Generation Of Lysophospholipids Research Articles

VpdC is a ubiquitin-activated phospholipase effector that regulates Legionella vacuole expansion during infection

Intravacuolar pathogens need to gradually expand their surrounding vacuole to accommodate the growing number of bacterial offspring during intracellular replication. Here we found that Legionella pneumophila controls vacuole expansion by fine-tuning the generation of lysophospholipids within the vacuolar membrane. Upon allosteric activation by binding to host ubiquitin, the type IVB (Dot/Icm) effector VpdC converts phospholipids into lysophospholipids which, at moderate concentrations, are known to promote membrane fusion but block it at elevated levels by generating excessive positive membrane curvature. Consequently, L. pneumophila overproducing VpdC were prevented from adequately expanding their surrounding membrane, trapping the replicating bacteria within spatially confined vacuoles and reducing their capability to proliferate intracellularly. Quantitative lipidomics confirmed a VpdC-dependent increase in several types of lysophospholipids during infection, and VpdC production in transiently transfected cells caused tubulation of organelle membranes as well as mitochondria fragmentation, processes that can be phenocopied by supplying cells with exogenous lysophospholipids. Together, these results demonstrate an important role for bacterial phospholipases in vacuolar expansion.

The role of the small intestine in modulating metabolism and inflammation in atherosclerosis and cancer.

To discuss recent findings on the importance of the small intestine in modulating metabolism and inflammation in atherosclerosis and cancer. Integrin β7 natural gut intraepithelial T cells modulated metabolism and accelerated atherosclerosis in mice. Reducing the generation of lysophospholipids in the small intestine mimicked bariatric surgery and improved diabetes. Enterocyte-specific knockdown of stearoyl-CoA desaturase-1 significantly improved dyslipidemia in LDL receptor null (Ldlr) mice fed a Western diet. Adding a concentrate of tomatoes transgenic for the apolipoprotein A-I mimetic peptide 6F to the chow of wild-type mice altered lipid metabolism in the small intestine, preserved Notch signaling and reduced tumor burden in mouse models. The phospholipid-remodeling enzyme Lpcat3 regulated intestinal stem cells and progenitor cells by stimulating cholesterol biosynthesis; increasing cholesterol in the diet or through genetic manipulation promoted tumorigenesis in Apc mice. The small intestine is important for regulating metabolism and inflammation in animal models of both atherosclerosis and cancer.

Tracing the Thermal History of Seafood Products through Lysophospholipid Analysis by Hydrophilic Interaction Liquid Chromatography⁻Electrospray Ionization Fourier Transform Mass Spectrometry.

Low temperature treatments commonly applied to seafood products have been shown to influence their phospholipid (PL) profile through enzymatic hydrolysis. In the present study, the generation of lysophospholipids (LPL) resulting from this process was systematically investigated for selected, commercially relevant seafood products, namely oysters, clams, octopuses, and shrimps. These products were subjected to thermal treatments like refrigeration or freezing after being purchased as fresh, defrozen, or frozen products depending on the case. The coupling between hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization with high resolution/accuracy Fourier transform mass spectrometry (ESI-FTMS) was exploited to evaluate the PL profile of the cited products, especially the incidence of LPL related to the two main PL classes of seafood products—phosphatidylcholines (PC) and phosphatidylethanolamines (PE)—in the lipid extracts. The lyso forms of PE (LPE) were found to be generally more sensitive than those of PC (LPC) to thermal treatments, usually exhibiting a significant increase upon prolonged refrigeration at 4 °C in all types of investigated products except European flat oysters. Moreover, the distinction between fresh and frozen or defrozen products could be achieved in the case of octopuses and shrimps, respectively.

