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At least 1,423 records · Page 79Linked to original sources
PubMed1986

Dibutyryl cyclic AMP decreases the rate of lipoprotein lipase synthesis in cultured adipocytes.

The regulation of avian lipoprotein lipase by dibutyryl cyclic AMP in cultured adipocytes was studied with quantitative and specific methods for the measurements of enzyme catalytic activity, enzyme protein mass, and immunoadsorption of labeled enzyme. Incubation of adipocytes in 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline results in a time-dependent decrease in cell lipoprotein lipase catalytic activity. The activity is decreased by 70% in 4 h and over 90% by 12 h. The decrease in cellular catalytic activity is due to a decrease in both enzyme content and enzyme catalytic efficiency. 4 h after exposure of adipocytes to cAMP, enzyme protein was decreased from 3.58 +/- 0.5 to 1.92 +/- 0.1 ng/dish and specific activity from 15.1 +/- 2.1 to 8.4 +/- 1.1 nmol/ng. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in lipoprotein lipase activity was half-maximal at less than 25 microM dibutyryl cyclic AMP. The rate of lipoprotein lipase synthesis was estimated by measuring the incorporation of L-[35S]methionine into enzyme protein during 30 min. A method for the quantitative immunoadsorption of lipoprotein lipase from cell lysates was developed. Utilizing this immunoadsorption technique, the rate of incorporation of L-[35S]methionine into lipoprotein lipase was 0.0026 +/- 0.002%, when expressed as a percentage of that incorporated into total trichloroacetic acid-precipitable counts. By 2 h after exposure of adipocytes to 0.5 mM dibutyryl cAMP, the relative synthesis rate had already decreased to 64 +/- 4% of the control rate. After 16 h the synthesis rate was 43.2 +/- 13.8% of the control rate. The observed decreased synthesis rate could account for most of the decreased cellular enzyme content and diminished enzyme secretion rate.

Adipose Tissue↗
PubMed1986

Hydrolysis of lipid monolayers and the substrate specificity of hepatic lipase.

The substrate specificities of the phospholipase and triglyceridase activities of purified rat liver hepatic lipase were compared using lipid monolayers so that the substrates were presented to the enzyme in a controlled physical state. The rate of hydrolysis of 14C-labeled lipid at constant surface pressure in the presence of hepatic lipase and fatty acid-free bovine serum albumin at 33 degrees C was determined by monitoring the decrease of surface radioactivity. In monolayers of sphingomyelin/cholesterol (2:1, mol/mol) containing either 1 mol% triacylglycerol, 1 mol% phosphatidylethanolamine, or 10 and 20 mol% phosphatidylcholine, hepatic lipase clearly showed a preference for unsaturated over saturated lipids. In addition, with a sphingomyelin/cholesterol (2:1) monolayer containing 1 mol% of lipid substrate, hepatic lipase showed the following preference: triolein = dioleoylphosphatidylethanolamine much greater than dioleoylphosphatidylcholine; the respective rates of hydrolysis were 15.3 +/- 1.2, 14.9 +/- 0.8, and 0.5 +/- 0.1 mumol fatty acid produced/h per mg hepatic lipase. Overall, it appears that when comparing rates of hydrolysis of molecules within a given lipid class, hydrocarbon chain interactions are important. However, when comparing different lipid classes such as phosphatidylcholines and phosphatidylethanolamines, it is apparent that the polar group has a significant influence on the rate of hydrolysis. The rate of [14C]triolein hydrolysis, when mixed at surface concentrations of up to 2 mol% in a sphingomyelin/cholesterol (2:1) monolayer, was significantly faster than when triolein was present in a 1-oleyl-2-palmitylphosphatidylcholine monolayer; the rates of hydrolysis were 47.7 +/- 5.4 and 8.9 +/- 0.8 mumol fatty acid produced/h per mg hepatic lipase, respectively. The monolayer physical state and the miscibility of the substrate in the inert matrix influence the presentation of the substrate to the enzyme, thereby affecting the hydrolysis rate.

