Diacylglycerol Lipase-Alpha Regulates Hippocampal-Dependent Learning and Memory Processes in Mice.

Diacylglycerol lipase-α (DAGL-α), the principal biosynthetic enzyme of the endogenous cannabinoid 2-arachidonylglycerol (2-AG) on neurons, plays a key role in CB1 receptor-mediated synaptic plasticity and hippocampal neurogenesis, but its contribution to global hippocampal-mediated processes remains unknown. Thus, the present study examines the role that DAGL-α plays on LTP in hippocampus, as well as in hippocampal-dependent spatial learning and memory tasks, and on the production of endocannabinoid and related lipids through the use of complementary pharmacologic and genetic approaches to disrupt this enzyme in male mice. Here we show that DAGL-α gene deletion or pharmacological inhibition disrupts LTP in CA1 of the hippocampus but elicits varying magnitudes of behavioral learning and memory deficits in mice. In particular, DAGL-α-/- mice display profound impairments in the Object Location assay and Morris Water Maze (MWM) acquisition engaging in nonspatial search strategies. In contrast, WT mice administered the DAGL-α inhibitor DO34 show delays in MWM acquisition and reversal learning, but no deficits in expression, extinction, forgetting, or perseveration processes in this task, as well as no impairment in Object Location. The deficits in synaptic plasticity and MWM performance occur in concert with decreased 2-AG and its major lipid metabolite (arachidonic acid), but increases of a 2-AG diacylglycerol precursor in hippocampus, PFC, striatum, and cerebellum. These novel behavioral and electrophysiological results implicate a direct and perhaps selective role of DAGL-α in the integration of new spatial information.SIGNIFICANCE STATEMENT Here we show that genetic deletion or pharmacologic inhibition of diacylglycerol lipase-α (DAGL-α) impairs hippocampal CA1 LTP, differentially disrupts spatial learning and memory performance in Morris water maze (MWM) and Object Location tasks, and alters brain levels of endocannabinoids and related lipids. Whereas DAGL-α-/- mice exhibit profound phenotypic spatial memory deficits, a DAGL inhibitor selectively impairs the integration of new information in MWM acquisition and reversal tasks, but not memory processes of expression, extinction, forgetting, or perseveration, and does not affect performance in the Objection Location task. The findings that constitutive or short-term DAGL-α disruption impairs learning and memory at electrophysiological and selective in vivo levels implicate this enzyme as playing a key role in the integration of new spatial information.

Intestinal vitamin D receptor modulates lipid metabolism, adipose tissue inflammation and liver steatosis in obese mice.

OBJECTIVE: Hypovitaminosis D is common in the obese population and patients suffering from obesity-associated disorders such as type 2 diabetes and fatty liver disease, resulting in suggestions for vitamin D supplementation as a potential therapeutic option. However, the pathomechanistic contribution of the vitamin D-vitamin D receptor (VDR) axis to metabolic disorders is largely unknown. METHODS: We analyzed the pathophysiological role of global and intestinal VDR signaling in diet-induced obesity (DIO) using global Vdr-/- mice and mice re-expressing an intestine-specific human VDR transgene in the Vdr deficient background (Vdr-/- hTg). RESULTS: Vdr-/- mice were protected from DIO, hepatosteatosis and metabolic inflammation in adipose tissue and liver. Furthermore, Vdr-/- mice displayed a decreased adipose tissue lipoprotein lipase (LPL) activity and a reduced capacity to harvest triglycerides from the circulation. Intriguingly, all these phenotypes were partially reversed in Vdr-/- hTg animals. This clearly suggested an intestine-based VDR activity on systemic lipid homeostasis. Scrutinizing this hypothesis, we identified the potent LPL inhibitor angiopoietin-like 4 (Angptl4) as a novel transcriptional target of VDR. CONCLUSION: Our study suggests a VDR-mediated metabolic cross-talk between gut and adipose tissue, which significantly contributes to systemic lipid homeostasis. These results have important implications for use of the intestinal VDR as a therapeutic target for obesity and associated disorders.

Anti-Obesity Effect of Allium hookeri Leaf Extract in High-Fat Diet-Fed Mice.

