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Pancreatic adaptation to dietary lipids is mediated by changes in lipase mRNA.

Lipase activity, rates of biosynthesis of lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) and amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) as well as concentrations of their corresponding mRNAs were measured in the pancreatic tissue of rats fed isocaloric and isoprotein diets with inverse changes in the amounts of lipids and carbohydrates. A control diet (3% sunflower oil--62% starch) and three lipid-rich diets (10% sunflower oil--46.2% starch, 25% sunflower oil--12.5% starch and 30% sunflower oil--1.25% starch) were fed to rats for 10 days. Ingestion of the 10% lipid diet already resulted in a 1.4-fold increase in lipase activity while a 2.4-fold increase was observed with the other 2 high-lipid low-carbohydrate diets. Similarly, 1.3- and 3.1-fold increases in the total rate of protein synthesis were measured in pancreatic lobules of rats fed 10 and 25% or 30% lipid diets, respectively, as compared with control animals. While absolute lipase synthesis showed an important increase during the dietary manipulation (1.7- and 5.9-fold, respectively), amylase synthesis was significantly lower (1.1- and 1.5-fold, respectively). The level of lipase mRNA, as measured by dot-blot hybridization with the corresponding specific cDNA, showed a 2.2-fold increase (10% lipid diet) and a 3.9-fold increase (25% lipid diet), whereas the level of amylase mRNA showed only 1.1- and 1.3-fold increases under the same experimental conditions. These data demonstrated that protein-specific synthesis rates more accurately reflected pancreatic adaptive states than tissue levels of enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗

L-type lipase activity in ovaries of superovulated rats. Relation to cholesterol homeostasis.

The impact of lowering the ovarian L(iver)-type lipase activity on cholesterol homeostasis in the ovaries was studied in superovulated rats. L-type lipase activity increased rapidly after injection with chorionic gonadotrophin (HCG) (day 0), its activity remained high between days 3 and 8. During this period plasma progesterone and 20 alpha-hydroxyprogesterone were raised. The ovarian content of unesterified cholesterol remained constant during this period while cholesterol esters increased. Lowering of the L-type lipase activity by in vivo treatment with anti-liver lipase (ALLA) during 4-5 h did not affect plasma hormones or ovarian cholesterol contents. However, de novo cholesterol synthesis in the ovaries was significantly increased by about 40%. After pretreatment of the rats with aminogluthetimide, ALLA administration led to a 250% increase in de novo cholesterol synthesis in the unesterified cholesterol fraction, but was without effect on plasma hormones and on the ovarian cholesterol content. Administration of the cholesterol synthesis inhibitor Simvastatin led to a 25% lowering in ovarian cholesterol synthesis without effect on plasma hormones or ovarian cholesterol content. Additional administration of ALLA affected only the plasma progesterone (-30%). These results indicate that L-type lipase is involved in ovarian cholesterol homeostasis.

Aminoglutethimide↗

Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization.

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol. The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.

Amino Acid Sequence↗

Some properties of pancreatic lipase in Salmo gairdnerii Rich.:Km, effects of bile salts and Ca2+, gel filtrations.

1. The Michaelis constant (Km) of the trout pancreatic lipase is 1.3 x 10(-6) M tributyrin, calculated by the interface concentration of the emulsion. This value is lower than that of porcine pancreatic lipase. 2. The lipase hydrolyses tributyrin in a Ca2+ free medium. Conversely, Ca2+ is essential for the lipolysis of triolein. The cation might be an effector of the reaction, but it seems to remove the inhibition of the enzyme by its product. 3. The curves of the lipase activity according to bile salt concentration seem to suggest the existence of a colipase, that we have not evidenced yet by direct procedures. 4. The apparent molecular weight of the lipase seems to be lower in the trout than in the species studied so far.

Animals↗

Cloning, characterization, and expression in Streptomyces lividans 66 of an extracellular lipase-encoding gene from Streptomyces sp. M11.