Animals↗
PubMed1986

Enrichment of apolipoprotein B-48 in the LDL density class following in vivo inhibition of hepatic lipase.

In order to explore the in vivo function of hepatic lipase, rats were injected with goat anti-rat hepatic lipase serum which produced a complete and specific inhibition of heparin-releasable hepatic lipase. In the fasting rats, protein, phospholipid and free cholesterol expressed as either mass or percent weight increased significantly in low-density lipoprotein (LDL) and high-density lipoprotein 2 (HDL-2) fractions. These three constituents were not affected in the VLDL and HDL-3 lipoproteins. In the fat-loaded (1 ml corn oil) rat, 6 h post inhibition of hepatic lipase triacylglycerol, phospholipid and free cholesterol concentrations in the d less than 1.006 fraction were 2.5-fold higher in the inhibited animals than in the control rats. The composition of the d less than 1.006 fraction was also affected. Expressed as percent mass, protein was lower (5.2 +/- 1.2 vs. 10.3 +/- 1.5, P less than 0.001) as was cholesteryl ester (1.7 +/- 0.7 vs. 2.6 +/- 0.4, P less than 0.01); triacylglycerol was elevated (77.2 +/- 4.0 vs. 72.6 +/- 2.4, P less than 0.025), as was free cholesterol (3.0 +/- 0.6 vs. 2.4 +/- 0.2, P less than 0.025). Overall, inhibition lowered the ratio of surface-to-core constituents suggesting a larger mean particle diameter. SDS-polyacrylamide gel electrophoresis showed the intermediate- and low-density lipoprotein from treated rats to be significantly enriched in apolipoprotein B-48. In the LDL fraction, apolipoprotein B-48 accounted for 62 +/- 14% of the total apolipoprotein B in the inhibited rats, vs. 12 +/- 2% in the control rats. The above results support the previously described in vivo function of hepatic lipase as a phospholipase. In addition, the results demonstrate a role of hepatic lipase in the catabolism of chylomicrons. Since removal of apolipoprotein B-48-containing lipoproteins is dependent upon apolipoprotein E, their appearance in the LDL fraction implies a masking of apolipoprotein E-binding determinants.

Animals↗
PubMed1987

Purification and characterization of a lipase from Staphylococcus aureus.

An extracellular lipase from Staphylococcus aureus (strain FN 37) was purified to homogeneity. A cell-free culture broth was subjected to ammonium sulphate precipitation, and the lipase was isolated from the resuspended pellet by adsorption chromatography on octyl-Sepharose. The purification was 957-fold, and the recovery of the octyl-Sepharose chromatography was about 100%. The specific activity of the purified lipase was 546 mU of lipase activity per micrograms protein. The purity of the final product was documented by SDS-polyacrylamide gel electrophoresis in which a homogeneous protein band of 43 kDa was found. In gel chromatography on Sephadex G-200 the lipase eluted as a homogeneous peak with an apparent molecular mass of 110 kDa, suggesting that the lipase may exist as an oligomer in physiological media. Analysis of the amino-acid composition revealed a predominance of polar, non-charged amino acids, with serine accounting for 24 mol% of the amino-acid residues.

Amino Acids↗
PubMed1987

Bovine pregastric lipase: a model for the human enzyme with respect to properties relevant to its site of action.

Preduodenal lipolysis is considered to promote efficient lipid digestion in the neonatal period. The lipase(s) responsible may be of pregastric or gastric origin depending upon the species. We have previously reported on purification and molecular characterization of a pregastric lipase from calf. Antibodies to this bovine enzyme crossreact with a protein of similar size in human gastric contents and also inhibit its lipolytic activity. Since the bovine and human enzymes also have similar kinetic properties, the view is favoured that the bovine enzyme can be used as a model for physiological studies relevant to human neonates. In contrast to the lipases operating in the small intestine pregastric lipase has the unique property of initiating the hydrolysis of human milk fat globule triacylglycerol. In order to do this no cofactor is required. Pregastric lipase was stable at low pH and had an acid-pH optimum. Furthermore, it was extremely resistant to pepsin. In contrast, pancreatic proteinases, i.e. trypsin and chymotrypsin, inactivated the enzyme. The rate of inactivation was increased in the presence of bile salts which by themselves could inhibit enzyme activity. Thus, pregastric lipase is ideally suited for activity in the stomach but will not, under healthy conditions, contribute to lipid digestion in the duodenum.