Allium hookeri has been widely cultivated and used as a vegetable and medicine in Asia, but its anti-obesity effects have not been previously reported. In this study, the effects of a leaf extract of A. hookeri on obesity were investigated by administering a high-fat diet (HFD) to mice. Male Institute of Cancer Research mice (n = 32; 5 weeks old) were randomly divided into four groups: normal-diet group, HFD group, HFD containing 200 mg/kg/day A. hookeri leaf extract (HFD-A1), and HFD containing 400 mg/kg/day A. hookeri leaf extract (HFD-A2). A. hookeri leaf extract was orally administered daily for 4 weeks. We found that the body weight gain and organ tissue weights of mice in the HFD-A1 and HFD-A2 groups were significantly lower compared with those of mice in the HFD group. Administration of A. hookeri leaf extract also significantly decreased the size of the epididymal adipose tissue (AT). Serum levels of triglyceride (TG), total cholesterol, low-density lipoprotein cholesterol, and the atherogenic index were significantly lower in the HFD-A1 and HFD-A2 groups than in the HFD group. The TG and total cholesterol levels in the hepatic, epididymal, and mesenteric ATs of the HFD-A2 group were significantly lower than the levels in the HFD group. In addition, mRNA levels of liver fatty acid synthase and lipoprotein lipase were decreased in the A. hookeri leaf extract groups compared with those of the HFD group. These results demonstrate that intake of A. hookeri leaf may have beneficial effects for suppressing obesity-related disease.

Computational analysis and enzyme assay of inhibitor response to disease single nucleotide polymorphisms (SNPs) in lipoprotein lipase.

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of the triglyceride (TG) core of circulating TG-rich lipoproteins, chylomicrons, and very low-density lipoproteins. The enzyme has been established as an efficacious and safe therapeutic target for the management of obesity. Here, a systematic profile of the lipase inhibitor response of three anti-obesity agents (Orlistat, Lipstatin, and Cetilistat) to clinical LPL missense mutations arising from disease single nucleotide polymorphisms (SNPs) was established by integrating complex structure modeling, virtual mutagenesis, molecular dynamics (MD) simulations, binding energy analysis, and radiolabeled TG hydrolysis assays. The profile was then used to characterize the resistance and sensitivity of systematic mutation-inhibitor pairs. It is suggested that the Orlistat and Lipstatin have a similar response profile to the investigated mutations due to their homologous chemical structures, but exhibit a distinct profile to that of Cetilistat. Most mutations were predicted to have a modest or moderate effect on inhibitor binding; they are located far away from the enzyme active site and thus can only influence the binding limitedly. A number of mutations were found to sensitize or cause resistance for lipase inhibitors by directly interacting with the inhibitor ligands or by indirectly addressing allosteric effect on enzyme active site. Long-term MD simulations revealed a different noncovalent interaction network at the complex interfaces of Orlistat with wild-type LPL as well as its sensitized mutant H163R and resistant mutant I221T.

A novel live cell assay to measure diacylglycerol lipase α activity.

Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.

A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase α (DAGL-α) in the CNS. Selective inhibitors of DAGL-α will provide valuable insights in the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-α, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-α activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-α activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-α. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-α inhibitors.

Chemical approaches to therapeutically target the metabolism and signaling of the endocannabinoid 2-AG and eicosanoids.

The endocannabinoid system, most popularly known as the target of the psychoactive component of marijuana, Δ(9)-tetrahydrocannabinol (THC), is a signaling network that modulates a diverse range of physiological processes including nociception, behavior, cognitive function, appetite, metabolism, motor control, memory formation, and inflammation. While THC and its derivatives have garnered notoriety in the eyes of the public, the endocannabinoid system consists of two endogenous signaling lipids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide), which activate cannabinoid receptors CB1 and CB2 in the nervous system and peripheral tissues. This review will focus on the recent efforts to chemically manipulate 2-AG signaling through the development of inhibitors of the 2-AG-synthesizing enzyme diacylglycerol lipase (DAGL) or the 2-AG-degrading enzyme monoacylglycerol lipase (MAGL), and assessing the therapeutic potential of DAGL and MAGL inhibitors in pain, inflammation, degenerative diseases, tissue injury, and cancer.

