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.

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.

Phosphorylation of the oncofetal variant of the human bile salt-dependent lipase. identification of phosphorylation site and relation with secretion process.

In this paper, we report, for the first time, the localization of the phosphorylation site of the fetoacinar pancreatic protein (FAPP), which is an oncofetal variant of the pancreatic bile salt-dependent lipase. Using Chinese hamster ovary (CHO) cells transfected with the cDNA encoding FAPP, we radiolabeled the enzyme with (32)P, and then the protein was purified by affinity chromatography on cholate-immobilized Sepharose column and submitted to a CNBr hydrolysis. Analysis of peptides by high pressure liquid chromatography, associated with the radioactivity profile, revealed that the phosphorylation site is located at threonine 340. Site-specific mutagenesis experiments, in which the threonine was replaced by an alanine residue, were used to invalidate the phosphorylation of FAPP and to study the influence of the modification on the activity and secretion of the enzyme. These studies showed that CHO cells, transfected with the mutated cDNA of FAPP, kept all of their ability to synthesize the protein, but the loss of the phosphorylation motif prevented the release of the protein in the extracellular compartment. However, the mutated enzyme, which was sequestrated in the transfected CHO cells, remains active on bile salt-dependent lipase substrates.

Phosphorylation of the rat pancreatic bile-salt-dependent lipase by casein kinase II is essential for secretion.

Bile-salt-dependent lipase (BSDL, EC 3.1.1.-) is an enzyme expressed by the pancreatic acinar cells and secreted as a component of the pancreatic juice of all examined species. During its secretion route BSDL is associated with intracellular membranes. This association allows the complete glycosylation of the enzyme or participates in the inhibition of the enzyme activity, which can deleterious for the acinar pancreatic cell. Thereafter, the human BSDL is phosphorylated by a serine/threonine protein kinase and released from intracellular membranes. In the present study, we show that the rat pancreatic BSDL, expressed by AR4-2J cells used as a model, is phosphorylated by a protein kinase that is insensitive to inhibitors of protein kinases A, C or G and that the phosphorylation process is favoured by okadaic acid (an inhibitor of protein phosphatases 1 and 2A). However, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), which is a specific inhibitor of casein kinase II, abolishes the phosphorylation in vitro of BSDL within micro- somes of AR4-2J pancreatic cells. We showed further that the alpha-subunit of casein kinase II co-locates with BSDL within the lumenal compartment of the Golgi. Genistein, which perturbs the trans-Golgi network, also inhibits the phosphorylation of BSDL, suggesting that this post-translational modification of BSDL probably occurred within this cell compartment. The inhibition of the phosphorylation of BSDL by DRB also decreases the rate at which the enzyme is secreted. Under the same conditions, the rate of alpha-amylase secretion was not modified. These data strongly suggest that phosphorylation is a post-translational event, which appears to be essential for the secretion of BSDL.

Immunodetection and molecular cloning of a bile-salt-dependent lipase isoform in HepG2 cells.

In this article, we report the nucleotide sequence of the cDNA encoding an isoform of bile-salt-dependent lipase (BSDL) expressed by human hepatoma cells. The BSDL is a 100-kDa glycoprotein normally expressed by the human pancreas. Using a polyclonal antibody raised against an internal peptide located between Ile(327) and Glu(350) of the human pancreatic BSDL, we have immunodetected an isoform of human pancreatic BSDL, with an apparent molecular mass of about 62 kDa. This isoform of BSDL was mainly associated with the cytosol of a human hepatoma cell line (HepG2), the remaining protein being found in the microsome fraction. In addition, esterolytic activity on p-nitrophenyl hexanoate measured in microsomes and cytosol appeared very low and was weakly stimulated by bile salts, such as taurocholate. In contrast to human pancreatic BSDL, which is secreted as a component of pancreatic juice, this isoform appeared to be retained in the HepG2 cells. Reverse transcription, followed by PCR and amplification, performed on RNA extracted from HepG2 cells using specific primers hybridizing to the sequence coding for the entire normal human pancreatic BSDL, allowed us to amplify a 1. 7-kb transcript that appeared to be 0.5 kb shorter than the transcript of the pancreatic enzyme (2.2 kb). From the sequence of the transcript thus obtained, a protein with a molecular mass of 62 kDa might be predicted, which is in good agreement with the size of the isoform of BSDL immunodetected in HepG2 cells. The N-terminal amino-acid sequence, deduced from the 1.7-kb transcript sequence, matched that of the pancreatic BSDL. However, the C-terminal domain appeared truncated, bearing only a single mucin-like sequence compared with sixteen for the human pancreatic BSDL. The actual intracellular function of this human BSDL hepatoma isoform remains to be elucidated.