Animals↗
PubMed1990

Acid lipase in rat intestinal mucosa: physiological parameters.

Previous studies have shown that up to a half of infused triacylglycerol does not exit the intestine via lymphatics. This suggests the presence of a mucosal lipase which could provide fatty acids for potential transport via the portal vein. The present study describes an acid-active lipase in rat intestinal mucosa. Acid lipase was assayed using a glyceryl tri[14C]oleate emulsion (pH 5.8). Mucosal homogenates were differentially centrifuged to yield cellular organelles and cytosol. Cells were sequentially released from villi using citrate and EDTA. The enzyme was found to be most active in the proximal quarter intestine and in the upper third of villi. Its greatest activity was in the lysosomal fraction. Esophageal diversion demonstrated that lingual lipase was not the precursor of the mucosal acid lipase. Bile salts stimulated activity 3- to 5-fold, but other neutral or anionic detergents were inhibitory. Of the detergents tested, taurocholate at super critical micellar concentrations could restore activity only with SDS. Sepharose 6B chromatography suggested that the enzyme partitioned into an SDS and taurocholate mixed micelle. We conclude that mucosal acid lipase is a distinct, intrinsic enzyme of the intestinal mucosa. It is predominantly lysosomal in origin. The location of its greatest activity in the villus tips of the proximal intestine suggests that it is potentially involved in mucosal triacylglycerol disposal.

Acid Phosphatase↗
PubMed1991

Milk lipid digestion in the neonatal dog: the combined actions of gastric and bile salt stimulated lipases.

Intragastric lipolysis may be particularly important for the digestion of milk lipid since milk fat globules are resistant to pancreatic lipase without prior disruption; milk bile salt stimulated lipase (BSSL) may supplement further intestinal hydrolysis. Previous information on gastric lipolysis has been based primarily on in vitro studies using artificial lipid emulsions containing a single component fatty acid and have focused on the preferential release of medium-chain fatty acids. The actual contribution of these enzymes to overall fat digestion in vivo on natural substrates has rarely been studied, however. The neonatal dog is an excellent model in the study of lipid digestion because, like the human, milk lipids are high in long-chain unsaturated fatty acids, milk contains BSSL and gastric lipase is the predominant lipolytic enzyme acting in the stomach. We used a combination of in vivo studies with in vitro incubations to investigate digestion of milk lipid by gastric and milk (BSSL) lipases in the suckling dog. In the first 4 weeks postpartum, 14-41% and 42-60% of milk triacylglycerol was hydrolyzed to primarily diacylglycerol and free fatty acid (FFA) in the first 30 and 60 min in the stomach, respectively. Milk lipid contained high levels (63%) of long-chain unsaturated fatty acids, which were preferentially released as FFA during in vivo gastric lipolysis, consistent with the actions and stereospecificity of gastric lipase. While levels of hydrolysis in gastric aspirates were significantly different (by age and time in stomach) at the start of in vitro studies, total hydrolysis in all incubation systems plateaued at about 65%, suggesting product inhibition by the long-chain FFA, but to a much lesser degree than previously expected from in vitro studies. The magnitude of in vivo intragastric lipolysis was 3- to 6-times greater than that predicted by in vitro assays using either milk lipid or labeled emulsion as substrate, respectively. Prior exposure to intragastric lipolysis resulted in 30% hydrolysis by BSSL compared to 5% hydrolysis without prior exposure. We suggest that previous in vitro studies have largely underestimated the actual degree of intragastric lipolysis that can occur and its activity on long-chain fatty acids; this study indicates the importance of the combined mechanisms of gastric lipase and BSSL to fat digestion in the suckling neonate.

