Lipase Secretion Research Articles

17beta-estradiol enhances the flux of cholesterol through the cholesteryl ester cycle in human macrophages.

Estrogens have been shown to have many positive effects on the function of arterial wall, and recent evidence suggest that 17beta-estradiol has a direct action in reducing the accumulation of cholesteryl ester in macrophages. The mechanisms underlying the effects of 17beta-estradiol on foam cell formation, however are poorly understood. The aim of this study is to investigate the role of 17beta-estradiol in the regulation of the cholesteryl ester cycle and cholesterol efflux in human macrophages. In addition, the influence of 17beta-estradiol on apolipoprotein E (apoE) and lipoprotein lipase (LDL) secretion by the cells was also tested. Human Monocyte Derived Macrophages (HMDM), matured in the presence or the absence of 17beta-estradiol, were loaded with [3H]-cholesteryl ester-labeled-acetyl LDL (low density lipoprotein) and the efflux of radioactivity into the medium was measured. The effect of 17beta-estradiol on cellular activities of acyl coenzyme A: cholesterol acyl transferase (ACAT), and both neutral and acid cholesteryl ester hydrolase (CEH) and the secretion of apoE and LDL into the medium, were also studied. The results indicate that 17beta-estradiol induces an increase in the amount of labeled cholesterol released from the cells and, the data obtained from the measurements of ACAT and CEH activities showed that, in estrogen-treated HMDM, the cholesteryl ester cycle favors the hydrolysis of lipoprotein cholesterol by CEH in comparison with its acylation by ACAT. In particular, for the first time a strong enhancement of neutral and acid CEH in human macrophages by 17beta-estradiol, was demonstrated. ApoE and LDL secretion increased during the maturation of monocytes to macrophages, and was not modified by 17beta-estradiol. In contrast, loading the cells with cholesterol by incubation in the presence of acetylated or oxidized LDL produced an increase in the levels of apoE secreted by both estrogen-treated and control macrophages. The activity of LPL found in the cell medium, on the other hand, in lipid loaded cells tended to be increased only in estrogen treated macrophages, suggesting that the effects of estrogen on unloaded macrophages are different from those produced on lipid-loaded macrophages. On the whole, the present findings suggest that one of the mechanisms by which 17beta-estradiol acts to reduce cholesterol accumulation in macrophages is by increasing reverse cholesterol transport through the enhancement of the cholesteryl ester cycle, so that the generation of intracellular unesterified cholesterol for excretion from the cells is favored.

Effects of arachidonic and docosahexaenoic acids on secretion and degradation of bile salt-dependent lipase in AR4-2J cells

In this study we demonstrated that two polyunsaturated fatty acids, arachidonic acid (AA, n–6) and docosahexaenoic acid (DHA, n–3), modulate the secretion of bile salt-dependent lipase (BSDL) by pancreatic AR4-2J cells. The effects of AA and DHA were also compared with that of the monounsaturated fatty acid, oleic acid (OA). Our results showed that the chronic treatment of cells with AA or DHA, that did not affect the biosynthesis rate of BSDL, similarly decreased the amount of secreted BSDL and perturbed the intracellular partitioning of the enzyme, whereas OA had no effect. Particularly, AA and DHA induced the retention of the enzyme in microsomes and lowered its content in the cell cytosol. We have further shown that AA treatment decreased the ubiquitination of the protein, and consequently diminished its export toward the cytosol, a result that might explain the retention of BSDL in microsomes and correlated with membrane phospholipids alteration. The retained protein was further degraded by a nonproteasomal pathway that likely involves ATP-dependent endoplasmic reticulum proteases. These findings concerning the regulation of the pancreatic BSDL secretion by two polyunsaturated acids, AA and DHA, might be of physiological importance in the plasmatic and cellular cholesterol homeostasis. —Le Petit-Thévenin, J., N. Bruneau, A. Nganga, D. Lombardo, and A. Vérine. Effects of arachidonic and docosahexaenoic acids on secretion and degradation of bile salt-dependent lipase in AR4-2J cells.

Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae.

