Phosphatidyl choline fatty acid remodeling in the hepatic cell nuclei

This study was performed to determine whether fatty acids incorporated into liver cell nuclei phosphatidylcholine (PtdCho) could be remodeled in the isolated nuclear. For this reason, rat liver cell nuclei were incubated in vitro with [1-14C]20:4n-6-CoA. PtdCho molecular species with the highest specific activity had an unsaturated fatty acid at sn-1 and sn-2 positions (20:4-20:4>18:2-20:4>18:1-20:4). 16:0-20:4 and 18:0-20:4 PtdChos showed a minor specific activity. When labeled nuclei were reincubated in the absence of labeled substrate with the addition of cytosol, ATP and CoA, the specific activity of 20:4-20:4, 18:2-20:4 and 18:1-20:4 species decreased, while that of 16:0-20:4 and 18:0-20:4 increased. In conclusion, the asymmetric fatty acid distribution of saturated fatty acids at sn-1 position, and unsaturated fatty acids at sn-2 position of nuclear PtdCho molecular species was re-established by an acyl-CoA-dependent remodeling process.     

LYCAT, a homologue of C. elegans acl-8, acl-9, and acl-10, determines the fatty acid composition of phosphatidylinositol in mice

Mammalian phosphatidylinositol (PI) has a unique fatty acid composition in that 1-stearoyl-2-arachidonoyl species is predominant. This fatty acid composition is formed through fatty acid remodeling by sequential deacylation and reacylation. We recently identified three Caenorhabditis elegans acyltransferases (ACL-8, ACL-9, and ACL-10) that incorporate stearic acid into the sn-1 position of PI. Mammalian LYCAT, which is the closest homolog of ACL-8, ACL-9, and ACL-10, was originally identified as a lysocardiolipin acyltransferase by an in vitro assay and was subsequently reported to possess acyltransferase activity toward various anionic lysophospholipids. However, the in vivo role of mammalian LYCAT in phospholipid fatty acid metabolism has not been well elucidated. In this study, we generated LYCAT-deficient mice and demonstrated that LYCAT determined the fatty acid composition of PI in vivo. LYCAT-deficient mice were outwardly healthy and fertile. In the mice, stearoyl-CoA acyltransferase activity toward the sn-1 position of PI was reduced, and the fatty acid composition of PI, but not those of other major phospholipids, was altered. Furthermore, expression of mouse LYCAT rescued the phenotype of C. elegans acl-8 acl-9 acl-10 triple mutants. Our data indicate that LYCAT is a determinant of PI molecular species and its function is conserved in C. elegans and mammals.     

Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata.

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid ('capric acid') in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.     

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.     

The effect of sn-2 fatty acid substitution on phospholipase C enzyme activities.

The human monocyte cell line U937 expresses phospholipase A2 and phospholipase C activities and produces eicosanoids. The phospholipase C (PLC) activity exhibits substrate preference for phosphatidyl-choline (PC), rather than phosphatidylinositol or phosphatidylethanolamine. In order to characterize the PLC activity found in these cells, the effects of substitution of the sn-2 fatty acid on this activity were examined. PC substrates with palmitic acid (PC-2P), oleic acid (PC-2O), arachidonic acid (PC-2A) and linoleic acid (PC-2L) at the sn-2 position were used. The sn-1 fatty acid was palmitic acid. PC-2L and PC-2A with the longer-chain less-saturated fatty acids linoleic acid and arachidonic acid esterified at sn-2 were found to be better substrates for PLC activity than PC-2P or PC-2O in these cells. This preference was maintained even when substrate phospholipid was solubilized in non-ionic, anionic, cationic and zwitterionic amphiphiles. Furthermore, when a 500-fold excess of 1,2-diolein or 1,2-dipalmitin was added to the reaction, the specificity of the PLC activity for PC-2A and PC-2L remained unchanged. When similar experiments were performed with phosphatidylinositol as a substrate, we did not observe any effect when the sn-2 position was altered. These data show that the fatty acid constituent at the sn-2 position affects the observed PLC activity when phosphatidylcholine, but not phosphatidylinositol, is used as a substrate by these cells.     