Aging↗
PubMed1995

Fatty acid selectivity of a lipase purified from Vernonia galamensis seed.

Vernonia galamensis is an annual herb whose seed oil contains high levels of an epoxy fatty acid, vernolic (cis-12,13-epoxy cis-9-octadecenoic) acid. The seed also contains lipase activity in the dormant state. A lipase was purified from the seed and its substrate specificity studied in isooctane. The lipase shows pronounced selectivity for the native triacylglycerol, trivernolin. The rate of hydrolysis of triolein, the corresponding non epoxy triacylglycerol, is only 3% of that of trivernolin. In the acidolysis of tricaprylin using a mixture of fatty acids, the Vernonia lipase also showed selectivity for vernolic acid. Michaelis-Menten kinetics of the hydrolysis of triacylglycerols revealed that the observed high selectivity of the Vernonia lipase for trivernolin was mainly due to a higher Vmax for trivernolin. The Vmax value for the hydrolysis of trivernolin was 5 times higher than that for triolein. This novel substrate specificity is an adaptation by the seed lipase to the triacylglycerols of the seed oil that contain up to 80% vernolic acid.

Epoxy Compounds↗
PubMed1984

Characterization of triglyceride lipase activities in rat skeletal muscle.

Triglyceride lipase activity was determined in particulate and soluble fractions from rat skeletal muscle homogenates. The fractions exhibited an acid (pH 5,0) optimum with an impressive enhancement in the combined P17 /100 fraction. Methylamine inhibited this acid lipase activity. A further lipase was observed with maximal activity at pH 7,0 and only a small enhancement in the combined P17 /100 fraction and inhibition by diethyl p-nitrophenyl-phosphate but not by protamine sulfate. Lipoprotein lipase activity was identified by the following in vitro criteria: Stimulation of activity by serum, maximal activity at alkaline pH (pH 8,5 - 9,0) and inhibition of activity by NaCl and protamine sulfate. There was a definite enhancement of lipoprotein lipase activity in the combined P17 /100 fraction after the lipase activity has been washed out from the capillary bed with heparin.

Animals↗
PubMed1988

Substrate specificity of Staphylococcus aureus (TEN5) lipases with isomeric oleoyl-sn-glycerol ethers as substrates.

For the first time fully protected substrates with only one hydrolyzable ester bond have been used to analyze the substrate specificity of microbial lipases. In these substrates the ester is attached to the glycerol molecule in a precisely defined position. The use of three different substituents generates chirality and thus allows the analysis of positional specificities of individual lipases. Therefore, these new substrates have been used to study the enzymatic activities of two closely related lipases isolated from Staphylococcus aureus (TEN5) designated the 44 and 43 kDa lipase. The lipases, especially the 44 kDa molecule, show a high specificity for the hydrolysis of the ester in the sn-1 position (S-configuration), which is hydrolyzed by a factor of ten faster than that in the sn-3 position. In addition, the study demonstrates for the first time that the rate of hydrolysis of a fatty acid ester attached to the sn-2 position of glycerol by microbial lipases depends on the configuration of the substrate molecule.

Glyceryl Ethers↗
PubMed1988

Human preduodenal lipase is entirely of gastric fundic origin.

Lipase activity was measured in supernatant homogenates from various anatomic regions in the upper part of the human digestive tract of two organ donors. It is shown unambiguously that lipase activity occurs only in the fundic mucosa of the stomach, whereas no significant activity takes place in the antral, pharyngeal, or lingual areas, including the circumvallate papillae. In adults, the potential activity of human gastric lipase, as measured using tributyrin as substrate, amounts to 20% of its pancreatic counterpart. Lipase activity was also determined on human gastric biopsy samples taken during gastrofibroscopy tests on healthy adults. These results confirmed the finding that a lipolytic activity of gastric origin occurs uniformly and only in the fundic mucosa. Triacylglycerol hydrolysis is associated with a genuine gastric lipase activity that is clearly distinct from the classical esterase observed using p-nitrophenyl acetate as substrate. Lipase activity decreases significantly with age: it ranges on average from 4700 U/g of fresh mucosa in subjects aged up to 50 yr to 700 U/g of fresh mucosa in persons over 60 yr of age.

