Does the neurotropic action of adrenocorticotrophic hormone involve a lipolytic step?

The stimulatory effect of adrenocorticotrophic hormone (ACTH)-related synthetic peptides on the hydrolysis of emulsified trioleoylglycerol by a rat brain lipase was studied. The ACTH effect was related to the net positive charge associated with the basic amino acid residues at position 15-18 in the ACTH sequence, as well as to the presence of the NH2-terminal amino acid residues at position 1-2. The ACTH effect on lipolysis was markedly reduced when lipids were partially removed from the enzyme preparation by extraction with chloroform/acetone. Full restoration of the stimulatory effect was obtained upon addition of phosphatidylcholine (2 mg/ml) to the lipolytic medium. Striking similarities between the structure-activity pattern for the stimulatory effect of ACTH on brain lipase and that described for the receptor-mediated actions of ACTH on adrenal and fat cells suggest that the ACTH effect might involve recognition of a binding site associated with the brain enzyme. Complete log concentration response curves obtained with four ACTH analogs may also be regarded as simulating hormone-receptor interaction. These findings are discussed in relation to the possibility that ACTH may have a neurohormonal role via lipase-catalyzed changes in the lipid matrix of membranes.

In vitro effect of adrenocorticotrophic hormone on the pH profile of tri- and diester lipase activities from rat brain.

The influence of adrenocorticotrophic hormone (ACTH) on the hydrolysis of tri- and dioleoylglycerol by a cerebral lipase was studied using a synaptosomal preparation from rat brain as source of enzyme. When ACTH was added to the lipolytic medium, it caused a marked shift of the optimum pH values of catalysis towards alkaline values. In the pH range 5.8-6.5, these shifts resulted in reaction rates 5- to 20-fold higher, depending on the experimental conditions, than those measured without hormone. The ACTH effect was dependent on the NH2-terminal sequence extending through the amino acid residues 15-18. The results suggest that the hormonal influence is specific and mediated through a colipase-like effect.

Enhanced activity of brain lipase in the presence of adrenocorticotrophic hormone. Comparison with a colipase effect.

The characteristics of the pH-dependent stimulatory influence of adrenocorticotrophic hormone (ACTH) on lipolysis (Arnaud, J., Nobili, O. and Boyer, J. (1980) Biochim, Biophys. Acta 617, 524-528) were investigated further. ACTH enhanced 7-30 times the rates of hydrolysis of emulsified trioleoylglycerol by a rat brain lipase, when added to the medium both before and after the enzyme. When lipase activity was inhibited by sodium taurocholate, ACTH fully reversed the inhibition at bile salt concentration up to 2 mM. The reactivation process followed a sharp S-shaped pattern, leveling off at about 10(-4) M ACTH. With and without bile salt, the stimulatory effect of ACTH culminated at pH 5.75, and was dependent on the presence of trace amount of a water-insoluble solvent in the substrate emulsion. Taken together, the results suggest that ACTH acts at the lipid-water interface in facilitating the enzyme-substrate interaction. The relevance of the hormonal influence to a colipase-like effect is discussed.

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.

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.

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.

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.

Decreased acylation of phosphatidylcholine in diabetic rat erythrocytes.

We investigated the effects of streptozocin-induced diabetes on composition and metabolism of rat erythrocyte lipids. Diabetes produced no change in contents of cholesterol, total phospholipids, and proportions of phosphatidylcholine and phosphatidylethanolamine in phospholipids. The acylation of total phospholipids with palmitic, oleic, or arachidonic acids was decreased (P less than .01) in intact erythrocytes from diabetic versus control animals. This anomaly was underlaid by a decrease (P less than .01) in acylation of phosphatidylcholine, whereas phosphatidylethanolamine was unaffected. The impaired acylation of phosphatidylcholine was unchanged in vitro by insulin or coenzyme A but was restored to control values by ATP and by insulin treatment of the diabetic rats. We conclude that diabetes specifically alters the acylation of at least phosphatidylcholine in rat erythrocyte, an effect that might modify the remodeling of erythrocyte phospholipids and thereby the membrane function.

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.

Stimulation of brain lipase activity by polyamines. Comparison with the effect of ACTH.

Polyamines, as well as ACTH, strongly stimulate at pH 5.75 triacylglycerol lipase (TAGL) activity from rat brain. Whether the activating potency is expressed in terms of molar concentration or amount of positive charges, polyarginine, polylysine, spermine and spermidine exhibit, in this order, decreasing potencies. By contrast to other lipases, heparin (25 micrograms/ml) inhibits brain TAGL. Polyarginine, polylysine and spermine reverse the heparin-dependent inhibition and further stimulate TAGL activity above basal values; spermidine is much less potent. In the presence of heparin, ACTH has the greatest stimulating effect, being 1.6-fold and 3.3-fold more potent than polyarginine and polylysine, respectively. Taken together, the data suggest that polybasic effectors modify the interaction of TAGL with its substrate, resulting in increased levels of TAGL activity. In the presence of heparin, the enzyme charge density is mandatory for determining the stimulation process. Such cationic interactions appear to be specific of brain TAGL and should be considered in assessing any direct neuro-hormonal role to ACTH or physiological polyamines in brain.

