Metabolic turnover of myelin glycerophospholipids

The apparent half life for metabolic turnover of glycerophospholipids in the myelin sheath, as determined by measuring the rate of loss of label in a myelin glycerophospholipid following radioactive precursor injection, varies with the radioactive precursor used, age of animal, and time after injection during which metabolic turnover is studied. Experimental strategies for resolving apparent inconsistencies consequent to these variables are discussed. Illustrative data concerning turnover of phosphatidylcholine (PC) in myelin of rat brain are presented. PC of the myelin membrane exhibits heterogeneity with respect to metabolic turnover rates. There are at least two metabolic pools of PC in myelin, one with a half life of the order of days, and another with a half life of the order of weeks. To a significant extent biphasic turnover is due to differential turnover of individual molecular species (which differ in acyl chain composition). The two predominant molecular species of myelin PC turnover at very different rates (16:0, 18:1 PC turning over several times more rapidly than 18:0, 18:1 PC). Therefore, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Possible mechanisms for differential turnover of molecular species are discussed, as are other factors that may contribute to a multiphasic turnover of glycerophospholipids.Special issue dedicated to Dr. Marjorie Lees.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Differential effects of conjugated linoleic acid isomers on macrophage glycerophospholipid metabolism

Conjugated linoleic acids (CLA) are dietary fatty acids. Whereas cis-9,trans-11-(c9,t11)-CLA can be found in meat and dairy products, trans-9,trans-11-(t9,t11)-CLA is a constituent of vegetable oils. Previous studies showed that these two isomers activate different nuclear receptors and, thus, expression of genes related to lipid metabolism. Here we show that these CLA isomers are differentially elongated and desaturated in primary monocyte-derived macrophages isolated from healthy volunteers by using gas chromatography-mass spectrometry (GC-MS). We further demonstrate that c9,t11-CLA incorporates in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species and activates de novo glycerophospholipid synthesis by quantitative electrospray ionization-tandem mass spectrometry (ESI-MS/MS). c9,t11-CLA leads to strong shifts of the species profiles to PC 18:2/18:2 and PE 18:2/18:2, which are due to de novo synthesis and fatty acid remodeling. In contrast, t9,t11-CLA is preferentially bound to neutral lipids, including triglycerides and cholesterol esters. Taken together our results show that c9,t11-CLA and t9,t11-CLA have differential effects on PC and PE metabolism. Moreover, these data demonstrate that the structure of fatty acids not only determines their incorporation into lipid classes but also modulates the kinetics of lipid metabolism, particularly PC synthesis.     

Hydration of Escherichia coli lipids: Deuterium T1 relaxation time studies of phosphatidylglycerol,phosphatidylethanolamine and phosphatidylcholine

The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic $\text{1,2-}\text{dioleoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ in H₂O/²H₂O mixtures (9:1, v/v) were investigated with ²H-NMR. Comparison of the ²H₂O spin lattice relaxation time ($\text{T}{}_1$) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H₂O systems. Two distinct hydration regions could be discerned in the $\text{T}{}_1$ relaxation time profile. (1) A minimum of 11–16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of $\text{T}{}_1\text{≈ 90}\text{ms}$. (2) Additional water was found to be in exchange with the primary hydration shell. The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with $\text{T}{}_1\text{= 500}\text{ms}$ and hydration water with $\text{T}{}_1\text{= 80–120}\text{ms}$. Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer. However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.     

Comparative study on the properties of saturated phosphatidylethanolamine and phosphatidylcholine bilayers: Barrier characteristics and susceptibility to phospholipase A2 degradation

Comparative studies on bilayer systems of saturated phosphatidylcholines and phosphatidylethanolamines revealed a maximum in ionic permeability in phosphatidylcholine bilayers at the temperature of the gel to liquid-crystalline phase transition but such an increase in permeability was not detectable in bilayers of phosphatidylethanolamine. Furthermore, it was found that at the phase transition temperature the phosphatidylcholine bilayers are subject to rapid hydrolysis by pancreatic phospholipase A₂ whereas phosphatidylethanolamine bilayers are not. These differences are discussed in view of detailed information on the molecular organization in the gel and liquid crystalline phases of the two phospholipid classes.     

Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in ras-transformed NIH 3T3 fibroblasts

Ras-transformation of cells is accompanied by an increase of the level of diacylglycerol (DAG), which participates in the signal transduction pathways. DAG could be generated from phospholipids either by activation of phospholipase C or by a more complex pathway involving phospholipase D and phosphatidate phosphohydrolase. To clarify which phospholipids produce DAG and which pathways are involved, we examined the DAG generating enzyme activities, using phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as substrates. The study showed that the breakdown of PC and more markedly of PE by phospholipases C and D was stimulated in membranes from ras-transformed cells. Phosphatidate phosphohydrolase activity was also elevated in oncogene-expressing cells. The increase in glycerol uptake was most pronounced in cells given PE, followed by PC. The fatty acid analysis revealed apparent similarities between the acyl chains of PE and DAG only in the transformed cells. These findings suggest that PE is a source of DAG in ras-fibroblasts but does not rule out the role of PC in DAG production, due to the activation of the PC-specific phospholipases C and D.     

LPGAT1 controls the stearate/palmitate ratio of phosphatidylethanolamine and phosphatidylcholine in sn-1 specific remodeling

Most mammalian phospholipids contain a saturated fatty acid at the sn-1 carbon atom and an unsaturated fatty acid at the sn-2 carbon atom of the glycerol backbone group. While the sn-2 linked chains undergo extensive remodeling by deacylation and reacylation (Lands cycle), it is not known how the composition of saturated fatty acids is controlled at the sn-1 position. Here, we demonstrate that lysophosphatidylglycerol acyltransferase 1 (LPGAT1) is an sn-1 specific acyltransferase that controls the stearate/palmitate ratio of phosphatidylethanolamine (PE) and phosphatidylcholine. Bacterially expressed murine LPGAT1 transferred saturated acyl-CoAs specifically into the sn-1 position of lysophosphatidylethanolamine (LPE) rather than lysophosphatidylglycerol and preferred stearoyl-CoA over palmitoyl-CoA as the substrate. In addition, genetic ablation of LPGAT1 in mice abolished 1-LPE:stearoyl-CoA acyltransferase activity and caused a shift from stearate to palmitate species in PE, dimethyl-PE, and phosphatidylcholine. Lysophosphatidylglycerol acyltransferase 1 KO mice were leaner and had a shorter life span than their littermate controls. Finally, we show that total lipid synthesis was reduced in isolated hepatocytes of LPGAT1 knockout mice. Thus, we conclude that LPGAT1 is an sn-1 specific LPE acyltransferase that controls the stearate/palmitate homeostasis of PE and the metabolites of the PE methylation pathway and that LPGAT1 plays a central role in the regulation of lipid biosynthesis with implications for body fat content and longevity.     

Regulation of transformed state by calpastatin via PKCepsilon in NIH3T3 mouse fibroblasts.

Ca(2+)-activated neutral protease calpain is ubiquitously expressed and may have pleiotropic biological functions. We have previously reported that repeated treatment of NIH3T3 mouse fibroblasts with the calpain inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) resulted in the induction of transformed foci [T. Hiwasa, T. Sawada, and S. Sakiyama (1990) Carcinogenesis 11, 75-80]. To elucidate further the effects of calpain in malignant transformation of NIH3T3 cells, calpastatin, an endogenous specific inhibitor of calpain, was expressed in NIH3T3 cells by transfection with cDNA. G418-selected calpastatin-expressing clones showed a significant increase in the anchorage-independent growth ability. A similar increase in cloning efficiency in soft agar medium was also observed in calpain small-subunit-transfected clones. On the other hand, reduced expression of calpastatin achieved by transfection with calpastatin antisense cDNA in Ha-ras-transformed NIH3T3 (ras-NIH) cells caused morphological reversion as well as a decrease in anchorage-independent growth. When NIH3T3 cells were treated with ALLN for 3 days, cell growth was stimulated by approximately 10%. This growth stimulation by ALLN was not observed in ras-NIH cells, but recovered by expression of a dominant negative form of protein kinase C (PKC)epsilon but not by that of PKCalpha. Western blotting analysis showed that an increase in PKCepsilon was much more prominent than that of PKCalpha in NIH3T3 cells after treatment with ALLN. These results are concordant with the notion that calpain suppresses malignant transformation by predominant degradation of PKCepsilon.     

