via modifying photomultiplier tube gain from wells containing 1 µM thioglycerol only. Final DMSO concentration in all wells was kept at 2% (v/v). Another control to measure endogenous ABHD6 activity was also set up using cell extract prepared from empty vector-transfected 293T cells. Activity was calculated from the linear portion of the curve after background correction. In order to compare the overall sensitivity of ABHD6 assays using the artificial substrate 1-S-arachidonoylthioglycerol and the natural substrate 1-palmitoylglycerol, extracts from hABHD6-overexpressing 293T cells were used, and extracts from untransfected 293T cells were used as control. The assay system in a final volume of 100 μl contained 10 µg protein of extracts, 50 mM Tris-Cl pH 7.2, without or with 5 μM WWL70 and either 5 µM 1-S-arachidonoylthioglycerol or 5 or 50 µM 1-palmitoylglycerol. Assay with 1-S-arachidonoylthioglycerol was described above. Incubations with 1-palmitoylglycerol were at 37°C for 30 min with constant mixing. Reactions were terminated by 5 µl 70% perchloric acid, and after complete mixing, pH was brought to 5–7 using 2 M KOH. Tubes were kept on ice 30 min and centrifuged for 10 min at 10,000 g. Supernatants were processed for measuring glycerol, released during hydrolysis of 1-palmitoylglycerol, by radiometric method (24.Bradley D.C. Kaslow H.R. Radiometric assays for glycerol, glucose, and glycogen.Anal. Biochem. 1989; 180: 11-16Crossref PubMed Scopus (108) Google Scholar). Purified human CES1 (hCES1) was used to assay CES1 activity. The assay conditions were similar to those described above for ABHD6, with potassium phosphate buffer adjusted to pH 7.5 and an enzyme concentration of 60 ng/well purified hCES1. 1-S-arachidonoylthioglycerol was used as a substrate, and the release of thioglycerol at 37°C was measured with ThioGlo-1. CES1 shows robust activity with many carboxyl esters including 1-MAG. Assay was performed using the FAAH inhibitor screening assay kit from Cayman Chemicals according to the manufacturer's protocol. Release of the fluorescent product, 7-amino-4-methylcoumarin (AMC), from the hydrolysis of AMC-arachidonoyl amide, mediated by FAAH at 37°C, was recorded using a plate reader with an excitation filter of 340–360 nm and an emission filter of 450–465 nm. Inhibitors were preincubated for 30 min at indicated concentrations with the enzyme prior to the addition of substrate to initiate the reaction. Change in fluorescence with time was taken as enzyme activity and the extent of inhibition was calculated from the initial rates. Positive control for FAAH inhibition was used using JZL195. Final DMSO concentration was kept at 2% (v/v) in the assay. Percentage of inhibition was calculated from lipase activity at initial rates measured using enzyme extracts treated with the inhibitors relative to the activity measured with vehicle-treated extracts. IC50 values were determined using Prism version 5.01 (GraphPad Software, San Diego, CA) based on percentage of inhibition values. Results shown represent two to three separate experiments with triplicate observations. Lipases are important in the metabolism of various lipids and thus play a critical role in the cell from membranogenesis to the generation of several signaling molecules. Several assays have been described before for different lipase enzymes, but many of them are cumbersome and use radioactive or custom-made substrates not available to all investigators. We have developed simplified and reliable assays of different lipases using recombinant human and mouse enzymes either in the form of cell extracts or purified enzymes. Even though the assays described here cannot be used directly for measuring the corresponding endogenous enzyme activities as many lipases have overlapping substrate specificities, they are adaptable for high-throughput screening of larger libraries of compounds. They may also be adapted for measuring endogenous lipase activity in case a highly specific inhibitor is available to distinguish its activity from other lipases. As we describe below, many such "specific" inhibitors previously described are indeed not specific. Using whole cell extracts from 293T cells overexpressing hATGL (Fig. 1A) or mouse ATGL, this enzyme activity could be assayed with EnzChek lipase substrate. We noticed that it is necessary to let the fluorescence stabilize for the first 15 to 20 min (not shown). The actual enzyme activity is recorded only after this initial stabilization, which is reflected by steady increase in fluorescence for at least 45 min. ATGL activity was almost completely inhibited by the panlipase inhibitor orlistat (Fig. 1B). Similar results were noticed with mouse ATGL, also expressed in 293T cells (data not shown). We also examined the need for added ATGL activator, CGI58 (3.Young S.G. Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis.Genes Dev. 2013; 27: 459-484Crossref PubMed Scopus (243) Google Scholar), for measuring ATGL activity. Coexpression of CGI58 along with ATGL or addition of separately expressed CGI58 to ATGL assay had no significant effect on the measured ATGL activity (not shown), which suggested that under the present assay conditions, ATGL is fully active. This also simplifies the assay procedure significantly. This assay, which does not involve any radioactive materials or reaction product extraction, could be easily adapted to high-throughput screening for the discovery of selective inhibitors of hATGL. This assay could also be adapted for measuring ATGL activity in various tissues where the enzyme is well expressed. hHSL exists in multiple isoforms (25.Lampidonis A.D. Rogdakis E. Voutsinas G.E. Stravopodis D.J. The resurgence of Hormone-Sensitive Lipase (HSL) in mammalian lipolysis.Gene. 2011; 477: 1-11Crossref PubMed Scopus (141) Google Scholar), and in the present study, we used the long form (118.3 kDa), which is available as purified recombinant enzyme commercially. We also used extracts of cells overexpressing either hHSL (118.3 kDa form; Fig. 1A) or mouse HSL (87.3 kDa), for assessing inhibitor sensitivity. hHSL could be rapidly assayed in 96-well format, using 1-S-arachidonoylthioglycerol, an analog of 1-MAG, as substrate (Fig. 1C). Similar results were obtained with the mouse enzyme (not shown). Both human and mouse HSL enzymes were inhibited by Cay10499, although to different degrees (see below). This assay is adapt