The role of the prosequence of Rhizopus oryzae lipase (ROL) with a preprosequence was analyzed by an expression system using Saccharomyces cerevisiae. When the mature portion of ROL (mROL) fused to the pre-alpha-factor leader sequence was expressed, secretion of active mROL was not observed. However, when mROL was synthesized together with the prosequence in trans (individually and coincidentally), secretion of active mROL was observed. The results indicate that the prosequence of ROL helped correct folding of mROL and its subsequent secretion from the yeast cells, and that physical linkage (cis) of the prosequence to the mature region was not prerequisite. From the expression of the ROL mutants with deletions at the N-terminal end of the prosequence together with mROL in trans, the residues from 20 to 37 in the prosequence were essential for the secretion, and those from 38 to 57 were essential for the formation of the active ROL and might play a role as an intramolecular chaperone. The results using the fragment of the prosequence confirmed that these residues (20-57) were significant for in vivo folding and secretion of active mROL.

Pancreatic enzyme replacement therapy.

Malabsorption due to severe pancreatic exocrine insufficiency is one of the most important late features of chronic pancreatitis. Generally, steatorrhea is more severe and occurs several years prior to malabsorption of other nutrients because synthesis and secretion of lipase are impaired more rapidly, its intraluminal survival is shorter, and the lack of pancreatic lipase activity is not compensated for by nonpancreatic mechanisms. Patients suffer not only from nutritional deficiencies but also from increased nutrient delivery to distal intestinal sites, causing symptoms by profound alteration of upper gastrointestinal secretory and motor functions. Adequate nutrient absorption requires delivery of sufficient enzymatic activity into the duodenal lumen simultaneously with meal nutrients. The following recommendations are based on modern therapeutic concepts: 25,000 to 40,000 units of lipase per meal using pH-sensitive pancreatin microspheres, with dosage increases, compliance checks, and differential diagnosis in case of treatment failure. Still, in most patients, lipid digestion cannot be completely normalized by current standard therapy, and future developments are needed to optimize treatment.

N-acyl-L-homoserine lactone-mediated regulation of the lip secretion system in Serratia liquefaciens MG1.

The analysis of Serratia liquefaciens MG1 'luxAB insertion mutants that are responsive to N-butanoyl-L-homoserine lactone revealed that expression of lipB is controlled by the swr quorum-sensing system. LipB is part of the Lip exporter, a type I secretion system, which is responsible for the secretion of extracellular lipase, metalloprotease, and S-layer protein.

Impaired pancreatic secretion in severely malnourished patients is a consequence of primary pancreatic dysfunction

Severe undernutrition has been associated with reduced secretions of gastric acid and pancreatic enzymes. This may be the result of an impaired gut mucosal response to food and primary gastric parietal and pancreatic acinar cell secretory dysfunction as a consequence of the poor nutritional state. To investigate the relative contributions of these factors, severely undernourished patients underwent enteral-meal–stimulated (ES; n = 7) or intravenous hormone (pentagastrin and cholecystokinin-8)–stimulated (HS; n = 12) gastric acid and pancreatic enzyme secretion before and after a period of nutritional support. Results were evaluated in comparison with normal healthy control subjects (ES = 7, HS = 10). In the control subjects, enteral-meal and cholecystokinin-8 stimulation resulted in similar outputs of the pancreatic enzymes amylase (2213 versus 2305 U/h), lipase (84.93 versus 118.6 U/h), and trypsin (498.9 versus 341.4 U/h), whereas acid output was significantly lower in the ES group (10.90 versus 25.53 mEq/h; P < 0.01). Compared with controls, malnourished groups had significantly reduced secretions of amylase (ES = 870.1 U/h, HS = 686.5 U/h; P < 0.02), lipase (ES = 30.68 U/h, HS = 25.96 U/h; P < 0.02), and trypsin (ES = 175.6 U/h, HS = 109.3 U/h; P < 0.01). The response to enteral-meal or CCK-8 stimulation was comparable. Gastric acid was similarly reduced in the undernourished patients (ES = 4.39 mEq/h, HS = 5.04 mEq/h; P < 0.01). After refeeding, secretion of amylase (ES = 2351 U/h, HS = 2228 U/h) and lipase (ES = 58.83 U/h, HS = 84.91 U/h) improved to levels not significantly different from controls, whereas trypsin (ES = 226.4 U/h, HS = 213.1 U/h; P < 0.03) and acid secretion (ES = 3.52 mEq/h, HS = 11.85 mEq/h; P < 0.01) remained significantly impaired. Severe undernutrition was associated with primary gastric parietal and pancreatic acinar cell dysfunction, which, at least in the case of pancreatic enzymes, appeared to be the determining factor controlling secretion in these patients.

