Specific Lipase Inhibitor Research Articles

Characterization of an acid inducible lipase Rv3203 from Mycobacterium tuberculosis H37Rv

The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif ‘GXSXG’. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as compared to normal whereas no expression was observed under nutrient and oxidative stress conditions. Therefore, detailed characterization of Rv3203 was done by gene cloning and its further expression and purification as his-tagged protein in microbial expression system. The enzyme was purified to homogeneity by affinity chromatography. It demonstrated broad substrate specificity and preferentially hydrolyzed p-nitrophenyl myristate. The purified enzyme demonstrated an optimum activity at pH 8.0 and temperature 50 °C. The specific activity, K m and V max of enzyme was determined to be 21.29 U mg(-1) protein, 714.28 μM and 62.5 μmol ml(-1) min(-1), respectively. The pH stability assay and circular dichroism spectroscopic analysis revealed that Rv3203 protein is more stable in acidic condition. Tetrahydrolipstatin, a specific lipase inhibitor and RHC80267, a diacylglycerol lipase inhibitor abolished the activity of this enzyme. The catalytic triad residues were determined to be Ser50, Asp180 and His203 residues by site-directed mutagenesis.

Two cutinase‐like proteins secreted byMycobacterium tuberculosisshow very different lipolytic activities reflecting their physiological function

Cutinases are extracellular enzymes that are able to degrade cutin, a polyester protecting plant leaves and many kinds of lipids. Although cutinases are mainly found in phytopathogenic fungi or bacteria, 7 genes related to the cutinase family have been predicted in the genome of Mycobacterium tuberculosis. These genes may encode proteins that are involved in the complex lipid metabolism of the bacterium. Here, we report on the biochemical characterization of two secreted proteins of M. tuberculosis, Rv1984c and Rv3452, belonging to the cutinase family. Although their amino acid sequence shows 50% identity with that of the well-characterized cutinase from Fusarium solani pisi, and a high level of homology has been found to exist between these two enzymes, they show distinct substrate specificities. Rv1984c preferentially hydrolyzes medium-chain carboxylic esters and monoacylglycerols, whereas Rv3452 behaves like a phospholipase A(2), and it is able to induce macrophage lysis. The tetrahydrolipstatin inhibitor, a specific lipase inhibitor, abolishes the activity of both enzymes. Site-directed mutagenesis was performed to identify the catalytic triad of Rv1984c. Structural models for Rv1984c and Rv3452 were built, based on the crystal structure of F. solani cutinase, with a view to investigating the contribution of specific residues to the substrate specificity. Our findings open new prospects for investigating the physiological roles of cutinase-like proteins in the lipid metabolism and virulence of M. tuberculosis.

Role of nonesterified fatty acids in necrotizing pancreatitis: an in vivo experimental study in rats.

In acute pancreatitis, nonesterified fatty acids (NEFA) might be released by lipase and cause tissue necrosis by their detergent properties, but this has not been established in vivo. To measure the release of NEFA in the blood stream, pancreatic tissue, and peritoneal cavity during taurocholate-induced acute necrotizing pancreatitis in rats. Ascites and blood were repeatedly sampled; after 24 hours, pancreatic lesions were scored, and NEFA were measured in the pancreas. The effects of a specific lipase inhibitor (Tetrahydrolipstatin [THL]) were also studied. A slight transient increase (22%) of NEFA concentration was observed in systemic circulation but did not parallel the time course of lipase activity, arguing against an intravascular production of NEFA by circulating lipase. Pancreatic NEFA did not differ between rats with pancreatitis and control rats. NEFA in ascites increased to threefold the basal value immediately after taurocholate and decreased rapidly thereafter, whereas lipase increased later in ascites and remained elevated during the 24-hour duration of the experiment. Lipase inhibition by THL neither modified the early increase of NEFA in ascites, nor altered the macroscopic, enzymatic, and histologic evolution of pancreatitis. This in vivo study does not confirm the hypothetical role of NEFA produced by pancreatic lipase in the necrotic process and its systemic complications, up to now mainly suggested on the basis of ex vivo experiments.

Current pharmacological approaches to the treatment of obesity.

Although comprehensive obesity treatment programmes were shown to induce weight loss and to improve risk factors and comorbidities, the weight reduction is moderate and most patients will rapidly regain weight. For these reasons, drugs have been developed or are in development to support and maintain weight loss. At present, two drugs are available for the adjunct treatment of obesity. Sibutramine is a centrally acting inhibitor of noradrenaline and serotonine reuptake, thereby decreasing caloric intake and increasing energy expenditure. Orlistat is a specific lipase inhibitor that impairs fat absorption, thereby reducing fat uptake. Both drugs have been found to be effective and safe in a number of clinical studies for up to two years. The current experience with these drugs raises questions related to the long-term efficacy with particular reference to cardiovascular end-points. In addition, other current and future pharmacological principles for weight reduction are discussed. There is no doubt that an evidence-based rational pharmacological treatment of obesity is still in an early stage.

Obesity: principles of drug therapy

Obesity is a major global public health problem. In many instances, a combination of diet modification, increased physical activity and behavior therapy fail or are insufficient for sustained weight loss. In these situations, drug therapy may be helpful. However, drug treatment of obesity resulted in unexpected devastating events in recent years. In the late sixties, aminorex caused an epidemic of pulmonary hypertension with high mortality rates. Dexfenfluramine and phentermine were also associated with the development of pulmonary hypertension and with alarming reports of cardiac valvular abnormalities. Therefore, these drugs were withdrawn from the market. Newer drugs, like sibutramine, a serotonin and norepinephrine reuptake inhibitor, and orlistat, a specific lipase inhibitor, reduce body weight significantly compared to placebo. In combination with a hypocaloric diet, weight loss of three to ten kilos can be achieved. Pharmacotherapy is limited to patients with a body mass index greater than 30 kg/m2, if non-pharmacological treatment programs have failed. The drugs should be prescribed under strict medical surveillance only.