Caenorhabditis elegans mboa-7, a member of the MBOAT family, is required for selective incorporation of polyunsaturated fatty acids into phosphatidylinositol

Phosphatidylinositol (PI) is a component of membrane phospholipids, and it functions both as a signaling molecule and as a compartment-specific localization signal in the form of polyphosphoinositides. Arachidonic acid (AA) is the predominant fatty acid in the sn-2 position of PI in mammals. LysoPI acyltransferase (LPIAT) is thought to catalyze formation of AA-containing PI; however, the gene that encodes this enzyme has not yet been identified. In this study, we established a screening system to identify genes required for use of exogenous polyunsaturated fatty acids (PUFAs) in Caenorhabditis elegans. In C. elegans, eicosapentaenoic acid (EPA) instead of AA is the predominant fatty acid in PI. We showed that an uncharacterized gene, which we named mboa-7, is required for incorporation of PUFAs into PI. Incorporation of exogenous PUFA into PI of the living worms and LPIAT activity in the microsomes were greatly reduced in mboa-7 mutants. Furthermore, the membrane fractions of transgenic worms expressing recombinant MBOA-7 and its human homologue exhibited remarkably increased LPIAT activity. mboa-7 encodes a member of the membrane-bound O-acyltransferase family, suggesting that mboa-7 is LPIAT. Finally, mboa-7 mutants had significantly lower EPA levels in PI, and they exhibited larval arrest and egg-laying defects.     

A novel Delta9 acyl-lipid desaturase, DesC2, from cyanobacteria acts on fatty acids esterified to the sn-2 position of glycerolipids

Acyl-lipid desaturases are enzymes that convert a C-C single bond into a C=C double bond in fatty acids that are esterified to membrane-bound glycerolipids. Four types of acyl-lipid desaturase, namely DesA, DesB, DesC, and DesD, acting at the Delta12, Delta15, Delta9, and Delta6 positions of fatty acids respectively, have been characterized in cyanobacteria. These enzymes are specific for fatty acids bound to the sn-1 position of glycerolipids. In the present study, we have cloned two putative genes for a Delta9 desaturase, designated desC1 and desC2, from Nostoc species. The desC1 gene is highly similar to the desC gene that encodes a Delta9 desaturase that acts on C18 fatty acids at the sn-1 position. Homologues of desC2 are found in genomes of cyanobacterial species in which Delta9-desaturated fatty acids are esterified to the sn-2 position. Heterologous expression of the desC2 gene in Synechocystis sp. PCC 6803, in which a saturated fatty acid is found at the sn-2 position, revealed that DesC2 could desaturate this fatty acid at the sn-2 position. These results suggest that the desC2 gene is a novel gene for a Delta9 acyl-lipid desaturase that acts on fatty acids esterified to the sn-2 position of glycerolipids.     

Effect of a fish oil diet on the composition of rat neutrophil lipids and the molecular species of choline and ethanolamine glycerophospholipids

When rats were fed a corn oil versus a corn oil-fish oil diet the overall phospholipid content and composition as well as the subclass distribution of the choline- and ethanolamine-containing glycerophospholipids from neutrophils were not altered. The serine-containing glycerophospholipids were characterized by high levels of stearic and oleic acids. When fish oil was added to the diet it replaced some of the arachidonate in both the inositol- and the serine-containing glycerophospholipids. In the corn oil-fed animals, 25.2 and 33.6 mole %, respectively, of the molecular species of 1,2-diacyl- and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine contained arachidonate. The values for 1,2-diacyl and 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine were, respectively, 41 and 55.8 mole %. When half of the 5% corn oil in the diet was replaced by fish oil, there was a 53, 38, 27, and 25% reduction, respectively, in the level of arachidonate in these four lipid subclasses. The amount of 5,8,11,14,17-eicosapentaenoic acid incorporated into these four subclasses was always less than the decline in arachidonic acid. This was due, in part, to the acylation of small amounts of 22-carbon (n-3) acids into these lipids. Molecular species analysis demonstrated that 5,8,11,14,17-eicosapentaenoic acid paired with the same components at the sn-1 position, and in the same ratio, as did arachidonic acid. The amounts of 16- and 18-carbon saturated and unsaturated fatty acid at the sn-2 position were not altered by dietary change. Collectively, these findings suggest that 5,8,11,14,17-eicosapentaenoic and arachidonic acids are metabolized in a similar way by neutrophils. These studies also support the concept that neutrophils contain two metabolic pools of phospholipids. One pool is altered by dietary fat change while the pool containing 16- and 18-carbon acids is resistant to change when fish oil is included in the diet.     