Mobilization of arachidonic acid from phosphatidylethanolamine fraction to phosphatidylcholine fraction in platelets

Rabbit platelets rapidly incorporated methyl groups of [³H] methionine to phosphatidylcholine (PC). Rabbit platelets also incorporated [³H]choline to PC, but the rate of incorporation was far lower than that of [³H]methionine. Further fractionation of labeled PC revealed that a considerable amount of arachidonyl PC was synthesized via the N-methylation pathway. Thrombin stimulation resulted in a release of arachidonic acid from PC, and not from phosphatidylethanolamine (PE). These observations suggest that the N-methylation pathway plays an important role in the intracellular mobilization of arachidonic acid from the PE fraction to the PC fraction, this fraction being more sensitive to the hydrolysis with phospholipase A₂ during platelet activation.     

Structure and function of lysosomal phospholipase A2: identification of the catalytic triad and the role of cysteine residues

Lysosomal phospholipase A2 (LPLA2) is an acidic phospholipase that is highly expressed in alveolar macrophages and that may play a role in the catabolism of pulmonary surfactant. The primary structure found in LCAT is conserved in LPLA2, including three amino acid residues potentially required for catalytic activity and four cysteine residues. LPLA2 activity was measured in COS-7 cells transfected with c-myc-conjugated mouse LPLA2 (mLPLA2) or mutated LPLA2. Single alanine substitutions in the catalytic triad resulted in the elimination of LPLA2 activity. Four cysteine residues (C65, C89, C330, and C371), conserved between LPLA2 and LCAT, were replaced with alanine. Quadruple mutations at C65, C89, C330, and C371, double mutations at C65 and C89, and a single mutation at C65 or C89 resulted in the elimination of activity. Double mutations at C330 and C371 and a single mutation at C330 or C371 resulted in a partial reduction of activity. Thus, the presence of a disulfide bond between C330 and C371 is not required for LPLA2 activity. We propose that one disulfide bond between C65 and C89 and free cysteine residues at C330 and C371 and the triad, serine-198, aspartic acid-360, and histidine-392, are required for the full expression of mLPLA2 activity.     

Genetic diseases of the Kennedy pathways for membrane synthesis

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.     

The effect of orthovanadate on phosphoinositide metabolism in NIH 3T3 fibroblasts.

Orthovanadate is an agent known to stimulate cell growth and mimic insulin action. The effects of this compound on phosphoinositides in NIH 3T3 cells were examined. Both 100 and 1000 microM orthovanadate were found to increase the cellular content of inositol phosphate secondary to the activation of phosphatidylinositol-specific phospholipase C (PtdIns-PLC). The time course, dependence on orthovanadate concentration, and sensitivity to the isoflavone genistein were similar for orthovanadate-induced accumulation of inositol phosphate and protein tyrosine phosphate, indicating that there is a correlation between cellular protein tyrosine phosphate levels and PtdIns-PLC activity. Increased phosphatidylinositol phosphate (PtdInsP) content also occurred when cells were incubated with orthovanadate and appeared to result from the activation of PtdIns kinase. This effect was not correlated with cellular protein tyrosine phosphate content. Hence, orthovanadate is shown to affect phosphoinositide metabolism at a minimum of two sites by both tyrosine phosphate-dependent and -independent mechanisms.     