Kinetic Properties and Redox Regulation of Recombinant Calcium-Independent Phospholipase A2γ Reconstituted In Liposomes

Calcium-independent phospholipase A2γ (iPLA2γ) catalyses the hydrolysis of fatty acids from phospholipids with the consequent generation of lysophospholipids. iPLA2γ is targeted to peroxisomes and mitochondria and results from our laboratory have shown that the activity of mitochondrial iPLA2γ is increased by a redox-sensitive process [1]. Here we aimed to characterize the properties of isolated recombinant iPLA2γ and test the hypothesis that iPLA2γ is activated by thiol oxidation directly. We expressed His-tagged human iPLA2γ using HepG2 or HEK293 cell lines and reconstituted recombinant purified iPLA2γ into liposomes composed of L-α-Phosphatitylcholine and cardiolipin. Using selected specific fluorescent probes and GC/MS analysis, we observed H2O2-induced release of fatty acids in liposomes containing purified recombinant iPLA2γ but not in liposomes lacking the enzyme. The H2O2-activated enzyme released free fatty acids with a specific activity of about 20 nmol.min-1.mg-1. The activity increased several fold in the presence of bovine serum albumin or in mixed micelles containing non-ionic detergent, indicating a possible inhibition of iPLA2γ by the products of its enzymatic activities. H2O2 activated iPLA2γ with a half-maximal constant corresponding to about 1.5 mol H2O2 per mol of enzyme and the active enzyme was fully inhibited by thiol reducing compounds. These results indicate that the regulation of iPLA2γ is dependent on reversible oxidation of accessible cysteine residues and also on a feedback regulation by either fatty acids or lysophospholipids, which is consistent with the physiological participation of iPLA2γ in cellular redox signaling.Supported by the grant GA15-02051S to M.J. from the Grant Agency of the Czech Republic.1. Jaburek M, Ježek J, Zelenka J, Ježek P. Antioxidant activity by a synergy of redox-sensitive mitochondrial phospholipase A2 and uncoupling protein-2 in lung and spleen. Int. J. Biochem & Cell Biol 45, 816-825, 2013

Lysophospholipid and fatty acid inhibition of pulmonary surfactant: Non-enzymatic models of phospholipase A 2 surfactant hydrolysis

Secretory A 2 phospholipases (sPLA 2) hydrolyze surfactant phospholipids cause surfactant dysfunction and are elevated in lung inflammation. Phospholipase-mediated surfactant hydrolysis may disrupt surfactant function by generation of lysophospholipids and free fatty acids and/or depletion of native phospholipids. In this study, we quantitatively assessed multiple mechanisms of sPLA 2-mediated surfactant dysfunction using non-enzymatic models including supplementation of surfactants with exogenous lysophospholipids and free fatty acids. Our data demonstrated lysophospholipids at levels ≥10 mol% of total phospholipid (i.e., ≥10% hydrolysis) led to a significant increase in minimum surface tension and increased the time to achieve a normal minimum surface tension. Lysophospholipid inhibition of surfactant function was independent of the lysophospholipid head group or total phospholipid concentration. Free fatty acids (palmitic acid, oleic acid) alone had little effect on minimum surface tension, but did increase the maximum surface tension and the time to achieve normal minimum surface tension. The combined effect of equimolar free fatty acids and lysophospholipids was not different from the effect of lysophospholipids alone for any measurement of surfactant function. Surfactant proteins did not change the percent lysophospholipids required to increase minimum surface tension. As a mechanism that causes surfactant dysfunction, depletion of native phospholipids required much greater change (equivalent to >80% hydrolysis) than generation of lysophospholipids. In summary, generation of lysophospholipids is the principal mechanism of phospholipase-mediated surfactant injury in our non-enzymatic models. These models and findings will assist in understanding more complex in vitro and in vivo studies of phospholipase-mediated surfactant injury.

Regulation of intracellular calcium levels by polyunsaturated fatty acids, arachidonic acid and docosahexaenoic acid, in astrocytes: possible involvement of phospholipase A2