Acyl-CoA dependent acylation of phospholipids in the chloroplast envelope

Acyl-CoAs are substrates for acyl lipid synthesis in the endoplasmic reticulum. In addition, they may also be substrates for lipid acylation in other membranes. In order to assess whether lipid acylation may have a role in plastid lipid metabolism, we have studied the incorporation of radiolabelled fatty acids from acyl-CoAs into lipids in isolated, intact pea chloroplasts. The labelled lipids were phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol and free fatty acids. With oleoyl-CoA, the fatty acid was incorporated preferably into the sn-2 position of PC and the acylation activity mainly occurred in fractions enriched in inner chloroplast envelope. Added lysoPC stimulated the activity. With palmitoyl-CoA, the fatty acid was incorporated primarily into the sn-1 position of PG and the reaction occurred at the surface of the chloroplasts. As chloroplast-synthesized PG generally contains 16C fatty acids in the sn-2 position, we propose that the acylation of PG studied represents activities present in a domain of the endoplasmic reticulum or an endoplasmic reticulum-derived fraction that is associated with chloroplasts and maintains this association during isolation. This domain or fraction contains a discreet population of lipid metabolizing activities, different from that of bulk endoplasmic reticulum, as shown by that with isolated endoplasmic reticulum, acyl-CoAs strongly labelled phosphatidic acid and phosphatidylethanolamine, lipids that were never labelled in the isolated chloroplasts.     

Effects of various numbers and positions of cis double bonds in the sn-2 acyl chain of phosphatidylethanolamine on the chain-melting temperature

In an attempt to investigate systematically the effects of various single and multiple cis carbon-carbon double bonds in the sn-2 acyl chains of natural phospholipids on membrane properties, we have de novo synthesized unsaturated C20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains were semi-synthesized by acylation of C20-lysophosphatidylcholine with unsaturated C20 fatty acids followed by phospholipase D-catalyzed base-exchange reaction in the presence of excess ethanolamine. The gel-to-liquid crystalline phase transitions of these 15 mixed-chain PE, in excess H2O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C20-saturated/sn-2 C20-unsaturated PE were simulated by molecular mechanics calculations. It is shown that the successive introduction of cis double bonds into the sn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature, Tm, of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position of cis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel Tm diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds on Tm can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristic Tm profiles exhibited by different series of mixed-chain PE with increasing degree of unsaturation can be interpreted in terms of structural changes associated with acyl chain unsaturation.     

Triglyceride metabolism in 3T3-L1 cells. An in vivo 13C NMR study.

