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.

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.

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.

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.

Estrogen hormones and lipid metabolism. Effect of ethynyl-estradiol on liver lipases.

Lipase activities assayed in liver subcellular fractions from rats given ethynyl-estradiol are lowered, while triglycerides increase and cholesterol decreases in blood. Triester lipase in microsomes is the most altered activity. Such enzymatic changes might play a role in the development of lipid abnormalities in blood of women taking estrogens as oral contraceptives.

Human erythrocyte monoester lipase: characterization and radiochemical assay of the cell-bound enzyme in normal subjects.

A monoester lipase (MEL) activity (EC 3.1.1.3) is described in human red blood cells (RBC). The lipase acts as a cell-bound enzyme and is able to exert its catalytic activity in vitro toward an exogenously added emulsified substrate. The enzyme activity, which appears to be confined to the cell membrane, is inhibited by Triton X-100. The MEL activity of human RBC is assayed using intact RBC as the enzyme source, with an emulsion of ethyl [3H]oleate (2 mM) as the substrate. The optimum pH for the reaction is 7.8 at 37 C. Lipolytic rates are monitored by quantitation of the amount of [3H]oleic acid released during 20 min of incubation after extraction by means of a liquid-liquid partition system. Suspensions of purified RBC obtained from 161 healthy adult subjects had a MEL activity of 1022 +/- 134 microunits/10(12) RBC (mean +/- SD), with a normal range (+/- 2 SD) between 754-1290 microunits. The individual activity values varied from 733-1490 microunits. The median of the 161 subjects was 1010 microunits/10(12) RB. There was no significant difference between the mean activities of RBC samples from men and women. MEL activity in RBC from the cord blood of 16 normal infants was found to be 43% higher than that in adults, with an average activity of 1458 +/- 174 microunits (mean +/- SD).

Increased monoester lipase activity in red blood cells during hyperthyroidism.

Human red blood cells (RBC) contain a monoester lipase (MEL) activity which is tightly associated with the cell membrane and has its active site externally oriented, as inferred from the ability of the intact cell to hydrolyse an exogenously added lipid substrate. Membrane-bound MEL activity was measured by a radiochemical assay in intact RBC from 29 untreated hyperthyroid patients. The mean (+/- S.D.) MEL level was higher (P less than 0.01) in these patients (1220 +/- 212 mu./10(12) RBC) than in the control group (1010 +/- 120 mu./10(12) RBC). There was no difference between men and women. The increase in MEL values was associated with significantly (P less than 0.001) decreased values of mean cellular volume and mean cellular haemoglobin content. A continued study of 13 patients, who became euthyroid with treatment, showed a normalization of the MEL values in RBC. The increased lipolytic potency of RBC represents a new characteristic of hyperthyroid patients. Further exploration of the possible diagnostic or prognostic implications of this enzymatic change seems warranted.

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.

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.

Evolution of plasma levels of apolipoprotein B, cholesterol and triglycerides in women during long-term oral contraception.

Apolipoprotein B (apo B) was measured by a sensitive and specific double antibody radioimmunoassay in plasma from 15 normal women and 47 women taking oral contraceptives. The plasma apo B concentration in normal women was 0.90 +/- 0.10 g/l (mean +/- S.D.) whereas the overall value for all women taking contraceptives was 0.69 +/- 0.19 g/l. The maximum decrease (p less than 0.001) occurred in women investigated within three months after the beginning of the steroid intake, whose mean apo B value was 0.60 +/- 0.12 g/l (n = 12); decreases found after 3--24 months of contraception were of lesser magnitude. Apo B decreases were associated with significant decreases (p less than 0.01) in total plasma cholesterol, although the latter regained normal values after one year of contraception. Apo B and cholesterol values were in the normal range in women investigated after two years of steroid intake. An inverse trend was observed for plasma triglycerides, which showed a mean increase of 29% (p less than 0.01) after three months of steroid intake, and then stayed elevated without significant change with time.

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.

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.

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.

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.

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.

[Plasma DHA, DHAS and cortisol levels after injection of beta 1-24 corticotrophin beta 1-24 ACTH in normal adult women (author's transl)]. / Modifications de concentrations de déhydroepiandrostérone, déhydroepiandrostérone sulfate et cortisol dans le plasma de la femme adulte normale après stimulation par la corticotrophine.

Plasma DHA, DHAS and cortisol levels were measured by R.I.A. before and after intravenous and intramuscular injection of beta 1-24 ACTH. In 7 normal adult women, plasma cortisol and DHA levels rose sharply in response to beta 1-24 ACTH (0,25 mg) i. v injection; in opposition, plasma DHAS levels did not change significantly 30 and 120 minutes after the injection. In 9 normal adults women, the i. m injection of Zn beta 1-24 ACTH (1 mg) caused a significant increase in both plasma cortisol, DHA and DHAS levels 8 and 24 hours after the injection.