A gene encoding an extracellular lipase from Streptomyces sp. M11 was cloned in the high-copy-number vector pIJ486, using S. lividans 66 as host. A 28-kDa protein was secreted by S. lividans carrying pB13, which harbors a 6-kb insert, and identified as the product of the cloned gene. Comparison of the N-terminal amino acid (aa) sequence of the purified extracellular lipase with the nucleotide (nt) sequence of the lip gene revealed the presence of a 48 aa long signal peptide. The nucleotide sequence also revealed the presence of a motif, Gly-His-Ser-Met-Gly, similar to the one found surrounding the active-site Ser in other lipases. The gene is most likely monocistronic. Subcloning experiments indicated that another gene might be required for high-level expression, since subcloning of the structural gene alone resulted in diminished extracellular lipase activity. The lipase gene promoter was identified by S1 mapping experiments, and found to be similar to other Streptomyces vegetative promoters.

Amino Acid Sequence↗

Extracellular lipase of Pseudomonas aeruginosa: biochemical characterization and effect on human neutrophil and monocyte function in vitro.

Lipase was isolated from P. aeruginosa by ultrafiltration of sterile-filtered culture supernatant. Gel filtration on Sepharose 4B yielded a broad peak corresponding to a molecular mass range of 100 to 1000 kDa. Electron microscopy of a negatively stained lipase preparation after Sepharose 4B chromatography revealed spherical particles with diameters ranging from 5 to 20 nm. Biochemical characterization and SDS polyacrylamide gel electrophoresis suggested that these particles consisted of protein and carbohydrate including lipopolysaccharide with the major enzyme activity being lipase. Various concentrations of this lipase preparation were preincubated with human peripheral blood neutrophils and monocytes. The chemotaxis and chemiluminescence of these cells were then determined. It was shown that lipase inhibited the monocyte chemotaxis and chemiluminescence, whereas it had no or very little effect on neutrophils. The inhibitory effect was concentration dependent and was abolished by heat treatment of the enzyme at 100 degrees C. Since monocytes are one of the important cells of the host defence system the inhibition of the function of these cells may contribute to the pathogenesis of infections caused by P. aeruginosa.

Cell Survival↗

Inhibition of lipases by phosphonates.

Ethyl hexylchlorophosphonate and analogues thereof were investigated as inhibitors of lipases. Both microbial and mammalian lipases were irreversibly inhibited. The inhibition could be monitored by p-nitrophenol release from the corresponding ethyl p-nitrophenyl hexylphosphonate inhibitor. Quantitative analysis of the data indicated that a 1:1 lipase-inhibitor complex was formed during inhibition. Enantioselective inhibition was found for the lipases derived from Candida antarctica and Rhizomucor miehei using pure enantiomers of ethyl p-nitrophenyl hexylphosphonate as inhibitors. Using the same inhibitor, reversed enantioselectivity was found for the protease alpha-chymotrypsin as compared to the two lipases.

Animals↗

High-yield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5.

An organic solvent-tolerant S5 lipase was purified by affinity chromatography and anion exchange chromatography. The molecular mass of the lipase was estimated to be 60 kDa with 387 purification fold. The optimal temperature and pH were 45 degrees C and 9.0, respectively. The purified lipase was stable at 45 degrees C and pH 6-9. It exhibited the highest stability in the presence of various organic solvents such as n-dodecane, 1-pentanol, and toluene. Ca2+ and Mg2+ stimulated lipase activity, whereas EDTA had no effect on its activity. The S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride. It showed random positional specificity on the thin-layer chromatography.

Calcium↗

Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase.