13C nuclear magnetic resonance spectroscopy has been used to study triglyceride metabolism in 3T3-L1 cells incubated with [1-13/14C] acetate, myristate, palmitate, stearate, or oleate. Labeled cells embedded in agarose filaments were perfused in a specially fitted NMR tube within the spectrometer magnet. Incubation of 3T3-L1 cells with a specific fatty acid enriched the cellular triglycerides with that fatty acid; the NMR signal observed in the carbonyl region of the cell spectrum was due in large part to that fatty acid. NMR data demonstrated that cellular enzymes preferentially esterified saturated fatty acids at the glyceride sn-1,3 position and unsaturated fatty acids at the sn-2 position. cellular triglyceride hydrolysis by hormone-sensitive lipase was monitored by measuring the decrease in the integrated intensities of resonances arising from fatty acyl carbonyls esterified at glycerol carbons sn-1,3 and sn-2. Under basal conditions, the time courses were first-order, and the average rates were 0.14% of signal/min at both carbonyl positions. Under isoproterenol stimulated conditions, these rates were still first-order and increased 6.4-fold at the sn-1,3 position and 2.4-fold at the sn-2 position. The observation that the hydrolysis time courses were first-order suggested that only a small amount of cellular triglyceride was available to hormone-sensitive lipase, supporting the view that lipolytic enzymes operate at lipid surfaces where only small amounts of neutral lipid may be soluble. Attempts to correlate the measured rates with the rates of hydrolysis at the sn-1,3 and sn-2 positions were hindered by the fact that the chemical shifts of the carbonyl carbons of the diglyceride hydrolysis product did not overlie those of the triglyceride. Analysis of hydrolysis kinetics revealed that hormone-sensitive lipase exhibited little preference for a particular esterified fatty acid under basal conditions; however, under stimulated conditions, the enzyme exhibited a preference for certain triglyceride species.     

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.     

The occurrence of polyenoic very long chain fatty acids with greater than 32 carbon atoms in molecular species of phosphatidylcholine in normal and peroxisome-deficient (Zellweger''s syndrome) brain.

The n-6 tetra- and pentaenoic fatty acids with carbon chain lengths greater than 32 found in normal brain are located predominantly in a separable species of phosphatidylcholine. A similar phospholipid is found in increased amounts in the brain of peroxisome-deficient (Zellweger's syndrome) patients, but the fatty acid composition differs in that penta- and hexaenoic derivatives predominate. Our data strongly suggest that the polyenoic very long chain fatty acids are confined to the sn-1 position of the glycerol moiety, while the sn-2 position is enriched in saturated, monounsaturated and polyunsaturated fatty acids with less than 24 carbon atoms. It is postulated that these unusual molecular species of phosphatidylcholine may play some, as yet undefined, role in brain physiology.     

Properties of the binding sites for the sn-1 and sn-2 acyl chains on the phosphatidylinositol transfer protein from bovine brain

We have studied the properties of the fatty acyl binding sites of the phosphatidylinositol transfer protein (PI-TP) from bovine brain, by measuring the binding and transfer of pyrenylacyl-containing phosphatidylinositol (PyrPI) species and pyrenylacyl-containing phosphatidylcholine (PyrPC) species as a function of the acyl chain length. The PyrPI species carried a pyrene-labeled acyl chain of variable length in the sn-2 position and either palmitic acid [C(16)], palmitoleic acid [C(16:1)], or stearic acid [C(18:1)] in the sn-1 position. Binding and transfer of the PI species increased in the order C(18) less than C(16) less than C(16:1), with a distinct preference for those species that carry a pyrenyloctanoyl [Pyr(8)] or a pyrenyldecanoyl [Pyr(10)] chain. The PyrPC species studied consisted of two sets of positional isomers: one set contained a pyrenylacyl chain of variable length and a C(16) chain, and the other set contained an unlabeled chain of variable length and a Pyr(10) chain. The binding and transfer experiments showed that PI-TP discriminates between positional isomers with a preference for the species with a pyrenylacyl chain in the sn-1 position. This discrimination is interpreted to indicate that separate binding sites exist for the sn-1 and sn-2 acyl chains. From the binding and transfer profiles it is apparent that the binding sites differ in their preference for a particular acyl chain length. The binding and transfer vs chain length profiles were quite similar for C(16)Pyr(x)PC and C(16)Pyr(x)PI species, suggesting that the sn-2 acyl chains of PI and PC share a common binding site in PI-TP.     