Pathological conditions in the brain, such as ischemia, trauma and seizure are accompanied by increased levels of free n-6 and n-3 polyunsaturated fatty acids (PUFA), mainly arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3). A neuroprotective role has been suggested for PUFA. For investigation of the potential molecular mechanisms involved in neuroprotection by PUFA, we studied the regulation of the concentration of intracellular Ca2+ ([Ca2+]i) in rat brain astrocytes. We evaluated the presence of extracellular PUFA and the release of intracellular PUFA. Interestingly, only the constitutive brain PUFA AA and DHA, but not eicosapentaenoic acid (EPA) had prominent effects on intracellular Ca2+. AA and DHA suppressed [Ca2+]i oscillation, inhibited store-operated Ca2+ entry, and reduced the amplitudes of Ca2+ responses evoked by agonists of G protein-coupled receptors. Moreover, prolonged exposure of astrocytes to AA and DHA brought the cells to a new steady state of a moderately elevated [Ca2+]i level, where the cells became virtually insensitive to external stimuli. This new steady state can be considered as a mechanism of self-protection. It isolates disturbed parts of the brain, because AA and DHA reduce pathological overstimulation in the tissue surrounding the damaged area. In inflammation-related events, frequently AA and DHA exhibit opposite effects. However, in astrocytes AA and DHA exerted comparable effects on [Ca2+]i. Extracellularly added AA and DHA, but not EPA, were also able to induce the release of [3H]AA from prelabeled astrocytes. Therefore, we also suggest the involvement of phospholipase A2 activation and lysophospholipid generation in the regulation of intracellular Ca2+ in astrocytes.

Impact of a Soluble Phospholipase A2 Inhibitor on Inhaled Allergen Challenge in Subjects with Asthma

The possible roles of secretory phospholipases A2 (sPLA2) in asthma include the release of arachidonic acid from cellular membranes, generation of lysophospholipids, sPLA2-mediated activation of cPLA2 with increased leukotriene production, and surfactant degradation. LY333013 is a potent inhibitor of sPLA2. This study examined the impact of two doses of LY333013 vs. placebo on allergen-induced bronchoconstriction following inhaled allergen challenge in atopic asthmatics. Fifty subjects were randomly assigned to treatment, and 40 subjects completed the study. A double-blind, placebo-controlled, random order, crossover study design was used. LY333013 had no impact on the primary outcome variables of the areas under the FEV1 response curve early (0–3 hours) (AUCearly) and late (3–8 hours) (AUClate) following inhaled allergen challenge. No significant drug-related adverse effects were observed. The response to inhaled allergen challenge was reproducible and confirms the utility of this technique as a model in which to screen compounds for further testing in asthmatic patients.

Lysophospholipid generation and phosphatidylglycerol depletion in phospholipase A2-mediated surfactant dysfunction

Pulmonary surfactant's complex mixture of phospholipids and proteins reduces the work of breathing by lowering alveolar surface tension during respiration. One mechanism of surfactant damage appears to be the hydrolysis of phospholipid by phospholipases activated in the inflamed lung. Humans have several candidate secretory phospholipase A(2) (sPLA(2)) enzymes in lung cells and infiltrating leukocytes that could damage extracellular surfactant. We considered two mechanisms of surfactant disruption by five human sPLA(2)s, including generation of lysophospholipids and the depletion of specific phospholipids. All five sPLA(2)s studied ultimately caused surfactant dysfunction. Each enzyme exhibited a different pattern of hydrolysis of surfactant phospholipids. Phosphatidylcholine, the major phospholipid in surfactant and the greatest potential source for generation of lysophospholipids, was susceptible to hydrolysis by group IB, group V, and group X sPLA(2)s, but not group IIA or IID. Group IIA hydrolyzed both phosphatidylethanolamine and phosphatidylglycerol, whereas group IID was active against only phosphatidylglycerol. Thus, with groups IB and X, the generation of lysophospholipids corresponded with surfactant dysfunction. However, hydrolysis of and depletion of phosphatidylglycerol had a greater correlation with surfactant dysfunction for groups IIA and IID. Surfactant dysfunction caused by group V sPLA(2) is less clear and may be the combined result of both mechanisms.

A novel mechanism for the store-operated calcium influx pathway.

Activation of store-operated channels (SOCs) and capacitative calcium influx are triggered by depletion of intracellular calcium stores. However, the exact molecular mechanism of such communication remains unclear. Recently, we demonstrated that native SOC channels can be activated by calcium influx factor (CIF) that is produced upon depletion of calcium stores, and showed that Ca(2+)-independent phospholipase A(2) (iPLA(2)) has an important role in the store-operated calcium influx pathway. Here, we identify the key plasma-membrane-delimited events that result in activation of SOC channels. We also propose a novel molecular mechanism in which CIF displaces inhibitory calmodulin (CaM) from iPLA(2), resulting in activation of iPLA(2) and generation of lysophospholipids that in turn activate soc channels and capacitative calcium influx. Upon refilling of the stores and termination of CIF production, CaM rebinds to iPLA(2), inhibits it, and the activity of SOC channels and capacitative calcium influx is terminated.