On the similarity of triacylglycerol and acylcholesterol lipases in rat brain.

Previously, we described a similar stimulating effect of adrenocorticotrophic hormone (ACTH) and ACTH-related synthetic peptides on triacylglycerol (TAGL) and acylcholesterol (ACL) lipase activities of rat brain. In present study, TAGL and ACL activities from rat brain were further investigated and compared through the use of tri-[3H]oleoylglycerol and [14C]oleoylcholesterol, respectively, as the substrates. A number of specific properties proved to be similar for both activities: (a) comparable rates of activity decay were observed upon heat treatment, (b) similar reaction kinetics as determined at different substrate concentrations with and without ACTH (10(-4) M), (c) both activities showed a common susceptibility to the presence of sodium taurocholate (5 mM) or trace amounts (5 microliter/ml) of organic solvent in the assay medium, (d) the distributions of the two activities in various brain regions were comparable. Taken together, these data, along with the similar activability by ACTH, suggest a common structural basis for TAGL and ACL activities in rat brain.

Changes in pattern of phospholipid acylation during in vivo aging of rat red blood cells.

We have investigated the patterns of incorporation of stearic, oleic, and linoleic acids into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of intact red blood cells of differing age isolated by centrifugation on discontinuous density gradient. Acylation rates of PC and PE elicited marked declines from the reticulocyte to the young erythrocyte stage followed by minimal changes of acylating potency in older cells; this biphasic decay pattern was similar with the three fatty acids. Molar acylation rates were higher for PC than for PE in reticulocytes, whereas they were comparable in erythrocytes. PC served as preferred fatty acid acceptor in circulating red blood cells, a function which was largely accounted for by PC contained in the small percentage of circulating reticulocytes. On a per cell basis, this function of PC was due to the cumulative effects of higher molar acylation rates in reticulocytes and higher content in PC over PE in the red blood cell membrane. Acylation rates in PC and PE increased with the number of unsaturated bonds in the acylating fatty acid, regardless of cell age.

Estrogen modulates phospholipid acylation in red blood cells: relationship to cell aging.

Ethinyl estradiol administered in vivo to female rats resulted in a mild anemia with a 120% increase in reticulocytosis. Consistent with a previous study, the red blood cell cholesterol-to-phospholipid molar ratio was decreased by 25%, whereas fatty acyl incorporation was significantly increased into phosphatidylethanolamine (PE) and not into phosphatidylcholine (PC), the major acyl acceptor in red blood cells. Analysis of this estrogen-dependent acylation increase as a function of cell age indicated that it was not expressed in reticulocytes but in erythrocytes and was associated with cell aging. Estrogen was further shown to increase the red blood cell susceptibility to peroxidation generated by incubation with H2O2. Altogether, the results suggest that estrogen indirectly increases phospholipid acylation in red blood cells by decreasing protection against oxidative damage, thereby favoring the action of endogenous phospholipases against oxidized substrates. This occurs predominantly in PE of oldest cells because 1) PE, being more unsaturated than PC, is more sensitive to oxidation, and 2) susceptibility to oxidation increases with cell age.

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.

Ethynylestradiol alters lipid composition and phosphatidylethanolamine metabolism in red blood cells.

Treatment of female rats with ethinylestradiol at a dose of 60 micrograms/rat, daily for 21 days, produced marked changes in red blood cell lipids. Cholesterol was decreased by 22% and total phospholipids were increased by 13%, resulting in a 31% decrease in the cholesterol to phospholipid ratio. The mass distribution of phosphatidylcholine and phosphatidylethanolamine relative to total phospholipids was unchanged. Whereas control red cells incorporated preferentially fatty acids in phosphatidylcholine, ethinylestradiol stimulated their incorporation specifically in phosphatidylethanolamine, where increases occurred with palmitic acid (+75%), oleic acid (+68%) and arachidonic acid (+31%). Incorporation in phosphatidylcholine was unaffected with any of the 3 fatty acids. The stimulation of fatty acid incorporation in phosphatidylethanolamine is likely to reflect an estrogen-dependent increase in turnover rate of fatty acids in this phospholipid. Such alterations in lipid composition and fatty acid incorporation in red cell phospholipids may have significant effects on membrane function.

Altered acylation of erythrocyte phospholipids in alcoholism.

The composition and metabolism of erythrocyte lipids were studied in 10 chronic alcoholic patients within 48 hr after discontinuation of alcohol intake and in 10 nonalcoholic control subjects. Chronic alcoholism produced no change in contents of cholesterol, total phospholipids, and proportions of phosphatidylcholine and phosphatidylethanolamine in erythrocyte phospholipids. The mean values of the rates of acylation of phosphatidylcholine and phosphatidylethanolamine with oleic acid were increased respectively by 59% (p less than 0.001) and 38% (p less than 0.05) as compared with the controls. There was no correlation between acylation rates and mean cellular volumes. Increases in acylation rates normalized over several weeks after alcohol withdrawal and were not related to a direct effect of alcohol on the intact erythrocyte, suggesting that these alterations result from ethanol-induced changes in the membrane during erythrocyte formation. The increased rates of acylation might modify the remodeling of the lipid matrix and thereby the membrane function.