Role of lipase in the regulation of postprandial gastric acid secretion and emptying of fat in humans: a study with orlistat, a highly specific lipase inhibitor

BACKGROUND AND AIMSTo investigate the importance of lipase on gastric functions, we studied the effects of orlistat, a potent and specific inhibitor of lipase, on postprandial gastric acidity and gastric...

Role of lipase in the regulation of gastric secretion and gastric emptying in humans: A study with orlistat, a highly specific lipase inhibitor: [formula omitted], J. Borovicka, G. Gutmann, C. Güzelhan, D. Hartmann, M. Fried. Gastroenterology, University Hospitals Zurich and Lausanne, and Hoffmann-LaRoche, Basel, Switzerland

Role of lipase in the regulation of gastric secretion and gastric emptying in humans: A study with orlistat, a highly specific lipase inhibitor: [formula omitted], J. Borovicka, G. Gutmann, C. Güzelhan, D. Hartmann, M. Fried. Gastroenterology, University Hospitals Zurich and Lausanne, and Hoffmann-LaRoche, Basel, Switzerland

Mode of action of tetrahydrolipstatin: a derivative of the naturally occurring lipase inhibitor lipstatin

Tetrahydrolipstatin is a specific lipase inhibitor derived from lipstatin, a lipid produced by Streptomyces toxytricini. In addition to pancreatic lipase, it is shown in the present study that tetrahydrolipstatin also inhibits human gastric lipase, carboxyl ester lipase (cholesterol esterase) of pancreatic origin and the closely related bile-salt-stimulated lipase of human milk. It does not inhibit the exocellular lipase from Rhizopus arrhizus or a lipase recently isolated from Staphylococcus aureus. In the presence of a water-insoluble substrate, such as tributyrin, the inhibition has the characteristics of an irreversible inactivation of the uncompetitive type, thus indicating that an enzyme · substrate · inhibitor complex is formed, which cannot undergo further reaction to yield the normal product. This reaction probably takes place at the aqueous/oil interface of the substrate. In aqueous solution, in the absence of substrate, the inhibition of carboxyl ester lipase by tetrahydrolipstatin has the characteristics of being reversible, and finally becomes of a temporary nature analogues to the trypsin-trypsin inhibitor system. It is suggested that an enzyme-inhibitor complex of an acyl-enzyme type is formed that is slowly hydrolysed, with water as the final acceptor, leaving an intact enzyme and an inactive form of the inhibitor. The enzyme thus consumes the inhibitor, which undergoes a chemical conversion, as indicated by a change in mobility in an appropriate thin-layer Chromatographic system, indicating an increase in hydrophilicity. Evidence is presented that the reaction product is an acid and that the functional group of tetrahydrolipstatin is the β-lactone reacting with the active site of the enzyme.

Tumor promoting phorbol diesters: Substrates for diacylglycerol lipase

Enzyme activity in rat serum was examined utilizing the potent tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA) and various glycerolipids as substrates. The serum activity was specific for hydrolysis of the long chain tetradecanoate moiety of TPA, hydrolyzed mono- and diacylglycerols, but was not effective against triacylglycerols, cholesterylesters, or phospholipids. Heating the enzyme preparation at 56°C for 1 min was dually effective in reducing the hydrolysis of both TPA and dioleoylglycerol by 83–86% of control levels. The potent diacylglycerol lipase inhibitor, RHC 80267, inhibited the hydrolysis of TPA in the 0.2–1.0 μM range and was also a potent blocker of monoacyl- and diacylglycerol hydrolysis. In substrate competition studies, exogenous unlabeled TPA was added to the [ 14C]dioleoylglycerol-containing reaction mixture, however, this produced an approximate 3-fold stimulation of [ 14C]dioleoylglycerol hydrolysis. Although we have not established whether the hydrolysis of TPA and diacylglycerol is the work of one enzyme, the effectiveness of the specific lipase inhibitor, RHC 80267, demonstrates that diacylglycerol lipase can utilize TPA as substrate, a finding never befofe documented. This point is of interest in light of the theory that phorbol esters act by mimicry of the natural lipid mediator, diacylglycerols.

Influence of colipase on the turbidimetric determination of pancreatic lipase catalytic activity.

The influence of colipase on the turbidimetric measurement of the catalytic activity of pure human pancreatic lipase (EC 3.1.1.3) and of sera from pancreatitis patients was studied. A deoxycholate-stabilized triolein emulsion served as substrate. It was found that the activity of the pure, colipase-free lipase is strongly inhibited by deoxycholate, and can be blocked completely if normal serum, pure human albumin, or the globulin fraction of normal serum is present. The inhibition by serum is competitive. This finding largely excludes the existence of a specific lipase inhibitor in human serum and explains the non-linear response of activity to the amount of serum added, a frequently observed problem with various turbidimetric lipase methods. A high molar excess of colipase (greater than 250-fold) completely abolishes the inhibition of lipase, irrespective of the inhibitory factor studied. Sera of pancreatitis patients, when measured turbidimetrically without addition of colipase, exhibit elevated lipase activity only if they contain colipase. However, the activity measured is not a function of the serum lipase concentration alone but of the molar ratio of colipase to lipase. Since this ratio varies considerably and is usually too low to ensure complete activation of lipase, erroneously low or even false negative results are obtained. For this reason it is strongly recommended that an excess of colipase is used in turbidimetric lipase assays. It therefore also appears important to study the influence of the serum colipase level on non-turbidimetric lipase methods.