Cooperative effects of ethanol and protein kinase C activators on phospholipase-D-mediated hydrolysis of phosphatidylethanolamine in NIH 3T3 fibroblasts.

In a previous study, ethanol was shown to enhance the stimulatory effect of phorbol 12-myristate 13-acetate (PMA), a prominent activator of protein kinase C (PKC), on phospholipase-D (PLD)-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts (Kiss et al. (1991) Eur. J. Biochem. 197, 785-790). Here, the mechanism and possible significance of ethanol-stimulated PtdEtn hydrolysis was further studied. In [14C]ethanolamine-labeled NIH 3T3 fibroblasts, 10 mM ethanol enhanced PMA-induced hydrolysis of PtdEtn 1.5-2.0-fold during a 2.5-15-min incubation period. Other alcohols, including glycerol, methanol, and 1-propanol, also enhanced PMA-induced PtdEtn hydrolysis. Of the other PLD activators tested, ethanol potentiated the PKC-dependent stimulatory effect of bombesin but failed to alter the apparently PKC-independent stimulatory effect of serum. Pretreatment of [14C]ethanolamine-labeled fibroblasts with 200 mM ethanol for 20 min resulted in increased (approx. 2-fold) hydrolysis of [14C]PtdEtn in isolated membranes. In membranes from ethanol-treated, but not from untreated, cells, PMA further enhanced (approx. 1.5-fold) the production of [14C]ethanolamine. Ethanol exerted none of the above stimulatory effects on phosphatidylcholine hydrolysis. These results suggest that the specific stimulatory action of ethanol on PLD-mediated PtdEtn hydrolysis can occur in vivo and may involve increased binding of a regulatory PKC-isoform to membranes.     

The effect of indoleacetic acid on phospholipid metabolism in pea stems

Indole-3-acetic acid (IAA) was found to stimulate stem elongation but inhibit the incorporation of [¹⁴C]choline into phosphatidylcholine within 1 h     

Synthesis of molecular species of phosphatidylcholine and phosphatidylethanolamine by germinating soya bean

The composition and metabolism of phosphatidylcholines and phosphatidylethanomines of germinating soya bean Glycine max have been examined. Both phospholipids have a very similar fatty acid composition and distribution, with saturated acids located at the 1- position. The fatty acid composition and relative amounts of individual molecular species of the two phospholipids were also very similar. The relative amounts of the species were in the order tetraenoic pentaenoic trienoic = dienoic = monoenoic. In contrast, the labelling of the molecular species from choline Me[¹⁴C] or ethanolamine [2-¹⁴C] showed considerable differences. Phosphatidylethanolamine-[¹⁴C] showed 58% label in trienoic, 17% in tetraenoic, 18% in pentaneoic and 5% in dienoic species 48 hr after germination. The equivalent figures for phosphatidylcholine-[¹⁴C] were 37, 34, 13 and 15% respectively. An increase in labelling of the more unsaturated species was seen with time.     

Deuterated phospholipids as raman spectroscopic probes of membrane structure: Phase diagrams for the dipalmitoyl phosphatidylcholine(and its d62 derivative)-dipalmitoyl phosphatidylethanolamine system

Raman spectroscopic techniques have been used to construct phase diagrams for the binary phospholipid systems, DPPC-d₆₂/DPPE and DPPC/DPPE (DPPC, dipalmitoyl phosphatidylcholine; DPPE, dipalmitoyl phosphatidylethanolamine). For the former, the half-width of the C-²H stretching modes of the deuterated component near 2100 cm^?1 serves as an indicator of phospholipid fluidity. The phase behavior is described semi-quantitatively using regular solution theory with the following non-ideality parameters:

$\text{ρ}{}_0{}^{\mathrm{(1)}}\text{=0.75}\text{kcal/mol and}\text{ρ}{}_0{}^{\mathrm{(s)}}\text{=1.05}\text{kcal/mol}$