Impairment of bile salt-dependent lipase secretion in AR4-2J rat pancreatic cells induces its degradation by the proteasome

Bile salt-dependent lipase (BSDL, EC 3.1.1.13) is a lipolytic enzyme normally secreted by the pancreatic acinar cell. Co- and post-translational modifications, such as N- and O-linked glycosylation, regulate the secretion of this enzyme; therefore it was of first importance to determine the behaviour of BSDL under conditions that impaired its secretion. Using AR4-2J pancreatic cells as model, we showed, particularly when BSDL secretion is impaired, that proteasome inhibitors increased the amount of intracellular BSDL, suggesting that the proteasome is involved in the degradation of this protein. This was strengthened by the detection of ubiquitinated BSDL and of degradation product. Our results suggested that both ubiquitination and degradation of the enzyme occurred at the level of the cytosolic side of microsome membranes. ATP hydrolysis appears essential in ubiquitinated BSDL association with membranes and degradation. Furthermore, under normal secretory conditions, we have shown that a fraction of ubiquitinated BSDL is neither O-glycosylated nor N-glycosylated, suggesting that the N-glycosylation-deficient proteasome substrate does not reach the Golgi and could be degraded by the ER-associated degradation machinery. However, another fraction of ubiquitinated BSDL that is deficient in O-glycosylation, carries out endoglycosidase H-insensitive N-linked glycans, meaning that a second system, that detects abnormal BSDL molecules, could also operate at the level of the Golgi compartment. Consequently, it appears that impairment of BSDL secretion consecutive to secretion inhibition or to a deficient glycosylation leads to the proteasome-ubiquitin-dependent degradation of the protein. Therefore, this pathway is part of the quality control involved in BSDL secretion.

Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase.

Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed in Escherichia coli, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P. aeruginosa. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.

Production of native and recombinant lipases by Candida rugosa: a review.

The yeast Candida rugosa produces multiple lipase isoenzymes sharing high sequence homology but with some differences in their catalytic properties. The regulation of C. rugosa lipase (CRL) synthesis and secretion in C. rugosa obeys a complex pattern. Fermentation processes for both wild-type and mutant C. rugosa strains are available for lipase production. Native CRL preparations have been extensively used for biotransformations. However, their inherent mixture of isoforms with variable profiles complicates interpretation and brings into question the reproducibility achieved between preparations. Although heterologous CRLs gene expression had been hampered owing to a nonuniversal codon usage, recent advances have made heterologous CRLs available. This will expand and improve the industrial utility of CRLs even further. The purpose of this review is to provide a summary of the recent advances on the production of native and recombinant lipases by C. rugosa.

Phenotypic characterization of Trichophyton rubrum isolates from two geographic locations in Brazil.

To characterize possible Trichophyton rubrum phenotypes, which circulate in two Brazilian localities, we tested 53 isolates of this dermatophyte for their ability to assimilate several carbon sources, for keratinase, proteinase, phospholipase, lipase and desoxiribonuclease (DNase) secretions, and for their susceptibility to the antifungals fluconazole, ketoconazole and itraconazole. For each method, the isolates were submitted to similarity analysis and the methods were evaluated for their discriminatory indexes. None of the isolates were capable of assimilating arabinose, dulcitol, lactose, melibiose, ribose and xylose, while all of the isolates assimilated maltose, sucrose and sorbitol. However, adonitol, cellobiose, dextrin, erythritol, fructose, galactose, inulin, mannitol, mannose, raffinose, rhamnose and trehalose were assimilated by some isolates but not by others. All isolates secreted keratinase and DNase, while none secreted phospholipase. Proteinase and lipase were secreted only by some isolates. All but four isolates were resistant to fluconazole, most of them were sensitive to ketoconazole and all were sensitive to itraconazole. Carbohydrate assimilation was the method that presented the highest discriminatory index, and also the method that displayed the largest number of biotypes. Taken together, these data suggest that significant phenotypic variations exist among T. rubrum isolates. They seem to occur independently from their geographic origins.