Adult↗
PubMed1994

Pancreatic triglyceride lipase and colipase: insights into dietary fat digestion.

Dietary fats have an impact on health and disease. A pancreatic exocrine protein, pancreatic triglyceride lipase, is essential for the efficient digestion of dietary fats. This enzyme requires another pancreatic exocrine protein, colipase, for full activity in the gut lumen. In addition to its importance in fat digestion, pancreatic triglyceride lipase has potential applications in medical therapy, medical diagnostics, and industry. This potential stimulated interest in lipases; radiograph during the last few years, studies applying the technologies of molecular biology and radiograph crystallography greatly increased our knowledge about pancreatic triglyceride lipase and colipase protein structure, enzyme mechanism, and gene structure. This review focuses on these recent advances and discusses models for the kinetic properties of pancreatic triglyceride lipase and for the interaction of pancreatic triglyceride lipase with colipase.

Amino Acid Sequence↗
PubMed1984

Some properties of acid lipase in the defatted liver from the laying hen (Gallus domesticus).

Acid lipase activity was found in the defatted liver from the laying hen, but little neutral or alkaline lipase activity was observed in the liver. Most of acid lipase was in the insoluble fraction of the defatted liver, and the enzyme was solubilized by sonication at 9 kHz for 50 min in a slightly alkaline solution. The lipase showed its maximum activity at pH 5.0, 38 degrees C. It was stable below 40 degrees C and over the pH range from 4.0 to 9.0. Detergents, serum of the laying hen and the soluble fraction from the defatted liver homogenate from the laying hen markedly inhibited the lipase activity. The lipase solubilized by sonication was large in molecular mass, suggesting that the preparation formed colloidal particles.

Animals↗
PubMed1985

Triacylglycerol lipase in bovine erythrocytes.

Acid lipase activity was found in the bovine erythrocyte ghosts, but little neutral or alkaline lipase activity was observed in the erythrocytes. The membrane-bound lipase showed a remarkable activity in the ghosts only after hemolysis. The membrane-bound lipase showed its maximum activity at pH 4.5, 38 degrees C, and it was stable below 40 degrees C. The hydrolysis rate was linear with time up to 60 min, and was proportional to the amount of enzyme up to 0.4 mg protein. The bound lipase was activated markedly by bovine serum albumin and slightly by octyl-glycoside. The lipase was remarkably inhibited by bovine serum.

Animals↗
PubMed1989

Schistosoma mansoni: female-dependent lipid secretion in males and corresponding changes in lipase activity.

Eight to 10-week old Schistosoma mansoni males from unisexual infections were examined histochemically for neutral lipids and lipase activity. In addition, in situ fixed pairs were examined for lipase activity. Neutral lipid content of males from unisexual infections was variable and lipase activity was minimal. Following 1 h incubation at 37 degrees C in Earle's balanced salt solution (EBSS) in which females had previously been maintained for 1 h, males showed moderate increase in lipid content and diminished lipase activity. In contrast, unisexually developed males incubated for 1 h with females from bisexual infections showed increased lipid accumulation and lipase activity. Unisexually developed males incubated for 1 h in EBSS showed both lipid accumulation and release from the dorsal surface. Worm-pairs fixed in situ showed greater lipase activity in females than in males. These observations suggest that a factor(s) released by females affects the physiology of males.

Animals↗
PubMed1989

Comparison of lipoprotein lipase activity in heart myocytes and perfused hearts.

