Expression and function of monoacylglycerol lipase in mouse ß-cells and human islets of Langerhans.

Elements of the endocannabinoid system (ECS) are expressed by islet endocrine cells and activation of CB1 and CB2 cannabinoid receptors regulates insulin secretion from mouse and human ß-cells. The current study aimed to investigate the expression and function, in mouse and human ß-cells, of monoacylglycerol lipase (MGL), an enzyme that facilitates degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). We found that MGL mRNA is expressed by MIN6 ß-cells, mouse islets, human islets and enriched human islet ß-cells, and immunohistochemistry indicated that MGL localisation in human islets is consistent with its expression by some ß- and -α-cells. Blockade of MGL activity with the pharmacological inhibitor URB602 led to increased [Ca(2+)](i )and enhanced insulin secretion from MIN6 ß-cells, and MGL inhibition also elevated insulin and glucagon secretion from isolated human islets in vitro. These data imply a stimulatory role for endogenous 2-AG in islets that is amplified when its degradation is blocked.

Time-dependent expression and distribution of monoacylglycerol lipase during the skin-incised wound healing in mice.

The study investigated the expression of monoacylglycerol lipase (MGL) during the skin-incised wound healing in mice and applicability of the time-dependent expression of MGL to wound age determination by immunofluorescent staining, Western blotting, and real-time PCR. Furthermore, cell types were identified by double immunofluorescence. A total of 45 BALB/c male mice were used in this study. After a 1.5-cm-long incision in the central dorsum skin, mice were killed at intervals ranging from 6 h to 14 days, followed by the sampling of wound margin. In the control, there was a low-level expression of MGL in the epidermis, hair follicles, and glandulae sebaceae. In the injured skin, MGL immunoreactivity was mainly detected in the neutrophils, macrophages, and myofibroblasts. Morphometrically, the average ratios of MGL-positive cells were more than 50% at 5 and 7 days post-wounding, whereas it was <50% at the other posttraumatic intervals. By Western blotting analysis, the average ratio of MGL protein expression was highest at 5 days after injury, which had a ratio of >2.30. Similarly, the relative quantity of MGL mRNA expression maximized at posttraumatic 5 days in comparison with control as detected by real-time PCR, with an average ratio of >2.54. In conclusion, MGL expression is detected in neutrophils, macrophages, and myofibroblasts and significantly up-regulated, suggesting that it may play roles in response to inflammation during skin-incised wound healing. From the viewpoint of forensic pathology, MGL detection is applicable to skin wound age determination.

Monoacylglycerol lipase promotes progression of hepatocellular carcinoma via NF-κB-mediated epithelial-mesenchymal transition.

BACKGROUND: Monoacylglycerol lipase (MAGL), a critical lipolytic enzyme, has emerged as a key regulator of tumor progression, yet its biological function and clinical significance in hepatocellular carcinoma (HCC) is still unknown. METHODS: In this study, we used a tissue microarray containing samples from 170 HCC patients to evaluate the expression of MAGL and its correlation with other clinicopathologic characteristics. In addition, we investigated the regulating effects of MAGL on various HCC lines. Finally, we identified the NF-κB signaling pathway participated in MAGL-mediated epithelial-mesenchymal transition (EMT) using HCC cell lines with different metastatic potentials. RESULTS: The expression of MAGL was significantly higher in HCC tumors than in matched peritumor tissues. Specifically, high MAGL expression was found in tumors with larger tumor size, microvascular invasion, poor differentiation, or advanced TNM stage. In addition, the clinical prognosis for the MAGLhigh group was markedly poorer than that for the MAGLlow group in the 1-, 3-, and 5-year overall survival times and recurrence rates of HCC patients. MAGL expression was an independent prognostic factor for both survival and recurrence after curative resection. Furthermore, the upregulation of MAGL in HCC cells promoted cell growth and invasiveness abilities, and accompanied by EMT. In contrast, downregulation of MAGL obviously inhibited these characteristics. Moreover, further investigations verified that MAGL facilitates HCC progression via NF-κB-mediated EMT process. CONCLUSIONS: Our findings demonstrate MAGL could promote HCC progression by the induction of EMT and suggest a potential therapeutic target, as well as a biomarker for prognosis, in patients with HCC.