Isolation and Analysis of Lipase-Overproducing Mutants of Serratia marcescens.

We have isolated a lipase-overproducing mutant, GE14, from Serratia marcescens 8000 after three rounds of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene (lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the lipA gene without an amino acid substitution. Promoter analysis using lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the S. marcescens lipase.

Isolation and analysis of lipase-overproducing mutants of Serratia marcescens

We have isolated a lipase-overproducing mutant, GE14, from Serratia marcescens 8000 after three rounds of N-methyl- N′-nitro- N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene ( lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the lipA gene without an amino acid substitution. Promoter analysis using lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the S. marcescens lipase.

Effects of Lansoprazole on Human Gastric Lipase Secretion and Intragastric Lipolysis in Healthy Human Volunteers

Background: Lansoprazole is a potent proton-pump inhibitor (PPI) of parietal cells, which reduces the secretion of gastric acid. Although human gastric lipase (HGL) is produced only by the chief cells of the stomach, the possibility that interactions may occur between lansoprazole and HGL has never been addressed so far in humans. The aim of this study was therefore to quantify the effects of lansoprazole on HGL secretion and intragastric lipolysis during the ingestion of test meals by healthy human volunteers. Methods: Six healthy volunteers were intubated twice with a gastric and a duodenal tube, before ingesting a standard liquid test meal alone (–PPI experiments) and after 7 days of lansoprazole treatment (+PPI experiments). The HGL concentration was assessed in gastric and duodenal samples by measuring the lipase activity using a pH-stat, and the lipolysis products were quantified by performing thin layer chromatography. The level of intragastric lipolysis was defined as the percentage acyl chains released from the meal triglycerides. The pyloric outputs of HGL and lipolysis products were calculated, based on the use of a non-absorbable marker added to the meal. Results: The pH of the gastric contents was significantly higher in the +PPI experiments than in the –PPI experiments (p < 0.05), since mean values of 4.3 ± 2.5 and 2.2 ± 1.6, respectively, were recorded at the end of the gastric emptying of the meal. The HGL concentrations recorded during the meal were found to be higher in the experiments with lansoprazole (p < 0.05) than in those without lansopra- zole, but the HGL secretion levels (–PPI: 15.4 ± 8.0 mg; +PPI: 19.0 ± 7.4 mg) and the intragastric lipolysis (–PPI: 24.0 ± 8.0%; +PPI: 23.6 ± 6.8%) were not significantly affected by lansoprazole (p > 0.05 in both cases). Conclusion: Lansoprazole affected neither the HGL secretion nor the intragastric lipolysis levels, although an increase was observed in the intragastric pH at the end of the gastric emptying of the meal. The HGL concentration increased, however, due to the decrease in the acid secretion process, resulting in less diluted gastric contents.

Parallel secretion of pancreatic phospholipase A(2), phospholipase A(1), lipase, and colipase in children with exocrine pancreatic dysfunction.

The cosecretion of pancreatic lipase and colipase are important in normal fat digestion. As adsorption of phosphatidylcholine to the lipid substrate interferes with lipase activity, hydrolysis to lysophosphatidylcholine with subsequent desorption is also essential for fat digestion. There are some data regarding the secretion of pancreatic phospholipases in normal adults but none in children or patients with pancreatic disease. In the present study, we aimed a) to develop an accurate fast assay method to measure phospholipase A(2) and b) to determine the secretion rate of pancreatic phospholipase A(2) and whether it is cosecreted with lipase and colipase in children with exocrine pancreatic dysfunction. Nine male patients aged 0.5 to 16 y (seven with cystic fibrosis, two with malabsorption) underwent pancreatic stimulation tests. Their colipase and lipase secretion rates were measured by titrimetric methods and phospholipase A(2) and A(1) by phosphorus magnetic resonance spectroscopy ((31)P NMR). It was found that the phospholipases, colipase, and lipase were absent in the two patients with pancreatic insufficiency. In patients with normal absorption, there were marked inter-and intrasubject variations of lipase, colipase, and phospholipase secretion rates that were consistent with the degree of exocrine pancreatic dysfunction. However, in the three 20-min stimulation periods of the pancreatic function test, pancreatic phospholipase is cosecreted with lipase and colipase, and average colipase and phospholipase A(2) secretion rates follow a similar or parallel pattern. These findings are consistent with the important role of pancreatic phospholipases in intestinal phospholipid hydrolysis leading to the desorption of phospholipids from the lipid substrate and enhancing lipid hydrolysis and phospholipid absorption.