Chicken pancreatic lipase (CPL) was purified from delipidated pancreas. Pure CPL was obtained after ammonium sulphate fractionation, then DEAE-cellulose, Sephacryl S-200 gel filtration, and FPLC Mono-Q Sepharose columns. The pure lipase is a glycosylated monomer having a molecular mass of about 50kDa. The 23 N-terminal amino acid residues of CPL were sequenced. The sequence is similar to those of avian and mammalian pancreatic lipases. CPL presents the interfacial activation phenomenon tested with tripropionin or vinyl ester. When CPL was inhibited by synthetic detergent (TX-100) or amphipathic protein (BSA), simultaneous addition of bile salts and colipase was required to restore the full CPL activity. In the absence of colipase and bile salts, CPL was unable to hydrolyse tributyrin emulsion. This enzyme can tolerate, more efficiently than HPL, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate in the absence of bile salts and colipase. The CPL activity, under these conditions, was linear whereas that of HPL decreased rapidly. Anti-TPL polyclonal antibodies cross-reacted specifically with CPL. The gene encoding the mature CPL was cloned and sequenced. The deduced amino acid sequence of the mature lipase shows a high degree of homology with the mammalian pancreatic lipases. A 3D structure model of CPL was built using the HPL structure as template. We have concluded that a slight increase in the exposed hydrophobic residues on the surface of CPL, as compared to HPL, could be responsible for a higher tolerance to the presence of long-chain free fatty acids at the lipid/water interface.

Amino Acid Sequence↗

Exploring the specific features of interfacial enzymology based on lipase studies.

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

Adsorption↗

Lipoprotein lipase is expressed by glomerular mesangial cells.

Lipoprotein lipase expressed by the vasculature plays a key role in atherogenesis by enhancing the binding and uptake of lipoproteins and, thereby, leading to the formation of lipid-loaded foam cells. Hyperlipidemia also accelerates the progression of glomerular diseases and addition of exogenous lipoprotein lipase to mesangial cells has been shown to lead to an enhanced binding of lipoproteins to these cells. Despite such potential importance, the expression of endogenous lipoprotein lipase by cells of the glomeruli has, as yet, not been investigated. We show here for the first time that mesangial cells, but not epithelial cells, express lipoprotein lipase. The minimal lipoprotein lipase gene promoter was active in mesangial cells and inhibited by interferon-gamma, which is known to suppress its expression.

Animals↗

The acetates of p-nitrophenyl alpha-L-arabinofuranoside--regioselective preparation by action of lipases.

All possible di-O-acetates and mono-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were prepared by chemoenzymatic way using lipases. The 2,3-di-O-acetate was obtained in 90% yield by deacetylation of the primary acetyl group of per-O-acetylated p-nitrophenyl alpha-L-arabinofuranoside by Candida cylindracea lipase (CCL) or Candida rugosa lipase (LAY). The 2,5- and 3,5-di-O-acetates were obtained by acetylation of p-nitrophenyl alpha-L-arabinofuranoside by Pseudomonas cepacia lipase (LPS-30) in organic solvents. The 5-O-acetate was regioselectively synthesised in 95% yield by acetylation of p-nitrophenyl alpha-L-arabinofuranoside catalysed by porcine pancreas lipase. Finally, the 2- and 3-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were obtained in two steps. The enzymatic di-O-acetylation of p-nitrophenyl alpha-L-arabinofuranoside by LPS-30 was followed by enzymatic hydrolysis of the primary acetyl group by CCL or LAY.

Acetates↗

Esterase and lipase in camel tick Hyalomma dromedarii (Acari: Ixodidae) during embryogenesis.

Esterase and lipase activity showed significant changes during embryogenesis of camel tick Hyalomma dromedarii. From the elution profile of chromatography on DEAE-cellulose, six forms of H. dromedarii esterase (El to EVI) can be distinguished. Esterase EIII was purified to homogeneity after chromatography on Sepharose 6B. The molecular mass of esterase EIII was 45 kDa for the native enzyme and represented a monomer of 45 kDa by SDS-PAGE. Esterase EIII had an acidic pI at 5.3. Lipase activity was detected in the same DEAE-cellulose peaks (LI to LVI) of H. dromedarii esterases. The highest lipase activity was exhibited by lipase LIII. Esterase EIII and lipase LIII were compared with respect to Michaelis constant, substrate specificity, temperature optimum, heat stability, pH optimum, effect of metal ions and inhibitors. This study suggests that H. dromedarii lipolytic enzymes may play a central role in the interconversion of lipovitellins during embryogenesis.

Animals↗

Integration of purification with immobilization of Candida rugosa lipase for kinetic resolution of racemic ketoprofen.