Effects of ethanol on the expression and secretion of bile salt-dependent lipase by pancreatic AR4-2J cells.

The mechanisms by which ethanol administration alters pancreatic function are unknown. We have evaluated the effects of chronic ethanol treatment on secretion of a digestive enzyme: the bile salt-dependent lipase (BSDL), by the rat pancreatic cell line AR4-2J (as a model). We report that ethanol (50-300 mM) in culture medium induced a rise, in secreted and intracellular BSDL, that was a function of the duration of treatment and of the ethanol concentration. This effect was not abolished by pyrazole, which suggests a direct effect of ethanol. We have further established that the increase of BSDL activity was due to an enhanced biosynthesis of the enzyme consecutive to a major steady-state level of mRNA encoding BSDL. Also, the subcellular localization showed a specific accumulation of BSDL in the cytosolic fraction of cells chronically treated with ethanol. Given the enzymatic properties of BSDL, all these data could have some physiological consequences regarding the digestive function, plasma lipid metabolism and intracellular cholesterol homeostasis.

An intracellular role for pancreatic bile salt-dependent lipase: evidence for modification of lipid turnover in transfected CHO cells.

Pancreatic bile salt-dependent lipase (BSDL) hydrolyzes cholesteryl esters, triglycerides and phospholipids. BSDL is also capable of transferring free fatty acid to cholesterol. BSDL has been detected in many cells including fetal and tumor cells, hepatocytes, macrophages and eosinophils and in tissues such as adrenal glands and testes. The enzyme may be secreted or located within subcellular compartments such as the endoplasmic reticulum or the cytosol. Although the role of the secreted enzyme is well documented, that of the intracellular form(s) is still hypothetical. In the present study, we addressed the effects of BSDL on cell lipid metabolism. For that purpose, the cDNA of rat BSDL was transfected into CHO K1 cells (CHO K1-BSDL clone) which were then loaded with [3H]oleic acid. The results demonstrate that the transfected BSDL is secreted; in spite of that, a large fraction of catalytically active BSDL is found in cell lysate. The lipid metabolism of transfected cells is affected and BSDL induces an enhanced incorporation of [3H]oleic acid in cholesteryl esters whereas fatty acid incorporation in phosphatidylcholine is decreased. These effects were particularly important in the cytosol of transfected cells where transfected BSDL preferentially locates. These data suggested that BSDL could be implicated in the cycle of the cellular homeostasis of cholesterol which is particularly affected in tumoral cells leading to cholesteryl ester storage within cytosolic lipid droplets.

Pancreatic lipase-related protein type I: a specialized lipase or an inactive enzyme.

The existence of pancreatic lipase-related protein 1 (PLRP1) in vertebrates has been postulated based on the screening of pancreatic cDNA libraries from different species. In this paper, we report the presence of variable amounts of PLRP1 relative to colipase-dependent lipase (PL) in adults from several species. Only a very low lipase activity could be detected for native or recombinant PLRP1 using a large variety of substrates and conditions. Interestingly, this activity is dependent on the presence of bile salts and colipase and PLRP1 is shown to possess the same affinity as PL for colipase. Modelling investigations revealed some interesting differences between PLRP1 and PL, notably concerning substitutions in the C-terminal domain which might affect the bending motion of this domain relative to the N-terminal domain in PLRP1. The potential impact of these differences on the lack of lipase activity of PLRP1 was investigated using chimeric proteins designed by C-terminal domain exchange between dog PLRP1 and horse PL. Analysis of the catalytic properties of the chimera clearly indicated that the C-terminal domain exchange neither inactivates the horse enzyme nor results in an active dog PLRP1. From these findings, it can be concluded that the PLRP1 C-terminal domain is fully functional with respect to colipase binding. The lack of lipase activity or the still undetermined function of PLRP1 is likely to result mainly from particular features of the N-terminal domain.