Mitochondrial glycerol-3-phosphate acyltransferase-deficient mice have reduced weight and liver triacylglycerol content and altered glycerolipid fatty acid composition

Microsomal and mitochondrial isoforms of glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyze the committed step in glycerolipid synthesis. The mitochondrial isoform, mtGPAT, was believed to control the positioning of saturated fatty acids at the sn-1 position of phospholipids, and nutritional, hormonal, and overexpression studies suggested that mtGPAT activity is important for the synthesis of triacylglycerol. To determine whether these purported functions were true, we constructed mice deficient in mtGPAT. mtGPAT(-/-) mice weighed less than controls and had reduced gonadal fat pad weights and lower hepatic triacylglycerol content, plasma triacylglycerol, and very low density lipoprotein triacylglycerol secretion. As predicted, in mtGPAT(-/-) liver, the palmitate content was lower in triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. Positional analysis revealed that mtGPAT(-/-) liver phosphatidylethanolamine and phosphatidylcholine had about 21% less palmitate in the sn-1 position and 36 and 40%, respectively, more arachidonate in the sn-2 position. These data confirm the important role of mtGPAT in the synthesis of triacylglycerol, in the fatty acid content of triacylglycerol and cholesterol esters, and in the positioning of specific fatty acids, particularly palmitate and arachidonate, in phospholipids. The increase in arachidonate may be functionally significant in terms of eicosanoid production.     

The reactivity of plasma phospholipids with lecithin:cholesterol acyltransferase is decreased in fish oil-fed monkeys

The size of low density lipoproteins (LDL) is strongly correlated with LDL cholesteryl ester (CE) content and coronary artery atherosclerosis in monkeys fed cholesterol and saturated fat. African green monkeys fed 11% (weight) fish oil diets have smaller LDL and less CE per LDL particle than lard-fed animals. We hypothesized that this might be due to a lower plasma lecithin:cholesterol acyltransferase (LCAT) activity in fish oil-fed animals. Using recombinant particles made of egg yolk lecithin-[14C]cholesterol-apoA-I as exogenous substrate, we found no difference in plasma LCAT activity (27 versus 28 nmol CE formed per h/ml) of fish oil- versus lard-fed animals, respectively; furthermore, no diet-induced difference in immunodetectable LCAT was found. However, plasma phospholipids from fish oil-fed animals were over 4-fold enriched in n-3 fatty acids in the sn-2 position compared to those of lard-fed animals. Additionally, the proportion of n-3 fatty acid-containing CE products formed by LCAT, relative to the available n-3 fatty acid in the sn-2 position of phospholipids, was less than one-tenth of that for linoleic acid. The overall rate of LCAT-catalyzed CE formation with phospholipid substrates from fish oil-fed animals was lower (5-50%) than with phospholipid substrates from lard-fed animals. These data show that n-3 fatty acids in phospholipids are not readily utilized by LCAT for formation of CE; rather, LCAT preferentially utilizes linoleic acid for CE formation. The amount of linoleic acid in the sn-2 position of plasma phospholipids is reduced and replaced with n-3 fatty acids in fish oil-fed animals. As a result, LCAT-catalyzed plasma CE formation in vivo is likely reduced in fish oil-fed animals contributing to the decreased cholesteryl ester content and smaller size of LDL particles in the animals of this diet group.     

Human neutrophils incorporate arachidonic acid and saturated fatty acids into separate molecular species of phospholipids

The incorporation of radiolabeled arachidonic acid and saturated fatty acids into choline-linked phosphoglycerides (PC) of rabbit and human neutrophils was investigated by resolving the individual molecular species by reversed-phase high performance liquid chromatography. PC from neutrophils incubated with a mixture of [3H]arachidonic acid and [14C]stearic or [14C]palmitic acid contains both radiolabels; however, double labeling of individual molecular species is minimal. After labeling for 2 h, the [3H]arachidonate is distributed almost equally between diacyl and 1-O-alkyl-2-acyl species, but it is incorporated into diacyl species containing unlabeled stearate or palmitate at the sn-1 position. In contrast, labeled saturated fatty acids are incorporated only into diacyl species and contain predominantly oleate and linoleate at the sn-2 position. Labeled linoleate is not incorporated into ether-linked species, but is found in the same species as labeled stearate. The findings suggest that mechanisms exist in neutrophils for specific shunting of exogenous arachidonic acid into certain phospholipid molecular species and support the concept that the 1-O-alkyl-2-arachidonoyl species may be a functionally segregated pool of arachidonic acid within the PC of neutrophils.     

