Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors

Abstract

is also an important source of several metabolic signals. As an integral part of the glycerolipid/fatty acid cycle ( 1 ), lipolysis produces lipid signals that modify cellular functions such as glucose-stimulated insulin secretion ( 2 ) and metabolic pathways and also alter transcription of various genes ( 1, 3 ). 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, 3 ). Finally MAG is hydrolyzed by either the classical MAG lipase (MAGL) or the recently described ␣ / ␤ -hydrolase domain 6 (ABHD6) to glycerol and FFA ( 4-6 ). 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) ␣ or ␤ ( 7 ), 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 ( 1,3,8 ), and the regulation of insulin secretion in pancreatic ␤ -cells ( 9 ). HSL was shown to be important in adipose lipid metabolism ( 10 ) and in the regulation of glucose-stimulated insulin secretion ( 11,12 ). MAGL, which hydrolyzes the endocannabinoid Abstract 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, ␣ / ␤ -hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purifi ed enzymes. We observed that many of the reported inhibitors lack specifi city. 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 specifi city of the many earlier described lipase inhibitors.

Connecting pancreatic islet lipid metabolism with insulin secretion and the development of type 2 diabetes

“…63 When the time course of changes in TG and DAG species in response to glucose was determined by lipidomics, it is implicated that lipolysis of TG produces DAG with 16:0 and 18:0 fatty acid chains in beta cells after glucose exposure. 66 Beta cell-specific knockout and knockdown of the ATGL gene and mRNA, respectively, in mice and INS1 cells have been associated with TG accumulation and impaired insulin secretion, supporting the notion that ATGL is the major TG lipase in beta cells and that sustained suppression of TG lipolysis negatively affects insulin secretion. 6,65 As orlistat suppresses multiple neutral lipases, it has yet to be determined which lipase plays a primary role in supporting GSIS in beta cells.…”

“…6,65 As orlistat suppresses multiple neutral lipases, it has yet to be determined which lipase plays a primary role in supporting GSIS in beta cells. 66 Beta cell-specific knockout and knockdown of the ATGL gene and mRNA, respectively, in mice and INS1 cells have been associated with TG accumulation and impaired insulin secretion, supporting the notion that ATGL is the major TG lipase in beta cells and that sustained suppression of TG lipolysis negatively affects insulin secretion. 67,68 In support of 1-MAG's role in promoting exocytosis, islets from mouse insulin promoter (MIP)-Cremediated ATGL knockout mice contained less 16:0 and 18:0 MAG and showed lower GSIS, which is partly recovered by the presence of 1-MAG.…”

JAK Inhibitors Suppress Colon Cancer Cachexia-Associated Anorexia and Adipose Wasting in Mice

“…CAY10499 is a commercially available pan inhibitor of these lipases. 20,21 To evaluate if cytokine-stimulated adipocyte lipolysis is required for the induction of cyto/adipokine mRNA expression, we performed quantitative RT-PCR for mRNA expression of differentiated adipocytes that were treated with either vehicle, isoproterenol, wild-type rLIF, mutant rLIF K159A, or rIL-6 in the absence or presence of lipase inhibitor CAY10499. Isoproterenol is a β-adrenergic agonist that enhances lipolysis by increasing cAMP stimulating the phosphorylation and activation of the lipase HSL.…”

“…The average values for day 0 for the PBS with vehicle, PBS with tofacitinib, rLIF with vehicle, and rLIF with tofacitinib were as follows: body weight(27, 25.4, 26.7, and 26.8 g), fat mass (3.6, 3.4, 3.7, and 3.6 g) and lean mass(19, 18, 18.7, and 19 g), respectively. The average values for day 0 for the PBS with vehicle, PBS with ruxolitinib, rLIF with vehicle, and rLIF with ruxolitinib were as follows: body weight(26.5, 28, 26.4, and 27 g), fat mass (2.7, 3.0, 3.0, and 2.8 g) and lean mass(20,21,20, and 21 g), respectively. Data is shown as dot plots with mean ± SEM (A and E) or each value represents the mean ± SEM (B-D,F-H) of four (A-D) or three (E-H) mice.…”

Therapy-induced lipid uptake and remodeling underpin ferroptosis hypersensitivity in prostate cancer

“…Indeed, development of ferroptosis hypersensitivity, as measured by RSL3-induced cell death, was significantly suppressed in LNCaP (Fig. 5G) and C4-2B cells (data not shown) when Enz was combined with non-toxic doses of inhibitors directed against lipase activity (Orlistat and CAY10499) [50] or FA delta-6 desaturation (SC26196) [51]. Co-inhibition of the critical lipogenesis transcription factor SREBF1 with Fatostatin [52] or ACACA (acetyl-CoA to malonyl-CoA conversion) and SCD1 (FA desaturation) with dual inhibitor TOFA [53] showed no or only limited reduction in ferroptosis sensitivity (Fig.…”