Identification of small molecules that selectively inhibit diacylglycerol lipase-α activity.

Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization.

A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects.

BACKGROUND AND PURPOSE: The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and ß is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. EXPERIMENTAL APPROACH: Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. KEY RESULTS: Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLα, and selectivity (IC50 > 10 µM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥ 10 µM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 µM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 µM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg·kg⁻¹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. CONCLUSIONS AND IMPLICATIONS: O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.

DAGLß inhibition perturbs a lipid network involved in macrophage inflammatory responses.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLß. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLß in living systems. Optimized inhibitors showed high selectivity for DAGLß over other serine hydrolases, including DAGLα (∼60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb(-/-) mice, we show that DAGLß inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2. We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLß is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.

Wedelolactone inhibits adipogenesis through the ERK pathway in human adipose tissue-derived mesenchymal stem cells.

Wedelolactone is an herbal medicine that is used to treat septic shock, hepatitis and venom poisoning. Although in differentiated and cancer cells, wedelolactone has been identified as anti-inflammatory, growth inhibitory, and pro-apoptotic, the effects of wedelolactone on stem cell differentiation remain largely unknown. Here, we report that wedelolactone inhibits the adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Wedelolactone reduced the formation of lipid droplets and the expression of adipogenesis-related proteins, such as CCAAT enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein aP2 (aP2). Wedelolactone mediated this process by sustaining ERK activity. In addition, inhibition of ERK activity with PD98059 resulted in reversion of the wedelolactone-mediated inhibition of adipogenic differentiation. Taken together, these results indicate that wedelolactone inhibits adipogenic differentiation through ERK pathway and suggest a novel inhibitory effect of wedelolactone on adipogenic differentiation in hAMSCs.

Lapacho tea (Tabebuia impetiginosa) extract inhibits pancreatic lipase and delays postprandial triglyceride increase in rats.

Earlier work in our laboratory indicated that ethanolic extracts of Tabebuia impetiginosa, Arctium lappa L., Calendula officinalis, Helianthus annuus, Linum usitatissimum and L. propolis, inhibit pancreatic lipase in vitro. In a follow-up study we assessed their effects on plasma triglycerides in rats fed on a fatty meal. Extracts, orlistat or only ethanol were given orally to the rats together with the test meal and the rate of increase of postprandial triglycerides was assessed over 4 h. Clearing of the triglycerides from the blood compartment was abolished by inhibiting lipoprotein lipase with Triton WR-1339. Our results showed that out of all the extracts, the bark of Tabebuia impetiginosa led to a significant delay in the postprandial increase of plasma triglycerides. However, lapachol, which is contained in the bark of Tabebuia impetiginosa and soluble in ethanol, had no lipase inhibitory effect in vitro and hence this substance did not seem to mediate the pertinent effect.

Hypolipidemic effect of Pleurotus eryngii extract in fat-loaded mice.

Pleurotus eryngii water extract (PEE), which showed the most significant inhibitory activity against pancreatic lipase in vitro among eight edible mushrooms, was investigated to determine the mechanism of its anti-lipase activity in vitro and its hypolipidemic effect in fat-loaded mice. The inhibitory effects of mushroom extracts on pancreatic lipase activity were examined using 4-methylumbelliferyl oleate (4-MUO) or trioleoylglycerol emulsified with lecithin, gum arabic or Triton X-100 as a substrate. For in vivo experiments, blood samples were taken after oral administration of corn oil and [(3)H]trioleoylglycerol with or without PEE to food-deprived mice. PEE inhibited hydrolysis of 4-MUO and trioleoylglycerol emulsified with lecithin or Triton X-100, but not that of trioleoylglycerol emulsified with gum arabic. PEE suppressed the elevations of plasma and chylomicron triacylglycerol levels after oral administration of corn oil, but had no effect on lipoprotein lipase activity. [(3)H]Trioleoylglycerol absorption was also decreased by administration of PEE. The results of in vitro studies suggest that PEE may prevent interactions between lipid emulsions and pancreatic lipase. The hypolipidemic effect of PEE in fat-loaded mice may be due to low absorption of fat caused by the inhibition of pancreatic lipase.