Cellular function of calcium-independent phospholipase A2.

The catalytic activity of calcium-independent phospholipase A2 (iPLA2), which is classified as a group VI PLA2, is regulated by protein kinase C, calmodulin, and others such as reactive oxygen species. Numerous findings have shown that iPLA2 is involved in stimulus-induced arachidonic acid release and lysophospholipid generation, although the participation is dependent upon the cell type and stimulus. The catalytic action of iPLA2 is known to be responsible for phospholipid remodeling as a housekeeping function. However, it has been widely accepted that arachidonic acid and lysophospholipid generated by iPLA2 act as a signaling molecule in cellular functions. Those include eicosanoid production, glucose-induced insulin secretion, Fas-induced apoptosis, cellular proliferation, membrane traffic in fusion, contribution to myocardial ischemia, and others. In this review, the functional role of iPLA2 in cellular responses upon stimulation is the focus.

Isolation of human small intestinal brush border membranes using polyethylene glycol and effect of exposure to various oxidants in vitro.

This study presents a method of brush border membrane (BBM) preparation from the human small intestine using polyethylene glycol (PEG) precipitation and also looks at the effect of in vitro oxidant exposure on structural and functional alterations in the membrane. Isolated BBM were relatively pure as judged by 10- to 14-fold enrichment of marker enzymes with less than 1% contamination by other subcellular organelles. These membranes showed uphill transport of glucose and lipid analysis showed a cholesterol-phospholipid (C/P) ratio of 1.19. Isolated BBM were found to be susceptible to superoxide generated by xanthine oxidase (XO), resulting in lipid and protein oxidation along with altered glucose uptake. Superoxide exposure also resulted in phospholipid alterations, especially generation of lyso phospholipids. These changes were prevented by inhibiting XO by allopurinol or scavenging superoxide by superoxide dismutase (SOD). Other oxidants studied did not have significant affect on these membranes. These studies suggest that PEG can be used for preparation of BBM from the human small intestine and these membranes undergo structural and functional alterations on exposure to superoxide.

Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type-II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 protein interaction.

Lyso-phospholipids exert a major injurious effect on lung cell membranes during Acute Respiratory Distress Syndrome (ARDS), but the mechanisms leading to their in vivo generation are still unknown. Intratracheal administration of LPS to guinea pigs induced the secretion of type II secretory phospholipase A2 (sPLA2-II) accompanied by a marked increase in fatty acid and lyso-phosphatidylcholine (lyso-PC) levels in the bronchoalveolar lavage fluid (BALF). Administration of LY311727, a specific sPLA2-II inhibitor, reduced by 60% the mass of free fatty acid and lyso-PC content in BALF. Gas chromatography/mass spectrometry analysis revealed that palmitic acid and palmitoyl-2-lyso-PC were the predominant lipid derivatives released in BALF. A similar pattern was observed after the intratracheal administration of recombinant guinea pig (r-GP) sPLA2-II and was accompanied by a 50-60% loss of surfactant phospholipid content, suggesting that surfactant is a major lung target of sPLA2-II. In confirmation, r-GP sPLA2-II was able to hydrolyze surfactant phospholipids in vitro. This hydrolysis was inhibited by surfactant protein A (SP-A) through a direct and selective protein-protein interaction between SP-A and sPLA2-II. Hence, our study reports an in vivo direct causal relationship between sPLA2-II and early surfactant degradation and a new process of regulation for sPLA2-II activity. Anti-sPLA2-II strategy may represent a novel therapeutic approach in lung injury, such as ARDS.