Pancreatic bile salt-dependent lipase activity in serum of normolipidemic patients.

Bile salt-dependent lipase (BSDL, E.C. 3.1.1.-) is a digestive enzyme secreted by the pancreatic acinar cell. Once in the duodenum, the enzyme, upon activation by primary bile salts, hydrolyzes dietary lipid esters such as cholesteryl esters and lipid-soluble vitamin esters. This enzyme is partially transferred from the duodenum or pancreas to the circulation where it has been postulated to exert a systemic action on atheroma-generating oxidized-low density lipoprotein (LDL). In the present study, sera from 40 healthy normolipidemic volunteers were used to investigate the possible linkage between circulating BSDL, lipids, and lipoproteins. We showed, firstly, that pancreatic-like BSDL activity can be detected in these serums. Secondly, BSDL activity increased significantly with the level of LDL-cholesterol and was also positively linked to the serum concentration of Apo B100 and Apo A-I. Thirdly, we also established that BSDL was associated with LDL, in part by a specific interaction with Apo B100, while no interaction was found with Apo A-I. No linkage with other recorded parameters (triglycerides, phospholipids, and high density lipoprotein-cholesterol) was detected. Because an increase in LDL-cholesterol represents an important risk factor for atheroma, the concomitant increase in BSDL, which can metabolize atherogenic LDL, suggests for the first time that this circulating enzyme may exert a positive effect against atherosclerosis.

Selective modulation of membrane sphingomyelin fatty acid turnover by nigericin. A study in the rat reticulocyte.

Exposure of rat reticulocytes to Nigericin produced a selective modulation of fatty acid incorporation into sphingomyelin (SM) of the cell membrane, via changes in SM acylation kinetics. At physiological fatty acid concentration, Nigericin accelerated 8-fold SM acylation by decreasing the apparent K(m) for oleate from 14.7 microM to 2.0 microM. The response was diminished in high K(+)-containing media, suggesting an effect of Nigericin as K+ transporter. This constitutes a novel piece of evidence for the important role of ions in SM metabolism.

Purification and properties of a monoacylglycerol lipase in human erythrocytes.

A membrane-bound monoacylglycerol lipase (MAGL) activity, previously demonstrated in intact human erythrocytes [Boyer, Somma, Vérine, L'Hôte, Finidori, Merger and Arnaud (1981) J. Clin. Endocrinol. Metab. 53, 143-148], has now been purified to apparent homogeneity by a five-step procedure involving solubilization in CHAPS and sequential chromatographies on Sephacryl S-400, DEAE-Trisacryl, Zn(2+)-chelating Sepharose and Superose 12 columns. The purified protein has a molecular mass of 68 +/- 2 kDa, as determined by SDS/PAGE and gel filtration, suggesting that the enzyme behaves as a monomer. The concentration-dependence of MAGL activity with monooleoylglycerol, the preferred substrate showed kinetics typical of an interfacial lipolytic enzyme displaying optimal activity on emulsified substrate particles; apparent Km values were 0.27 mM and 0.49 mM for the sn-1(3)- and sn-2-isomers respectively. MAGL had no, or negligible, activity towards tri-oleoylglycerol, di-oleoylglycerol, oleoylcholesterol, oleoyl-CoA and phosphatidylcholine; it was inhibited by di-isopropylfluorophosphate, PMSF and diethyl p-nitrophenyl phosphate, suggesting that MAGL is a serine hydrolase. MAGL activity was not modified by bile salt or apolipoprotein C-II, whereas a dose-dependent inhibition was observed with apolipoprotein A-I.