Antiobesity action of a daidzein derivative on male obese mice induced by a high-fat diet.

Emerging evidence suggests that consumption or supplementation of foods rich in iso flavones may has a beneficial effect on obesity and glucose levels in animals and humans. It has been demonstrated that genistein, the main component of iso fl avones, could significantly reduce body weight and fat pad size. However, there is no evidence as to whether daidzein, which is also a main component of isoflavones, has the same effect. We hypothesize that LRXH609 (Dzd; a daidzein derivative) also has an antiobesity effect. In this study, we investigate the effects of Dzd on body weight, adipose tissue, blood, and liver lipid levels in obese mice fed a high-fat diet. Activities of pancreatic lipase and lipoprotein lipase, as well as lipolysis, were verified to clarify the potential mechanism of the daidzein. The results indicate that Dzd can significantly reduce body and fat pad weight and ameliorate the high-fat diet-induced hyperlipoidemia. We also found that Dzd inhibits the activity of pancreatic lipase and lipoprotein lipase in a dose-dependent manner, inhibits the differentiation of rat preadipocytes, and stimulates lipolysis by activating hormone-sensitive lipase.

Endocannabinoid chemical biology: a tool for the development of novel therapies.

The identification of the major psychoactive constituent of Cannabis and marijuana, Delta(9)-tetrahydrocannabinol, opened the way first to the cloning of the G-protein-coupled cannabinoid CB(1) and CB(2) receptors, and then to the isolation and characterisation of their endogenous agonists, the endocannabinoids. Considerable progress has been made in the characterisation of pathways and enzymes for the biosynthesis and degradation of anandamide and 2-arachidonoylglycerol, the two best-known endocannabinoids, as well as of endocannabinoid-related molecules, such as the N-acylethanolamines, which, as in the case of N-palmitoylethanolamine and N-oleoylethanolamine, may interact with other receptor types. However, it is still not fully understood how other plant cannabinoids, of which cannabidiol is the most studied representative, exert their pharmacological effects. Together with these issues, this first review article on the endocannabinoids describes the synthetic pharmacological tools that have been designed so far to interact with the proteins of the 'endocannabinoid system' and that can potentially be used as templates for the development of new therapies.

Inhibitory effect of morinda citrifolia L. On lipoprotein lipase activity.

Efficacy of Morinda citrifolia L. leaf (MLE) and fruit extracts (MFE) in inhibiting lipoprotein lipase (LPL) was determined in vitro. The result of the study showed that the highest inhibition on the LPL activity was exhibited by MLE (66%+/- 2.1%), which is significantly higher than that demonstrated by MFE (54.5%+/- 2.5%), green tea extract (GTE) (54.5%+/- 2.6%), and catechin (43.6%+/- 6.1%). Percent of LPL inhibition increase with concentration of the extracts. Quantitative analysis of the extracts revealed the presence of high levels of (+)-catechin at 63.5 +/- 17 and 53.7 +/- 5.7 mg/g in MLE and MFE, respectively, although not as high as that found in GTE (530.6 +/- 42 mg/g). Appreciable amount of epicatechin was found in all extracts tested, while rutin was only found in MLE and MFE. The study suggested that both leaf and fruit of M. citrifolia may be used as antiobesity agents in body weight management.

Anti-obesity effects of conjugated linoleic acid, docosahexaenoic acid, and eicosapentaenoic acid.

Obesity has become a prevailing epidemic throughout the globe. Effective therapies for obesity become attracting. Food components with beneficial effects on "weight loss" have caught increasing attentions. Conjugated linoleic acid (CLA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) belong to different families of polyunsaturated fatty acids (PUFA). However, they have similar effects on alleviating obesity and/or preventing from obesity. They influence the balance between energy intake and expenditure; and reduce body weight and/or fat deposition in animal models, but show little effect in healthy human subjects. They inhibit key enzymes responsible for lipid synthesis, such as fatty acid synthase and stearoyl-CoA desaturase-1, enhance lipid oxidation and thermogenesis, and prevent free fatty acids from entering adipocytes for lipogenesis. PUFA also exert suppressive effects on several key factors involved in adipocyte differentiation and fat storage. Despite their similar effects and shared mechanisms, they display differences in the regulation of lipid metabolism. Moreover, DHA and EPA exhibit "anti-obesity" effect as well as improving insulin sensitivity, while CLA induces insulin resistance and fatty liver in most cases. A deeper and more detailed investigation into the complex network of anti-obesity regulatory pathways by different PUFA will improve our understanding of the mechanisms of body weight control and reduce the prevalence of obesity.