Effect of Ethanol on Intracellular Vesicular Transport from Golgi to the Apical Cell Membrane: Role of Phosphatidylinositol 3‐Kinase and Phospholipase A2 in Golgi Transport Vesicles Association and Fusion with the Apical Membrane

The study of ethanol effects on intracellular transport and membrane biogenesis in rat hepatocytes revealed that, during synthesis of transport vesicles, the cytosolic phosphatidylinositol 3-kinase incorporated into the membrane of Golgi transport vesicles and a portion of the vesicular phosphatidylinositol was phosphorylated to phosphatidylinositol 3-phosphate. Association of the enzyme with Golgi transport vesicles and the transport to the apical portion of the cell membrane was not affected by 0 to 120 mM ethanol, but was dependent on the presence of the p85 subunit of the phosphatidylinositol 3-kinase. In the presence of ATP-enriched cytosol and calcium ions, association of Golgi transport vesicles with the apical membrane was followed by phospholipase A2-specific hydrolysis of phosphatidylinositol 3-phosphate and incorporation of the transport vesicle membrane into the apical membrane. Association of Golgi transport vesicles with apical membranes was not affected by preincubation of the cell membrane or Golgi transport vesicles with 0 to 120 mM ethanol, but was inhibited when the p85 phosphatidylinositol 3-kinase was incorporated into the membrane before incubation with Golgi transport vesicles. The fusion of Golgi transport vesicles with the apical membrane and generation of lysophosphatidylinositol 3-phosphate and arachidonate was inhibited with EGTA or after depletion of ATP from cytosol. Results of these studies provide evidence that phosphatidylinositol 3-kinase and phospholipase A2 activities are crucial for the final step of exocytotic transport. The process consists of two stages. First, the p85 subunit of phosphatidylinositol 3-kinase is involved in the specific association of the vesicle with membrane receptor, and that is followed by phospholipase A2-specific lysophospholipid generation, perturbation of the membranes, and fusion of the transport vesicle membrane with the apical membrane. Addition of ethanol to the in vitro transport system decreased production of Golgi transport vesicles, but had no effect on their association with apical membrane or fusion with the membrane.

Coenzyme A-dependent cleavage of membrane phospholipids in several rat tissues: ATP-independent acyl-CoA synthesis and the generation of lysophospholipids.

Substantial amounts of acyl-CoA were formed when microsomes from several rat tissues were incubated with varying concentrations of free CoA and bovine serum albumin even in the absence of ATP and Mg2+. For instance, 86 nmol of acyl-CoA was produced when microsomes (5 mg protein) were incubated with 300 microM CoA for 30 min. It was calculated that 1.8% of total fatty acyl residues were converted to acyl-CoA during the incubation. No appreciable amount of acyl-CoA was formed from free fatty acid or from boiled microsomes under the same experimental conditions. These observations indicate that acyl-CoA is formed from microsomal lipids by an enzyme activity distinct from previously known long-chain fatty acyl-CoA synthetase. The apparent Km value for CoA and Vmax were 180 microM and 20 nmol/30 min per mg protein, respectively. We found that several species of acyl-CoA such as arachidonoyl-CoA were preferentially synthesized through the reaction and that several types of phospholipids actually act as acyl donors in the formation of acyl-CoA. Phosphatidylinositol and phosphatidylcholine appear to be preferred substrates. We confirmed that lysophosphatidylinositol and lysophosphatidylcholine were generated along with the formation of acyl-CoA. It seems very likely that CoA-mediated cleavage of phospholipids/ATP-independent acyl-CoA synthesis is implicated in the metabolism of certain types of fatty acyl residues of membranous phospholipids in mammalian cells.

Effects of a fish-oil and vegetable-oil formula on aggregation and ethanolamine-containing lysophospholipid generation in activated human platelets and on leukotriene production in stimulated neutrophils

The effects of consuming a liquid formula containing either fish oil enriched in omega-3 fatty acids or vegetable oil enriched in oleic acid was evaluated in 20 male subjects randomly allocated into two groups over a 42-d period. A decrease in collagen-induced aggregation by using washed platelet suspensions was found in both groups after nutritional supplementation. A considerable rise in omega-3 and a decrease in omega-6 fatty acids occurred in the platelet phospholipid with fish-oil consumption. The degree of eicosapentaenoic acid (EPA, 20:5n-3) enrichment (fish-oil group) was dramatically greater in the ether-containing plasmenylethanolamine (13.5 mol% of fatty acids; mol% of fatty acids = moles per 100 moles of total fatty acids) than in phosphatidylethanolamine (2.8 mol%) or phosphatidylcholine (2.9 mol%). Neither treatment significantly influenced the agonist-induced accumulation of lysoplasmenylethanolamine as derived via phospholipase A2 hydrolysis of plasmenylethanolamine. HPLC measurements of eicosanoid production in A23187-stimulated neutrophils revealed a considerable decrease in the formation of arachidonic acid-derived leukotriene B4 (LTB4), by 41%, and 5-HETE (5-hydroxyeicosatetraenoic acid), by 30%, in the fish-oil group along with the appearance of the corresponding EPA-derived products [LTB5 and 5-HEPE (5-hydroxyeicosapentaenoic acid)]. No such alterations in the formation of lipoxygenase products were found with the vegetable oil treatment.