This study was initiated to compare lipoprotein lipase activity in isolated heart myocytes and the heparin residual compartment of perfused hearts from adult rats. Heart lipoprotein lipase activity was divided into two fractions by 1 min of heparin perfusion. Heparin-residual and myocyte lipoprotein lipase activity were lower in hearts obtained from fasted compared to fed rats. In each case, the myocyte enzyme activity was 55 to 60% of heparin-residual levels. The difference between myocyte and heparin-residual activity may be a consequence of the time and treatment required to isolate cells in that long-term in vitro exposure of heart tissue to heparin also reduces residual activity. In vivo treatment with endotoxin decreased both heparin-residual and myocyte lipoprotein lipase activities; whereas, colchicine administration increased both activities compared to saline injected rats. In this latter experiment heart heparin-residual and myocyte lipoprotein lipase activities were positively correlated (r = 0.90). The results indicate that in the mature heart intracellular lipoprotein lipase activity is primarily associated with myocytes.

Animals↗
PubMed1990

Inhibition of diacylglycerol metabolism in isolated cardiac myocytes by U-57 908 (RHC 80267), a diacylglycerol lipase inhibitor.

U-57 908 (RHC 80267) inhibited diacylglycerol (DG) lipase activity in soluble and microsomal subcellular fractions from cardiac myocytes isolated from adult rat hearts; half-maximal inhibition was observed at a concentration of 3.5 microM. Monoacylglycerol lipase activity was much less sensitive to inhibition, but U-57 908 reduced lipoprotein lipase activity in cardiac myocytes with the same sensitivity as observed for DG lipase. DG kinase activity was not inhibited by U-57 908. DG metabolism by intact cardiac myocytes was studied in incubations with a cell-permeable DG analog, [3H]-dioctanoylglycerol (diC8). DiC8 was mainly metabolized by conversion to mono-octanoylglycerol (monoC8) and glycerol (lipase pathway); much less radioactivity was incorporated into the triacylglycerol and total phospholipid fractions. U-57 908 reduced the loss of radioactivity from the exogenous diC8 substrate, with a corresponding decline in the formation of radiolabelled monoC8 and glycerol. The incorporation of radioactivity into phospholipids was slightly reduced, but triacylglycerol synthesis from diC8 was increased in the presence of U-57 908. Therefore, U-57 908 is an effective inhibitor of DG metabolism by the lipase pathway in intact cardiac myocytes.

Animals↗
PubMed1988

Regulation of lipoprotein lipase in different rat tissues.

In rats maintained on a fat-free diet, hepatic production of very low density lipoprotein (VLDL) fell by only 18% after 20 hours of fasting (as judged by rates of triglyceride accumulation in plasma following administration of Triton WR1339). Infusion of heparin into fed rats caused a rapid accumulation in plasma of two distinct lipase activities in approximately equal amounts: heparin releasable lipoprotein lipase (HR-LPL) and heparin releasable triglyceride lipase (HR-TGL). Fasting reduced the former by 67%, but the latter by only 20 to 25%. To gain insight into the effects of nutritional status on the interorgan distribution of these enzymes, experiments were designed for the perfusion of liver and heart by standard procedures, and of epididymis, gastrocnemius muscle (red fiber), and psoas muscle (white fiber) by micro techniques. After addition of heparin, perfusates were analyzed for each species of lipase. The objective was to selectively monitor the capillary-bound, physiologically functional lipase without significant contamination by intracellular enzyme. HR-TGL was found only in liver, where it represented the major lipase activity; it was only minimally affected by fasting. In epididymis HR-LPL fell by 85% in fasted animals and, remarkably, rebounded almost to ad libitum fed values after only two hours of refeeding. In both heart and gastrocnemius muscle, directional changes in HR-LPL were exactly reciprocal to those seen in epididymis. By contrast, HR-LPL of psoas muscle was unresponsive to fasting. Qualitatively, the data were in keeping with documented nutritionally-induced shifts in tissue disposal of VLDL-triglycerides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