The two processes for the partial purification and for the immobilization of a crude lipase preparation (Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity (E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5 mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.

Adsorption↗

Stereoselective lipases from Burkholderia sp., cloning and their application to preparation of methyl (R)-N-(2,6-dimethylphenyl)alaninate, a key intermediate for (R)-Metalaxyl.

Two microbial strains (referred to as MC 16-3 and 99-2-1) that produce extracellular lipases were isolated from soil samples and identified as Burkholderia species. The lipases were partially purified by isopropyl alcohol precipitation and gave molecular weight of 33kDa. The lipases were characterized in terms of stereoselectivity with racemic methoxyethyl (R,S)-N-(2,6-dimethylphenyl)alaninate and the genes encoding the proteins have been identified by homology alignment of lipases reported belonging to I.2 subfamily and their complete DNA sequences were determined. The lipases will be useful for the preparation of methyl (R)-N-(2,6-dimethylphenyl)alaninate, a key intermediate for the synthesis of (R)-Metalaxyl, which is one of the best-selling fungicides.

Alanine↗

Screening of some medicinal plants for anti-lipase activity.

In order to find new pancreatic lipase (triacylglycerol lipase, EC 3.1.1.3) inhibitors from natural sources, 75 medicinal plants belonging to different families were screened for their anti-lipase activity, using a radioactive method. On evaluating the results, methanolic extracts of three plants namely, Eriochloa villosa (Thunb.) Kunth, Orixa japonica Thunb. and Setaria italica (L.) Palib., exhibited strong in vitro anti-lipase activity (above 80%). These plants will be studied further to elucidate the structure and chemical properties of the active compound responsible for anti-lipase action.

Animals↗

Estrogen regulation of adipose tissue lipoprotein lipase--possible mechanism of body fat distribution.

OBJECTIVE: The purpose of this study was to evaluate the regulation of lipoprotein lipase activity, protein mass, and messenger ribonucleic acid by estradiol. STUDY DESIGN: Premenopausal women not taking exogenous sex steroids had transdermal 17 beta-estradiol and placebo patches placed in the gluteal region during the early follicular phase of the menstrual cycle. Adipose biopsies were performed from beneath the patches. Adipose tissue lipoprotein lipase activity was determined by a radiometric assay, protein mass was determined by enzyme-linked immunosorbent assay, and messenger ribonucleic acid level was determined by Northern analysis. Comparisons between the treated and placebo sides were analyzed by nonparametric statistics. RESULTS: Adipose tissue from beneath the 17 beta-estradiol patch had significantly decreased lipoprotein lipase activity and extracellular protein mass than did adipose tissue from beneath the placebo patch. There was no difference in lipoprotein lipase messenger ribonucleic acid levels. CONCLUSION: Estrogen decreases lipoprotein lipase activity by a posttranscriptional modification of protein levels. A hypothesis of sex steroid regulation of body fat distribution is proposed.

Adipose Tissue↗

Impaired secretion of heart lipoprotein lipase in cyclophosphamide-treated rabbit.

Cyclophosphamide administration into fasted rabbits induces a hypertriglyceridaemia and a defect in vascular lipoprotein lipase. Heart LPL activity was more than 50% decreased after antimitotic treatment in fasted animals. The tissue distribution of lipoprotein lipase activity was followed in heart using recycling perfusion. Cyclophosphamide administration resulted in a profound decline in the heparin-releasable lipoprotein lipase activity, concordant with a higher recovery in the residual heart tissue. The effects were more pronounced in fasted than in fed animals. In agreement, the proportion of neosynthesized [35S]methionine-labelled lipoprotein lipase released by heparin was decreased by 50% following antimitotic treatment. The lipolysis of very low density lipoprotein-labelled triacylglycerols was found 2.5-fold reduced in hearts from cyclophosphamide-treated rabbits as compared to controls. These results suggest that a defective secretion of lipoprotein lipase may contribute to the poor expression of lipolytic activity in the vascular bed and to the occurrence of hypertriglyceridaemia during cyclophosphamide treatment.

Animals↗

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