Stimulation of receptor-coupled phospholipase A 2 by interferon-γ

The biomolecular mechanisms that mediate signal transduction by type 11 (γ) interferon (IFN) are poorly understood. IFN-γ is a potent growth inhibitory cytokine also endowed with antiviral, immunomodulatory, and differentiating activities on various cell targets, including neural cells. IFN-γ induced a rapid and transient activation of phospholipase A 2 in LAN-5, a human neuroblastoma cell line. A consequence of phospholipase A 2 activation was the release of arachidonic acid and the generation of lysophospholipids from membrane phospholipids. Treatment of pre-labeled LAN-5 cells with a receptor-saturating concentration of IFN-γ led to a time-dependent release of [ 3H]arachidonic acid into the culture media and generation or [ 32P]lysophosphatidylcholine. Pretreatment of cultures with the phospholipase A 2 inhibitor, bromophenacyl bromide, markedly inhibited both [ 3H]arachidonic acid release and lysophosphatidylcholine production induced by IFN-γ treatment. Pretreatment of LAN-5 cells with nordihydroguaiaretic acid, a lipoxygenase inhibitor, or with indomethacin, a cyclooxygenase inhibitor, amplified the release of[ 3H]arachidonic acid and production of lysophosphatidylcholine induced by non-saturating concentrations of IFN-γ. In parallel, and with the same time-dependent effect, a significant decrease in phosphatidylcholine labeling was observed in IFN-γ-treated cells, further indicating that a potential signal transduction mechanism of IFN-γ is the hydrolysis of membrane phosphatidylcholine by phospholipase A 2.

Function of intracellular phospholipase a 2 in vectorial transport of apoproteins from er to golgi

1. 1. The cytosolic fraction required in in vitro reconstituted intracellular transport of mucus glycoprotein apopeptide (apomucin) was isolated and its potential as transport supporting factor assessed by the quantitation of the gastric apomucin transferred to Golgi. 2. 2. The experiments with the fraction promoting transport and delivery of apomucin to Golgi revealed that the active protein has the property of phospholipase A 2 (PLA 2) which assists ER vesicles fusion with Golgi. 3. 3. The ability of the 76 kDa PLA 2 to hydrolyze phospholipids and to support transport and fusion of ER vesicles with Golgi was abolished by phosphorylation and regained following dephosphorylation. 4. 4. The data provide evidence that 76 kDa intracellular PLA 2 is responsible for the fusion of ER-transport vesicles with Golgi. The process of fusion is accomplished by generation of lysophospholipids in fusing membranes.

Effect of lysophospholipids, arachidonic acid and other fatty acids on regulation of Ca 2+ transport in permeabilized pancreatic islets

The immediate reaction products of PLA 2-mediated hydrolysis of phospholipids were tested for their ability to induce Ca 2+ mobilization from internal stores in permeabilized ob ob mouse pancreatic islets. Lysophospholipids and unsaturated fatty acids increased the free Ca 2+ concentration in the incubation medium of permeabilized ob ob mouse pancreatic islets. The potency of the lysophospholipids decreased in the following order: lysophosphatidylcholine = lysophosphatidylglycerol ⪢ lysophosphatidylinositol > lysophosphatidylserine ⪢ lysophosphatidylethanolamine. Arachidonic acid and palmitoleic acid had a potency comparable to lysophosphatidylinositol, while palmitic acid was ineffective. The Ca 2+-mobilizing effect of inositol-1,4,5-trisphosphate (IP 3) in permeabilized islet cells was additive to the lysophospholipid effect, indicating different sites of action. Both Ca 2+-mobilizing effects were counteracted by the polyamine spermine, while the presence of Mg 2+ shifted the Ca 2+ concentrations to higher levels. Since not only an activation of a phospholipase C but also an activation of a phospholipase A 2 with subsequent generation of lysophospholipids and free fatty acids is reported to occur in glucose-induced insulin secretion, the interaction of the phospholipase C reaction product IP 3 with a lysophospholipid or an unsaturated fatty acid may affect the extent and duration of the rise in the free cytoplasmic Ca 2+ concentration responsible for initiation of insulin secretion.