trans-10,cis-12 Conjugated linoleic acid inhibits lipoprotein lipase but increases the activity of lipogenic enzymes in adipose tissue from hamsters fed an atherogenic diet.

The aim of the present work was to investigate the effects of trans-10,cis-12 conjugated linoleic acid (CLA) on the activity and expression of lipogenic enzymes and lipoprotein lipase (LPL), as well as on the expression of transcriptional factors controlling these enzymes, in adipose tissue from hamsters, and to evaluate the involvement of these changes in the body fat-reducing effect of this CLA isomer. Thirty male hamsters were divided into three groups and fed atherogenic diets supplemented with 0 (linoleic group), 5 or 10 g trans-10,cis-12 CLA/kg diet, for 6 weeks. Body and adipose tissue weights, food intake and serum insulin were measured. Total and heparin-releasable LPL and lipogenic enzyme activities (acetyl-CoA carboxylase (ACC); fatty acid synthase (FAS); glucose-6-phosphate dehydrogenase (G6PDH); and malic enzyme (ME)) were assessed. ACC, FAS, LPL, sterol regulatory element-binding proteins (SREBP-1a), SREBP-1c and PPARgamma mRNA levels were also determined by real-time PCR. CLA did not modify food intake, body weight and serum insulin level. CLA feeding reduced adipose tissue weight, LPL activity and expression, and increased lipogenic enzyme activities, despite a significant reduction in ACC and FAS mRNA levels. The expression of the three transcriptional factors analysed (SREBP-1a, SREBP-1c and PPARgamma) was also reduced. These results appear to provide a framework for partially understanding the reduction in body fat induced by CLA. Inhibition of LPL activity seems to be an important mechanism underlying body fat reduction in hamsters. Further research is needed to better characterize the effects of CLA on lipogenesis and the role of these effects in CLA action.

Development of the first potent and specific inhibitors of endocannabinoid biosynthesis.

Enzymes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases alpha and beta (DAGLalpha and beta) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLalpha to screen new synthetic substances as DAGLalpha inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLalpha and MAGL activities were assessed by using sn-1-[14C]-oleoyl-2-arachidonoyl-glycerol and 2-[3H]-arachidonoylglycerol as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat) (IC50 approximately 60 nM), the most potent inhibitors of DAGLalpha were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC50 = 500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC50 = 160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat N-acylphosphatidyl-ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB1 and CB2 receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl-O-p-nitro-phenyl oleylphosphonate] inhibited both DAGLalpha and MAGL with similar potencies (IC50 = 0.8-0.1 and 3.7-3.2 microM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.

Inhibitory effects of grape seed extract on lipases.

OBJECTIVE: The aim of the present study was to assess the effects of grape seed extract (GSE) on the fat-metabolizing enzymes pancreatic lipase, lipoprotein lipase, and hormone-sensitive lipase in vitro and evaluate its potential application as a treatment for obesity. METHODS: Crushed grape seeds were extracted in ethanol, and the extract was assayed for the measurement of inhibitory effects on pancreatic lipase and lipoprotein lipase activities and on lipolysis of 3T3-L1 adipocytes. RESULTS: The GSE rich in bioactive phytochemicals showed inhibitory activity on the fat-metabolizing enzymes pancreatic lipase and lipoprotein lipase, thus suggesting that GSE might be useful as a treatment to limit dietary fat absorption and the accumulation of fat in adipose tissue. The observed reduction in intracellular lipolytic activity of cultured 3T3-L1 adipocytes may reduce the levels of circulating free fatty acids that have been linked to insulin resistance in obese patients. CONCLUSION: The GSE rich in compounds that inhibit lipases may provide a safe, natural, and cost-effective weight control treatment.