Synthesis and Preclinical Evaluation of Sulfonamido-based [(11)C-Carbonyl]-Carbamates and Ureas for Imaging Monoacylglycerol Lipase.

Monoacylglycerol lipase (MAGL) is a 33 kDa member of the serine hydrolase superfamily that preferentially degrades 2-arachidonoylglycerol (2-AG) to arachidonic acid in the endocannabinoid system. Inhibition of MAGL is not only of interest for probing the cannabinoid pathway but also as a therapeutic and diagnostic target for neuroinflammation. Limited attempts have been made to image MAGL in vivo and a suitable PET ligand for this target has yet to be identified and is urgently sought to guide small molecule drug development in this pathway. Herein we synthesized and evaluated the physiochemical properties of an array of eleven sulfonamido-based carbamates and ureas with a series of terminal aryl moieties, linkers and leaving groups. The most potent compounds were a novel MAGL inhibitor, N-((1-(1H-1,2,4-triazole-1-carbonyl)piperidin-4-yl) methyl)-4-chlorobenzenesulfonamide (TZPU; IC50 = 35.9 nM), and the known inhibitor 1,1,1,3,3,3-hexafluoropropan-2-yl 4-(((4-chlorophenyl)sulfonamido) methyl)piperidine-1-carboxylate (SAR127303; IC50 = 39.3 nM), which were also shown to be selective for MAGL over fatty acid amide hydrolase (FAAH), and cannabinoid receptors (CB1 & CB2). Both of these compounds were radiolabeled with carbon-11 via [(11)C]COCl2, followed by comprehensive ex vivo biodistribution and in vivo PET imaging studies in normal rats to determine their brain permeability, specificity, clearance and metabolism. Whereas TZPU did not show adequate specificity to warrant further evaluation, [(11)C]SAR127303 was advanced for preliminary PET neuroimaging studies in nonhuman primate. The tracer showed good brain permeability (ca. 1 SUV) and heterogeneous regional brain distribution which is consistent with the distribution of MAGL.

A comparison of the lipolytic activities in liver perfusates and liver plasma membranes from rats.

We have undertaken a study to resolve the conflicting reports on the substrate specificity of the lipolytic enzyme(s) released by heparin from liver and report the following: (1) Heparin perfusates from liver contain an enzyme(s) capable of degrading triacylglycerol, diacylglycerophosphorylethanolamine and monoacylglycerol, whereas a heparin-solubilized fraction from liver plasma membranes hydrolyzes diacylglycerophosphorylethanolamine and monoacylglycerol only; (2) The lipolytic activities for the two sources behave differently on gel filtration but have the same behavior on heparin-Sepharose affinity chromatography; (3) Treatment of the preparation from the plasma membrane with Triton X-100 followed by heparin-Sepharose affinity chromatography produces forms of the enzyme(s) that now have activity on triacylglycerol This study suggests that the enzyme(s) from the two sources may be the same and that some change occurs when the enzyme is released from the intact liver.

Highly sensitive active-site titration of lipase in microscale culture media using fluorescent organophosphorus ester.