Phospholipase-induced modulation of dolichyl-phosphomannose synthase activity.

Rat liver dolichyl-phosphomannose synthase is optimally active when the enzyme is reconstituted with lipids that prefer a nonlamellar macroscopic organization in isolation, such as phosphatidylethanolamine (PE), but the enzyme is only negligibly active in the presence of lipids that normally form stable bilayers, such as phosphatidylcholine (PC) [Jensen, J.W., & Schutzbach, J.S. (1985) Eur. J. Biochem. 153, 41-48]. We now report that the activity of the synthase can be modulated by incorporating diacylglycerol and lysophosphatidylcholine into the lipid matrix. Enzyme activity in PC bilayers was stimulated by the presence of diacylglycerol, a lipid that has a conical dynamic molecular shape and disrupts bilayer stability. In PC/diacylglycerol mixtures the apparent Km for dolichyl-P was 30-fold lower than the apparent Km for the polyprenol acceptor in PC membranes. Enzyme activity was also stimulated when diacylglycerol was generated in situ by incubation of PC vesicles with phospholipase C. In contrast, the activity of enzyme reconstituted in PE dispersions, or in PE/PC bilayers, was markedly inhibited by the presence of lysophospholipids. Enzyme activity was also reduced by the in situ generation of lysophospholipids in PE/PC vesicles by incubation with phospholipase A2. Since lysophospholipids and diacylglycerols arise in vivo as products of phospholipid metabolism, modulation of enzyme activity by these compounds may represent a potential regulatory mechanism for the synthesis of oligosaccharide lipids.

Lack of specificity of melittin as a probe for insulin release mediated by endogenous phospholipase A 2 or lipoxygenase

The effects of the basic polypeptide melittin on islet phospholipid degradation and insulin release were studied in static incubations of intact rat islets as a possible model of endogenous phospholipase A 2 (PLA 2) activation. Melittin (2 μg/ml) increased [ 3H]-arachidonic acid ([ 3H]-AA) release from prelabeled islets (at 1.7 mM glucose) to 371% of basal values. Concomitantly, melittin induced degradation of islet phospholipids labeled with [ 3H]-AA or [ 14C]-stearic acid, and led to the accumulation of stearate-labeled (but not AA-labeled) lysophosphatidylcholine (LPC, 605% of control). These findings suggested, for the first time in intact rat islets, the presence and activation of a PLA 2. Under identical conditions, melittin initiated insulin secretion (at 1.7 mM glucose) in a manner that represented stimulation of physiologic exocytosis—that is, it was dose-dependent, reversible (albeit slowly), unassociated with impairment of other physiologic islet processes (i.e. the response to 16.7 mM glucose after removal of the drug) and inhibitable by reduced ambient temperature. The effect of melittin seemed to be independent of extracellular Ca 2+ influx or mobilization of intracellular Ca 2+ stores but was blocked by nickel or lanthanum, indirectly suggesting that the effects of this cationic amphiphile might involve a superficial pool of Ca 2+. Unexpectedly, melittin-induced insulin release (at least at low glucose concentrations) was not greatly or consistently altered by a battery of inhibitors of endogenous PLA 2 or of enzymes affecting AA oxygenation. Furthermore, significant contamination by bee venom PLA 2 of the commercially-available melittin preparation was found, and insulin release could be induced by pure bee venom PLA 2, probably through the generation of lysophospholipids. Although estimates of the amount of PLA 2 in the melittin preparation suggested that such contamination was insufficient to explain at least some of the islet phospholipid hydrolysis and insulin release caused by melittin, we conclude that this agent does not serve as a specific probe of the role of endogenous PLA 2 or of AA lipoxygenation in hormone release.