Diabetic state induces lipid loading and altered expression and secretion of lipoprotein lipase in human monocyte-derived macrophages

Non-insulin-dependent diabetes mellitus (NIDDM) is frequently associated with macroangiopathies and coronary heart diseases. Lipoprotein lipase (LPL), an enzyme known to undergo significant functional alterations in diabetic state, is also a potential atherogenic protein. Since, to the best of our knowledge, there are no data concerning LPL secreted by macrophages of NIDDM patients we conducted a study to assess the expression and activity of LPL secreted by monocyte-derived macrophages from NIDDM patients with cardiovascular complications versus cardiovascular patients without diabetes (controls). Isolated cells from NIDDM patients, after 7 days in culture in the presence of 20% autologous serum, readily exhibit a foam cell phenotype, in contrast to the cells from controls. Macrophages were mainly loaded with triglycerides, whose cellular amount was well correlated to triglyceridemia of NIDDM subjects. Concomitantly, macrophages from NIDDM patients displayed a ∼six-fold decrease of mRNA expression and a ∼two-fold reduction of the activity of secreted LPL, as compared to control cells. These data suggest that in complicated diabetic state, macrophage loading leading to foam cell formation is accelerated, at least in part, due to a diminished expression and activity of LPL. These observations add and extend the data that may explain the occurrence of accelerated atherogenesis and of the atherosclerotic complications associated with diabetes.

Fats Infused Intraduodenally Affect the Postprandial Secretion of the Exocrine Pancreas and the Plasma Concentration of Cholecystokinin but Not of Peptide YY in Growing Pigs

In pigs, the spontaneous secretion of the exocrine pancreas and the release of cholecystokinin (CCK) and peptide YY (PYY) after intraduodenal infusion of fully saturated synthetic fats differing in chain length was studied. Growing pigs (n = 6) were prepared with pancreatic duct catheters, duodenal T-cannulas and catheters placed in the jugular vein. The pigs were fed 2 g/100 g body twice daily. Beginning with the morning feeding, a medium-chain triglyceride (MCT: glycerol tricaprylate), a long-chain triglyceride (LCT: glycerol tristearate) or saline was infused at a rate of 0.1 g/100 g body. Pancreatic juice was collected, beginning 1 h preprandially until 3 h postprandially. Blood samples were obtained 15 min preprandially and 15, 45, 90 and 150 min postprandially. The infusion of MCT evoked a change in the trend of the curve for the volume of secretion of pancreatic juice, lipase and colipase concentrations and outputs. The trend of the curve did not change over time for CCK and PYY. Differences between the trends of the curves for the saline and MCT treatment were observed for volume of secretion, protein output, lipase content and output, trypsin and colipase output. Differences in the trends of the curves between MCT and LCT were obtained for the outputs of protein, lipase and colipase. Plasma CCK levels were lower as a result of the MCT treatment compared with the saline and LCT treatments. The results suggest an immediate, distinguished response of the porcine exocrine pancreas to fats differing in chain length.