Platelet-derived growth factor stimulates synthesis of 1,2-diacylglycerol from monoacylglycerol in Balb/c 3T3 cells.

1,2-Diacylglycerol (1,2,-DAG) plays an important role in the protein kinase C-mediated signal-transduction system. Several reports have shown that 1,2-DAG is generated through various pathways other than classical phospholipid hydrolysis. We observed a rapid incorporation of [3H]myristate into 1,2-DAG in platelet-derived-growth-factor (PDGF)-treated Balb/c 3T3 cells. [14C]Palmitate was similarly incorporated into 1,2-DAG. The effect of PDGF, which was inhibited by cycloheximide, became maximal after 60 min treatment with PDGF, and disappeared 300 min after removal of PDGF. Treatment with triacylglycerol lipase revealed that labelled saturated fatty acid was incorporated into the sn-1 position. PDGF barely stimulated incorporation of [3H]glycerol or [14C]glucose into 1,2-DAG. Incorporation of [3H]myristate into 1,2-DAG preceded that into triacyglycerol and phospholipids. These results suggest that synthesis of 1,2-DAG from monoacylglycerol is enhanced in PDGF-treated cells. However, there is no significant difference in the activity of monoacylglycerol acyltransferase measured in vitro in quiescent and PDGF-treated cells. The reason for this discrepancy is discussed.     

Synthesis of saturated phosphatidylcholine and phosphatidylglycerol by freshly isolated rat alveolar type II cells

Saturated phosphatidylcholine and phosphatidylglycerol are important components of pulmonary surface active material, but the relative contributions of different pathways for the synthesis of these two classes of phospholipids by alveolar type II cells are not established. We purified freshly isolated rat type II cells by centrifugal elutriation and incubated them with [1-14C]palmitate as the sole exogenous fatty acid in one series of experiments or with [9,10-3H]palmitate, mixed fatty acids (16:0, 18:1 and 18:2), and [U-14C]glucose in another series of experiments. Type II cells readily incorporated [1-14C]palmitate into saturated phosphatidic acid (55-59% of total phosphatidic acid), saturated diacylglycerol (82-87% of total diacylglycerol), saturated phosphatidylcholine (69-76% of total phosphatidylcholine), and saturated phosphatidylglycerol (55-59% of total phosphatidylglycerol). Saturated phosphatidic acid, diacylglycerol and phosphatidylglycerol were nearly equally labeled in the sn-1 and sn-2 positions, whereas saturated phosphatidylcholine was preferentially labeled in the sn-2 position. With [9,10-3H]palmitate and [U-14C]glucose, the labeling patterns of phosphatidic acid, diacylglycerol and phosphatidylglycerol were similar to each other but different from that of phosphatidylcholine. The glucose label was found predominantly in the unsaturated phosphatidylcholines at early times (3-10 min) and in the saturated phosphatidylcholines at later times (30-90 min). Similarly, the 3H/14C ratio was very high in saturated phosphatidylcholine and always above that in saturated diacylglycerol. We conclude that freshly isolated type II cells synthesize saturated phosphatidic acid, diacylglycerol, phosphatidylcholine and phosphatidylglycerol and that under our in vitro conditions the deacylation-reacylation pathway is important for the synthesis of saturated phosphatidylcholine but is less important for the synthesis of saturated phosphatidylglycerol. By the assumptions stated in the text during the pulse chase experiment de novo synthesis of saturated phosphatidylcholine from saturated diacylglycerol accounted for 25% of the total synthesis of saturated phosphatidylcholine.     

Distribution of distinct arachidonoyl-specific and non-specific isoenzymes of diacylglycerol kinase in baboon (Papio cynocephalus) tissues.

We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.