The fluorescent organophosphorus esters, diethyl 4-methylumbelliferyl phosphate (1), ethyl hexyl 4-methylumbelliferyl phosphate (2) and ethyl 4-methylumbelliferyl heptylphosphonate (3) have been synthesized and evaluated as a sensitive active-site titrant of lipase. The phosphorus esters 1, 2 and 3 inactivated the lipase from Pseudomonas aeruginosa (LPL-312) with a second-order rate constant for enzyme inactivation (k(on)) of 1.8, 32 and 5600 s(-1) M(-1), respectively. The long-chain phosphonate 3 turned out to be the most potent inactivator of the lipase to release a stoichiometric amount of highly fluorescent 4-methylumbelliferone (4MU) as a leaving group. By using the phosphate 3 as an active-site titrant, the low concentration (4.5 nM) of the active lipase was titrated successfully. The highly sensitive active-site titration with 3 enabled the direct determination of the concentration of the active lipase expressed in a microscale culture medium. Although the expression level differed significantly from one culture to another, the titrated concentration of the active lipase was proportional to the apparent activity for all the independent cultures. The molecular activity calculated for the expressed lipase was found to be the same as that of the purified lipase. The present active-site titration method is widely applicable to the biocatalytic engineering of lipases such as directed evolution, site-directed mutagenesis, chemical modification and immobilization.

Heterogeneity of carboxylesterases in rat liver cells.

Rat liver cells were separated into parenchymal cells (PC), Kupffer cells (KC) and endothelial cells (EC). The distribution of carboxylesterases (EC 3.1.1.1) between these cell types was investigated by PAGE and chromatogenic substrate staining, and compared with the results for total liver preparation and individual isoenzymes isolated by chromatofocusing. All of the liver carboxylesterase isoenzymes could be detected in the PC, whereas in both KC and EC only those with isoelectric point (pI) 6.4/6.2 could be detected. Use of carboxylesterase inhibitors like bis-(4-nitrophenyl)phosphate and paraoxon, and organophosphorus compound hydrolase inhibitors like 4-hydroxymercuribenzoate and EDTA confirmed that these esterases were of the carboxylesterase type.

Spectrophotometric determination of lipases, lysophospholipases, and phospholipases.

Spectrophotometric techniques for determining the activities of lipases, lysophospholipases, and phospholipases are reviewed. These methods involve the use of thioester substrate analogs as well as omega-nitrophenyl derivatives of the corresponding lipids. The most promising results are obtained with the thioester substrate analogs. Mono- and diacylglycerol lipases are assayed by using rac-1-S-decanoyl-1-mercapto-2,3-propanediol and rac-1,2-S,O-didecanoyl-1-mercapto-2,3-propanediol, respectively. Phospholipases A1 and A2 are determined by using rac-1,2-S,O-didecanoyl-3-phosphocholine-1-mercapto-2,3-propanediol and 2-hexadecanoylthio-1-ethyl-phosphocholine, respectively. Lysophospholipases are measured by using 2-hexadecanoylthio-1-ethyl-phosphocholine. Phospholipase C is assayed with rac-1-S-phosphocholine-2,3-O-didecanoyl-1-mercapto-2,3-propanediol. Thioester substrate analog assay procedures are more rapid, sensitive, convenient, continuous, and less expensive than the classical radiochemical techniques.

Modificações no óleo da castanha do Pará (Bertholletia excelsa) / Modification in the Brazil nut (Bertholletia excelsa) oil

Óleo da castanha do Pará (Bertholletia excelsa), extraído por prensagem das sementes a frio, foi submetido às modificações de fracionamento, hidrogenação, interesterificação e misturas com as gorduras obtidas na hidrogenação. Na hidrogenação o óleo foi submetido a três reações em reator Parr de 1 L, catalisador de níquel (Pricatt 9920-06 - Unichema), e sob as seguintes condições: 175ºC, 3 atm, 60 min (GH1), 150ºC, 1 atm, 30 min (GH2) e 125ºC, 1 atm, 30 min (GH3). O teor de acido esteárico aumentou de 11,6 por cento (óleo original) a 56,7 por cento, 16,3 por cento e 15,3 por cento em GH1, GH2 e GH3, respectivamente. O ácido oléico, representando 34,5 por cento no óleo original descresceu em GH1 a 25,9 por cento e aumentou em GH2 e GH3 a 59,9 por cento e 53,8 por cento, respectivamente...