The use of deuterated phospholipids as one component of a binary mixture permits direct evaluation of the conformation of a particular component in the mixture throughout the phase separation region. The approach is demonstrated with the help of a simple model correlating the half-width of the symmetric C-²H stretching mode with the fraction of DPPC-d₆₂ hydrocarbon chains in the liquid crystalline state.The effect of chain perdeuteration on the phase behavior of DPPC with DPPE is evaluated by comparison of the phase diagram of the DPPC-d₆₂/DPPE system with that of DPPC-DPPE. The latter has been constructed previously from both probe and calorimetric techniques, and is created from the Raman spectroscopic data using the $\text{I(}\text{1130}\text{1100}\text{)}$ ratio to characterize the transgauche population ratio in non-deuterated hydrocarbon chains. A reasonable fit to the phase behavior is obtained using:

$\text{ρ}{}_0{}^{\mathrm{(1)}}\text{=0.85}\text{kcal/mol and}\text{ρ}{}_0{}^{\mathrm{(s)}}\text{=0.90}\text{kcal/mol}$

The similarities of the non-ideality parameters in the two phase diagrams indicate that the effect of perdeuteration on the phase behavior of DPPC is not extensive. The use of deuterated phospholipids as essentially unperturbed components of a model membrane system is justified.     

Effects of acclimation temperature on membrane phospholipids in the flesh fly Sarcophaga similis

Cold acclimation is a well‐known strategy for enhancing cold tolerance in ectotherms including insects. Nevertheless, information on the physiological mechanisms underpinning this phenomenon is still limited. Biological membrane integrity is critical for insects to perform at low temperatures, and an advantage is conferred on those insects that can adjust the composition of their membrane phospholipids. Such changes contribute to homeoviscous adaptation, a process that allows membranes to maintain a liquid–crystalline (fluid) state at low temperatures. Here we investigated phospholipids in the flesh fly Sarcophaga similis acclimated to various temperatures. Significant differences were observed in the composition of their fatty acyl chains: flies acclimated to low temperatures showed a higher proportion of palmitic and oleic acids at the expense of palmitoleic acid. Other fatty acids (stearic, linoleic, linolenic, arachidonic, eicosapentaenoic acids) were not significantly changed. The degree of unsaturation decreased in cold‐acclimated flies, but the difference was quite small. The weighted average chain length and number of double bonds were unchanged among flies acclimated to different temperatures. As temperatures decreased, the percentage of phosphatidylethanolamine increased to twice that of phosphatidylcholine. We discuss the role of these phospholipid changes in cold acclimation.     

Nuclear and cytosolic calcium levels in NIH 3T3 fibroblasts

The spatial distribution of intracellular calcium in resting NIH 3T3 fibroblasts loaded with Fura-2 has been studied by digital image analysis. Calibration parameters were determined separately for the nucleus and the cytosol to take into account possible differences in the physico-chemical properties of the two compartments and were found not to differ significantly. The apparent resting calcium concentration in these cells was found to be significantly lower in the nucleus than in the cytoplasm; however, this difference appears to be an artefact arising from the presence in the cytoplasm of regions with higher calcium levels. Application of thapsigargin, to block active uptake of calcium into these compartments, substantially eliminated the differences between nuclear and cytosolic calcium concentrations. These observations indicate that nuclear and cytosolic calcium are in equilibrium in the resting fibroblasts and argue against the existence of diffusional barriers between these two compartments.     

Ca2+-induced phase separation in phosphatidylserine,phosphatidylethanolamine and phosphatidylcholine mixed membranes

Ca²⁺-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidyl-ethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94–100% at 23°C and 84–88% at 40°C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74–85% at 23°C and 61–79% at 40°C. Ca²⁺ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was $\text{1.4 · 10}{}^{\mathrm{?7}}$ M for phosphatidylserine-phosphatidylethanolamine and $\text{1.2 · 10}{}^{\mathrm{?6}}$ M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca²⁺ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17°C) and phosphatidylcholine (?15°C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca²⁺-chelated solid-phase phosphatidylserine domain.