The specific activities of human digestive lipases measured from the in vivo and in vitro lipolysis of test meals

The lipolytic potential of digestive lipases in vivo has always been deduced so far from their in vitro activities under nonphysiologic conditions. In the present study, the specific activities of human gastric lipase (HGL) and pancreatic lipase (HPL) were measured on dietary triglycerides (TGs) during test meal lipolysis. Healthy human volunteers ingested a liquid or solid meal. The specific activities of HGL and HPL were estimated from the lipase and free fatty acid (FFA) outputs at the postpyloric and duodenal levels, respectively. Based on the in vivo data, lipolysis was also performed in vitro by mixing the meal either with gastric juice and subsequently with pancreatic juice and bile or with purified HGL and HPL. FFAs were measured by thin-layer chromatography, and the specific activities of HGL and HPL were expressed as micromoles of FFA per minute per milligram of lipase. In vitro, the specific activities on the liquid meal TGs were 32 (gastric juice) and 34 (pure lipase) micromol x min(-1) x mg(-1) with HGL and 47 (pancreatic juice) and 43 (pure lipase) micromol x min(-1). mg(-1) with HPL. The specific activities on the solid meal TGs were 33 (gastric juice) and 32 (pure lipase) micromol x min(-1) x mg(-1) with HGL and 12 (pancreatic juice) and 15 (pure lipase) micromol x min(-1) x mg(-1) with HPL. The in vivo values obtained were in the same range. The secretory lipase outputs were 21.6+/-14.5 mg HGL and 253.5+/-95.5 mg HPL with the liquid test meal and 15.2+/-5.1 mg HGL and 202.9+/-96.1 mg HPL with the solid test meal. The specific activities of HGL and HPL on meal TGs were much lower than those measured in vitro under optimized assay conditions (1300-8000). However, these low specific activities are enough for the meal TGs to be completely lipolysed, given the amounts of HGL and HPL secreted during a meal.

Secretion of hepatic lipase by perfused liver and isolated hepatocytes

Hepatic lipase is found in liver and in adrenal glands and ovaries. Because in adult rats, neither adrenals nor ovaries synthesize this enzyme, it is assumed that the liver is the origin of their hepatic lipase. Our aim was to study the secretion of hepatic lipase by the liver. We observed that plasma of both fed and fasted rats contained hepatic lipase activity. This activity was significantly correlated with that in the liver. Isolated livers, perfused with heparin-free medium, secreted fully active hepatic lipase to the perfusate. The addition of heparin resulted in a rapid and larger release of hepatic lipase to the perfusate. In isolated hepatocytes, heparin did not affect the secretion of hepatic lipase mass, although it increased the stability of the enzyme activity. To study the degradation of hepatic lipase by hepatocytes, protein synthesis was blocked with cycloheximide, and both secreted and intracellular hepatic lipases were analyzed by Western blotting. We observed that the amount of hepatic lipase secreted equaled the decrease of intracellular mass. The total mass of the enzyme (inside and outside the cells) remained constant, at least for 90 min. In the next experiment, 0.7 nM 125I-hepatic lipase was added to hepatocyte suspensions, and the appearance of trichloracetic acid-soluble products was analyzed. Only 12% of the radioactivity added was associated with the cells after 90 min of incubation, and less than 2% of the hepatic lipase added was degraded. Although the association was decreased in the presence of heparin, the amount of 125I-hepatic lipase degraded was not affected. Taking all these results into account, we propose a model for the continuous secretion of hepatic lipase by the liver.

The use of a low fat diet in the treatment of acute pancreatitis

Purpose: A low fat diet hypothetically stimulates less pancreatic lipase secretion than a diet with a higher fat content, yet very little data exists on whether the use of a low fat diet during the oral refeeding of patients with acute pancreatitis improves recovery from the disease.

Effect of different carbon sources on lipase production by Candida rugosa.

Different carbon sources affecting growth and lipase production in Candida rugosa were studied by using batch cultures on defined medium. Carbohydrates and acids non-related to fats did not induce lipase production. The highest yields of enzyme were obtained with lipids or fatty acids as carbon sources. Tween 80 stimulated lipase biosynthesis and secretion outside the cell. Combinations of two types of substrates, carbohydrates and fatty acids, did not improve lipase production, and in some cases, their consumption was produced in a sequential pattern. Glucose presented a repressing effect on lipase production. Moreover, glucose was found to be effective in stimulating lipase secretion by cells with a high level of cell-bound lipase activity because of their previous growth in oleic acid.