Differential in vitro effects of ethanol on glycerolipid acylation and biosynthesis in rat reticulocytes.

Earlier reports have shown that, in human and rat red blood cells (RBC), ethanol modulates acylation reaction sin several membrane glycerolipid components. Little is known, however, about the kinetics and the mechanisms involved in the acylation changes. In the present study, we show that short-term in vitro exposure of intact rat reticulocytes to ethanol differentially modifies within minutes the incorporation of [3H]oleic acid in glycerolipids. A concentration-dependent inhibition of acyl incorporation was measured in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). This effect did not involve inhibition of the corresponding acyltransferase activities and is likely to be due to ethanol-dependent decreases in phospholipase activities. In contrast, ethanol markedly stimulated [3H]oleic acid incorporation in phosphatidic acid (PA), diacylglycerol (DG) and, to a lesser extent, in triacylglycerol (TG). To determine the mechanisms of the latter increases, reticulocytes were pulsed with [14C]glycerol and assayed as a function of time for labeled biosynthetic precursors and products. We observed a very close correlation between time courses and amplitudes of the ethanol stimulation of acylation and biosynthesis reactions, suggesting that stimulation of acylation in PA, DG and TG is causally related at least partly to their increased biosynthesis. Further studies revealed that increases in glycerolipid acylation and biosynthesis in reticulocytes were: (a) readily reversible upon ethanol withdrawal; (b) detectable for clinically relevant concentration (50 mM) of ethanol; and (c) associated with concomitant increases in cell resistance to hemolysis. These changes may be relevant to the development of tolerance to ethanol.

Reversible inhibition by ethanol of Mg(2+)-dependent phosphatidate phosphohydrolase: an in vitro study in the rat reticulocyte.

By using a tracer method, we demonstrate that short-term in vitro exposure of intact rat reticulocytes to ethanol elicits a biphasic response of cell-bound Mg(2+)-dependent phosphatidate phosphohydrolase (PAP). An initial concentration-dependent (200-750 mM) activity decrease is rapidly (< 10 min) followed by reversal of the inhibition in the presence of ethanol, suggesting the development of a cell resistance to the inhibitory agent. Addition to the cell suspension of propranolol (100 microM), a known PAP inhibitor, does elicit PAP inhibition but unlike ethanol, inhibition is not followed by a return with time to control value. Ethanol-induced inhibition of cell-bound PAP was also demonstrated in cell-free extracts, where the Mg(2+)-dependent activity was decreased both in the particulate and soluble fractions. In the intact cells, the transient PAP inhibition occurs in concomitance with an overall increase in total glycerolipid biosynthesis, which is constant over 60-min incubation. We suggest that the biphasic mode of response to ethanol of Mg(2+)-dependent PAP activity may play a role in the mechanism of membrane adaptation to ethanol, and thereby to the pathogenesis of alcoholism.

Triacylglycerol in biomembranes.

A better knowledge of the biochemical and biophysical properties of cell membranes has revealed fundamental concepts concerning the regulation of cell functions by intrinsic components of the lipid matrix. Membrane lipids exhibit high chemical heterogeneity, with hundreds of distinct chemical species; studies of structure-function relationships have unraveled new roles for an increasing number of these lipids as determinants of membrane structure, anchors for membrane-associated proteins or signalling agents. Recent observations have confirmed triacylglycerol (TG) as a quantitatively minor intrinsic membrane component which seems to play a specific role in important metabolic events such as cell stimulation or transformation and metastatic processes. The rapid turnover of the acyl chains into TG of cell membranes suggests an active metabolism. In the plasma membrane, TG appears to be implicated in the generation of transient non-bilayer domains suspected to be associated with specific cellular events. This paper summarizes the current information on TG metabolism and focuses on the potential role of this neutral lipid species on the structure and function of cell membranes.

Norethindrone alters apolipoprotein E concentration and isoform expression in rat plasma high-density lipoproteins.

The administration of norethindrone, a synthetic progestin, to female rats caused marked decreases in the cholesterol and protein contents of high-density lipoproteins (HDL). A 84% decrease in the total apolipoprotein (apo) E of HDL was associated with a 80% decrease in the proportions of apoE isoforms E1 and E2, whereas E3 and E4 were unchanged. This appears to be the first demonstration of hormonally-induced modification of the apoE phenotype.

Effects of ethanol on phospholipid acylation in rat erythrocytes: a model for a biochemical approach to membrane adaptation.

Challenging intact erythrocytes from naive rats with ethanol resulted in dose-dependent decreases in rates of acylation of phosphatidylcholine and phosphatidylethanolamine. In erythrocytes from ethanol-treated animals, the responses were of lesser magnitude, indicating a lower sensitivity to ethanol. This relative resistance, typical of the state of tolerance, was not associated with increased baseline rates of acylation of PC and PE, nor with changes in fatty acid specificity of acylation reactions. Taken together, the data suggest that (1) intact rat erythrocytes represent a reliable and easily reproducible model for studying biochemical correlates of the adaptive response to ethanol; (2) phospholipid acylation reactions are implicated in the initial sensitivity and subsequent acquisition of tolerance to ethanol in membrane erythrocytes; (3) on the basis of the measured acylation reactions, rat erythrocytes appear to develop tolerance, but not dependence, to ethanol.

Effect of ethanol intake on lipoprotein lipase activity in rat heart.

We examined the effects of the in vivo administration of ethanol on lipolytic activities assayed in rat post-heparin heart effluents, that hydrolyse tri-, di- and monoacylglycerol. Properties of triacylglycerol lipase (TAGL) are typical of lipoprotein lipase (LPL) whereas diacylglycerol (DAGL) and monoacylglycerol (MAGL) lipase activities hydrolyse sequentially the products of LPL action. After 15 days of ethanol intake, TAGL, DAGL and MAGL activities in post-heparin heart effluents were decreased respectively by 25, 38 and 22%; after 30 days, the decreases amounted to 81, 79 and 71%. After 30 days, but not after 15 days, ethanol increased the levels of triacylglycerol in plasma. Ethanol intake concomitantly decreased TAGL and DAGL activities in post-heparin effluents and in heart tissue extracts, whereas MAGL activity was decreased only in the latter extracts. We conclude that ethanol intake causes a marked impairment in heart LPL and in two closely-related heparin-releasable activities, seemingly by altering the production of a catalytically active enzyme. A distinct heparin-unreleasable MAGL appears to exist in heart, that could be ethanol-insensitive. Overall, the results suggest that a LPL-related alteration in fatty acid supply could contribute to the toxicity of ethanol in heart.

Acute ethanol treatment induces a bimodal response of phospholipid acylation rates in rat red blood cells.

A single intraperitoneal injection of ethanol (4 g/kg) in rats elicited a bimodal response of acylation rates in phosphatidylcholine and phosphatidylethanolamine of intact red blood cells. Within an initial period, ethanol inhibited acylation rates. The inhibition then reversed, leading to increased values which persisted as long as ethanol was present in plasma. Acylation rates were not correlated to ethanol concentrations in plasma. We suggest that red cells first desensitize to, then overcompensate for the inhibitory effect of ethanol on acylation reactions. These adaptive changes may be one of the events mediating membrane tolerance to ethanol.

Lipoprotein lipase in rat heart--I. Characterization of tri-, di- and monoacylglycerol lipase activities in post-heparin effluents.

1. The lipolytic activities that sequentially hydrolyze tri-, di- and monoacylglycerol in rat post-heparin heart effluents were examined. 2. Properties of triacylglycerol lipase (TAGL) activity were typical of lipoprotein lipase. Diacylglycerol lipase (DAGL) behaved similarly to TAGL, suggesting that both activities refer to the same catalytic entity. 3. Differences, particularly in thermal stability, between TAGL and DAGL activities on one hand, and monoacylglycerol lipase (MAGL) activity on the other, may reflect different intrinsic molecular properties. 4. TAGL, DAGL and MAGL activities could not be separated by physical means and appeared to belong to a single unit at the same site on the capillary wall.